DETAILED ACTION
Status of the Application
Claims 1-2, 5-6, 9, 11-14, 16, 19-21, 25-26, 28-29, 34-38 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment of claims 1-2, 5-6, 9, 13-14, 16, 19, 28, addition of claims 36-38, cancellation of claim 4, and amendments to the specification as submitted in a communication filed on 4/1/2026 is acknowledged.
Applicant elected with traverse Group 1, claims 2, 4, 6, 9, 11-14, 16, 28, 35, drawn in part to a recombinant microorganism that expresses a Fe-Fe dependent dehydrogenase from C. reinhardtii, wherein said microorganism has a genetic modification which promotes utilization of carbon via the pentose phosphate pathway by reducing or inhibiting the activity or levels of one or more endogenous proteins, and further elected reducing or inhibiting the activity or levels of endogenous phosphofructokinase and glycerate mutase, in a communication filed on 8/18/2025.
New claims 36-38 are directed to the elected invention. Claims 5, 19-21, 25-26, 29, 34 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 8/18/2025. Claim 1 (linking) and claims 2, 6, 9, 11-14, 16, 28, 35-38 are at issue and will be examined to the extent they encompass the elected invention.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Specification
The previous objection to the title of the invention as being non-descriptive is hereby withdrawn by virtue of Applicant’s amendment.
The previous specification is objected to due to the recitation of “pgmA” (page 29, third full paragraph) is hereby withdrawn by virtue of Applicant’s amendment.
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
Claims 1-2, 6, 9, 11-14, 16, 28, 35 remain rejected and new claims 36-38 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention. New grounds of rejection are necessitated by amendment.
Claim 1 (claims 2, 6, 9, 11-14, 16, 28, 35-38 dependent thereon) is indefinite in the recitation of “…microorganism comprises exogenous nucleic acids encoding proteins for enabling the microorganism to produce hydrogen, wherein the proteins for enabling the microorganism….comprise an Fe-Fe dependent hydrogenase that is a HydA dehydrogenase…or a functionally equivalent homolog thereof having at least 80% identity to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 6, wherein the proteins for enabling the microorganism to produce hydrogen comprise the assembly proteins HydEF and HydG or functionally equivalent homologs thereof, wherein the nucleic acids are operably linked…for enabling expression of the nucleic acid sequences in the microorganism…” for the following reasons.
It is unclear as to how a Fe-Fe dependent hydrogenase can be a dehydrogenase. Moreover, the claim recites “wherein the proteins for enabling the microorganism to produce hydrogen comprise…HydA dehydrogenase…wherein the proteins for enabling the microorganism to produce hydrogen comprise the assembly proteins HydEF and HydG..”, thus defining the proteins for enabling the microorganism to produce hydrogen twice using different proteins. Therefore, it is unclear if the intended limitation is “wherein the proteins for enabling the microorganism to produce hydrogen comprise HydA dehydrogenase, HydEF and HydG” or if the intended limitation is “wherein the proteins for enabling the microorganism to produce hydrogen comprise (i) HydA dehydrogenase, or (ii) HydEF and HydG”.
In addition, the terms “HydA”, “HydEF” and “HydG” are unclear for the following reasons. These terms appear to be generic and not limited to a specific organism. While the gene/protein nomenclature used may be appropriate for a protein encoded by the C. reinhardtii hydA, hdyE, hydF, or hydG gene, the use of this nomenclature for a protein of identical function from another organism may not be accurate. As known in the art, genes encoding proteins of identical function in two different organisms may use different designations. See the abstract of Sousa et al. previously provided. As such, the use of gene/protein terminology which is applicable to some organisms and not to others as used herein is confusing since one cannot determine if by using this nomenclature, the claim is limiting the organism from which this protein derives to those that use the same nomenclature. If Applicant wishes to use the recited terminology in the claims, it is suggested that the claim be amended to clearly indicate the organism associated with the specific gene/protein designation.
The term “functionally equivalent homologs” is unclear without indicating the specific function that should be equivalent. A protein can have more than one function. For example, an enzyme can have binding activity, antibody eliciting activity, enzymatic activity, etc. Therefore, in the absence of the specific function, the term is meaningless. Furthermore, an amino acid sequence is the graphical representation of the order in which amino acids are arranged in a polypeptide. A polypeptide is a molecule. Therefore, while a protein can have % sequence identity to another protein, a protein cannot have identity to an amino acid sequence. Also, the term “for enabling expression of the nucleic acid sequences in the microorganism” is unclear because there is no antecedent basis for the nucleic acid sequences in the microorganism. It is noted that nucleic acid sequences are graphical representations of the order in which nucleotides are arranged in a nucleic acid molecule. Therefore, while a microorganism can comprise nucleic acids and express nucleic acids, it is unclear as to how a microorganism can express nucleic acid sequences.
For examination purposes, it will be assumed that the claim requires the recombinant microorganism to comprise nucleic acids encoding one or more proteins that enable a microorganism to produce hydrogen, wherein one of said proteins is a Fe-Fe hydrogenase, or an assembly protein for enabling maturation and activation of a hydrogenase, wherein said nucleic acids are operably linked to a promoter. Correction is required.
Claim 2 is indefinite in the recitation of “…wherein the exogenous nucleic acid sequences encode a ferredoxin NADP reductase (FNR) and a ferredoxin protein, or functionally equivalent homologs having at least 80% identity to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 8 and 9, and/or wherein the nucleic acid sequences encode at least….” for the following reasons. There is no antecedent basis for “the exogenous nucleic acid sequences” or “the nucleic acid sequences”. The term “functionally equivalent homologs” is unclear without indicating the specific function that should be equivalent. A protein can have more than one function. For example, an enzyme can have binding activity, antibody eliciting activity, enzymatic activity, etc. Therefore, in the absence of the specific function, the term is meaningless. In addition, an amino acid sequence is the graphical representation of the order in which amino acids are arranged in a polypeptide. A polypeptide is a molecule. Therefore, while a protein can have % sequence identity to another protein, a protein cannot have identity to a sequence. For examination purposes, it will be assumed that the recombinant microorganism requires in part a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 8, or a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 9. Correction is required.
Claim 9 is indefinite in the recitation of “wherein the HydA protein or a functionally equivalent homolog has at least 80% identity to an HydA from a microorganism selected from the group consisting of Chlamydomonas reinhardtii…and Peptoclostridum bifermentas” for the following reasons. The term “HydA” appears to be generic and not limited to a specific organism. While the gene/protein nomenclature used may be appropriate for a protein encoded by the C. reinhardtii hydA gene, the use of this nomenclature for a protein of identical function from another organism may not be accurate. See discussion of the teachings of Sousa et al. in the prior Office action. As such, the use of gene/protein terminology which is applicable to some organisms and not to others as used herein is confusing since one cannot determine if by using this nomenclature, the claim is limiting the organism from which this protein derives to those that use the same nomenclature. The term “or a functionally equivalent homolog” is unclear without indicating the specific function that should be equivalent. A protein can have more than one function. For example, an enzyme can have binding activity, antibody eliciting activity, enzymatic activity, etc. Therefore, in the absence of the specific function, the term is meaningless. Furthermore, the term “has 80% identity to an HydA from a microorganism selected from the group consisting of….” is unclear because one cannot determine the property/characteristic that has the recited % identity. Even if one assumes that the term refers to “sequence identity”, it is noted that such numerical limitation is meaningless in the absence of the specific sequence identifier that should be used to determine % sequence identity. An organism can have more than one protein having the same enzymatic activity, wherein each of these proteins has a different amino acid sequence. Therefore, while a protein can have 80% sequence identity to protein X of organism A, the same protein may have 70% sequence identity to protein Y of organism A, wherein protein X and protein Y are from the same organism and have the same enzymatic activity. The same protein can be included or excluded from the scope of the claim depending on the reference sequence used to determine % sequence identity. For examination purposes, claim 9 will be interpreted as a duplicate of claim 1. Correction is required.
Claim 12 is indefinite in the recitation of “wherein the exogenous nucleic acid sequences are…” for the following reasons. There is no antecedent basis for the exogenous nucleic acid sequences. In addition, a nucleotide sequence is a graphical representation of the order in which nucleotides are arranged in a nucleic acid molecule. A polynucleotide construct is a nucleic acid molecule. Therefore, it is unclear as to how sequences are provided by a polynucleotide construct. For examination purposes, it will be assumed that the claim requires exogenous nucleic acids in a single polynucleotide construct. Correction is required.
Claim 13 is indefinite in the recitation of “wherein the exogenous nucleic acid sequences…” because there is no antecedent basis for the exogenous nucleic acid sequences. Claim 13 will be interpreted as a duplicate of claim 1. Correction is required.
Claim 14 is indefinite in the recitation of “…or are functionally equivalent homologs thereof having at least 80% identity to the amino acid sequence encoded by the nucleic acid sequences of SEQ ID NO: 2, 4, 8 and 9, respectively…” for the following reasons. The term “functionally equivalent homologs” is unclear without indicating the specific function that should be equivalent. A protein can have more than one function. For example, an enzyme can have binding activity, antibody eliciting activity, enzymatic activity, etc. Therefore, in the absence of the specific function, the term is meaningless. In addition, an amino acid sequence is the graphical representation of the order in which amino acids are arranged in a polypeptide. A polypeptide is a molecule. Therefore, while a protein can have % sequence identity to another protein, a protein cannot have identity to a sequence. For examination purposes, it will be assumed that the recombinant microorganism requires in part a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 2, a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 4, a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 8, and a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 9. Correction is required.
Claim 16 is indefinite in the recitation of “….genetic modification that partially or completely excises the nucleic acid encoding one or mor of tis genes pfkA, pps, gpmA/gpmM, edd and eda…” for the following reasons. As known in the art, a gene is a nucleic acid. While nucleic acids encode proteins, nucleic acids do not encode genes. In addition, the terms “pfkA”, “pps”, “gpmA”, “gpmM”, “edd” and “eda” are unclear for the following reasons. These terms appears to be generic and not limited to a specific organism. While the gene nomenclature used may be appropriate for E. coli genes, the use of this nomenclature for genes encoding proteins of identical function in other organisms may not be accurate. See the teachings of Sousa et al. previously discussed. As such, the use of gene terminology which is applicable to some organisms and not to others as used herein is confusing since one cannot determine if by using this nomenclature, the claims are limiting the organism from which these genes derive to those that use the same nomenclature. If Applicant wishes to use the recited terminology in the claims, it is suggested that the claims be amended to clearly indicate the organism associated with the specific gene designation. If the intended limitation is a genetic modification that partially or completely delete genes encoding the recited enzymes, the claim should be amended accordingly. Correction is required.
Claim 36 is indefinite in the recitation of “HydA protein is….or has at least 80% sequence identity to the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 6” for the following reasons. As explained above, an amino acid sequence is the graphical representation of the order in which amino acids are arranged in a polypeptide. A polypeptide is a molecule. Therefore, while a protein can have % sequence identity to another protein, a protein cannot have identity to a sequence. In addition, it is noted that claim 1 already requires the limitation has at least 80% sequence identity to the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 6”. Therefore, it is unclear as to how this limitation further limits claim 36, which depends from claim 1. For examination purposes, no patentable weight will be given to the term “has at least 80% sequence identity to the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 6”. Correction is required.
Claim 37 is indefinite in the recitation of “HydA protein is….or has at least 80% sequence identity to the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 6 and wherein the HydEF and HydG are from …or are functionally equivalent homologs of the HydEF…having at least 80% identity the amino acid sequence encoded by the nucleic acid sequences of SEQ ID NO: 2 and 4, respectively; and wherein the microorganism further comprises nucleic acid sequences encoding a ferredoxin…reductase…or functionally equivalent homologs thereof having at least 80% identity to the amino acid sequence encoded by the nucleic acid sequences of SEQ ID NO: 8 and 9” for the following reasons.
As explained above, an amino acid sequence is the graphical representation of the order in which amino acids are arranged in a polypeptide. A polypeptide is a molecule. Therefore, while a protein can have % sequence identity to another protein, a protein cannot have identity to a sequence. In addition, it is noted that claim 1 already requires the limitation has at least 80% sequence identity to the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 6”. Therefore, it is unclear as to how this limitation further limits claim 36, which depends from claim 1. The term “functionally equivalent homologs” is unclear without indicating the specific function that should be equivalent. A protein can have more than one function. For example, an enzyme can have binding activity, antibody eliciting activity, enzymatic activity, etc. Therefore, in the absence of the specific function, the term is meaningless. For examination purposes, no patentable weight will be given to the term “or has at least 80% sequence identity to the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO: 6”. It will be assumed that the claim requires in part the recombinant microorganism to comprise a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 2, a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 4, a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 8, and a nucleic acid encoding a variant of the polypeptide encoded by the polynucleotide of SEQ ID NO: 9. Correction is required.
Claim 38 is indefinite in the recitation of “wherein the nucleic acid construct comprises the genes cssA, cscB and sp genes…respectively” for the following reasons. The terms “cssA”, “cscB” and “sp” are unclear for the following reasons. These terms appears to be generic and not limited to a specific organism. While the gene nomenclature used may be appropriate for E. coli genes, the use of this nomenclature for genes encoding proteins of identical function in other organisms may not be accurate. As known in the art, genes encoding proteins of identical function in two different organisms may use different designations. See teachings of Sousa et al. previously discussed. As such, the use of gene/protein terminology which is applicable to some organisms and not to others as used herein is confusing since one cannot determine if by using this nomenclature, the claims are limiting the organism from which these genes derive to those that use the same nomenclature. If Applicant wishes to use the recited terminology in the claims, it is suggested that the claims be amended to clearly indicate the organism associated with the specific gene designation. For examination purposes, it will be assumed that claim 38 is a duplicate of claim 28. Correction is required.
When amending the claims, applicant is advised to carefully review all examined claims and make the necessary changes to ensure proper antecedent basis and dependency.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
Claims 1-2, 6, 9, 11-14, 16, 28, 35 remain rejected and new claims 36-38 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
This rejection has been discussed at length in the prior Office action. It is maintained and further applied to new claims 36-38 for the reasons of record and those set forth below.
Applicant argues that the claims have been amended to require an Fe-Fe dependent hydrogenase or a functionally equivalent homolog thereof having at least 80% identity to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 6, and genetic modifications that reduce or inhibit the activity or levels of one or more endogenous proteins having specific enzymatic activity. Applicant further argues that claim 2 has been amended to define that the ferredoxin NADP-reductase and ferredoxin protein or functionally equivalent homologs thereof as having at least 80% identity to the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 8 and 9. Applicant also states that claim 6 has been amended to require the HydEF and HydG proteins to have at least 80% identity to the amino acid sequences encoded by the polynucleotides of SEQ ID NO: 2 and 4 and claim 9 has been amended to further limit the HydA protein.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of new claims 36-38. The Examiner acknowledges the amendments made to the claims. However, the Examiner disagrees with Applicant’s contention that the genus of enzymes and modifications recited in the claims is adequately described by the teachings of the specification and/or the prior art.
The claims as interpreted and amended require a genus of proteins having Fe-Fe hydrogenase activity and at least 80% sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NO: 6 as well as (i) proteins which are assembly proteins of an Fe-Fe hydrogenase having any structure or at least 80% sequence identity to polypeptides encoded by the nucleic acids of SEQ ID NO: 2 and 4, and (ii) a genus of ferredoxin NADP reductases and ferredoxin proteins having any structure or at least 80% sequence identity to polypeptides encoded by the nucleic acids of SEQ ID NO: 8 and 9 . In addition, the claims require a genus of unknown modifications that reduce or inhibit the activity or levels of endogenous proteins such as a phosphofructokinase or a glycerate mutase. The claims also require nucleic acids encoding proteins having any structure and function that would allow any microorganism to metabolize sucrose. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
The claims encompass a large genus of nucleic acids encoding proteins which are structurally unrelated or substantially unrelated. The protein encoded by the nucleic acid of SEQ ID NO: 2 has 1151 amino acids, the protein encoded by the nucleic acid of SEQ ID NO: 4 has 567 amino acids, the protein encoded by the nucleic acid of SEQ ID NO: 6 has 497 amino acids, the protein encoded by the nucleic acid of SEQ ID NO: 8 has 126 amino acids and the protein encoded by the nucleic acid of SEQ ID NO: 9 has 346 amino acids See alignments provided in Claim Rejections - 35 USC § 102 (AIA ) and Claim Rejections - 35 USC § 103 (AIA ).
