Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
This application is a 371 of PCT/EP2021/083188.
The amendment filed on June 25, 2026 has been entered.
Election/Restrictions
Applicant elected without traverse of Group I with a species election of (1) Bacillus subtilis as the gram-positive microorganism, (2) SEQ ID NO:7 as the polypeptide having adenylate isopentenyltransferase, (3) LOG (cytokinin riboside 5’-monophosphate phosphoribohydrolase) from C. glutamicum (gene Cgl2379, UniProt Q8NN34), SEQ ID NO:34, DXS (1-deoxy-D-xylulose-5-phospahte synthase) from B. subtilis, SEQ ID NO:63, PurA (adenylosuccinate synthase) from B. subtilis (gene purA), and CYP450 monooxygenase (cytochrome P450 monooxygenase) from Rhodococcus fascians SEQ ID NO:93 as the polypeptides whose expression or activity in increase, and (4) (a) purine nucleotide degradation pathway of purine nucleoside phosphorylase (PNP) encoded by deoD and adenosine phosphoribosyltransferase (APRT) encoded by apt and (b) guanosine monophosphate biosynthesis pathway of IMP dehydrogenase encoded by guaB and GMP synthetase encoded by guaA as the polypeptides whose expression or activity is decreased in the reply filed on December 19, 2025.
Claims 50-52 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on December 19, 2025.
Status of Claims
Claims 1, 35-39, and 41-52 are pending.
Claims 50-52 are withdrawn.
Claims 1, 35-39, and 41-49 are under examination.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on April 2, 2026 and June 23, 2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Response to Amendments/Arguments
Nucleotide and/or Amino Acid Sequence Disclosures
Applicant’s arguments, see page 7 of the Remarks, filed June 25, 2026, with respect to the sequences in the Specification have been fully considered and are persuasive. Applicant has submitted a new Sequence Listing of the amino acid sequences at page 29 of the Specification and amended the Speciation at page 29 to recite sequence identifiers for the amino acid sequences.
Specification
Applicant’s arguments, see page 7 of the Remarks, filed June 25, 2026, with respect to the Specification have been fully considered and are persuasive. The Specification has been amended to remove all embedded hyperlinks and/or other form of browser-executable codes. Therefore, the objection to the Specification has been withdrawn.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Withdrawn Rejections
Applicant’s arguments, see pages 7-9 of the Remarks, filed June 25, 2026, with respect to claims 1 and 34-49 have been fully considered and are persuasive. Claim 1 has been amended to incorporate limitations from claim 34 (Bacillaceae or Corynebacteriaceae) and claim 40 (increased protein expression of a polypeptide having 1-deoxy-D- xylulose-5-phosphate synthase activity compared to an otherwise identical bacterium that does not carry said modification). The references of Ma, Shumann, P58592, Q8NN34, P46373, Zhang, Ashihara, O34443, AAB72065, Wang, NP_387890, NP_388517, and Liu do not disclose a bacterium having increased protein expression of a polypeptide having 1-deoxy-D- xylulose-5-phosphate synthase activity compared to an otherwise identical bacterium that does not carry said modification. Therefore, the rejections of (I) claims 1, 34-36, 38, and 48, (II) claims 37, 39, 49, (III) claims 44-45, and (IV) claims 46-47 under 35 U.S.C. 103 have been withdrawn.
Amended Rejections
Claims 1, 35-36, 38, 41-43, and 48 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ma (Genetic engineering of cytokinins and their application to agriculture. Crit Rev Biotechnol. 2008;28(3):213-32. – form PTO-1449), Schumann (Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol. 2007;62:137-89 – cited previously on form PTO-892), Julsing (Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis. Appl Microbiol Biotechnol. 2007 Jul;75(6):1377-84 – form PTO-1449), Hecht (Enzyme-assisted preparation of isotope-labeled 1-deoxy-d-xylulose 5-phosphate. J Org Chem. 2001 Jun 1;66(11):3948-52 – cited previously on form PTO-892), and Wang (Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering. J Biotechnol. 2016 Aug 10;231:115-121. Epub 2016 May 24 – form PTO-1449).
Ma discloses that cytokinins are a class of plant hormones and are involved in plant growth and development (page 213, 1st paragraph). Ma discloses the cytokinin biosynthesis pathway in bacteria (FIG. 2 at page 215). Ma discloses that this pathway is called the de novo synthetic pathway (page 214, left column). Ma discloses that the initial step in the de novo biosynthesis of cytokinin is the formation of isopentenyladenosine 5’monophosphate (iPMP) from DMAPP and AMP, ADP or ATP, which is catalyzed by IPT (also known as adenylate isopentenyltransferase) (page 214 and FIG.2). Ma discloses that IPT is the rate-limiting enzyme and is likely the control point for cytokinin synthesis (page 214, right column).
Regarding claim 1, Ma discloses E. coli expressing IPT resulted in production of cytokinins iP and zeatin (page 215).
Regarding claim 38, Ma discloses a LOG gene that encodes a cytokinin-activating enzyme (cytokinin riboside 5'-monophosphate phosphoribohydrolase) that works in the final step of cytokinin biosynthesis that converts cytokinin nucleotide (iPMP, iPDP, iPTP,tZMP, tZDP and tZTP) to the nucleoside and the free-base form, which are biologically active (page 216, 3rd full paragraph).
Regarding claim 48, Ma discloses that co-expression of IPT and a P450 monooxygenase enabled yeast to produce trans-zeatin (page 216, 2nd full paragraph).
Ma does not disclose a gram-positive bacterium or B. subtilis expressing IPT, LOG, and P450 monooxygenase, DSX (1-deoxy-D-xylulose-5-phospahte synthase) and PurA.
However, production of heterologous enzymes in Bacillus subtilis, a Gram-positive bacterium, was well established in the art, as discussed below.
Regarding claims 35-36, Schumann discloses that B. subtilis has advantages over host cells, such as E. coli, because B. subtilis (1) is nonpathogenic, (2) secretes proteins efficiently, (3) it does not produce any endotoxin, (4) has been granted a GRAS status, (5) has no significant bias in codon usage, and (6) a great deal of vital information concerning its transcription and translation mechanisms, genetic manipulation, and large-scale fermentation has been acquired (page 139, top). Schuman discloses methods of increasing expressing heterologous enzymes in B. subtilis (Sections II and III).
Regarding claim 1, Julsing discloses DMADP (synonymous with DMAPP) is produced in the mevalonate pathway (page 1378, 1st full paragraph). DMAPP is the substrate of IPT (see Ma above). Julsing references Hecht for the teaching of a Bacillus subtilis DXS (page 1382, 1st full paragraph).
Regarding claim 41, Hecht discloses Bacillus subtilis DXS of accession D84432 having 100% sequence identity to the DXS of SEQ ID NO:63 of the instant application (page 3951, 3rd full paragraph and see the sequence alignment below).
Regarding claims 42-43, Wang discloses a Bacillus subtilis adenylsoccuiante synthase encoded by purA that catalyzes the first step of the conversion of IMP to AMP of the purine biosynthesis pathway in Bacillus subtilis (abstract, Section 2 and table 1). AMP is the substrate of IPT (see Ma above).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to increase expression of (I) IPT, LOG, and cytochrome P450 monooxygenase and (II) DXS and PurA in Bacillus subtilis. One having ordinary skill in the art would have been motivated to do so in order to (I) produce cytokinins, such as trans-zeatin, in a nonpathogenic and GRAS host cell and (II) to increase the precursors of cytokinin production and thereby increasing production of cytokinins, such as trans-zeatin, in a nonpathogenic and GRAS host cell. One having ordinary skill in the art would have had a reasonable expectation of success since Ma discloses cytokinin biosynthesis pathway in bacteria, E. coli expressing of a heterologous IPT, and expression of LOG and P450 monooxygenase in cytokinin biosynthesis, Schumann discloses increasing expression of heterologous enzymes in Bacillus subtilis, Julsing and Hecht disclose a Bacillus subtilis DXS, Wang discloses a Bacillus subtilis PurA, and Liu discloses a system for deletion of multiple genes in Bacillus subtilis.
Therefore, the above references render claims 1, 35-36, 38, 41-43, and 48 prima facie obvious.
Applicant's arguments filed June 25, 2026 have been fully considered but they are not persuasive.
Applicant has addressed all rejections together.
Applicant argues that although the additional cited references (P58592, Q8NN34, P46373, Julsing, Hecht, Wang, Zhang, Ashihara, 034443, AAB72065, NP_387890, NP_388517, and Liu) disclose particular enzymes and metabolic pathway components, none of the additional references, alone or in combination, teaches or suggests expressing IPT in combination with increased expression of DXS in a bacterium from the families Bacillaceae or Corynebacteriaceae.
This is not found persuasive. “The rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art”, see MPEP 2144. I. In the instant case, one having ordinary skill in the art would have been motivated to increase expression of (I) IPT, LOG, and cytochrome P450 monooxygenase and (II) DXS and PurA in Bacillus subtilis in order to (I) produce cytokinins, such as trans-zeatin, in a nonpathogenic and GRAS host cell, which includes Bacillus subtilis, and (II) to increase the precursors of cytokinin production and thereby increasing production of cytokinins, such as trans-zeatin, in a nonpathogenic and GRAS host cell.
Applicant argues that the present application demonstrates that the claimed combination produces unexpectedly high cytokinin titers (Examples 10 and 12-14 and Figures 9-12 of the specification as filed, Corynebacterium stationis expressing IPT and increased levels of DXS produces cytokinin titers of approximately 95-110 mg/L, while Bacillus subtilis expressing IPT and increased levels of DXS produces cytokinin titers of approximately 75-88 mg/L. In contrast, Escherichia coli expressing the same enzymes produces only approximately 2.5-3.5 mg/L cytokinin).
This is not found persuasive.
“Any differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected.”, see MPEP 716.02. “Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the “objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support.”, see MPEP 716.02(d). In the instant case, Examples 10 and 12-14 discloses cytokinin titers produced by (I) Bacillus subtilis having increased expression of IPT-LOG of SEQ ID NO:82 and DXS of SEQ ID NO:90 (Example 10), (II) Bacillus subtilis having increased expression of IPT-LOG of SEQ ID NO:82 or IPT fragment of SEQ ID NO:184, and DXS of SEQ ID NO:84 (Example 12), (III) Corynebacterium stationis having increased expression of IPT-LOG of SEQ ID NO:178 or IPT1 of SEQ ID NO:180, and DXS of SEQ ID NO:182 (Example 14), and (IV) E. coli having increased expression of IPT-LOG of SEQ ID NO:82 or IPT of SEQ ID NO:179 and DXS of SEQ ID NO:182. However, the instant claims are directed to any Bacillaceae or Corynebacteriaceae having increased expression of IPT, LOG, P450 monooxygenase, DXS, and PurA. Therefore, the results of Examples 10 and 12-14 are insufficient to rebut the prima facie case because the alleged unexpected results are not commensurate in scope with the claims. Further, the increased cytokinin titers is not unexpected because of the combined increased expression of (A) IPT, LOG, and P450 monooxygnease, involved in production of cytokinin from DMAPPP and AMP/ADP/ATP and (B) DXS and PurA, involved in production of precursors of DMAPPP and AMP. One having ordinary skill in the art would have reasonably expected an increase in cytokinin production because the increased expression of IPT, the rate-limiting enzyme of cytokinin synthesis, LOG, and P450 direct the carbon flow from DMAPP + AMP/ADP/ATP and increased expression of DXS and purA increases the carbon flow to DMAPP and AMP, precursors for cytokinin synthesis. Therefore, the asserted unexpected results are insufficient to rebut the prima facie case because the alleged unexpected results are not commensurate in scope with the claims and are not unexpected.
Hence the rejection has been maintained.
Claim(s) 37, 39, and 49 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ma (Genetic engineering of cytokinins and their application to agriculture. Crit Rev Biotechnol. 2008;28(3):213-32. – form PTO-1449), Schumann (Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol. 2007;62:137-89 – cited previously on form PTO-892), Julsing (Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis. Appl Microbiol Biotechnol. 2007 Jul;75(6):1377-84 – form PTO-1449), Hecht (Enzyme-assisted preparation of isotope-labeled 1-deoxy-d-xylulose 5-phosphate. J Org Chem. 2001 Jun 1;66(11):3948-52 – cited previously on form PTO-892), and Wang (Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering. J Biotechnol. 2016 Aug 10;231:115-121. Epub 2016 May 24 – form PTO-1449) as applied to claims 1, 35-36, 38, 41-43, and 48 above, and further in view of P58592 (UniProtKB/Swiss-Prot Database. May 8, 2019. – cited previously on form PTO-892), Q8NN34 (UniProtKB/TrEMBL Database. October 16, 2019 – cited previously on form PTO-892), and P46373 (UniProtKB/Swiss-Prot Database. May 8, 2019 – cited previously on form PTO-892).
Ma and Schumann do not teach the IPT recited in claim 37, the LOG recited in claim 39, and the cytochrome P450 monooxygenase recited in claim 49. However, all these enzymes were known in the art.