A polypeptide having 80% sequence identity with the protein encoded by the polynucleotide of SEQ ID NO: 6 allows for any combination of 100 amino acid modifications within said protein in view of the fact that the protein encoded by the polynucleotide of SEQ ID NO: 6 has 497 amino acids (100 = 0.2x497). The total number of variants of a polypeptide having a specific number of amino acid substitutions can be calculated from the formula N!x19A/(N-A)!/A!, where N is the length in amino acids of the reference polypeptide and A is the number of allowed substitutions. Thus, the total number of variants of the protein encoded by the polynucleotide of SEQ ID NO: 6 having at least 80% sequence identity to said protein that result from amino acid substitutions is 497!x19100/(497-100)!/100! or 7.83x10234 variants. Using the same formula, the total number of variants of the protein encoded by the polynucleotide of SEQ ID NO: 2 having at least 80% sequence identity to said protein that result from amino acid substitutions is 1151!x19231/(1151-231)!/231! or 3.0x10544 variants (231 = 0.2x1151), the total number of variants of the protein encoded by the polynucleotide of SEQ ID NO: 8 having at least 80% sequence identity to said protein that result from amino acid substitutions is 126!x1926/(126-26)!/26! or 1.11x1060 variants (26=0.2x126), the total number of variants of the protein encoded by the polynucleotide of SEQ ID NO: 4 having at least 80% sequence identity to said protein that result from amino acid substitutions is 567!x19114/(567-114)!/114! or 9.56x10267 variants (114=0.2x567), and the total number of variants of the protein encoded by the polynucleotide of SEQ ID NO: 9 having at least 80% sequence identity to said protein that result from amino acid substitutions is 346!x1970/(346-70)!/70! or 8.16x10163 variants (70=0.2x346). The specification and the prior art are silent with regard to the structural features required in proteins having Fe-Fe hydrogenase activity, ferredoxins, ferredoxin NADP reductases, or Fe-Fe hydrogenase assembly proteins. Also, the specification and the prior art are silent with regard to the structural features required in any protein that enables any microorganism to metabolize sucrose. There is no structure/function correlation provided that would allow one of skill in the art to determine from an essentially infinite number of variants, those more likely to have the recited activity. Furthermore, while one could argue that the few species disclosed are representative of the structure of all the members of the genus, it is noted that the art teaches several examples of how even highly structurally homologous polypeptides can have different enzymatic activities. See the teachings of Witkowski et al., Tang et al. and Seffernick et al. previously discussed. Therefore, since minor structural differences may result in changes affecting function, and no additional information correlating structure with the desired functional characteristics has been provided, one cannot reasonably conclude that the few species disclosed are representative of the structure of all the proteins encoded by the nucleic acids recited in the claims.
With regard to the genus of genetic modifications that reduce or inhibit the activity or levels of one or more of the endogenous proteins recited in claim 1, it is noted that the genus of modifications required encompass not only those disclosed in the specification but also unknown modifications to achieve the recited reduction or inhibition. This encompasses, for example, genetic modifications to genes encoding proteins that control the expression of the recited endogenous proteins, structural modifications to promoters, the expression of proteins that could act as inhibitors of transcription, antisense molecules to block expression of the desired gene, or structural modifications to the coding region of a gene to alter the enzymatic activity of an enzyme. While the specification discloses disruption of the endogenous pfkA, gpmA and/or gpmM genes to increase carbon utilization via the pentose phosphate pathway, and to decrease the activity or level of an endogenous enzyme such as phosphofructokinase, or glycerate mutase, the specification is silent with regard to other genetic modifications that could be made to reduce the activity/level of the recited enzymes.
In view of the fact that the specification only discloses a very limited number of enzymes and proteins that can be used to produce hydrogen and metabolize sucrose, and a very limited number of genetic modifications to reduce or inhibit the activity or levels of an endogenous protein, one of skill in the art would not recognize from the disclosure that Applicant was in possession of the claimed invention.
Claims 1-2, 6, 9, 11-14, 16, 28, 35 remain rejected and new claims 36-38 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for an E. coli cell modified to disrupt the endogenous pfkA, gpmA and/or gpmB genes, wherein said E. coli cell has been transformed with a nucleic acid construct that comprises SEQ ID NO: 10, and wherein said cell has been transformed with the E. coli cscA gene, the E. coli cscB gene, and the L. mesenteroides gtfA gene, does not reasonably provide enablement for a microorganism that has been modified with any genetic modification to increase carbon utilization via the pentose phosphate pathway, including any genetic modification to reduce or inhibit the activity or levels of an endogenous phosphofructokinase and/or glycerate mutase, wherein said microorganism comprises nucleic acids encoding variants of the proteins encoded by the polynucleotides of SEQ ID NO: 2, 4, 6, 8 or 9, wherein said varaints can have any function, and wherein said microorganism can comprise nucleic acids encoding proteins having any structure and function that would allow the microorganism to metabolize sucrose. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
This rejection has been discussed at length in the prior Office action. It is maintained and further applied to new claims 36-38 for the reasons of record and those set forth below.
Applicant argues that the claims have been amended to require nucleic acids encoding specific proteins and a genetic modification that promotes utilization of carbon via the pentose phosphate pathway wherein said genetic modification reduces or inhibits the activity or levels of one or more endogenous proteins having specific enzymatic activity. Applicant further argues that one of skill in the art could easily identify HydA and HydEFG nucleic acids from the prior art for purposes of making a microorganism as recited. Applicant states that the specification provides guidance on the relevant sequences of these proteins. Applicant submits that the skilled person would readily be able to determine how to introduce a genetic modification to reduce or inhibit one or more of the enzymes recited in claim 1 using standard techniques in the art. Applicant states that the specification provides guidance on the relevant sequences and methods for the expression or activity of those proteins recited.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of new claims 36-38. The Examiner acknowledges the amendments made to the claims and the teachings of the prior art and the specification. However, the Examiner disagrees with Applicant’s contention that the entire scope of the enzymes and modifications recited in the claims is fully enabled by the teachings of the specification and/or the prior art.
It is reiterated herein that the claims as interpreted and amended require a genus of proteins having Fe-Fe hydrogenase activity and at least 80% sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NO: 6 as well as (i) proteins which are assembly proteins of an Fe-Fe hydrogenase having any structure or at least 80% sequence identity to polypeptides encoded by the nucleic acids of SEQ ID NO: 2 and 4, (ii) a genus of ferredoxin NADP reductases and ferredoxin proteins having any structure or at least 80% sequence identity to polypeptides encoded by the nucleic acids of SEQ ID NO: 8 and 9 . In addition, the claims require a genus of unknown modifications that reduce or inhibit the activity or levels of endogenous proteins such as a phosphofructokinase or a glycerate mutase. The claims also require nucleic acids encoding proteins having any structure and function that would allow any microorganism to metabolize sucrose. It is also noted that the claims are not limited to nucleic acids encoding Fe-Fe hydrogenases, ferredoxin, ferredoxin NADP reductases, or Fe-Fe hydrogenase assembly proteins in view of the fact that the nucleic acids recited can encode proteins having any function in view of the recitation of “functionally equivalent homologs”. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
The claims require nucleic acids encoding proteins of unknown structure and/or function. The specification fails to disclose how to use recombinant microorganisms that comprise nucleic acids that do not encode proteins that have Fe-Fe hydrogenase, ferredoxin, ferredoxin NADP reductase, and Fe-Fe hydrogenase assembly activity. Even if the argument is made that the claims require nucleic acids that encode proteins with a defined function and at least 80% sequence identity to the proteins encoded by the nucleic acids of SEQ ID NO: 2, 4, 6, 8, or 9, it is noted that the structural variants that meet the 80% sequence identity recited are essentially infinite. See calculation provided above. In the case of sucrose metabolism, the claims require nucleic acids encoding any protein associated with sucrose metabolism. The specification and the prior art are silent with regard to the structural features required in the proteins encoded by the required nucleic acids for such proteins to have the desired activity. There is no structure/function correlation that would allow one of skill in the art to determine which proteins are more likely to have the desired activity. Therefore, one of skill in the art would have to test an essentially infinite number of proteins to find those having the recited activity. With regard to the genus of modifications required, it is reiterated herein that the genetic modifications recited encompass, for example, genetic modifications to unknown genes encoding proteins that control the expression of the recited endogenous proteins, unknown structural modifications to promoters, the expression of unknown proteins that could act as inhibitors of transcription, unknown antisense molecules to block expression of the desired gene, or unknown structural modifications to the coding region of a gene to alter the enzymatic activity of an enzyme. In the absence of (a) a rational and predictable scheme for identifying which proteins and their genes are more likely to have the desired activity, (b) a correlation between structure and activity, and (c) a rational and predictable scheme for identifying those genetic modifications most likely to result in the desired decreased/inhibited activity/level of an enzyme in a microorganism, one of skill in the art would have to test an essentially infinite number of proteins, nucleic acids, and modifications to determine which genetic modifications would lead to the desired outcome, and which proteins have the desired activity. This is not deemed routine experimentation. Therefore, for the reasons of record and those set forth above, one cannot reasonably conclude that the entire scope of the claims is fully enabled by the teachings of the specification and/or the prior art.
Claim Rejections - 35 USC § 102 (AIA )
Claims 1-2, 6, 9, 11-12, 14 were rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Jones et al. (WO 2007/123258 11/1/2007; cited in the IDS).
Applicant has amended claim 1 to now require the recombinant microorganism to comprise a nucleic acid that encodes a protein having at least 80% sequence identity to the protein encoded by the polynucleotide of SEQ ID NO: 6. The polynucleotide of SEQ ID NO: 6 encodes the C. reinhardtiii Fe-Fe hydrogenase disclosed in GenBank accession No. AAG00591. See alignment below. The Fe-Fe dehydrogenase of the E. coli cell of Jones et al. is from C. acetobutylicum and is encoded by the polynucleotide disclosed by Gorwa et al. in GenBank accession No. U15277 (Table 2 of Jones et al.). The C. acetobutylicum Fe-Fe hydrogenase encoded by the hydA gene disclosed by Gorwa et al. has 37% sequence identity to the protein encoded by the polynucleotide of SEQ ID NO: 6 (37% = 184x100/497; protein encoded by the polynucleotide of SEQ ID NO: 6 has 497 amino acids). See alignment below. Therefore, this rejection is hereby withdrawn.
SEQ ID NO: 6
RESULT 1
Q9FYU1_CHLRE
ID Q9FYU1_CHLRE Unreviewed; 497 AA.
AC Q9FYU1;
DT 01-MAR-2001, integrated into UniProtKB/TrEMBL.
DT 01-MAR-2001, sequence version 1.
DT 28-JAN-2026, entry version 149.
DE SubName: Full=Fe-hydrogenase {ECO:0000313|EMBL:AAG00591.1};
DE EC=1.18.99.1 {ECO:0000313|EMBL:AAG00591.1};
DE SubName: Full=Iron-hydrogenase HydA1 {ECO:0000313|EMBL:AAL23572.1};
DE Flags: Precursor;
GN Name=hyd1 {ECO:0000313|EMBL:AAG00591.1};
GN Synonyms=hydA {ECO:0000313|EMBL:CAC80065.1}, hydA1
GN {ECO:0000313|EMBL:AAL23572.1};
GN ORFNames=CHLRE_03g199800v5 {ECO:0000313|EMBL:PNW85924.1};
OS Chlamydomonas reinhardtii (Chlamydomonas smithii).
OC Eukaryota; Viridiplantae; Chlorophyta; core chlorophytes; Chlorophyceae;
OC CS clade; Chlamydomonadales; Chlamydomonadaceae; Chlamydomonas.
OX NCBI_TaxID=3055 {ECO:0000313|EMBL:AAG00591.1};
RN [1] {ECO:0000313|EMBL:CAC80065.1}
RP NUCLEOTIDE SEQUENCE.
RA Kaminski A.U., Happe T.;
RT "Isolation and characterization of the hydA gene encoding the Fe-
RT hydrogenase of Chlamydomonas reinhardtii.";
RL Submitted (OCT-1998) to the EMBL/GenBank/DDBJ databases.
RN [2] {ECO:0000313|EMBL:AAG00591.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=C9 {ECO:0000313|EMBL:AAG00591.1};
RA Mets L.J.;
RT "The iron hydrogenase of Chlamydomonas reinhardtii has a single folding
RT domain containing an H-cluster catalytic center and lacking bound electron
RT carriers.";
RL Submitted (JUL-2000) to the EMBL/GenBank/DDBJ databases.
RN [3] {ECO:0000313|EMBL:CAC83731.1}
RP NUCLEOTIDE SEQUENCE.
RA Happe T., Kaminski A.;
RT "Isolation and characterization of the hydA gene encoding the Fe-
RT hydrogenase of Chlamydomonas reinhardtii.";
RL Submitted (FEB-2001) to the EMBL/GenBank/DDBJ databases.
RN [4] {ECO:0000313|EMBL:AAL23572.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=21gr {ECO:0000313|EMBL:AAL23572.1};
RA Forestier M., Zhang L., Plummer S., Ahmann D., Seibert M., Ghirardi M.L.;
RT "Two putative Fe-only hydrogenases cloned from Chlamydomonas reinhardtii
RT are coexpressed in cells undergoing anaerobiosis.";
RL Submitted (SEP-2001) to the EMBL/GenBank/DDBJ databases.
RN [5] {ECO:0000313|EMBL:PNW85924.1, ECO:0000313|Proteomes:UP000006906}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=CC-503 {ECO:0000313|Proteomes:UP000006906}, and CC-503 cw92 mt+
RC {ECO:0000313|EMBL:PNW85924.1};
RX PubMed=17932292; DOI=10.1126/science.1143609;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Cardol P., Cerutti H., Chanfreau G., Chen C.L.,
RA Cognat V., Croft M.T., Dent R., Dutcher S., Fernandez E., Fukuzawa H.,
RA Gonzalez-Ballester D., Gonzalez-Halphen D., Hallmann A., Hanikenne M.,
RA Hippler M., Inwood W., Jabbari K., Kalanon M., Kuras R., Lefebvre P.A.,
RA Lemaire S.D., Lobanov A.V., Lohr M., Manuell A., Meier I., Mets L.,
RA Mittag M., Mittelmeier T., Moroney J.V., Moseley J., Napoli C.,
RA Nedelcu A.M., Niyogi K., Novoselov S.V., Paulsen I.T., Pazour G.,
RA Purton S., Ral J.P., Riano-Pachon D.M., Riekhof W., Rymarquis L.,
RA Schroda M., Stern D., Umen J., Willows R., Wilson N., Zimmer S.L.,
RA Allmer J., Balk J., Bisova K., Chen C.J., Elias M., Gendler K., Hauser C.,
RA Lamb M.R., Ledford H., Long J.C., Minagawa J., Page M.D., Pan J.,
RA Pootakham W., Roje S., Rose A., Stahlberg E., Terauchi A.M., Yang P.,
RA Ball S., Bowler C., Dieckmann C.L., Gladyshev V.N., Green P., Jorgensen R.,
RA Mayfield S., Mueller-Roeber B., Rajamani S., Sayre R.T., Brokstein P.,
RA Dubchak I., Goodstein D., Hornick L., Huang Y.W., Jhaveri J., Luo Y.,
RA Martinez D., Ngau W.C., Otillar B., Poliakov A., Porter A., Szajkowski L.,
RA Werner G., Zhou K., Grigoriev I.V., Rokhsar D.S., Grossman A.R.;
RT "The Chlamydomonas genome reveals the evolution of key animal and plant
RT functions.";
RL Science 318:245-250(2007).
RN [6] {ECO:0007829|PDB:3LX4}
RP X-RAY CRYSTALLOGRAPHY (1.97 ANGSTROMS) OF 57-497 IN COMPLEX WITH
RP IRON-SULFUR (4FE-4S).
RX PubMed=20418861; DOI=10.1038/nature08993;
RA Mulder D.W., Boyd E.S., Sarma R., Lange R.K., Endrizzi J.A.,
RA Broderick J.B., Peters J.W.;
RT "Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure
RT of HydA(DeltaEFG).";
RL Nature 465:248-251(2010).
RN [7] {ECO:0007829|PDB:2N0S}
RP STRUCTURE BY NMR OF 58-497 IN COMPLEX WITH IRON-SULFUR (4FE-4S).
RX PubMed=26010059; DOI=10.1002/cbic.201500130;
RA Rumpel S., Siebel J.F., Diallo M., Fares C., Reijerse E.J., Lubitz W.;
RT "Structural Insight into the Complex of Ferredoxin and [FeFe] Hydrogenase
RT from Chlamydomonas reinhardtii.";
RL ChemBioChem 16:1663-1669(2015).
RN [8] {ECO:0007829|PDB:4R0V}
RP X-RAY CRYSTALLOGRAPHY (2.29 ANGSTROMS) IN COMPLEX WITH IRON-SULFUR
RP (4FE-4S).
RX PubMed=25579778; DOI=10.1021/ja510169s;
RA Swanson K.D., Ratzloff M.W., Mulder D.W., Artz J.H., Ghose S., Hoffman A.,
RA White S., Zadvornyy O.A., Broderick J.B., Bothner B., King P.W.,
RA Peters J.W.;
RT "[FeFe]-hydrogenase oxygen inactivation is initiated at the H cluster 2Fe
RT subcluster.";
RL J. Am. Chem. Soc. 137:1809-1816(2015).
RN [9] {ECO:0000313|EMBL:PNW85924.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=CC-503 cw92 mt+ {ECO:0000313|EMBL:PNW85924.1};
RG Chlamydomonas Annotation Team;
RG JGI Annotation Team;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Grigoriev I.V., Rokhsar D.S.;
RT "WGS assembly of Chlamydomonas reinhardtii.";
RL Submitted (JUL-2017) to the EMBL/GenBank/DDBJ databases.
RN [10] {ECO:0007829|PDB:6GM5, ECO:0007829|PDB:6GM6}
RP X-RAY CRYSTALLOGRAPHY (1.45 ANGSTROMS) OF 57-497 IN COMPLEX WITH [4FE-4S]
RP CLUSTER.
RX PubMed=30413719; DOI=10.1038/s41467-018-07140-x;
RA Duan J., Senger M., Esselborn J., Engelbrecht V., Wittkamp F., Apfel U.P.,
RA Hofmann E., Stripp S.T., Happe T., Winkler M.;
RT "Crystallographic and spectroscopic assignment of the proton transfer
RT pathway in [FeFe]-hydrogenases.";
RL Nat. Commun. 9:4726-4726(2018).
RN [11] {ECO:0007829|PDB:6GL6}
RP X-RAY CRYSTALLOGRAPHY (1.80 ANGSTROMS) OF 57-497 IN COMPLEX WITH [4FE-4S]
RP CLUSTER.
RX PubMed=30545220; DOI=10.1021/jacs.8b11149;
RA Rodriguez-Macia P., Kertess L., Burnik J., Birrell J.A., Hofmann E.,
RA Lubitz W., Happe T., Rudiger O.;
RT "His-Ligation to the [4Fe-4S] Subcluster Tunes the Catalytic Bias of [FeFe]
RT Hydrogenase.";
RL J. Am. Chem. Soc. 141:472-481(2019).
CC -!- SIMILARITY: Belongs to the NARF family.
CC {ECO:0000256|ARBA:ARBA00006596}.
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DR EMBL; AF289201; AAG00591.1; -; mRNA.
DR EMBL; AY055755; AAL23572.1; -; mRNA.
DR EMBL; AJ012098; CAC80065.1; -; mRNA.
DR EMBL; AJ308413; CAC83731.1; -; Genomic_DNA.
DR EMBL; CM008964; PNW85924.1; -; Genomic_DNA.
DR RefSeq; XP_001693376.1; XM_001693324.1.
DR PDB; 2N0S; NMR; -; A=58-497.
DR PDB; 3LX4; X-ray; 1.97 A; A/B=57-497.
DR PDB; 4R0V; X-ray; 2.29 A; A/B=1-497.
DR PDB; 6GL6; X-ray; 1.80 A; A=57-497.
DR PDB; 6GM5; X-ray; 1.45 A; A=57-497.
DR PDB; 6GM6; X-ray; 1.61 A; A=57-497.
DR PDB; 6GM7; X-ray; 2.14 A; A=57-497.
DR PDBsum; 2N0S; -.
DR PDBsum; 3LX4; -.
DR PDBsum; 4R0V; -.
DR PDBsum; 6GL6; -.
DR PDBsum; 6GM5; -.
DR PDBsum; 6GM6; -.
DR PDBsum; 6GM7; -.
DR AlphaFoldDB; Q9FYU1; -.
DR SMR; Q9FYU1; -.
DR STRING; 3055.Q9FYU1; -.
DR PaxDb; 3055-EDP03402; -.
DR GeneID; 5718949; -.
DR Gramene; PNW85924; PNW85924; CHLRE_03g199800v5.
DR KEGG; cre:CHLRE_03g199800v5; -.
DR eggNOG; KOG2439; Eukaryota.
DR HOGENOM; CLU_018240_2_0_1; -.
DR OMA; AIMPCIA; -.
DR OrthoDB; 10253113at2759; -.
DR BioCyc; CHLAMY:MONOMER-16491; -.
DR BioCyc; MetaCyc:MONOMER-16491; -.
DR BRENDA; 1.12.7.2; 1318.
DR Proteomes; UP000006906; Chromosome 3.
DR GO; GO:0051539; F:4 iron, 4 sulfur cluster binding; IEA:UniProtKB-KW.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0016491; F:oxidoreductase activity; IEA:UniProtKB-KW.
DR Gene3D; 3.40.50.1780; -; 1.
DR Gene3D; 3.40.950.10; Fe-only Hydrogenase (Larger Subunit), Chain L, domain 3; 1.
DR InterPro; IPR050340; Cytosolic_Fe-S_CAF.
DR InterPro; IPR009016; Fe_hydrogenase.
DR InterPro; IPR004108; Fe_hydrogenase_lsu_C.
DR InterPro; IPR003149; Fe_hydrogenase_ssu.
DR PANTHER; PTHR11615; NITRATE, FORMATE, IRON DEHYDROGENASE; 1.
DR Pfam; PF02906; Fe_hyd_lg_C; 2.
DR Pfam; PF02256; Fe_hyd_SSU; 1.
DR SMART; SM00902; Fe_hyd_SSU; 1.
DR SUPFAM; SSF53920; Fe-only hydrogenase; 1.
PE 1: Evidence at protein level;
KW 3D-structure {ECO:0007829|PDB:2N0S, ECO:0007829|PDB:3LX4};
KW 4Fe-4S {ECO:0007829|PDB:3LX4, ECO:0007829|PDB:4R0V};
KW Iron {ECO:0007829|PDB:3LX4, ECO:0007829|PDB:4R0V};
KW Iron-sulfur {ECO:0007829|PDB:3LX4, ECO:0007829|PDB:4R0V};
KW Metal-binding {ECO:0007829|PDB:3LX4, ECO:0007829|PDB:4R0V};
KW Oxidoreductase {ECO:0000313|EMBL:AAG00591.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000006906}.
FT DOMAIN 430..489
FT /note="Iron hydrogenase small subunit"
FT /evidence="ECO:0000259|SMART:SM00902"
FT BINDING 170
FT /ligand="[4Fe-4S] cluster"
FT /ligand_id="ChEBI:CHEBI:49883"
FT /evidence="ECO:0007829|PDB:3LX4, ECO:0007829|PDB:4R0V"
FT BINDING 225
Alignment Scores:
Length: 497
Score: 2576.00 Matches: 497
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 85.8% Indels: 0
Gaps: 0
US-17-916-137-6 (1-1642) x Q9FYU1_CHLRE (1-497)
Qy 18 ATGTCCGCATTAGTACTTAAGCCCTGTGCGGCAGTATCCATCCGTGGCTCCTCGTGTCGC 77
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetSerAlaLeuValLeuLysProCysAlaAlaValSerIleArgGlySerSerCysArg 20
Qy 78 GCACGCCAAGTAGCCCCACGCGCTCCGCTTGCAGCTAGCACGGTTCGTGTCGCTCTTGCA 137
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 AlaArgGlnValAlaProArgAlaProLeuAlaAlaSerThrValArgValAlaLeuAla 40
Qy 138 ACCCTGGAGGCACCAGCGCGTCGTTTAGGAAACGTCGCCTGTGCCGCAGCGGCACCGGCC 197
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 ThrLeuGluAlaProAlaArgArgLeuGlyAsnValAlaCysAlaAlaAlaAlaProAla 60
Qy 198 GCAGAGGCACCCTTGTCTCACGTCCAGCAAGCACTGGCCGAACTGGCAAAGCCCAAAGAT 257
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaGluAlaProLeuSerHisValGlnGlnAlaLeuAlaGluLeuAlaLysProLysAsp 80
Qy 258 GACCCCACGCGTAAGCACGTTTGCGTTCAAGTCGCTCCCGCAGTGCGTGTCGCTATTGCT 317
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 AspProThrArgLysHisValCysValGlnValAlaProAlaValArgValAlaIleAla 100
Qy 318 GAAACCTTAGGGCTTGCGCCGGGCGCTACGACACCGAAACAATTAGCAGAAGGCCTGCGT 377
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 GluThrLeuGlyLeuAlaProGlyAlaThrThrProLysGlnLeuAlaGluGlyLeuArg 120
Qy 378 CGCTTGGGCTTTGACGAGGTTTTCGATACGCTGTTCGGGGCCGACTTGACGATCATGGAA 437
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ArgLeuGlyPheAspGluValPheAspThrLeuPheGlyAlaAspLeuThrIleMetGlu 140
Qy 438 GAGGGCTCAGAACTTTTGCACCGTCTGACGGAGCACTTGGAAGCACACCCGCACTCTGAT 497
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 141 GluGlySerGluLeuLeuHisArgLeuThrGluHisLeuGluAlaHisProHisSerAsp 160
Qy 498 GAGCCGCTGCCTATGTTTACCAGCTGCTGTCCTGGTTGGATCGCGATGCTGGAGAAATCA 557
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 GluProLeuProMetPheThrSerCysCysProGlyTrpIleAlaMetLeuGluLysSer 180
Qy 558 TACCCAGACCTTATCCCTTATGTAAGTTCTTGCAAATCCCCTCAAATGATGCTGGCTGCT 617
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 TyrProAspLeuIleProTyrValSerSerCysLysSerProGlnMetMetLeuAlaAla 200
Qy 618 ATGGTCAAATCGTATCTGGCGGAAAAAAAGGGGATTGCACCTAAAGATATGGTTATGGTA 677
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 201 MetValLysSerTyrLeuAlaGluLysLysGlyIleAlaProLysAspMetValMetVal 220
Qy 678 AGCATTATGCCATGCACACGCAAGCAGAGTGAGGCGGATCGTGATTGGTTTTGTGTCGAC 737
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 SerIleMetProCysThrArgLysGlnSerGluAlaAspArgAspTrpPheCysValAsp 240
Qy 738 GCGGACCCAACACTTCGCCAATTGGACCATGTGATCACGACCGTAGAGCTGGGGAATATT 797
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 AlaAspProThrLeuArgGlnLeuAspHisValIleThrThrValGluLeuGlyAsnIle 260
Qy 798 TTCAAAGAGCGTGGGATCAACCTTGCGGAATTACCCGAGGGGGAGTGGGACAACCCAATG 857
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 261 PheLysGluArgGlyIleAsnLeuAlaGluLeuProGluGlyGluTrpAspAsnProMet 280
Qy 858 GGGGTAGGCTCCGGTGCTGGAGTACTTTTTGGCACCACTGGAGGGGTAATGGAGGCGGCG 917
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 281 GlyValGlySerGlyAlaGlyValLeuPheGlyThrThrGlyGlyValMetGluAlaAla 300
Qy 918 CTGCGTACTGCATACGAATTATTTACTGGAACCCCCTTACCCCGCCTTTCGCTTTCGGAG 977
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LeuArgThrAlaTyrGluLeuPheThrGlyThrProLeuProArgLeuSerLeuSerGlu 320
Qy 978 GTGCGCGGCATGGATGGCATTAAAGAGACTAACATCACGATGGTACCGGCCCCTGGCAGC 1037
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 321 ValArgGlyMetAspGlyIleLysGluThrAsnIleThrMetValProAlaProGlySer 340
Qy 1038 AAGTTTGAAGAACTGTTAAAGCACCGCGCGGCAGCGCGTGCCGAGGCTGCTGCACACGGA 1097
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 341 LysPheGluGluLeuLeuLysHisArgAlaAlaAlaArgAlaGluAlaAlaAlaHisGly 360
Qy 1098 ACACCTGGTCCCTTGGCCTGGGACGGCGGCGCAGGGTTCACCTCGGAGGACGGGCGTGGG 1157
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 ThrProGlyProLeuAlaTrpAspGlyGlyAlaGlyPheThrSerGluAspGlyArgGly 380
Qy 1158 GGTATCACTCTTCGTGTGGCGGTAGCTAATGGCTTGGGAAACGCCAAAAAGTTGATCACA 1217
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 381 GlyIleThrLeuArgValAlaValAlaAsnGlyLeuGlyAsnAlaLysLysLeuIleThr 400
Qy 1218 AAGATGCAGGCGGGAGAGGCGAAGTATGACTTTGTAGAAATCATGGCGTGTCCAGCGGGA 1277
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 LysMetGlnAlaGlyGluAlaLysTyrAspPheValGluIleMetAlaCysProAlaGly 420
Qy 1278 TGCGTTGGGGGCGGCGGGCAGCCACGTTCCACCGATAAAGCAATTACCCAGAAGCGCCAG 1337
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 CysValGlyGlyGlyGlyGlnProArgSerThrAspLysAlaIleThrGlnLysArgGln 440
Qy 1338 GCTGCACTTTACAATCTGGACGAGAAGTCGACTCTGCGCCGCTCCCATGAAAACCCGTCT 1397
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 441 AlaAlaLeuTyrAsnLeuAspGluLysSerThrLeuArgArgSerHisGluAsnProSer 460
Qy 1398 ATCCGTGAGTTATACGACACTTATTTGGGTGAGCCCTTAGGGCACAAAGCACACGAACTT 1457
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 461 IleArgGluLeuTyrAspThrTyrLeuGlyGluProLeuGlyHisLysAlaHisGluLeu 480
Qy 1458 TTACATACTCACTATGTAGCTGGCGGGGTCGAGGAGAAGGATGAGAAAAAG 1508
|||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 LeuHisThrHisTyrValAlaGlyGlyValGluGluLysAspGluLysLys 497
Query = GenBank accession No. AAG00591 – protein encoded by SEQ ID NO: 6 of this application
Sbjct = protein encoded by GenBank accession No. U15277
NW Score Identities Positives Gaps
551 184/645(29%) 258/645(40%) 211/645(32%)
Query 1 MSALVL------------------------------KPCA--------AVSIRGSSCRAR 22
M ++L K C V + G RA
Sbjct 1 MKTIILNGNEVHTDKDITILELARENNVDIPTLCFLKDCGNFGKCGVCMVEVEGKGFRAA 60
Query 23 QVA--------------------------------------------------------- 25
VA
Sbjct 61 CVAKVEDGMVINTESDEVKERIKKRVSMLLDKHEFKCGQCSRRENCEFLKLVIKTKAKAS 120
Query 26 -PRAP---------------------------LAASTVRVALATLEA----PARRLGNV- 52
P +AA + +++ R +G V
Sbjct 121 KPFLPEDKDALVDNRSKAIVIDRSKCVLCGRCVAACKQHTSTCSIQFIKKDGQRAVGTVD 180
Query 53 -------------ACAAAAP-AAEAPLSHVQQALAELAKPKDDPTRKHVCVQVAPAVRVA 98
C A P AA SH+++ L PK KHV V +AP+VR A
Sbjct 181 DVCLDDSTCLLCGQCVIACPVAALKEKSHIEKVQEALNDPK-----KHVIVAMAPSVRTA 235
Query 99 IAETLGLAPGATTPKQLAEGLRRLGFDEVFDTLFGADLTIMEEGSELLHRLTEHLEAHPH 158
+ E + G +L LR LGFD+VFD FGAD+TIMEE +ELL R+
Sbjct 236 MGELFKMGYGKDVTGKLYTALRMLGFDKVFDINFGADMTIMEEATELLGRVK-------- 287
Query 159 SDEPLPMFTSCCPGWIAMLEKSYPDLIPYVSSCKSPQMMLAAMVKSYLAEKKGIAPKDMV 218
++ P PMFTSCCP W+ + + +P+L+ +SS KSPQ + K+Y GIAP+D+
Sbjct 288 NNGPFPMFTSCCPAWVRLAQNYHPELLDNLSSAKSPQQIFGTASKTYYPSISGIAPEDVY 347
Query 219 MVSIMPCTRKQSEADRDWFCVDADPTLRQLDHVITTVELGNIFKERGINLAELPEGEWDN 278
V+IMPC K+ EAD + ++ LR +D +TT EL + K+ I A+L +GE D
Sbjct 348 TVTIMPCNDKKYEADIPFMETNS---LRDIDASLTTRELAKMIKDAKIKFADLEDGEVDP 404
Query 279 PMGVGSGAGVLFGTTGGVMEAALRTAYELFTGTPLPRLSLSEVRGMDGIKETNITMVPAP 338
MG SGAG +FG TGGVMEAA+R+A + L + +EVRG GIKE + +
Sbjct 405 AMGTYSGAGAIFGATGGVMEAAIRSAKDFAENKELENVDYTEVRGFKGIKEAEVEIA--- 461
Query 339 GSKFEELLKHRAAARAEAAAHGTPGPLAWDGGAGFTSEDGRGGITLRVAVANGLGNAKKL 398
G L VAV NG N +
Sbjct 462 ------------------------------------------GNKLNVAVINGASNFFEF 479
Query 399 ITKMQAGEAKYDFVEIMACPAGCVGGGGQPR--STDKAITQKRQ---AALYNLDEKS-TL 452
+ + E +Y F+E+MACP GC+ GGGQP + D+ R+ + LYN D+ +
Sbjct 480 MKSGKMNEKQYHFIEVMACPGGCINGGGQPHVNALDRENVDYRKLRASVLYNQDKNVLSK 539
Query 453 RRSHENPSIRELYDTYLGEPLGHKAHELLHTHYVAGGVEEKDEKK 497
R+SH+NP+I ++YD+Y G+P AH+LLH Y K E
Sbjct 540 RKSHDNPAIIKMYDSYFGKPGEGLAHKLLHVKYTKDKNVSKHE-- 582
Claim Rejections - 35 USC § 103 (AIA )
Claim 28 was rejected under 35 U.S.C. 103 as being unpatentable over Jones et al. (WO 2007/123258 11/1/2007; cited in the IDS) in view of Bruschi et al. (Biotechnology Advances 30:1001-1010, 2012) and Penfold et al. (Biotechnology Letters 26:1879-1883, 2004; cited in the IDS).