Regarding claim 37, P58592 discloses an IPT having 100% sequence identity to the IPT of SEQ INO:7 (pages 1-2 and see the sequence alignment below).
Regarding claim 39, Q8NN34 discloses a cytokinin riboside 5'-monophosphate phosphoribohydrolase having 100% sequence identity to the LOG of SEQ ID NO:34 of the instant application (pages 1-2 and see the sequence alignment below).
Regarding claim 49, P46373 discloses a cytochrome P450 monooxygenase having 100% sequence identity to the cytochrome P450 monooxygenase of SEQ ID NO:93 of the instant application (pages 1-2 and see the sequence alignment below).
Therefore, in combing the above teachings, it would have been obvious to one having ordinary skill in the art at the time the claimed invention was effectively filed to replacing the IPT, LOG, and cytochrome P450 monooxygenase with other known IPT, LOG, and cytochrome P450 monooxygenase because one of ordinary skill in the art would have been able to carry out such a substitution, and the results were reasonably predictable. Further, the normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to substitute other known IPT, LOG, and cytochrome P450 monooxygenase. The rationale to support a conclusion that the claims would have been obvious is that the substitution of one known element (IPT, LOG, and cytochrome P450 monooxygenase) for another yields predictable results (catalyzing AMP + DMAPP to iPMP, catalyzing the final step of cytokinin biosynthesis, and catalyzing production of trans-zeatin) to one of ordinary skill in the art. Therefore, it would have been obvious to one of ordinary skill in the art to replace the prior art IPT, LOG, and cytochrome P450 monooxygenase with another known and available cytokinin biosynthesis enzyme, such as the IPT of P58592, LOG of Q8NN34, and cytochrome P450 monooxygenase of P46373. One having ordinary skill in the art would have had a reasonable expectation of success since Ma discloses cytokinin biosynthesis pathway in bacteria, E. coli expressing of a heterologous IPT, and expression of LOG and P450 monooxygenase in cytokinin biosynthesis, Schumann discloses increasing expression of heterologous enzymes in Bacillus subtilis, and IPT of P58592, LOG of Q8NN34, and cytochrome P450 monooxygenase of P46373 were known in the prior art.
Therefore, the above references render claims 1, 35-39, 41-43, and 48-49 prima facie obvious.
Applicant has addressed all rejections together, see above.
Hence the rejection has been maintained.
Claim(s) 44-45 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ma (Genetic engineering of cytokinins and their application to agriculture. Crit Rev Biotechnol. 2008;28(3):213-32. – form PTO-1449), Schumann (Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol. 2007;62:137-89 – cited previously on form PTO-892), Julsing (Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis. Appl Microbiol Biotechnol. 2007 Jul;75(6):1377-84 – form PTO-1449), Hecht (Enzyme-assisted preparation of isotope-labeled 1-deoxy-d-xylulose 5-phosphate. J Org Chem. 2001 Jun 1;66(11):3948-52 – cited previously on form PTO-892), and Wang (Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering. J Biotechnol. 2016 Aug 10;231:115-121. Epub 2016 May 24 – form PTO-1449) as applied to claims 1, 35-36, 38, 41-43, and 48 above, and further in view of Zhang (Adenine phosphoribosyl transferase 1 is a key enzyme catalyzing cytokinin conversion from nucleobases to nucleotides in Arabidopsis. Mol Plant. 2013 Sep;6(5):1661-72 – cited previously on form PTO-892), Ashihara (Purine nucleoside phosphorylases: properties, functions, and clinical aspects, Pharmacology & Therapeutics, Volume 88, Issue 3, 2000, Pages 349-425 – cited previously on form PTO-892), O34443 (GenPept Database. April 10, 2019 – form PTO), AAB72065 (GenBank Database. July 24, 2016 – cited previously on form PTO-892), and Liu (Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter. Microbiology (Reading). 2008 Sep;154(Pt 9):2562-2570 – cited previously on form PTO-892).
Ma and Schuman do not disclose decreasing expression or activity of endogenous adenosine phosphoribosyltransferase encoded by apt and endogenous purine nucleotide phosphorylase encoded by deoD, which are enzymes of the purine nucleotide degradation pathway.
Regarding claims 44-45, Zhang discloses that adenosine phosphoribosyltransferase catalyzes the cytoknin conversion from free bases (active form) to nucleotides and loss of adenosine phosphoribosyltransferase leads to excess accumulation of cytokinin bases (abstract). Ashihara discloses that nucleotide phosphorylase is involved in cytokinin metabolism (Section 3.4 at pages 99-100). O34443 discloses Bacillus subtilis adenosine phosphoribosyltransferase (APRT) encoded by apt (pages 1-2). AAB72065 discloses Bacillus subtilis purine nucleotide phosphorylase encoded by deoD (pages 1-2). Liu discloses a system for deletion of multiple genes in Bacillus subtilis (abstract page 2563, 2nd full paragraph).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to decrease expression or activity of apt and deoD in Bacillus subtilis expressing IPT, LOG, and cytochrome P450 monooxygenase. One having ordinary skill in the art would have been motivated to do so in order to increase production of cytokinin. One having ordinary skill in the art would have had a reasonable expectation of success since Ma discloses cytokinin biosynthesis pathway in bacteria, E. coli expressing of a heterologous IPT, and expression of LOG and P450 monooxygenase in cytokinin biosynthesis, Schumann discloses increasing expression of heterologous enzymes in Bacillus subtilis, Bacillus subtilis apt and deoD were known in the art, and Liu discloses a system for deletion of multiple genes in Bacillus subtilis.
Therefore, the above references render claims 1, 35-36, 38, 41-45, and 48 prima facie obvious.
Applicant has addressed all rejections together, see above.
Hence the rejection has been maintained.
Claim(s) 46-47 is/are rejected under 35 U.S.C. 103 as being unpatentable over Ma (Genetic engineering of cytokinins and their application to agriculture. Crit Rev Biotechnol. 2008;28(3):213-32. – form PTO-1449), Schumann (Production of recombinant proteins in Bacillus subtilis. Adv Appl Microbiol. 2007;62:137-89 – cited previously on form PTO-892), Julsing (Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis. Appl Microbiol Biotechnol. 2007 Jul;75(6):1377-84 – form PTO-1449), Hecht (Enzyme-assisted preparation of isotope-labeled 1-deoxy-d-xylulose 5-phosphate. J Org Chem. 2001 Jun 1;66(11):3948-52 – form PTO-892), and Wang (Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering. J Biotechnol. 2016 Aug 10;231:115-121. Epub 2016 May 24 – form PTO-1449) as applied to claims 1, 35-36, 38, 41-43, and 48 above, and further in view of Wang (Directed evolution of adenylosuccinate synthetase from Bacillus subtilis and its application in metabolic engineering. J Biotechnol. 2016 Aug 10;231:115-121. Epub 2016 May 24 – cited previously on form PTO-1449), NP_387890 (GenPept Database. Septemer 18, 2018 – cited previously on form PTO-892), NP_388517 (GenPept Database. September 18, 2018 – form PTO-892, and Liu (Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter. Microbiology (Reading). 2008 Sep;154(Pt 9):2562-2570 – cited previously on form PTO-892).
Ma and Schuman do not disclose decreasing expression or activity of endogenous IMP dehydrogenase encoded by guaB and endogenous GMP synthetase encoded by gauA, which are enzymes of the guanosine monophosphate biosynthesis.
Regarding claims 46-47, Wang discloses that inactivation of IMP dehydrogenase in Bacillus subtilis increases production of IMP, which is precursor to AMP, substrate of cytokinin synthesis (page 120, 3rd full paragraph and Fig. 1). Wang discloses that IMP is converted to XMP by guaB and XMP is converted to GMP by guaA (Fig. 1). NP_387890 discloses Bacillus subtilis IMP dehydrogenase encoded by guaA (pages 1-2). NP_388517 discloses Bacillus subtilis GMP synthetase encoded by guaA (pages 1-2). Liu discloses a system for deletion of multiple genes in Bacillus subtilis (abstract page 2563, 2nd full paragraph).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to decrease expression or activity of guaB and guaA in Bacillus subtilis. One having ordinary skill in the art would have been motivated to do so in order to increase production of cytokinin by increasing production of IMP and thereby AMP, precursors to cytokinin synthesis. One having ordinary skill in the art would have had a reasonable expectation of success since Ma discloses cytokinin biosynthesis pathway in bacteria and E. coli expressing of a heterologous IPT, Schumann discloses increasing expression of heterologous enzymes in Bacillus subtilis, Bacillus subtilis guaB and guaA were known in the art, and Liu discloses a system for deletion of multiple genes in Bacillus subtilis.
Therefore, the above references render claims 1, 35-36, 38, 41-43, and 46-48 prima facie obvious.
Applicant has addressed all rejections together, see above.
Hence the rejection has been maintained.
Conclusion
Claims 1, 35-39, and 41-52 are pending.
Claims 50-52 are withdrawn.
Claims 1, 35-39, and 41-49 are under examination.
THIS ACTION IS MADE FINAL. 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 nonprovisional extension fee (37 CFR 1.17(a)) 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 mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YONG D PAK whose telephone number is (571)272-0935. The examiner can normally be reached M-Th: 5:30 am - 3:30 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached on 408-918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/YONG D PAK/Primary Examiner, Art Unit 1652
Sequence alignment between the IPT of SEQ ID NO:7 of the instant application (“Qy”) and the IPT of P58592 (“Db”)
PT_RALN1
ID IPT_RALN1 Reviewed; 238 AA.
AC P58592;
DT 23-JAN-2002, integrated into UniProtKB/Swiss-Prot.
DT 23-JAN-2002, sequence version 1.
DT 09-APR-2025, entry version 116.
DE RecName: Full=Adenylate dimethylallyltransferase;
DE EC=2.5.1.27;
DE AltName: Full=Dimethylallyl transferase;
DE AltName: Full=Isopentenyl transferase;
DE AltName: Full=Trans-zeatin producing protein;
GN Name=tzs; OrderedLocusNames=RSp1225; ORFNames=RS03169;
OS Ralstonia nicotianae (strain ATCC BAA-1114 / GMI1000) (Ralstonia
OS solanacearum).
OG Plasmid megaplasmid Rsp.
OC Bacteria; Pseudomonadati; Pseudomonadota; Betaproteobacteria;
OC Burkholderiales; Burkholderiaceae; Ralstonia;
OC Ralstonia solanacearum species complex.
OX NCBI_TaxID=267608;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC BAA-1114 / GMI1000;
RX PubMed=11823852; DOI=10.1038/415497a;
RA Salanoubat M., Genin S., Artiguenave F., Gouzy J., Mangenot S., Arlat M.,
RA Billault A., Brottier P., Camus J.-C., Cattolico L., Chandler M.,
RA Choisne N., Claudel-Renard C., Cunnac S., Demange N., Gaspin C., Lavie M.,
RA Moisan A., Robert C., Saurin W., Schiex T., Siguier P., Thebault P.,
RA Whalen M., Wincker P., Levy M., Weissenbach J., Boucher C.A.;
RT "Genome sequence of the plant pathogen Ralstonia solanacearum.";
RL Nature 415:497-502(2002).
CC -!- FUNCTION: Transfers dimethylallyl groups to AMP as part of the
CC biosynthesis of cytokinin phytohormones. {ECO:0000250}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=dimethylallyl diphosphate + AMP = N(6)-
CC (dimethylallyl)adenosine 5'-phosphate + diphosphate;
CC Xref=Rhea:RHEA:15285, ChEBI:CHEBI:33019, ChEBI:CHEBI:57526,
CC ChEBI:CHEBI:57623, ChEBI:CHEBI:456215; EC=2.5.1.27;
CC -!- SIMILARITY: Belongs to the isopentenyl transferase family.
CC {ECO:0000305}.
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DR EMBL; AL646053; CAD18376.1; -; Genomic_DNA.
DR RefSeq; WP_011004509.1; NC_003296.1.
DR AlphaFoldDB; P58592; -.
DR SMR; P58592; -.
DR STRING; 267608.RSp1225; -.
DR EnsemblBacteria; CAD18376; CAD18376; RSp1225.
DR KEGG; rso:RSp1225; -.
DR PATRIC; fig|267608.8.peg.4715; -.
DR eggNOG; COG0324; Bacteria.
DR HOGENOM; CLU_1115409_0_0_4; -.
DR Proteomes; UP000001436; Plasmid megaplasmid Rsp.
DR GO; GO:0009824; F:AMP dimethylallyltransferase activity; IEA:UniProtKB-EC.
DR GO; GO:0009691; P:cytokinin biosynthetic process; IEA:UniProtKB-KW.
DR Gene3D; 1.10.287.890; Crystal structure of tRNA isopentenylpyrophosphate transferase (bh2366) domain; 1.
DR Gene3D; 3.40.50.300; P-loop containing nucleotide triphosphate hydrolases; 1.
DR InterPro; IPR027417; P-loop_NTPase.
DR InterPro; IPR002648; Tzs.
DR Pfam; PF01745; IPT; 1.