In view of Applicant’s amendment of claim 1, from which claim 28 depends, that now requires the recombinant microorganism to comprise a nucleic acid that encodes a protein having at least 80% sequence identity to the protein encoded by the polynucleotide of SEQ ID NO: 6, and the fact that the Fe-Fe hydrogenase of Jones et al. has 37% sequence identity to the protein encoded by the polynucleotide of SEQ ID NO: 6 (37% = 184x100/497; protein encoded by the polynucleotide of SEQ ID NO: 6 has 497 amino acids), this rejection is hereby withdrawn.
Claim 35 is rejected under 35 U.S.C. 103 as being unpatentable over Jones et al. (WO 2007/123258 11/1/2007; cited in the IDS) in view of Kumar et al. (Bioresource Technology 219:725-737, 2016).
In view of Applicant’s amendment of claim 1, from which claim 35 depends, that now requires the recombinant microorganism to comprise a nucleic acid that encodes a protein having at least 80% sequence identity to the protein encoded by the polynucleotide of SEQ ID NO: 6, and the fact that the Fe-Fe hydrogenase of Jones et al. has 37% sequence identity to the protein encoded by the polynucleotide of SEQ ID NO: 6 (37% = 184x100/497; protein encoded by the polynucleotide of SEQ ID NO: 6 has 497 amino acids), this rejection is hereby withdrawn.
Claims 1-2, 6, 9, 11-14, 16 remain rejected and new claims 36-37 are rejected under 35 U.S.C. 103 as being unpatentable over Edmonds (iGEM Registry of Standard Biological Parts, Part:BBa_K2300001; October 3, 2017) in view of Sekar et al. (Biotechnology for Biofuels 9:95, pages 1-11, 2016; cited in the IDS).
This rejection has been discussed at length in the prior Office action. It is maintained for the reasons of record and further applied to new claims 36-37 for the following reasons.
Applicant states that the instant invention provides a solution whereby recombinant cells that encode a HydA hydrogenase and comprise a genetic modification which promotes utilization of carbon via the pentose phosphate pathway provide an unexpected increase in rate and yield of hydrogen production. According to Applicant, neither Edmonds nor Sekar et al. teach or suggest methods using a HydA Fe-Fe hydrogenase. Applicant states that the selection of a HydA hydrogenase does not represent a routine selection and that there is no direct motivation from the recited references to select HydA from all possible Fe-Fe dehydrogenases. Applicant refers to Figure 1 of the specification as showing an increase in the production of hydrogen when the plasmid that comprises a gene encoding the Fe-Fe hydrogenase from C. reinhardtii was expressed, including the combination with a pfk deletion or with a gpmA deletion. Applicant states that the results disclosed in the specification show that when HydA hydrogenase is combined with a genetic modification to increase metabolism through the pentose phosphate pathway, there is a further unexpected increase, citing Figures 4 and 5 of the specification. Applicant submits that the invention produces a rate of hydrogen production that is higher than that of Sekar et al.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection or avoid the rejection of new claims 36-37. The Examiner acknowledges the teachings of the specification and those of the prior art. However, the Examiner disagrees with Applicant’s contention that the claimed invention is not obvious.
Claims 1-2, 6, 9, 11-14, 16, 36-37 are directed in part to a recombinant E. coli cell that has exogenous nucleic acids encoding a C. reinhardtii Fe-Fe dependent hydrogenase, a C. reinhardtii ferredoxin NADP reductase, C. reinhardtii ferredoxin, and C. reinhardtii proteins for enabling maturation and activation of the Fe-Fe dependent hydrogenase, wherein said exogenous nucleic acids are codon-optimized, wherein said exogenous nucleic acids are in a single construct, wherein said E. coli cell has a genetic modification to promote the utilization of carbon via the pentose phosphate pathway, wherein genetic modification is the reduction or inhibition of phosphofructokinase. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation. It is noted that the polynucleotide of SEQ ID NO: 6 encodes the C. reinhardtii Fe-Fe dependent hydrogenase HydA as evidenced by GenBank accession No. AAG00591. See alignment above. The polynucleotide of SEQ ID NO: 8 encodes the C. reinhardtii ferredoxin as evidenced by GenBank accession No. PNW73294. See alignment below. The polynucleotide of SEQ ID NO: 9 encodes the C. reinhardtii ferredoxin--NADP reductase as evidenced by GenBank accession No. PNW76801. See alignment below. The polynucleotide of SEQ ID NO: 2 encodes the C. reinhardtii Fe-Fe hydrogenase assembly protein HydEF as evidenced by GenBank accession No. AAS92601. See alignment below. The polynucleotide of SEQ ID NO: 4 encodes the C. reinhardtii Fe-Fe hydrogenase assembly protein HydG as evidenced by GenBank accession No. AAS92602. See alignment below.
SEQ ID NO: 8
RESULT 1
A8IV40_CHLRE
ID A8IV40_CHLRE Unreviewed; 126 AA.
AC A8IV40;
DT 04-DEC-2007, integrated into UniProtKB/TrEMBL.
DT 04-DEC-2007, sequence version 1.
DT 28-JAN-2026, entry version 101.
DE RecName: Full=Ferredoxin {ECO:0000256|RuleBase:RU364001};
GN ORFNames=CHLRE_14g626700v5 {ECO:0000313|EMBL:PNW73294.1};
OS Chlamydomonas reinhardtii (Chlamydomonas smithii).
OC Eukaryota; Viridiplantae; Chlorophyta; core chlorophytes; Chlorophyceae;
OC CS clade; Chlamydomonadales; Chlamydomonadaceae; Chlamydomonas.
OX NCBI_TaxID=3055 {ECO:0000313|EMBL:PNW73294.1, ECO:0000313|Proteomes:UP000006906};
RN [1] {ECO:0000313|EMBL:PNW73294.1, ECO:0000313|Proteomes:UP000006906}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=CC-503 {ECO:0000313|Proteomes:UP000006906};
RX PubMed=17932292; DOI=10.1126/science.1143609;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Cardol P., Cerutti H., Chanfreau G., Chen C.L.,
RA Cognat V., Croft M.T., Dent R., Dutcher S., Fernandez E., Fukuzawa H.,
RA Gonzalez-Ballester D., Gonzalez-Halphen D., Hallmann A., Hanikenne M.,
RA Hippler M., Inwood W., Jabbari K., Kalanon M., Kuras R., Lefebvre P.A.,
RA Lemaire S.D., Lobanov A.V., Lohr M., Manuell A., Meier I., Mets L.,
RA Mittag M., Mittelmeier T., Moroney J.V., Moseley J., Napoli C.,
RA Nedelcu A.M., Niyogi K., Novoselov S.V., Paulsen I.T., Pazour G.,
RA Purton S., Ral J.P., Riano-Pachon D.M., Riekhof W., Rymarquis L.,
RA Schroda M., Stern D., Umen J., Willows R., Wilson N., Zimmer S.L.,
RA Allmer J., Balk J., Bisova K., Chen C.J., Elias M., Gendler K., Hauser C.,
RA Lamb M.R., Ledford H., Long J.C., Minagawa J., Page M.D., Pan J.,
RA Pootakham W., Roje S., Rose A., Stahlberg E., Terauchi A.M., Yang P.,
RA Ball S., Bowler C., Dieckmann C.L., Gladyshev V.N., Green P., Jorgensen R.,
RA Mayfield S., Mueller-Roeber B., Rajamani S., Sayre R.T., Brokstein P.,
RA Dubchak I., Goodstein D., Hornick L., Huang Y.W., Jhaveri J., Luo Y.,
RA Martinez D., Ngau W.C., Otillar B., Poliakov A., Porter A., Szajkowski L.,
RA Werner G., Zhou K., Grigoriev I.V., Rokhsar D.S., Grossman A.R.;
RT "The Chlamydomonas genome reveals the evolution of key animal and plant
RT functions.";
RL Science 318:245-250(2007).
CC -!- FUNCTION: Ferredoxins are iron-sulfur proteins that transfer electrons
CC in a wide variety of metabolic reactions.
CC {ECO:0000256|RuleBase:RU364001}.
CC -!- COFACTOR:
CC Name=[2Fe-2S] cluster; Xref=ChEBI:CHEBI:190135;
CC Evidence={ECO:0000256|RuleBase:RU364001};
CC Note=Binds 1 [2Fe-2S] cluster. {ECO:0000256|RuleBase:RU364001};
CC -!- SUBCELLULAR LOCATION: Plastid, chloroplast
CC {ECO:0000256|RuleBase:RU364001}.
CC -!- SIMILARITY: Belongs to the 2Fe2S plant-type ferredoxin family.
CC {ECO:0000256|ARBA:ARBA00007874, ECO:0000256|RuleBase:RU364001}.
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DR EMBL; CM008975; PNW73294.1; -; Genomic_DNA.
DR RefSeq; XP_001692808.1; XM_001692756.2.
DR AlphaFoldDB; A8IV40; -.
DR SMR; A8IV40; -.
DR FunCoup; A8IV40; 480.
DR STRING; 3055.A8IV40; -.
DR GeneID; 5718285; -.
DR Gramene; PNW73294; PNW73294; CHLRE_14g626700v5.
DR KEGG; cre:CHLRE_14g626700v5; -.
DR HOGENOM; CLU_082632_1_1_1; -.
DR InParanoid; A8IV40; -.
DR OMA; ADCTIKA; -.
DR OrthoDB; 1885901at2759; -.
DR Proteomes; UP000006906; Chromosome 14.
DR GO; GO:0009507; C:chloroplast; IEA:UniProtKB-SubCell.
DR GO; GO:0051537; F:2 iron, 2 sulfur cluster binding; IEA:UniProtKB-KW.
DR GO; GO:0009055; F:electron transfer activity; IEA:InterPro.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0022900; P:electron transport chain; IEA:InterPro.
DR CDD; cd00207; fer2; 1.
DR FunFam; 3.10.20.30:FF:000014; Ferredoxin; 1.
DR Gene3D; 3.10.20.30; -; 1.
DR InterPro; IPR036010; 2Fe-2S_ferredoxin-like_sf.
DR InterPro; IPR001041; 2Fe-2S_ferredoxin-type.
DR InterPro; IPR006058; 2Fe2S_fd_BS.
DR InterPro; IPR012675; Beta-grasp_dom_sf.
DR InterPro; IPR010241; Fd_pln.
DR InterPro; IPR023383; Ferredoxin_transit_pept.
DR NCBIfam; TIGR02008; fdx_plant; 1.
DR PANTHER; PTHR43112; FERREDOXIN; 1.
DR PANTHER; PTHR43112:SF3; FERREDOXIN-2, CHLOROPLASTIC; 1.
DR Pfam; PF11591; 2Fe-2S_Ferredox; 1.
DR Pfam; PF00111; Fer2; 1.
DR SUPFAM; SSF54292; 2Fe-2S ferredoxin-like; 1.
DR PROSITE; PS00197; 2FE2S_FER_1; 1.
DR PROSITE; PS51085; 2FE2S_FER_2; 1.
PE 3: Inferred from homology;
KW 2Fe-2S {ECO:0000256|ARBA:ARBA00022714, ECO:0000256|RuleBase:RU364001};
KW Chloroplast {ECO:0000256|RuleBase:RU364001};
KW Electron transport {ECO:0000256|ARBA:ARBA00022982,
KW ECO:0000256|RuleBase:RU364001};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|RuleBase:RU364001};
KW Iron-sulfur {ECO:0000256|ARBA:ARBA00023014, ECO:0000256|RuleBase:RU364001};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723,
KW ECO:0000256|RuleBase:RU364001}; Plastid {ECO:0000256|RuleBase:RU364001};
KW Reference proteome {ECO:0000313|Proteomes:UP000006906};
KW Transport {ECO:0000256|ARBA:ARBA00022448, ECO:0000256|RuleBase:RU364001}.
SQ SEQUENCE 126 AA; 13232 MW; 155AFEA9E6F6E411 CRC64;
Alignment Scores:
Length: 126
Score: 648.00 Matches: 124
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 89.1% Indels: 0
Gaps: 0
US-17-916-137-8 (1-392) x A8IV40_CHLRE (1-126)
Qy 18 ATGGCGATGCGTTCCACATTTGCTGCGCGTGTCGGTGCCAAACCGGCAGTGCGTGGGGCG 77
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 3 MetAlaMetArgSerThrPheAlaAlaArgValGlyAlaLysProAlaValArgGlyAla 22
Qy 78 CGTCCCGCGTCGCGTATGTCTTGCATGGCCTATAAGGTTACACTGAAAACGCCATCAGGC 137
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 23 ArgProAlaSerArgMetSerCysMetAlaTyrLysValThrLeuLysThrProSerGly 42
Qy 138 GATAAAACAATCGAATGTCCGGCGGACACCTATATCCTGGATGCTGCGGAGGAAGCCGGG 197
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 43 AspLysThrIleGluCysProAlaAspThrTyrIleLeuAspAlaAlaGluGluAlaGly 62
Qy 198 CTGGACCTTCCCTATAGTTGCCGTGCGGGCGCCTGTTCATCCTGTGCTGGCAAAGTGGCA 257
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 63 LeuAspLeuProTyrSerCysArgAlaGlyAlaCysSerSerCysAlaGlyLysValAla 82
Qy 258 GCTGGAACGGTAGATCAATCTGATCAGTCCTTTCTGGATGACGCTCAGATGGGGAACGGA 317
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 83 AlaGlyThrValAspGlnSerAspGlnSerPheLeuAspAspAlaGlnMetGlyAsnGly 102
Qy 318 TTCGTCCTGACATGTGTCGCGTATCCTACAAGCGACTGCACTATCCAAACTCACCAAGAG 377
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 103 PheValLeuThrCysValAlaTyrProThrSerAspCysThrIleGlnThrHisGlnGlu 122
Qy 378 GAGGCTTTATAT 389
||||||||||||
Db 123 GluAlaLeuTyr 126
SEQ ID NO: 9
RESULT 1
A8J6Y8_CHLRE
ID A8J6Y8_CHLRE Unreviewed; 346 AA.
AC A8J6Y8;
DT 04-DEC-2007, integrated into UniProtKB/TrEMBL.
DT 04-DEC-2007, sequence version 1.
DT 28-JAN-2026, entry version 99.
DE RecName: Full=Ferredoxin--NADP reductase, chloroplastic {ECO:0000256|PIRNR:PIRNR000361};
DE Short=FNR {ECO:0000256|PIRNR:PIRNR000361};
DE EC=1.18.1.2 {ECO:0000256|PIRNR:PIRNR000361};
GN ORFNames=CHLRE_11g476750v5 {ECO:0000313|EMBL:PNW76801.1};
OS Chlamydomonas reinhardtii (Chlamydomonas smithii).
OC Eukaryota; Viridiplantae; Chlorophyta; core chlorophytes; Chlorophyceae;
OC CS clade; Chlamydomonadales; Chlamydomonadaceae; Chlamydomonas.
OX NCBI_TaxID=3055 {ECO:0000313|EMBL:PNW76801.1, ECO:0000313|Proteomes:UP000006906};
RN [1] {ECO:0000313|EMBL:PNW76801.1, ECO:0000313|Proteomes:UP000006906}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=CC-503 {ECO:0000313|Proteomes:UP000006906};
RX PubMed=17932292; DOI=10.1126/science.1143609;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Cardol P., Cerutti H., Chanfreau G., Chen C.L.,
RA Cognat V., Croft M.T., Dent R., Dutcher S., Fernandez E., Fukuzawa H.,
RA Gonzalez-Ballester D., Gonzalez-Halphen D., Hallmann A., Hanikenne M.,
RA Hippler M., Inwood W., Jabbari K., Kalanon M., Kuras R., Lefebvre P.A.,
RA Lemaire S.D., Lobanov A.V., Lohr M., Manuell A., Meier I., Mets L.,
RA Mittag M., Mittelmeier T., Moroney J.V., Moseley J., Napoli C.,
RA Nedelcu A.M., Niyogi K., Novoselov S.V., Paulsen I.T., Pazour G.,
RA Purton S., Ral J.P., Riano-Pachon D.M., Riekhof W., Rymarquis L.,
RA Schroda M., Stern D., Umen J., Willows R., Wilson N., Zimmer S.L.,
RA Allmer J., Balk J., Bisova K., Chen C.J., Elias M., Gendler K., Hauser C.,
RA Lamb M.R., Ledford H., Long J.C., Minagawa J., Page M.D., Pan J.,
RA Pootakham W., Roje S., Rose A., Stahlberg E., Terauchi A.M., Yang P.,
RA Ball S., Bowler C., Dieckmann C.L., Gladyshev V.N., Green P., Jorgensen R.,
RA Mayfield S., Mueller-Roeber B., Rajamani S., Sayre R.T., Brokstein P.,
RA Dubchak I., Goodstein D., Hornick L., Huang Y.W., Jhaveri J., Luo Y.,
RA Martinez D., Ngau W.C., Otillar B., Poliakov A., Porter A., Szajkowski L.,
RA Werner G., Zhou K., Grigoriev I.V., Rokhsar D.S., Grossman A.R.;
RT "The Chlamydomonas genome reveals the evolution of key animal and plant
RT functions.";
RL Science 318:245-250(2007).
CC -!- CATALYTIC ACTIVITY:
CC Reaction=2 reduced [2Fe-2S]-[ferredoxin] + NADP(+) + H(+) = 2 oxidized
CC [2Fe-2S]-[ferredoxin] + NADPH; Xref=Rhea:RHEA:20125, Rhea:RHEA-
CC COMP:10000, Rhea:RHEA-COMP:10001, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:33737, ChEBI:CHEBI:33738, ChEBI:CHEBI:57783,
CC ChEBI:CHEBI:58349; EC=1.18.1.2;
CC Evidence={ECO:0000256|ARBA:ARBA00047776,
CC ECO:0000256|PIRNR:PIRNR000361};
CC -!- COFACTOR:
CC Name=FAD; Xref=ChEBI:CHEBI:57692;
CC Evidence={ECO:0000256|ARBA:ARBA00001974};
CC -!- SUBCELLULAR LOCATION: Plastid, chloroplast
CC {ECO:0000256|PIRNR:PIRNR000361}.
CC -!- SIMILARITY: Belongs to the ferredoxin--NADP reductase type 1 family.
CC {ECO:0000256|ARBA:ARBA00008312, ECO:0000256|PIRNR:PIRNR000361}.
CC ---------------------------------------------------------------------------
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CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
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DR EMBL; CM008972; PNW76801.1; -; Genomic_DNA.
DR RefSeq; XP_001697352.1; XM_001697300.2.
DR AlphaFoldDB; A8J6Y8; -.
DR SMR; A8J6Y8; -.
DR FunCoup; A8J6Y8; 105.
DR STRING; 3055.A8J6Y8; -.
DR GeneID; 5722854; -.
DR Gramene; PNW76801; PNW76801; CHLRE_11g476750v5.
DR KEGG; cre:CHLRE_11g476750v5; -.
DR HOGENOM; CLU_053066_1_0_1; -.
DR InParanoid; A8J6Y8; -.
DR OMA; CHIIIEH; -.
DR OrthoDB; 1688044at2759; -.
DR BRENDA; 1.18.1.2; 1318.
DR Proteomes; UP000006906; Chromosome 11.
DR ExpressionAtlas; A8J6Y8; baseline and differential.
DR GO; GO:0009507; C:chloroplast; IEA:UniProtKB-SubCell.
DR GO; GO:0004324; F:ferredoxin-NADP+ reductase activity; IEA:UniProtKB-EC.
DR CDD; cd06208; CYPOR_like_FNR; 1.
DR FunFam; 3.40.50.80:FF:000008; Ferredoxin--NADP reductase, chloroplastic; 1.
DR Gene3D; 3.40.50.80; Nucleotide-binding domain of ferredoxin-NADP reductase (FNR) module; 1.
DR Gene3D; 2.40.30.10; Translation factors; 1.
DR InterPro; IPR017927; FAD-bd_FR_type.
DR InterPro; IPR001709; Flavoprot_Pyr_Nucl_cyt_Rdtase.
DR InterPro; IPR015701; FNR.
DR InterPro; IPR039261; FNR_nucleotide-bd.
DR InterPro; IPR035442; FNR_plant_Cyanobacteria.
DR InterPro; IPR001433; OxRdtase_FAD/NAD-bd.
DR InterPro; IPR017938; Riboflavin_synthase-like_b-brl.
DR PANTHER; PTHR43314; -; 1.
DR Pfam; PF00175; NAD_binding_1; 1.
DR PIRSF; PIRSF501178; FNR-PetH; 1.
DR PIRSF; PIRSF000361; Frd-NADP+_RD; 1.
DR PRINTS; PR00371; FPNCR.
DR SUPFAM; SSF52343; Ferredoxin reductase-like, C-terminal NADP-linked domain; 1.
DR SUPFAM; SSF63380; Riboflavin synthase domain-like; 1.
DR PROSITE; PS51384; FAD_FR; 1.
PE 3: Inferred from homology;
KW FAD {ECO:0000256|ARBA:ARBA00022827, ECO:0000256|PIRNR:PIRNR000361};
KW Flavoprotein {ECO:0000256|ARBA:ARBA00022630,
KW ECO:0000256|PIRNR:PIRNR000361};
KW NADP {ECO:0000256|ARBA:ARBA00022857, ECO:0000256|PIRNR:PIRNR000361};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000256|PIRNR:PIRNR000361};
KW Reference proteome {ECO:0000313|Proteomes:UP000006906}.
FT BINDING 125
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|PIRSR:PIRSR000361-1"
FT BINDING 145
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|PIRSR:PIRSR000361-1"
FT BINDING 205
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|PIRSR:PIRSR000361-1"
FT BINDING 267..268
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|PIRSR:PIRSR000361-1"
FT BINDING 277
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|PIRSR:PIRSR000361-1"
FT BINDING 305..306
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|PIRSR:PIRSR000361-1"
FT BINDING 344
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|PIRSR:PIRSR000361-1"
SQ SEQUENCE 346 AA; 38267 MW; CCBA155BF2A47399 CRC64;
Alignment Scores:
Length: 346
Score: 1839.00 Matches: 346
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 86.6% Indels: 0
Gaps: 0
US-17-916-137-9 (1-1189) x A8J6Y8_CHLRE (1-346)
Qy 18 ATGCAAACTGTTCGCGCTCCAGCAGCTTCAGGTGTTGCCACACGTGTCGCAGGTCGTCGT 77
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetGlnThrValArgAlaProAlaAlaSerGlyValAlaThrArgValAlaGlyArgArg 20
Qy 78 ATGTGTCGTCCGGTTGCGGCGACGAAGGCTTCCACGGCTGTTACCACAGACATGTCGAAG 137
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 MetCysArgProValAlaAlaThrLysAlaSerThrAlaValThrThrAspMetSerLys 40
Qy 138 CGCACTGTTCCAACCAAGTTAGAGGAAGGTGAAATGCCGTTGAACACGTACAGTAATAAA 197
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 ArgThrValProThrLysLeuGluGluGlyGluMetProLeuAsnThrTyrSerAsnLys 60
Qy 198 GCTCCGTTCAAGGCAAAGGTTCGTTCCGTGGAAAAAATCACAGGACCAAAAGCCACAGGT 257
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaProPheLysAlaLysValArgSerValGluLysIleThrGlyProLysAlaThrGly 80
Qy 258 GAGACGTGCCACATCATTATTGAAACCGAGGGGAAGATCCCGTTTTGGGAGGGACAATCG 317
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 GluThrCysHisIleIleIleGluThrGluGlyLysIleProPheTrpGluGlyGlnSer 100
Qy 318 TACGGTGTAATTCCGCCGGGGACCAAGATCAACTCTAAGGGAAAAGAAGTGCCTCATGGT 377
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 TyrGlyValIleProProGlyThrLysIleAsnSerLysGlyLysGluValProHisGly 120
Qy 378 ACTCGTCTTTATTCGATTGCTTCTAGTCGTTACGGAGATGACTTCGATGGTCAAACGGCA 437
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ThrArgLeuTyrSerIleAlaSerSerArgTyrGlyAspAspPheAspGlyGlnThrAla 140
Qy 438 TCGCTGTGTGTTCGCCGCGCGGTATACGTCGATCCAGAGACTGGAAAGGAGGACCCCGCG 497
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 141 SerLeuCysValArgArgAlaValTyrValAspProGluThrGlyLysGluAspProAla 160
Qy 498 AAAAAAGGACTGTGTAGTAACTTTTTGTGTGATGCCACACCAGGCACGGAAATTTCCATG 557
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 LysLysGlyLeuCysSerAsnPheLeuCysAspAlaThrProGlyThrGluIleSerMet 180
Qy 558 ACAGGGCCCACAGGAAAAGTATTGCTTCTGCCAGCAGACGCGAACGCGCCATTAATCTGT 617
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ThrGlyProThrGlyLysValLeuLeuLeuProAlaAspAlaAsnAlaProLeuIleCys 200
Qy 618 GTCGCAACGGGGACTGGAATCGCGCCTTTTCGCTCATTCTGGCGCCGTTGCTTCATCGAG 677
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 201 ValAlaThrGlyThrGlyIleAlaProPheArgSerPheTrpArgArgCysPheIleGlu 220
Qy 678 AATGTCCCAAGTTATAAGTTCACTGGCCTTTTCTGGTTGTTTATGGGTGTCGCTAACTCT 737
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 AsnValProSerTyrLysPheThrGlyLeuPheTrpLeuPheMetGlyValAlaAsnSer 240
Qy 738 GATGCTAAATTGTACGATGAGGAACTGCAAGCTATCGCAAAAGCGTATCCGGGACAATTC 797
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 AspAlaLysLeuTyrAspGluGluLeuGlnAlaIleAlaLysAlaTyrProGlyGlnPhe 260
Qy 798 CGCCTGGACTATGCCTTGTCTCGTGAACAAAATAATCGCAAGGGCGGGAAAATGTACATC 857
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 261 ArgLeuAspTyrAlaLeuSerArgGluGlnAsnAsnArgLysGlyGlyLysMetTyrIle 280
Qy 858 CAAGATAAGGTTGAAGAGTATGCCGACGAAATTTTCGATTTATTGGATAATGGGGCGCAC 917
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 281 GlnAspLysValGluGluTyrAlaAspGluIlePheAspLeuLeuAspAsnGlyAlaHis 300
Qy 918 ATGTACTTCTGCGGGTTAAAGGGGATGATGCCAGGCATCCAAGATATGTTAGAACGCGTT 977
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 MetTyrPheCysGlyLeuLysGlyMetMetProGlyIleGlnAspMetLeuGluArgVal 320
Qy 978 GCAAAAGAAAAGGGGCTGAACTACGAAGAGTGGGTCGAGGGGTTAAAGCACAAGAATCAA 1037
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 321 AlaLysGluLysGlyLeuAsnTyrGluGluTrpValGluGlyLeuLysHisLysAsnGln 340
Qy 1038 TGGCATGTTGAAGTCTAC 1055
||||||||||||||||||
Db 341 TrpHisValGluValTyr 346
SEQ ID NO: 2
RESULT 1
Q6PSL5_CHLRE
ID Q6PSL5_CHLRE Unreviewed; 1151 AA.
AC Q6PSL5;
DT 05-JUL-2004, integrated into UniProtKB/TrEMBL.
DT 05-JUL-2004, sequence version 1.
DT 28-JAN-2026, entry version 114.
DE SubName: Full=Fe-hydrogenase assembly protein {ECO:0000313|EMBL:AAS92601.1};
GN Name=HydEF {ECO:0000313|EMBL:AAS92601.1};
GN ORFNames=CHLRE_06g296750v5 {ECO:0000313|EMBL:PNW82842.1};
OS Chlamydomonas reinhardtii (Chlamydomonas smithii).
OC Eukaryota; Viridiplantae; Chlorophyta; core chlorophytes; Chlorophyceae;
OC CS clade; Chlamydomonadales; Chlamydomonadaceae; Chlamydomonas.
OX NCBI_TaxID=3055 {ECO:0000313|EMBL:AAS92601.1};
RN [1] {ECO:0000313|EMBL:AAS92601.1}
RP NUCLEOTIDE SEQUENCE.
RX PubMed=15082711; DOI=10.1074/jbc.M403206200;
RA Posewitz M.C., King P.W., Smolinski S.L., Zhang L., Seibert M.,
RA Ghirardi M.L.;
RT "Discovery of two novel radical S-adenosylmethionine proteins required for
RT the assembly of an active [Fe] hydrogenase.";
RL J. Biol. Chem. 279:25711-25720(2004).