DR PIRSF; PIRSF000507; IPT; 1.
DR SUPFAM; SSF52540; P-loop containing nucleoside triphosphate hydrolases; 1.
PE 3: Inferred from homology;
KW Cytokinin biosynthesis; Plasmid; Reference proteome; Transferase.
FT CHAIN 1..238
FT /note="Adenylate dimethylallyltransferase"
FT /id="PRO_0000216436"
SQ SEQUENCE 238 AA; 26526 MW; 95F1596E0BF2400C CRC64;
Query Match 100.0%; Score 1231; Length 238;
Best Local Similarity 100.0%;
Matches 238; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MPVRLYLIWGATTTGKTAQSVALARSAGAPVISLDRVQCCHELAVGSGRPSPSELLGTRR 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MPVRLYLIWGATTTGKTAQSVALARSAGAPVISLDRVQCCHELAVGSGRPSPSELLGTRR 60
Qy 61 EYLCEREVSRGVVSAAEANQLLLDKVARYATQERALILEGGSVSLINAMIRDARWSERGE 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 EYLCEREVSRGVVSAAEANQLLLDKVARYATQERALILEGGSVSLINAMIRDARWSERGE 120
Qy 121 WILRRIPVPGRAAFMAAARKRVREMLDPPPGQAGILDELQGLWGYPRNHAVLEDIDGYRQ 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 WILRRIPVPGRAAFMAAARKRVREMLDPPPGQAGILDELQGLWGYPRNHAVLEDIDGYRQ 180
Qy 181 IIRYANALQVPICRITSIDPNAKALLIERIAQEYWEHALWQEQEFLGIPASWMRADDA 238
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 IIRYANALQVPICRITSIDPNAKALLIERIAQEYWEHALWQEQEFLGIPASWMRADDA 238
Sequence alignment between the LOG of SEQ ID NO:34 of the instant application (“Qy”) and the LOG of Q8NN34 “Db”)
Q8NN34_CORGL
ID Q8NN34_CORGL Unreviewed; 195 AA.
AC Q8NN34; Q6M378;
DT 01-OCT-2002, integrated into UniProtKB/TrEMBL.
DT 01-OCT-2002, sequence version 1.
DT 05-FEB-2025, entry version 129.
DE RecName: Full=Cytokinin riboside 5'-monophosphate phosphoribohydrolase {ECO:0000256|RuleBase:RU363015};
DE EC=3.2.2.n1 {ECO:0000256|RuleBase:RU363015};
GN OrderedLocusNames=Cgl2379 {ECO:0000313|EMBL:BAB99772.1};
OS Corynebacterium glutamicum (strain ATCC 13032 / DSM 20300 / JCM 1318 / BCRC
OS 11384 / CCUG 27702 / LMG 3730 / NBRC 12168 / NCIMB 10025 / NRRL B-2784 /
OS 534).
OC Bacteria; Bacillati; Actinomycetota; Actinomycetes; Mycobacteriales;
OC Corynebacteriaceae; Corynebacterium.
OX NCBI_TaxID=196627 {ECO:0000313|EMBL:BAB99772.1, ECO:0000313|Proteomes:UP000000582};
RN [1] {ECO:0000313|Proteomes:UP000000582}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 13032 / DSM 20300 / BCRC 11384 / JCM 1318 / LMG 3730 /
RC NCIMB 10025 {ECO:0000313|Proteomes:UP000000582};
RX PubMed=12743753; DOI=10.1007/s00253-003-1328-1;
RA Ikeda M., Nakagawa S.;
RT "The Corynebacterium glutamicum genome: features and impacts on
RT biotechnological processes.";
RL Appl. Microbiol. Biotechnol. 62:99-109(2003).
RN [2] {ECO:0007829|PDB:5ITS}
RP X-RAY CRYSTALLOGRAPHY (2.30 ANGSTROMS).
RX PubMed=27507425; DOI=10.1038/srep31390;
RA Seo H., Kim S., Sagong H.Y., Son H.F., Jin K.S., Kim I.K., Kim K.J.;
RT "Structural basis for cytokinin production by LOG from Corynebacterium
RT glutamicum.";
RL Sci. Rep. 6:31390-31390(2016).
RN [3] {ECO:0007829|PDB:5ZBL}
RP X-RAY CRYSTALLOGRAPHY (2.30 ANGSTROMS) IN COMPLEX WITH AMP.
RX PubMed=29901273; DOI=10.1111/1462-2920.14287;
RA Seo H., Kim K.J.;
RT "Structural insight into molecular mechanism of cytokinin activating
RT protein from Pseudomonas aeruginosa PAO1.";
RL Environ. Microbiol. 20:3214-3223(2018).
CC -!- CATALYTIC ACTIVITY:
CC Reaction=9-ribosyl-trans-zeatin 5'-phosphate + H2O = trans-zeatin + D-
CC ribose 5-phosphate; Xref=Rhea:RHEA:48564, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:16522, ChEBI:CHEBI:78346, ChEBI:CHEBI:87947; EC=3.2.2.n1;
CC Evidence={ECO:0000256|RuleBase:RU363015};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=N(6)-(dimethylallyl)adenosine 5'-phosphate + H2O = N(6)-
CC dimethylallyladenine + D-ribose 5-phosphate; Xref=Rhea:RHEA:48560,
CC ChEBI:CHEBI:15377, ChEBI:CHEBI:17660, ChEBI:CHEBI:57526,
CC ChEBI:CHEBI:78346; EC=3.2.2.n1;
CC Evidence={ECO:0000256|RuleBase:RU363015};
CC -!- SIMILARITY: Belongs to the LOG family. {ECO:0000256|ARBA:ARBA00006763,
CC ECO:0000256|RuleBase:RU363015}.
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DR EMBL; BA000036; BAB99772.1; -; Genomic_DNA.
DR RefSeq; NP_601580.1; NC_003450.3.
DR RefSeq; WP_011015078.1; NC_006958.1.
DR PDB; 5ITS; X-ray; 2.30 A; A/B/C/D=1-195.
DR PDB; 5ZBL; X-ray; 2.30 A; A/B/C/D=1-195.
DR PDBsum; 5ITS; -.
DR PDBsum; 5ZBL; -.
DR AlphaFoldDB; Q8NN34; -.
DR SMR; Q8NN34; -.
DR STRING; 196627.cg2612; -.
DR KEGG; cgb:cg2612; -.
DR KEGG; cgl:Cgl2379; -.
DR PATRIC; fig|196627.13.peg.2314; -.
DR eggNOG; COG1611; Bacteria.
DR HOGENOM; CLU_058336_4_2_11; -.