RN [2] {ECO:0000313|EMBL:PNW82842.1, ECO:0000313|Proteomes:UP000006906}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=CC-503 {ECO:0000313|Proteomes:UP000006906}, and CC-503 cw92 mt+
RC {ECO:0000313|EMBL:PNW82842.1};
RX PubMed=17932292; DOI=10.1126/science.1143609;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Cardol P., Cerutti H., Chanfreau G., Chen C.L.,
RA Cognat V., Croft M.T., Dent R., Dutcher S., Fernandez E., Fukuzawa H.,
RA Gonzalez-Ballester D., Gonzalez-Halphen D., Hallmann A., Hanikenne M.,
RA Hippler M., Inwood W., Jabbari K., Kalanon M., Kuras R., Lefebvre P.A.,
RA Lemaire S.D., Lobanov A.V., Lohr M., Manuell A., Meier I., Mets L.,
RA Mittag M., Mittelmeier T., Moroney J.V., Moseley J., Napoli C.,
RA Nedelcu A.M., Niyogi K., Novoselov S.V., Paulsen I.T., Pazour G.,
RA Purton S., Ral J.P., Riano-Pachon D.M., Riekhof W., Rymarquis L.,
RA Schroda M., Stern D., Umen J., Willows R., Wilson N., Zimmer S.L.,
RA Allmer J., Balk J., Bisova K., Chen C.J., Elias M., Gendler K., Hauser C.,
RA Lamb M.R., Ledford H., Long J.C., Minagawa J., Page M.D., Pan J.,
RA Pootakham W., Roje S., Rose A., Stahlberg E., Terauchi A.M., Yang P.,
RA Ball S., Bowler C., Dieckmann C.L., Gladyshev V.N., Green P., Jorgensen R.,
RA Mayfield S., Mueller-Roeber B., Rajamani S., Sayre R.T., Brokstein P.,
RA Dubchak I., Goodstein D., Hornick L., Huang Y.W., Jhaveri J., Luo Y.,
RA Martinez D., Ngau W.C., Otillar B., Poliakov A., Porter A., Szajkowski L.,
RA Werner G., Zhou K., Grigoriev I.V., Rokhsar D.S., Grossman A.R.;
RT "The Chlamydomonas genome reveals the evolution of key animal and plant
RT functions.";
RL Science 318:245-250(2007).
RN [3] {ECO:0000313|EMBL:PNW82842.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=CC-503 cw92 mt+ {ECO:0000313|EMBL:PNW82842.1};
RG Chlamydomonas Annotation Team;
RG JGI Annotation Team;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Grigoriev I.V., Rokhsar D.S.;
RT "WGS assembly of Chlamydomonas reinhardtii.";
RL Submitted (JUL-2017) to the EMBL/GenBank/DDBJ databases.
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DR EMBL; AY582739; AAS92601.1; -; mRNA.
DR EMBL; CM008967; PNW82842.1; -; Genomic_DNA.
DR AlphaFoldDB; Q6PSL5; -.
DR SMR; Q6PSL5; -.
DR STRING; 3055.Q6PSL5; -.
DR PaxDb; 3055-EDP05198; -.
DR Gramene; PNW82842; PNW82842; CHLRE_06g296750v5.
DR eggNOG; ENOG502RA0G; Eukaryota.
DR HOGENOM; CLU_276519_0_0_1; -.
DR OMA; RRALEPW; -.
DR OrthoDB; 188276at2759; -.
DR BioCyc; CHLAMY:MONOMER-17780; -.
DR BioCyc; MetaCyc:MONOMER-17780; -.
DR Proteomes; UP000006906; Chromosome 6.
DR GO; GO:0005525; F:GTP binding; IEA:InterPro.
DR GO; GO:0051536; F:iron-sulfur cluster binding; IEA:UniProtKB-KW.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0016740; F:transferase activity; IBA:GO_Central.
DR CDD; cd01335; Radical_SAM; 1.
DR Gene3D; 3.40.50.11410; -; 1.
DR Gene3D; 3.40.50.11420; -; 1.
DR Gene3D; 3.20.20.70; Aldolase class I; 2.
DR Gene3D; 3.40.50.300; P-loop containing nucleotide triphosphate hydrolases; 1.
DR InterPro; IPR013785; Aldolase_TIM.
DR InterPro; IPR006638; Elp3/MiaA/NifB-like_rSAM.
DR InterPro; IPR006073; GTP-bd.
DR InterPro; IPR034422; HydE/PylB-like.
DR InterPro; IPR041606; HydF_dimer.
DR InterPro; IPR040644; HydF_tetramer.
DR InterPro; IPR027417; P-loop_NTPase.
DR InterPro; IPR007197; rSAM.
DR InterPro; IPR058240; rSAM_sf.
DR InterPro; IPR005225; Small_GTP-bd.
DR NCBIfam; TIGR00231; small_GTP; 1.
DR PANTHER; PTHR43726; 3-METHYLORNITHINE SYNTHASE; 1.
DR PANTHER; PTHR43726:SF1; BIOTIN SYNTHASE; 1.
DR Pfam; PF18128; HydF_dimer; 1.
DR Pfam; PF18133; HydF_tetramer; 1.
DR Pfam; PF01926; MMR_HSR1; 1.
DR Pfam; PF04055; Radical_SAM; 1.
DR PRINTS; PR00326; GTP1OBG.
DR SFLD; SFLDG01082; B12-binding_domain_containing; 1.
DR SFLD; SFLDG01060; BATS_domain_containing; 1.
DR SFLD; SFLDG01280; HydE/PylB-like; 1.
DR SFLD; SFLDS00029; Radical_SAM; 1.
DR SMART; SM00729; Elp3; 1.
DR SUPFAM; SSF52540; P-loop containing nucleoside triphosphate hydrolases; 1.
DR SUPFAM; SSF102114; Radical SAM enzymes; 1.
DR PROSITE; PS51918; RADICAL_SAM; 1.
PE 2: Evidence at transcript level;
KW Iron {ECO:0000256|ARBA:ARBA00023004};
KW Iron-sulfur {ECO:0000256|ARBA:ARBA00023014};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723};
KW Reference proteome {ECO:0000313|Proteomes:UP000006906};
KW S-adenosyl-L-methionine {ECO:0000256|ARBA:ARBA00022691}.
FT DOMAIN 140..405
FT /note="Radical SAM core"
FT /evidence="ECO:0000259|PROSITE:PS51918"
FT REGION 1..30
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT REGION 534..593
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT COMPBIAS 534..543
FT /note="Basic and acidic residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT COMPBIAS 555..567
FT /note="Basic and acidic residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT COMPBIAS 581..593
FT /note="Gly residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
SQ SEQUENCE 1151 AA; 121934 MW; 0052D0B0D67658B2 CRC64;
Alignment Scores:
Length: 1151
Score: 5947.00 Matches: 1151
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 93.1% Indels: 0
Gaps: 0
US-17-916-137-2 (1-3478) x Q6PSL5_CHLRE (1-1151)
Qy 18 ATGGCTCATAGTTTAAGCGCACATTCCCGTCAGGCCGGAGATCGCAAACTGGGCGCAGGT 77
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetAlaHisSerLeuSerAlaHisSerArgGlnAlaGlyAspArgLysLeuGlyAlaGly 20
Qy 78 GCGGCATCTAGCCGCCCATCATGTCCTTCTCGCCGCATTGTCCGCGTGGCGGCCCATGCT 137
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 AlaAlaSerSerArgProSerCysProSerArgArgIleValArgValAlaAlaHisAla 40
Qy 138 TCTGCGTCCAAGGCGACTCCCGATGTACCTGTTGACGATCTTCCTCCTGCGCATGCCCGT 197
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 SerAlaSerLysAlaThrProAspValProValAspAspLeuProProAlaHisAlaArg 60
Qy 198 GCAGCAGTAGCGGCCGCTAACCGTCGTGCGCGCGCTATGGCATCAGCGGAGGCCGCGGCA 257
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AlaAlaValAlaAlaAlaAsnArgArgAlaArgAlaMetAlaSerAlaGluAlaAlaAla 80
Qy 258 GAGACCCTGGGCGATTTCCTGGGCTTAGGGAAGGGGGGGCTTTCGCCGGGCGCAACCGCC 317
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 GluThrLeuGlyAspPheLeuGlyLeuGlyLysGlyGlyLeuSerProGlyAlaThrAla 100
Qy 318 AACCTTGATCGTGAGCAAGTATTAGGTGTGTTGGAGGCGGTCTGGCGTCGTGGAGACCTT 377
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 AsnLeuAspArgGluGlnValLeuGlyValLeuGluAlaValTrpArgArgGlyAspLeu 120
Qy 378 AATCTGGAGCGTGCGTTGTACAGCCATGCCAATGCGGTGACTAACAAATACTGTGGTGGG 437
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 AsnLeuGluArgAlaLeuTyrSerHisAlaAsnAlaValThrAsnLysTyrCysGlyGly 140
Qy 438 GGGGTCTACTATCGCGGCCTTGTGGAGTTCAGTAATATTTGCCAAAACGATTGCTCATAT 497
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 141 GlyValTyrTyrArgGlyLeuValGluPheSerAsnIleCysGlnAsnAspCysSerTyr 160
Qy 498 TGTGGGATTCGCAACAATCAAAAAGAAGTTTGGCGCTATACTATGCCGGTTGAAGAGGTG 557
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 CysGlyIleArgAsnAsnGlnLysGluValTrpArgTyrThrMetProValGluGluVal 180
Qy 558 GTCGAGGTAGCTAAGTGGGCGCTTGAAAACGGCATTCGCAACATTATGTTGCAAGGGGGT 617
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ValGluValAlaLysTrpAlaLeuGluAsnGlyIleArgAsnIleMetLeuGlnGlyGly 200
Qy 618 GAATTAAAGACAGAGCAACGCTTAGCCTACTTGGAGGCATGCGTCCGTGCGATTCGCGAG 677
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 201 GluLeuLysThrGluGlnArgLeuAlaTyrLeuGluAlaCysValArgAlaIleArgGlu 220
Qy 678 GAGACAACGCAGTTAGACCTTGAAATGCGCGCACGTGCCGCGAGTACGACAACAGCGGAA 737
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 GluThrThrGlnLeuAspLeuGluMetArgAlaArgAlaAlaSerThrThrThrAlaGlu 240
Qy 738 GCGGCAGCCAGTGCACAGGCAGACGCCGAAGCTAAACGCGGCGAGCCAGAATTGGGTGTG 797
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 AlaAlaAlaSerAlaGlnAlaAspAlaGluAlaLysArgGlyGluProGluLeuGlyVal 260
Qy 798 GTCGTATCCTTAAGCGTCGGAGAACTTCCTATGGAACAGTATGAGCGTTTGTTCCGCGCC 857
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 261 ValValSerLeuSerValGlyGluLeuProMetGluGlnTyrGluArgLeuPheArgAla 280
Qy 858 GGCGCCCGTCGCTATCTGATCCGCATTGAAACCTCGAATCCCGACCTGTACGCGGCACTT 917
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 281 GlyAlaArgArgTyrLeuIleArgIleGluThrSerAsnProAspLeuTyrAlaAlaLeu 300
Qy 918 CACCCGGAGCCGATGTCCTGGCACGCGCGTGTAGAATGCCTGCGCAACCTGAAGAAAGCT 977
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 HisProGluProMetSerTrpHisAlaArgValGluCysLeuArgAsnLeuLysLysAla 320
Qy 978 GGGTATATGTTGGGCACAGGAGTGATGGTCGGCTTGCCGGGACAAACCTTACACGACCTG 1037
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 321 GlyTyrMetLeuGlyThrGlyValMetValGlyLeuProGlyGlnThrLeuHisAspLeu 340
Qy 1038 GCTGGGGATGTCATGTTCTTTCGCGACATTAAAGCGGACATGATCGGTATGGGCCCCTTC 1097
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 341 AlaGlyAspValMetPhePheArgAspIleLysAlaAspMetIleGlyMetGlyProPhe 360
Qy 1098 ATTACGCAGCCTGGGACGCCTGCAACTGATAAGTGGACCGCGCTGTATCCGAACGCCAAT 1157
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 IleThrGlnProGlyThrProAlaThrAspLysTrpThrAlaLeuTyrProAsnAlaAsn 380
Qy 1158 AAGAACAGCCACATGAAATCTATGTTCGATCTGACCACTGCTATGAATGCACTTGTACGT 1217
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 381 LysAsnSerHisMetLysSerMetPheAspLeuThrThrAlaMetAsnAlaLeuValArg 400
Qy 1218 ATTACGATGGGGAACGTAAATATCAGTGCTACGACTGCATTACAAGCGATTATCCCCACT 1277
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 IleThrMetGlyAsnValAsnIleSerAlaThrThrAlaLeuGlnAlaIleIleProThr 420
Qy 1278 GGACGTGAAATTGCGCTTGAGCGCGGCGCAAATGTTGTCATGCCTATTTTAACGCCTACT 1337
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 GlyArgGluIleAlaLeuGluArgGlyAlaAsnValValMetProIleLeuThrProThr 440
Qy 1338 CAGTATCGCGAGTCCTATCAGTTATATGAGGGTAAGCCCTGCATTACGGATACTGCTGTT 1397
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 441 GlnTyrArgGluSerTyrGlnLeuTyrGluGlyLysProCysIleThrAspThrAlaVal 460
Qy 1398 CAGTGTCGTCGTTGCCTGGACATGCGTTTGCACTCCGTTGGCAAAACGTCTGCGGCCGGA 1457
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 461 GlnCysArgArgCysLeuAspMetArgLeuHisSerValGlyLysThrSerAlaAlaGly 480
Qy 1458 GTTTGGGGCGATCCTGCTTCGTTCTTGCATCCCATCGTTGGCGTCCCAGTCCCGCACGAC 1517
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 ValTrpGlyAspProAlaSerPheLeuHisProIleValGlyValProValProHisAsp 500
Qy 1518 TTGTCATCACCTGCTTTGGCCGCTGCTGCAAGTGCGGATTTTCACGAGGTTGGCGCAGGT 1577
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 501 LeuSerSerProAlaLeuAlaAlaAlaAlaSerAlaAspPheHisGluValGlyAlaGly 520
Qy 1578 CCCTGGAACCCCATCCGACTAGAGCGTCTGGTTGAAGTGCCGGACCGTTACCCTGACCCC 1637
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 521 ProTrpAsnProIleArgLeuGluArgLeuValGluValProAspArgTyrProAspPro 540
Qy 1638 GATAACCACGGACGCAAAAAGGCAGGAGCCGGGAAAGGAGGCAAGGCGCATGACTCTCAC 1697
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 AspAsnHisGlyArgLysLysAlaGlyAlaGlyLysGlyGlyLysAlaHisAspSerHis 560
Qy 1698 GACGATGGCGATCACGACGACCACCACCACCACCACGGAGCTGCCCCCGCGGGTGCAGCG 1757
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 561 AspAspGlyAspHisAspAspHisHisHisHisHisGlyAlaAlaProAlaGlyAlaAla 580
Qy 1758 GCTGGAAAGGGTACCGGTGCAGCTGCAATTGGTGGCGGAGCGGGGGCTAGCCGTCAACGC 1817
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 581 AlaGlyLysGlyThrGlyAlaAlaAlaIleGlyGlyGlyAlaGlyAlaSerArgGlnArg 600