DR OrthoDB; 9801098at2; -.
DR BioCyc; CORYNE:G18NG-11976-MONOMER; -.
DR Proteomes; UP000000582; Chromosome.
DR GO; GO:0005829; C:cytosol; IEA:TreeGrafter.
DR GO; GO:0102682; F:cytokinin riboside 5'-monophosphate phosphoribohydrolase activity; IEA:RHEA.
DR GO; GO:0000166; F:nucleotide binding; IEA:UniProtKB-KW.
DR GO; GO:0009691; P:cytokinin biosynthetic process; IEA:UniProtKB-UniRule.
DR Gene3D; 3.40.50.450; -; 1.
DR InterPro; IPR005269; LOG.
DR InterPro; IPR031100; LOG_fam.
DR NCBIfam; TIGR00730; Rossman fold protein, TIGR00730 family; 1.
DR PANTHER; PTHR31223; LOG FAMILY PROTEIN YJL055W; 1.
DR PANTHER; PTHR31223:SF70; LOG FAMILY PROTEIN YJL055W; 1.
DR Pfam; PF03641; Lysine_decarbox; 1.
DR SUPFAM; SSF102405; MCP/YpsA-like; 1.
PE 1: Evidence at protein level;
KW 3D-structure {ECO:0007829|PDB:5ITS, ECO:0007829|PDB:5ZBL};
KW Cytokinin biosynthesis {ECO:0000256|RuleBase:RU363015};
KW Hydrolase {ECO:0000256|RuleBase:RU363015};
KW Nucleotide-binding {ECO:0007829|PDB:5ZBL};
KW Reference proteome {ECO:0000313|Proteomes:UP000000582}.
FT BINDING 19
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 99
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 100
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 116
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 118
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 119
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 121
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 122
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
FT BINDING 125
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0007829|PDB:5ZBL"
SQ SEQUENCE 195 AA; 21491 MW; DE73FEDDB01AB348 CRC64;
Query Match 100.0%; Score 1018; Length 195;
Best Local Similarity 100.0%;
Matches 195; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MTSLFDAPTLQRVTVFTGSALGSSSLYTQAAQTLAKTAVDRGIDLVYGGGKVGLMGIVAD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTSLFDAPTLQRVTVFTGSALGSSSLYTQAAQTLAKTAVDRGIDLVYGGGKVGLMGIVAD 60
Qy 61 AFLESGGEAFGVITESLMKGELGHEKLTELEIVPDMHIRKRRMAELGDGFIAMPGGAGTL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AFLESGGEAFGVITESLMKGELGHEKLTELEIVPDMHIRKRRMAELGDGFIAMPGGAGTL 120
Qy 121 EELFEVWTWQQLGIHQKPVALYDVDGFWQPLLEMLEQMTQRGFIKRDFFECLIVESDPHA 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 EELFEVWTWQQLGIHQKPVALYDVDGFWQPLLEMLEQMTQRGFIKRDFFECLIVESDPHA 180
Qy 181 LLKAMQTWTPPAPKW 195
|||||||||||||||
Db 181 LLKAMQTWTPPAPKW 195
Sequence alignment between the cytochrome P450 monooxygenase of SEQ ID NO:93 of the instant application (“Qy”) and the cytochrome P450 monooxygenase of P46373 (“Db”)
FAS1_RHOFA
ID FAS1_RHOFA Reviewed; 399 AA.
AC P46373;
DT 01-NOV-1995, integrated into UniProtKB/Swiss-Prot.
DT 01-NOV-1995, sequence version 1.
DT 05-FEB-2025, entry version 97.
DE RecName: Full=Cytochrome P450 FAS1;
DE EC=1.14.-.-;
GN Name=fas1; Synonyms=CYP105E1;
OS Rhodococcoides fascians (Rhodococcus fascians).
OG Plasmid pFiD188.
OC Bacteria; Bacillati; Actinomycetota; Actinomycetes; Mycobacteriales;
OC Nocardiaceae; Rhodococcoides.
OX NCBI_TaxID=1828;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA].
RC STRAIN=D188;
RX PubMed=8169198; DOI=10.1128/jb.176.9.2492-2501.1994;
RA Crespi M., Vereecke D., Temmerman W., van Montagu M., Desomer J.;
RT "The fas operon of Rhodococcus fascians encodes new genes required for
RT efficient fasciation of host plants.";
RL J. Bacteriol. 176:2492-2501(1994).
CC -!- FUNCTION: May be involved in the biosynthesis of cytokinin
CC phytohormones and in host plant fasciation (leafy gall).
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413; Evidence={ECO:0000250};
CC -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250}.
CC -!- INDUCTION: During the interaction with host plants.
CC -!- SIMILARITY: Belongs to the cytochrome P450 family. {ECO:0000305}.
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DR EMBL; Z29635; CAA82741.1; -; Genomic_DNA.
DR PIR; A55578; A55578.
DR RefSeq; WP_015586131.1; NZ_NPFU01000019.1.
DR RefSeq; YP_007878704.1; NC_021080.1.
DR AlphaFoldDB; P46373; -.
DR SMR; P46373; -.
DR STRING; 1443905.GCA_000761075_00040; -.
DR eggNOG; COG2124; Bacteria.
DR GO; GO:0005737; C:cytoplasm; IEA:UniProtKB-SubCell.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR GO; GO:0004497; F:monooxygenase activity; IEA:UniProtKB-KW.
DR GO; GO:0016705; F:oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen; IEA:InterPro.
DR CDD; cd11030; CYP105-like; 1.
DR FunFam; 1.10.630.10:FF:000018; Cytochrome P450 monooxygenase; 1.
DR Gene3D; 1.10.630.10; Cytochrome P450; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR002397; Cyt_P450_B.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR036396; Cyt_P450_sf.
DR PANTHER; PTHR46696:SF1; CYTOCHROME P450 YJIB-RELATED; 1.
DR PANTHER; PTHR46696; P450, PUTATIVE (EUROFUNG)-RELATED; 1.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00359; BP450.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; Cytochrome P450; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 2: Evidence at transcript level;
KW Cytoplasm; Heme; Iron; Metal-binding; Monooxygenase; Oxidoreductase;
KW Plasmid.
FT CHAIN 1..399
FT /note="Cytochrome P450 FAS1"
FT /id="PRO_0000052231"
FT BINDING 349
FT /ligand="heme"
FT /ligand_id="ChEBI:CHEBI:30413"
FT /ligand_part="Fe"
FT /ligand_part_id="ChEBI:CHEBI:18248"
FT /note="axial binding residue"
FT /evidence="ECO:0000250"
SQ SEQUENCE 399 AA; 43596 MW; A66B6F3DF1286566 CRC64;
Query Match 100.0%; Score 2020; Length 399;
Best Local Similarity 100.0%;
Matches 399; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAGTADLPLEMRRNGLNPTEELAQVRDRDGVIPVGELYGAPAFLVCRYEDVRRIFADSNR 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAGTADLPLEMRRNGLNPTEELAQVRDRDGVIPVGELYGAPAFLVCRYEDVRRIFADSNR 60
Qy 61 FSNAHTPMFAIPSGGDVIEDELAAMRAGNLIGLDPPDHTRLRHILAAEFSVHRLSRLQPR 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 FSNAHTPMFAIPSGGDVIEDELAAMRAGNLIGLDPPDHTRLRHILAAEFSVHRLSRLQPR 120
Qy 121 IAEIVDSALDGLEQAGQPADLMDRYALPVSLLVLCELLGVPYADRDELRDRTARLLDLSA 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 IAEIVDSALDGLEQAGQPADLMDRYALPVSLLVLCELLGVPYADRDELRDRTARLLDLSA 180
Qy 181 SAEQRAVAQREDRRYMATLVTRAQEQPGDDLLGILARKIGDNLSTDELISIISLIMLGGH 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SAEQRAVAQREDRRYMATLVTRAQEQPGDDLLGILARKIGDNLSTDELISIISLIMLGGH 240
Qy 241 ETTASMIGLSVLALLHHPEQAAMMIEDPNCVNSGIEELLRWLSVAHSQPPRMAVTEVQIA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 ETTASMIGLSVLALLHHPEQAAMMIEDPNCVNSGIEELLRWLSVAHSQPPRMAVTEVQIA 300
Qy 301 GVTIPAGSFVIPSLLAANRDSNLTDRPDDLDITRGVAGHLAFGHGVHFCLGHSLARMTLR 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 GVTIPAGSFVIPSLLAANRDSNLTDRPDDLDITRGVAGHLAFGHGVHFCLGHSLARMTLR 360
Qy 361 TAVPAVLRRFPDLALSPSHDVRLRSASIVLGLEELQLTW 399
|||||||||||||||||||||||||||||||||||||||
Db 361 TAVPAVLRRFPDLALSPSHDVRLRSASIVLGLEELQLTW 399
Sequence alignment between the DXS of SEQ ID NO:63 of the instant application (“Qy”) and the DSX of Jusling/B. subtilis (“Db”)
DXS_BACSU
ID DXS_BACSU Reviewed; 633 AA.
AC P54523;
DT 01-OCT-1996, integrated into UniProtKB/Swiss-Prot.
DT 01-OCT-1996, sequence version 1.
DT 18-JUN-2025, entry version 154.
DE RecName: Full=1-deoxy-D-xylulose-5-phosphate synthase {ECO:0000255|HAMAP-Rule:MF_00315};
DE EC=2.2.1.7 {ECO:0000255|HAMAP-Rule:MF_00315};
DE AltName: Full=1-deoxyxylulose-5-phosphate synthase {ECO:0000255|HAMAP-Rule:MF_00315};
DE Short=DXP synthase {ECO:0000255|HAMAP-Rule:MF_00315};
DE Short=DXPS {ECO:0000255|HAMAP-Rule:MF_00315};
GN Name=dxs {ECO:0000255|HAMAP-Rule:MF_00315}; Synonyms=yqiE;
GN OrderedLocusNames=BSU24270;
OS Bacillus subtilis (strain 168).
OC Bacteria; Bacillati; Bacillota; Bacilli; Bacillales; Bacillaceae; Bacillus.
OX NCBI_TaxID=224308;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA].
RC STRAIN=168 / JH642;
RX PubMed=8969508; DOI=10.1099/13500872-142-11-3103;
RA Mizuno M., Masuda S., Takemaru K., Hosono S., Sato T., Takeuchi M.,
RA Kobayashi Y.;
RT "Systematic sequencing of the 283 kb 210 degrees-232 degrees region of the
RT Bacillus subtilis genome containing the skin element and many sporulation
RT genes.";
RL Microbiology 142:3103-3111(1996).
RN [2]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=168;
RX PubMed=9384377; DOI=10.1038/36786;
RA Kunst F., Ogasawara N., Moszer I., Albertini A.M., Alloni G., Azevedo V.,
RA Bertero M.G., Bessieres P., Bolotin A., Borchert S., Borriss R.,
RA Boursier L., Brans A., Braun M., Brignell S.C., Bron S., Brouillet S.,
RA Bruschi C.V., Caldwell B., Capuano V., Carter N.M., Choi S.-K.,
RA Codani J.-J., Connerton I.F., Cummings N.J., Daniel R.A., Denizot F.,
RA Devine K.M., Duesterhoeft A., Ehrlich S.D., Emmerson P.T., Entian K.-D.,
RA Errington J., Fabret C., Ferrari E., Foulger D., Fritz C., Fujita M.,
RA Fujita Y., Fuma S., Galizzi A., Galleron N., Ghim S.-Y., Glaser P.,
RA Goffeau A., Golightly E.J., Grandi G., Guiseppi G., Guy B.J., Haga K.,
RA Haiech J., Harwood C.R., Henaut A., Hilbert H., Holsappel S., Hosono S.,
RA Hullo M.-F., Itaya M., Jones L.-M., Joris B., Karamata D., Kasahara Y.,
RA Klaerr-Blanchard M., Klein C., Kobayashi Y., Koetter P., Koningstein G.,
RA Krogh S., Kumano M., Kurita K., Lapidus A., Lardinois S., Lauber J.,
RA Lazarevic V., Lee S.-M., Levine A., Liu H., Masuda S., Mauel C.,
RA Medigue C., Medina N., Mellado R.P., Mizuno M., Moestl D., Nakai S.,
RA Noback M., Noone D., O'Reilly M., Ogawa K., Ogiwara A., Oudega B.,
RA Park S.-H., Parro V., Pohl T.M., Portetelle D., Porwollik S.,
RA Prescott A.M., Presecan E., Pujic P., Purnelle B., Rapoport G., Rey M.,
RA Reynolds S., Rieger M., Rivolta C., Rocha E., Roche B., Rose M., Sadaie Y.,
RA Sato T., Scanlan E., Schleich S., Schroeter R., Scoffone F., Sekiguchi J.,
RA Sekowska A., Seror S.J., Serror P., Shin B.-S., Soldo B., Sorokin A.,
RA Tacconi E., Takagi T., Takahashi H., Takemaru K., Takeuchi M.,
RA Tamakoshi A., Tanaka T., Terpstra P., Tognoni A., Tosato V., Uchiyama S.,
RA Vandenbol M., Vannier F., Vassarotti A., Viari A., Wambutt R., Wedler E.,
RA Wedler H., Weitzenegger T., Winters P., Wipat A., Yamamoto H., Yamane K.,
RA Yasumoto K., Yata K., Yoshida K., Yoshikawa H.-F., Zumstein E.,
RA Yoshikawa H., Danchin A.;
RT "The complete genome sequence of the Gram-positive bacterium Bacillus
RT subtilis.";
RL Nature 390:249-256(1997).