Qy 1818 GTAGCAGGGGCTGCTGCCGCCTCTGCTCGTCTGTGTGCGGGAGCTCGCCGCGCTGGCCGT 1877
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 ValAlaGlyAlaAlaAlaAlaSerAlaArgLeuCysAlaGlyAlaArgArgAlaGlyArg 620
Qy 1878 GTTGTCGCGTCGCCATTGCGCCCAGCTGCCGCTTGCCGTGGTGTGGCCGTCAAGGCCGCA 1937
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 621 ValValAlaSerProLeuArgProAlaAlaAlaCysArgGlyValAlaValLysAlaAla 640
Qy 1938 GCTGCTGCTGCGGGTGAAGACGCTGGTGCTGGTACGTCTGGCGTAGGTTCAAATATTGTT 1997
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 641 AlaAlaAlaAlaGlyGluAspAlaGlyAlaGlyThrSerGlyValGlySerAsnIleVal 660
Qy 1998 ACGTCTCCCGGAATCGCTAGCACTACGGCACATGGTGTTCCTCGTATCAATATTGGGGTG 2057
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 ThrSerProGlyIleAlaSerThrThrAlaHisGlyValProArgIleAsnIleGlyVal 680
Qy 2058 TTCGGCGTGATGAATGCAGGAAAGTCTACACTTGTGAATGCTCTGGCGCAACAGGAAGCA 2117
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 681 PheGlyValMetAsnAlaGlyLysSerThrLeuValAsnAlaLeuAlaGlnGlnGluAla 700
Qy 2118 TGCATTGTTGACTCAACCCCCGGCACGACCGCAGATGTAAAAACAGTTTTGCTTGAGCTT 2177
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 701 CysIleValAspSerThrProGlyThrThrAlaAspValLysThrValLeuLeuGluLeu 720
Qy 2178 CATGCCCTTGGACCAGCCAAGTTGCTGGACACAGCCGGATTAGACGAAGTCGGTGGACTT 2237
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 HisAlaLeuGlyProAlaLysLeuLeuAspThrAlaGlyLeuAspGluValGlyGlyLeu 740
Qy 2238 GGGGATAAAAAGCGCCGTAAAGCCCTGAATACGCTGAAGGAGTGCGATGTTGCTGTGCTG 2297
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 741 GlyAspLysLysArgArgLysAlaLeuAsnThrLeuLysGluCysAspValAlaValLeu 760
Qy 2298 GTTGTTGACACTGATACGGCAGCCGCCGCAATCAAATCGGGACGCCTTGCTGAAGCCCTG 2357
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 761 ValValAspThrAspThrAlaAlaAlaAlaIleLysSerGlyArgLeuAlaGluAlaLeu 780
Qy 2358 GAATGGGAATCGAAAGTAATGGAACAGGCACACAAGTATAATGTCAGTCCTGTACTGCTT 2417
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 GluTrpGluSerLysValMetGluGlnAlaHisLysTyrAsnValSerProValLeuLeu 800
Qy 2418 CTGAATGTAAAATCACGCGGGCTGCCTGAAGCGCAGGCAGCTAGCATGCTTGAGGCGGTC 2477
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 801 LeuAsnValLysSerArgGlyLeuProGluAlaGlnAlaAlaSerMetLeuGluAlaVal 820
Qy 2478 GCTGGTATGCTTGACCCATCCAAGCAAATCCCTCGTATGTCGCTGGATTTAGCGTCCACC 2537
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 821 AlaGlyMetLeuAspProSerLysGlnIleProArgMetSerLeuAspLeuAlaSerThr 840
Qy 2538 CCCCTGCACGAGCGTAGTACGATTACGTCTGCATTCGTCAAGGAAGGAGCAGTGCGCAGT 2597
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 ProLeuHisGluArgSerThrIleThrSerAlaPheValLysGluGlyAlaValArgSer 860
Qy 2598 TCACGCTATGGGGCTCCTCTGCCGGGGTGTTTGCCCCGTTGGTCTCTTGGACGCAACGCC 2657
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 861 SerArgTyrGlyAlaProLeuProGlyCysLeuProArgTrpSerLeuGlyArgAsnAla 880
Qy 2658 CGCTTACTGATGGTAATCCCGATGGATGCCGAAACCCCAGGTGGACGTCTTCTTCGTCCT 2717
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 881 ArgLeuLeuMetValIleProMetAspAlaGluThrProGlyGlyArgLeuLeuArgPro 900
Qy 2718 CAAGCGCAAGTTATGGAGGAAGCAATCCGCCATTGGGCTACCGTGCTTTCCGTACGCCTG 2777
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 GlnAlaGlnValMetGluGluAlaIleArgHisTrpAlaThrValLeuSerValArgLeu 920
Qy 2778 GATTTAGACGCCGCACGTGGCAAATTGGGGCCCGAGGCCTGTGAGATGGAGCGTCAGCGT 2837
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 921 AspLeuAspAlaAlaArgGlyLysLeuGlyProGluAlaCysGluMetGluArgGlnArg 940
Qy 2838 TTCGACGGGGTAATTGCTATGATGGAACGTAATGACGGACCCACCCTGGTGGTGACAGAT 2897
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 941 PheAspGlyValIleAlaMetMetGluArgAsnAspGlyProThrLeuValValThrAsp 960
Qy 2898 TCTCAAGCGATCGATGTTGTACACCCTTGGACCCTGGACCGCTCATCAGGACGTCCGTTG 2957
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 SerGlnAlaIleAspValValHisProTrpThrLeuAspArgSerSerGlyArgProLeu 980
Qy 2958 GTTCCAATTACTACCTTTAGCATCGCTATGGCGTACCAACAAAACGGCGGACGTTTGGAC 3017
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 981 ValProIleThrThrPheSerIleAlaMetAlaTyrGlnGlnAsnGlyGlyArgLeuAsp 1000
Qy 3018 CCGTTTGTGGAAGGATTGGAAGCATTGGAGACGTTACAAGATGGGGATCGCGTTCTTATT 3077
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1001 ProPheValGluGlyLeuGluAlaLeuGluThrLeuGlnAspGlyAspArgValLeuIle 1020
Qy 3078 TCGGAAGCGTGTAATCATAACCGTATCACCTCCGCTTGCAACGACATTGGAATGGTGCAG 3137
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 SerGluAlaCysAsnHisAsnArgIleThrSerAlaCysAsnAspIleGlyMetValGln 1040
Qy 3138 ATCCCTAACAAGCTGGAGGCAGCACTTGGCGGGAAAAAGTTACAAATCGAACATGCGTTT 3197
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1041 IleProAsnLysLeuGluAlaAlaLeuGlyGlyLysLysLeuGlnIleGluHisAlaPhe 1060
Qy 3198 GGTCGCGAGTTTCCCGAGCTTGAGAGTGGGGGTATGGATGGATTGAAGTTAGCGATCCAT 3257
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1061 GlyArgGluPheProGluLeuGluSerGlyGlyMetAspGlyLeuLysLeuAlaIleHis 1080
Qy 3258 TGTGGAGGGTGTATGATCGACGCTCAGAAAATGCAGCAGCGTATGAAAGACTTGCATGAG 3317
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 CysGlyGlyCysMetIleAspAlaGlnLysMetGlnGlnArgMetLysAspLeuHisGlu 1100
Qy 3318 GCTGGGGTGCCGGTCACTAATTATGGGGTGTTTTTCTCTTGGGCAGCTTGGCCCGACGCC 3377
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1101 AlaGlyValProValThrAsnTyrGlyValPhePheSerTrpAlaAlaTrpProAspAla 1120
Qy 3378 CTTCGTCGCGCATTGGAACCGTGGGGAGTGGAGCCACCGGTAGGTACTCCTGCTACCCCC 3437
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1121 LeuArgArgAlaLeuGluProTrpGlyValGluProProValGlyThrProAlaThrPro 1140
Qy 3438 GCAGCCGCGCCTGCTACGGCAGCGTCCGGGGTA 3470
|||||||||||||||||||||||||||||||||
Db 1141 AlaAlaAlaProAlaThrAlaAlaSerGlyVal 1151
SEQ ID NO: 4
RESULT 1
Q6PSL4_CHLRE
ID Q6PSL4_CHLRE Unreviewed; 567 AA.
AC Q6PSL4;
DT 05-JUL-2004, integrated into UniProtKB/TrEMBL.
DT 05-JUL-2004, sequence version 1.
DT 28-JAN-2026, entry version 106.
DE SubName: Full=Fe-hydrogenase assembly protein {ECO:0000313|EMBL:AAS92602.1};
GN Name=HydG {ECO:0000313|EMBL:AAS92602.1};
GN ORFNames=CHLRE_06g296700v5 {ECO:0000313|EMBL:PNW82841.1};
OS Chlamydomonas reinhardtii (Chlamydomonas smithii).
OC Eukaryota; Viridiplantae; Chlorophyta; core chlorophytes; Chlorophyceae;
OC CS clade; Chlamydomonadales; Chlamydomonadaceae; Chlamydomonas.
OX NCBI_TaxID=3055 {ECO:0000313|EMBL:AAS92602.1};
RN [1] {ECO:0000313|EMBL:AAS92602.1}
RP NUCLEOTIDE SEQUENCE.
RX PubMed=15082711; DOI=10.1074/jbc.M403206200;
RA Posewitz M.C., King P.W., Smolinski S.L., Zhang L., Seibert M.,
RA Ghirardi M.L.;
RT "Discovery of two novel radical S-adenosylmethionine proteins required for
RT the assembly of an active [Fe] hydrogenase.";
RL J. Biol. Chem. 279:25711-25720(2004).
RN [2] {ECO:0000313|EMBL:PNW82841.1, ECO:0000313|Proteomes:UP000006906}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=CC-503 {ECO:0000313|Proteomes:UP000006906}, and CC-503 cw92 mt+
RC {ECO:0000313|EMBL:PNW82841.1};
RX PubMed=17932292; DOI=10.1126/science.1143609;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Cardol P., Cerutti H., Chanfreau G., Chen C.L.,
RA Cognat V., Croft M.T., Dent R., Dutcher S., Fernandez E., Fukuzawa H.,
RA Gonzalez-Ballester D., Gonzalez-Halphen D., Hallmann A., Hanikenne M.,
RA Hippler M., Inwood W., Jabbari K., Kalanon M., Kuras R., Lefebvre P.A.,
RA Lemaire S.D., Lobanov A.V., Lohr M., Manuell A., Meier I., Mets L.,
RA Mittag M., Mittelmeier T., Moroney J.V., Moseley J., Napoli C.,
RA Nedelcu A.M., Niyogi K., Novoselov S.V., Paulsen I.T., Pazour G.,
RA Purton S., Ral J.P., Riano-Pachon D.M., Riekhof W., Rymarquis L.,
RA Schroda M., Stern D., Umen J., Willows R., Wilson N., Zimmer S.L.,
RA Allmer J., Balk J., Bisova K., Chen C.J., Elias M., Gendler K., Hauser C.,
RA Lamb M.R., Ledford H., Long J.C., Minagawa J., Page M.D., Pan J.,
RA Pootakham W., Roje S., Rose A., Stahlberg E., Terauchi A.M., Yang P.,
RA Ball S., Bowler C., Dieckmann C.L., Gladyshev V.N., Green P., Jorgensen R.,
RA Mayfield S., Mueller-Roeber B., Rajamani S., Sayre R.T., Brokstein P.,
RA Dubchak I., Goodstein D., Hornick L., Huang Y.W., Jhaveri J., Luo Y.,
RA Martinez D., Ngau W.C., Otillar B., Poliakov A., Porter A., Szajkowski L.,
RA Werner G., Zhou K., Grigoriev I.V., Rokhsar D.S., Grossman A.R.;
RT "The Chlamydomonas genome reveals the evolution of key animal and plant
RT functions.";
RL Science 318:245-250(2007).
RN [3] {ECO:0000313|EMBL:PNW82841.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=CC-503 cw92 mt+ {ECO:0000313|EMBL:PNW82841.1};
RG Chlamydomonas Annotation Team;
RG JGI Annotation Team;
RA Merchant S.S., Prochnik S.E., Vallon O., Harris E.H., Karpowicz S.J.,
RA Witman G.B., Terry A., Salamov A., Fritz-Laylin L.K., Marechal-Drouard L.,
RA Marshall W.F., Qu L.H., Nelson D.R., Sanderfoot A.A., Spalding M.H.,
RA Kapitonov V.V., Ren Q., Ferris P., Lindquist E., Shapiro H., Lucas S.M.,
RA Grimwood J., Schmutz J., Grigoriev I.V., Rokhsar D.S.;
RT "WGS assembly of Chlamydomonas reinhardtii.";
RL Submitted (JUL-2017) to the EMBL/GenBank/DDBJ databases.
CC -!- COFACTOR:
CC Name=[4Fe-4S] cluster; Xref=ChEBI:CHEBI:49883;
CC Evidence={ECO:0000256|ARBA:ARBA00001966};
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DR EMBL; AY582740; AAS92602.1; -; mRNA.
DR EMBL; CM008967; PNW82841.1; -; Genomic_DNA.
DR RefSeq; XP_001691319.1; XM_001691267.1.
DR AlphaFoldDB; Q6PSL4; -.
DR STRING; 3055.Q6PSL4; -.
DR PaxDb; 3055-EDP05052; -.
DR GeneID; 5717047; -.
DR Gramene; PNW82841; PNW82841; CHLRE_06g296700v5.
DR KEGG; cre:CHLRE_06g296700v5; -.
DR eggNOG; ENOG502R3WT; Eukaryota.
DR HOGENOM; CLU_046249_0_0_1; -.
DR OMA; QIFQETY; -.
DR OrthoDB; 10261561at2759; -.
DR BioCyc; CHLAMY:MONOMER-17781; -.
DR BioCyc; MetaCyc:MONOMER-17781; -.
DR Proteomes; UP000006906; Chromosome 6.
DR GO; GO:0051539; F:4 iron, 4 sulfur cluster binding; IEA:UniProtKB-KW.
DR GO; GO:0003824; F:catalytic activity; IEA:InterPro.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0044272; P:sulfur compound biosynthetic process; IEA:UniProtKB-ARBA.
DR GO; GO:0042364; P:water-soluble vitamin biosynthetic process; IEA:UniProtKB-ARBA.
DR Gene3D; 3.20.20.70; Aldolase class I; 1.
DR InterPro; IPR013785; Aldolase_TIM.
DR InterPro; IPR010722; BATS_dom.
DR InterPro; IPR024007; FeFe-hyd_mat_HydG.
DR InterPro; IPR007197; rSAM.
DR InterPro; IPR058240; rSAM_sf.
DR InterPro; IPR034428; ThiH/NoCL/HydG-like.
DR NCBIfam; TIGR03955; rSAM_HydG; 1.
DR PANTHER; PTHR43583; 2-IMINOACETATE SYNTHASE; 1.
DR PANTHER; PTHR43583:SF2; THIAZOLE BIOSYNTHESIS PROTEIN; 1.
DR Pfam; PF06968; BATS; 1.
DR Pfam; PF04055; Radical_SAM; 1.
DR SFLD; SFLDG01060; BATS_domain_containing; 1.
DR SFLD; SFLDG01081; cleavage_of_the_Ca-Cb_bond_in; 1.
DR SFLD; SFLDF00319; Fe_hydrogenase_maturase_(HydG; 1.
DR SFLD; SFLDS00029; Radical_SAM; 1.
DR SMART; SM00876; BATS; 1.
DR SUPFAM; SSF102114; Radical SAM enzymes; 1.
PE 2: Evidence at transcript level;
KW 4Fe-4S {ECO:0000256|ARBA:ARBA00022485};
KW Iron {ECO:0000256|ARBA:ARBA00023004};
KW Iron-sulfur {ECO:0000256|ARBA:ARBA00023014};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723};
KW Reference proteome {ECO:0000313|Proteomes:UP000006906};
KW S-adenosyl-L-methionine {ECO:0000256|ARBA:ARBA00022691}.
FT DOMAIN 359..474
FT /note="Biotin and thiamin synthesis-associated"
FT /evidence="ECO:0000259|SMART:SM00876"
SQ SEQUENCE 567 AA; 63731 MW; 4A8A357E382A46CB CRC64;
Alignment Scores:
Length: 567
Score: 2972.00 Matches: 567
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 91.2% Indels: 0
Gaps: 0
US-17-916-137-4 (1-1850) x Q6PSL4_CHLRE (1-567)
Qy 18 ATGTCGGTCCCCCTACAGTGCAATGCAGGCCGTCTTTTGGCGGGCCAGCGCCCCTGCGGC 77
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MetSerValProLeuGlnCysAsnAlaGlyArgLeuLeuAlaGlyGlnArgProCysGly 20
Qy 78 GTCCGCGCGCGTCTGAACCGCCGTGTATGTGTTCCAGTGACCGCACACGGCAAAGCATCT 137
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 21 ValArgAlaArgLeuAsnArgArgValCysValProValThrAlaHisGlyLysAlaSer 40
Qy 138 GCCACGCGCGAATACGCTGGTGACTTTCTTCCAGGGACCACTATTAGTCATGCGTGGTCC 197
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 41 AlaThrArgGluTyrAlaGlyAspPheLeuProGlyThrThrIleSerHisAlaTrpSer 60
Qy 198 GTGGAGCGTGAAACTCATCACCGTTATCGTAATCCGGCCGAGTGGATTAACGAGGCTGCC 257
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ValGluArgGluThrHisHisArgTyrArgAsnProAlaGluTrpIleAsnGluAlaAla 80
Qy 258 ATCCACAAGGCGCTTGAAACGTCAAAGGCTGATGCTCAGGACGCAGGACGCGTGCGTGAG 317
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 81 IleHisLysAlaLeuGluThrSerLysAlaAspAlaGlnAspAlaGlyArgValArgGlu 100
Qy 318 ATTTTGGCGAAGGCTAAGGAAAAGGCTTTTGTTACTGAACATGCGCCTGTAAATGCGGAA 377
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 IleLeuAlaLysAlaLysGluLysAlaPheValThrGluHisAlaProValAsnAlaGlu 120
Qy 378 AGTAAATCCGAATTTGTACAAGGGCTTACCTTGGAGGAGTGCGCCACCTTAATTAACGTT 437
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 SerLysSerGluPheValGlnGlyLeuThrLeuGluGluCysAlaThrLeuIleAsnVal 140
Qy 438 GATTCTAATAATGTCGAGTTGATGAATGAAATCTTCGACACTGCGCTTGCTATCAAAGAA 497
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 141 AspSerAsnAsnValGluLeuMetAsnGluIlePheAspThrAlaLeuAlaIleLysGlu 160
Qy 498 CGTATCTACGGGAACCGCGTCGTGCTGTTCGCGCCCTTGTACATTGCTAATCATTGTATG 557
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 ArgIleTyrGlyAsnArgValValLeuPheAlaProLeuTyrIleAlaAsnHisCysMet 180
Qy 558 AACACTTGCACTTATTGTGCGTTTCGCTCAGCCAATAAGGGCATGGAACGCTCTATTCTG 617
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 AsnThrCysThrTyrCysAlaPheArgSerAlaAsnLysGlyMetGluArgSerIleLeu 200
Qy 618 ACAGACGATGACCTGCGTGAAGAAGTTGCCGCCTTGCAACGTCAAGGTCATCGCCGCATT 677
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 201 ThrAspAspAspLeuArgGluGluValAlaAlaLeuGlnArgGlnGlyHisArgArgIle 220
Qy 678 TTAGCCCTTACTGGAGAACATCCTAAATACACTTTCGACAATTTCTTGCATGCAGTCAAT 737
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 LeuAlaLeuThrGlyGluHisProLysTyrThrPheAspAsnPheLeuHisAlaValAsn 240
Qy 738 GTAATCGCTTCCGTGAAGACAGAGCCTGAAGGGTCCATTCGCCGTATCAATGTCGAAATC 797
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 ValIleAlaSerValLysThrGluProGluGlySerIleArgArgIleAsnValGluIle 260
Qy 798 CCACCACTTAGTGTCTCAGACATGCGCCGCCTTAAGAATACAGATTCAGTTGGCACGTTC 857
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 261 ProProLeuSerValSerAspMetArgArgLeuLysAsnThrAspSerValGlyThrPhe 280
Qy 858 GTTCTGTTTCAAGAGACATATCACCGCGACACATTCAAGGTAATGCATCCGTCAGGTCCA 917
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 281 ValLeuPheGlnGluThrTyrHisArgAspThrPheLysValMetHisProSerGlyPro 300
Qy 918 AAGAGCGACTTTGATTTTCGCGTCTTGACCCAGGACCGCGCCATGCGCGCAGGCTTGGAC 977
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LysSerAspPheAspPheArgValLeuThrGlnAspArgAlaMetArgAlaGlyLeuAsp 320
Qy 978 GATGTTGGTATTGGTGCTTTATTCGGGTTATATGATTACCGTTATGAAGTTTGCGCCATG 1037
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 321 AspValGlyIleGlyAlaLeuPheGlyLeuTyrAspTyrArgTyrGluValCysAlaMet 340
Qy 1038 TTAATGCATAGCGAGCATTTGGAACGTGAGTACAACGCAGGACCACATACTATCTCTGTT 1097
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 341 LeuMetHisSerGluHisLeuGluArgGluTyrAsnAlaGlyProHisThrIleSerVal 360
Qy 1098 CCCCGTATGCGCCCCGCGGACGGAAGCGAGCTTAGTATCGCGCCTCCTTATCCTGTTAAT 1157
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 ProArgMetArgProAlaAspGlySerGluLeuSerIleAlaProProTyrProValAsn 380
Qy 1158 GATGCTGATTTTATGAAATTAGTTGCGGTCCTTCGTATTGCTGTACCATACACAGGTATG 1217
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 381 AspAlaAspPheMetLysLeuValAlaValLeuArgIleAlaValProTyrThrGlyMet 400
Qy 1218 ATCCTTAGCACTCGTGAATCACCAGAAATGCGCTCGGCGCTTCTGAAGTGTGGGATGAGT 1277
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 IleLeuSerThrArgGluSerProGluMetArgSerAlaLeuLeuLysCysGlyMetSer 420
Qy 1278 CAAATGAGTGCCGGCTCGCGCACTGATGTAGGAGCCTATCACAAAGATCACACGTTAAGT 1337
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 GlnMetSerAlaGlySerArgThrAspValGlyAlaTyrHisLysAspHisThrLeuSer 440
Qy 1338 ACAGAGGCGAATTTGAGTAAGTTAGCTGGGCAGTTTACCTTGCAGGATGAACGTCCAACT 1397
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 441 ThrGluAlaAsnLeuSerLysLeuAlaGlyGlnPheThrLeuGlnAspGluArgProThr 460
Qy 1398 AATGAGATTGTTAAGTGGCTGATGGAGGAGGGCTATGTCCCGTCTTGGTGCACAGCTTGT 1457
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 461 AsnGluIleValLysTrpLeuMetGluGluGlyTyrValProSerTrpCysThrAlaCys 480
Qy 1458 TACCGTCAAGGTCGTACGGGCGAAGACTTCATGAACATCTGTAAGGCTGGTGACATTCAC 1517
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 TyrArgGlnGlyArgThrGlyGluAspPheMetAsnIleCysLysAlaGlyAspIleHis 500
Qy 1518 GATTTCTGTCATCCCAATAGTCTGCTTACGCTTCAAGAGTACCTTATGGATTATGCAGAT 1577
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 501 AspPheCysHisProAsnSerLeuLeuThrLeuGlnGluTyrLeuMetAspTyrAlaAsp 520
Qy 1578 CCAGACCTTCGTAAGAAAGGCGAGCAAGTAATTGCCCGTGAGATGGGACCGGACGCCTCT 1637
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 521 ProAspLeuArgLysLysGlyGluGlnValIleAlaArgGluMetGlyProAspAlaSer 540
Qy 1638 GAGCCGTTATCGGCGCAAAGCCGTAAGCGCCTTGAACGCAAGATGAAACAAGTATTGGAG 1697
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 GluProLeuSerAlaGlnSerArgLysArgLeuGluArgLysMetLysGlnValLeuGlu 560
Qy 1698 GGGGAGCACGATGTATATCTT 1718
|||||||||||||||||||||
Db 561 GlyGluHisAspValTyrLeu 567
With regard to the arguments that neither Edmonds nor Sekar et al. teach or suggest methods using a HydA Fe-Fe hydrogenase or that there is no direct motivation from the recited references to select HydA from all possible Fe-Fe dehydrogenases, it is noted that (i) the recombinant E. coli cell of Edmonds comprises the C. reinhardtii hyd1 gene that encodes a Fe-Fe dehydrogenase, the C. reinhardtii hydEF and hydG genes that encode maturation enzymes, a C. reinhardtii gene encoding a ferredoxin NADP-reductase and a C. reinhardtii gene encoding ferredoxin, and (ii) Edmonds teaches a method for the production of hydrogen by culturing said E. coli cell (page 2, last 7 lines-page 3, second full paragraph; Figures 3 and 4). Therefore, (a) the argument regarding motivation to select the C. reinhardtii HydA is irrelevant in the instant case because Edmonds teach a cell that comprises said C. reinhardtii HydA as well as other C. reinhardtii proteins which are required to make hydrogen, and (b) the argument that the cited references do not teach a method for producing hydrogen with the Fe-Fe hydrogenase does not apply in the instant case in view of the method to make hydrogen by culturing a cell that expresses the C. reinhardtii HydA specifically taught by Edmonds.
With regard to the argument that the results disclosed in the specification are unexpected and that the invention produces hydrogen at a rate higher than that of Sekar et al., it is noted that while the specific rates of hydrogen production disclosed in the specification are not taught by the cited prior art, there was a reasonable expectation of success in obtaining an increase in hydrogen production by combining the expression of the C. reinhardtii HydA with the modifications taught by Sekar et al. in the E. coli cell of Edmonds to activate the pentose phosphate pathway. One of skill in the art did not need to know the specific rates of hydrogen production disclosed in the specification to be motivated to combine the teachings of Edmonds and Sekar et al. because Sekar et al. specifically teach that the modifications recited in the claims made to an E. coli cell enhance the production of hydrogen. The motivation to make the recited modifications is specifically taught by Sekar et al.
As previously indicated, Sekar et al. teach the deletion of the pfkA gene encoding phosphofructokinase in a E. coli cell genetically modified to produce hydrogen (Abstract). Sekar et al. teach that deleting the pfkA gene, which encodes phosphofructokinase, allows diverting carbon flux to the pentose phosphate pathway (page2, right column, first full paragraph). Sekar et al. teach that overexpression of zwf (encodes glucose-6-phosphate-1-dehydrogenase; Figure 1) and gnd (encodes 6-phosphogluconate dehydrogenase; Figure 1) can activate the pentose phosphate pathway and that the EMP pathway must be down regulated to enhance the glycolytic flux through the pentose phosphate pathway (page 8, left column, first full paragraph). Sekar et al. teach increased hydrogen yields by deleting the pfkA gene (SH9*) as well as by deleting the pfkA gene and increasing the expression of zwf and/or gnd (Table 2). In addition, the advantages of increasing the carbon flux through the pentose phosphate pathway for the production of hydrogen in an E. coli cell that expresses an Fe-Fe hydrogenase and maturation factors for the Fe-Fe hydrogenase were well known in the art as evidenced by Jones et al. (WO 2007/123258; previously cited). See the teachings of Jones et al. previously discussed. While there is no absolute certainty that the modifications of Sekar et al. made to the E. coli cell of Edmonds would result in an increase in hydrogen production compared to the hydrogen produced by the E. coli cell of Edmonds lacking the modifications, it is noted that an obviousness analysis does not require absolute certainty but a reasonable expectation of success. In the instant case, the modifications found to enhance production of hydrogen were made in E. coli as taught by Sekar et al. Therefore, it would be reasonable to conclude that the same modifications made in an organism of the same species as that of Edmonds that produces the same product (i.e., hydrogen) would likely result in an increase in the production of hydrogen. As such, contrary to Applicant’s assertions, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claim 28 remains rejected and new claim 38 is rejected under 35 U.S.C. 103 as being unpatentable over Edmonds (iGEM Registry of Standard Biological Parts, Part:BBa_K2300001; October 3, 2017) in view of Sekar et al. (Biotechnology for Biofuels 9:95, pages 1-11, 2016; cited in the IDS), and further in view of Bruschi et al. (Biotechnology Advances 30:1001-1010, 2012) and Penfold et al. (Biotechnology Letters 26:1879-1883, 2004; cited in the IDS).
This rejection has been discussed at length in the prior Office action. It is maintained and further applied to new claim 38 for the reasons of record and those set forth below.
Applicant argues that the references by Bruschi et al., Penfold et al. do not cure the deficiencies of Edmonds et al. and Sekar et al. Therefore, claim 28 is not obvious over the cited prior art.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the rejection of claim 28 or avoid the rejection of claim 38. For the reasons extensively discussed above, the teachings of Edmonds et al. and Sekar et al. render the recombinant microorganism of claim 1 obvious. Claims 28 and 38 are directed in part to the recombinant E. coli cell of claim 1, wherein said recombinant E. coli cell further comprises nucleic acids that encode proteins that enable the recombinant microorganism to metabolize sucrose.. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation. As previously indicated, Bruschi et al. teach the chromosomal insertion of the E. coli cscA, cscB and cscK genes in a E. coli strain that does not utilize sucrose (page 1004, left column, last line, right column, lines 1-4; Figure 2). Bruschi et al. teach that E. coli-based bioprocesses usually utilize glucose but sucrose is emerging as a preferred carbon source relative to glucose (page 1001, Introduction). Bruschi et al. teach that the ability to engineer production strains for efficient sucrose utilization will allow substitution of sucrose, and cheap sucrose-containing substrates, for glucose in industrial fermentations (page 1009, right column, second full paragraph). Penfold et al. teach a hydrogen-producing E. coli cell transformed with a plasmid that comprises genes necessary for sucrose transport into the cell and its metabolism (Abstract). Penfold et al. teach that the advantage of being able to metabolize sucrose is found in that sucrose is a major constituent of many waste materials that could be sued as feedstock for hydrogen production processes (Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the E. coli cell of Edmonds and Sekar et al. with the genetic modifications of Bruschi et al. or Penfold et al. A person of ordinary skill in the art is motivated to add these genetic modifications for the benefit of having a hydrogen-producing E. coli that is able to use sucrose, a cheaper alternative to glucose. One of ordinary skill in the art has a reasonable expectation of success at further adding these modifications because the molecular biology techniques required to make these modifications are well known in the art as evidenced by Penfold et al. and Bruschi et al. Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claim 35 remains rejected under 35 U.S.C. 103 as being unpatentable over Edmonds (iGEM Registry of Standard Biological Parts, Part:BBa_K2300001; October 3, 2017) in view of Sekar et al. (Biotechnology for Biofuels 9:95, pages 1-11, 2016; cited in the IDS), and further in view of Kumar et al. (Bioresource Technology 219:725-737, 2016).
This rejection has been discussed at length in the prior Office action. It is maintained for the reasons of record and those set forth below.
Applicant argues that the reference by Kumar et al. does not cure the deficiencies of Edmonds et al. and Sekar et al. Therefore, claim 35 is not obvious over the cited prior art.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the rejection of claim 35. For the reasons extensively discussed above, the teachings of Edmonds et al. and Sekar et al. render the recombinant microorganism of claim 1 obvious. Claim 35 is directed in part to the recombinant E. coli cell of claim 1, wherein said microorganism is encapsulated. Kumar et al. teach different immobilization types for hydrogen-producing microorganisms (Abstract). Kumar et al. tech that encapsulation is one of the main ways to immobilize cells for the production of hydrogen (page 727, right column, last 5 lines). Kumar et al. teach that the development of cell immobilization technology for the production of biohydrogen aimed to improve hydrogen production rate and yield, to address key limitations associated with suspended cells, and to improve the stability of continuous hydrogen production (page 726, right column, last five lines). Kumar et al. teach that encapsulation gave a much better biohydrogen generation performance (page 730, left column, first full paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further encapsulate the E. coli cell of Edmonds and Sekar et al. A person of ordinary skill in the art is motivated to further encapsulate the E. coli cell of Edmonds and Sekar et al. for the benefit of improving hydrogen rate and yield, avoid the limitations of suspended cells, and improve stability of continuous biohydrogen production as taught by Kumar et al. One of ordinary skill in the art has a reasonable expectation of success at further encapsulating the cells of Edmonds and Sekar et al. because encapsulation is a well-known process as evidenced by Kumar et al. Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Conclusion
No claim is in condition for allowance.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a). A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
June 25, 2025
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