CC -!- FUNCTION: Catalyzes the acyloin condensation reaction between C atoms 2
CC and 3 of pyruvate and glyceraldehyde 3-phosphate to yield 1-deoxy-D-
CC xylulose-5-phosphate (DXP). {ECO:0000255|HAMAP-Rule:MF_00315}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=D-glyceraldehyde 3-phosphate + pyruvate + H(+) = 1-deoxy-D-
CC xylulose 5-phosphate + CO2; Xref=Rhea:RHEA:12605, ChEBI:CHEBI:15361,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:16526, ChEBI:CHEBI:57792,
CC ChEBI:CHEBI:59776; EC=2.2.1.7; Evidence={ECO:0000255|HAMAP-
CC Rule:MF_00315};
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000255|HAMAP-Rule:MF_00315};
CC Note=Binds 1 Mg(2+) ion per subunit. {ECO:0000255|HAMAP-Rule:MF_00315};
CC -!- COFACTOR:
CC Name=thiamine diphosphate; Xref=ChEBI:CHEBI:58937;
CC Evidence={ECO:0000255|HAMAP-Rule:MF_00315};
CC Note=Binds 1 thiamine pyrophosphate per subunit. {ECO:0000255|HAMAP-
CC Rule:MF_00315};
CC -!- PATHWAY: Metabolic intermediate biosynthesis; 1-deoxy-D-xylulose 5-
CC phosphate biosynthesis; 1-deoxy-D-xylulose 5-phosphate from D-
CC glyceraldehyde 3-phosphate and pyruvate: step 1/1. {ECO:0000255|HAMAP-
CC Rule:MF_00315}.
CC -!- SUBUNIT: Homodimer. {ECO:0000255|HAMAP-Rule:MF_00315}.
CC -!- SIMILARITY: Belongs to the transketolase family. DXPS subfamily.
CC {ECO:0000255|HAMAP-Rule:MF_00315}.
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DR EMBL; D84432; BAA12576.1; -; Genomic_DNA.
DR EMBL; AL009126; CAB14358.1; -; Genomic_DNA.
DR PIR; B69961; B69961.
DR RefSeq; NP_390307.1; NC_000964.3.
DR RefSeq; WP_003245985.1; NZ_OZ025638.1.
DR AlphaFoldDB; P54523; -.
DR SMR; P54523; -.
DR FunCoup; P54523; 450.
DR STRING; 224308.BSU24270; -.
DR PaxDb; 224308-BSU24270; -.
DR EnsemblBacteria; CAB14358; CAB14358; BSU_24270.
DR GeneID; 938609; -.
DR KEGG; bsu:BSU24270; -.
DR PATRIC; fig|224308.179.peg.2645; -.
DR eggNOG; COG1154; Bacteria.
DR InParanoid; P54523; -.
DR OrthoDB; 9803371at2; -.
DR PhylomeDB; P54523; -.
DR BioCyc; BSUB:BSU24270-MONOMER; -.
DR BioCyc; MetaCyc:BSU24270-MONOMER; -.
DR UniPathway; UPA00064; UER00091.
DR Proteomes; UP000001570; Chromosome.
DR GO; GO:0005829; C:cytosol; IBA:GO_Central.
DR GO; GO:0008661; F:1-deoxy-D-xylulose-5-phosphate synthase activity; IBA:GO_Central.
DR GO; GO:0000287; F:magnesium ion binding; IEA:UniProtKB-UniRule.
DR GO; GO:0030976; F:thiamine pyrophosphate binding; IEA:UniProtKB-UniRule.
DR GO; GO:0052865; P:1-deoxy-D-xylulose 5-phosphate biosynthetic process; IEA:UniProtKB-UniPathway.
DR GO; GO:0019288; P:isopentenyl diphosphate biosynthetic process, methylerythritol 4-phosphate pathway; IBA:GO_Central.
DR GO; GO:0016114; P:terpenoid biosynthetic process; IEA:UniProtKB-UniRule.
DR GO; GO:0009228; P:thiamine biosynthetic process; IEA:UniProtKB-UniRule.
DR CDD; cd02007; TPP_DXS; 1.
DR CDD; cd07033; TPP_PYR_DXS_TK_like; 1.
DR FunFam; 3.40.50.920:FF:000002; 1-deoxy-D-xylulose-5-phosphate synthase; 1.
DR FunFam; 3.40.50.970:FF:000030; 1-deoxy-D-xylulose-5-phosphate synthase; 1.
DR Gene3D; 3.40.50.920; -; 1.
DR Gene3D; 3.40.50.970; -; 2.
DR HAMAP; MF_00315; DXP_synth; 1.
DR InterPro; IPR005477; Dxylulose-5-P_synthase.
DR InterPro; IPR029061; THDP-binding.
DR InterPro; IPR009014; Transketo_C/PFOR_II.
DR InterPro; IPR005475; Transketolase-like_Pyr-bd.
DR InterPro; IPR020826; Transketolase_BS.
DR InterPro; IPR033248; Transketolase_C.
DR InterPro; IPR049557; Transketolase_CS.
DR NCBIfam; TIGR00204; dxs; 1.
DR NCBIfam; NF003933; PRK05444.2-2; 1.
DR PANTHER; PTHR43322; 1-D-DEOXYXYLULOSE 5-PHOSPHATE SYNTHASE-RELATED; 1.
DR PANTHER; PTHR43322:SF5; 1-DEOXY-D-XYLULOSE-5-PHOSPHATE SYNTHASE, CHLOROPLASTIC; 1.
DR Pfam; PF13292; DXP_synthase_N; 1.
DR Pfam; PF02779; Transket_pyr; 1.
DR Pfam; PF02780; Transketolase_C; 1.
DR SMART; SM00861; Transket_pyr; 1.
DR SUPFAM; SSF52518; Thiamin diphosphate-binding fold (THDP-binding); 2.
DR SUPFAM; SSF52922; TK C-terminal domain-like; 1.
DR PROSITE; PS00801; TRANSKETOLASE_1; 1.
DR PROSITE; PS00802; TRANSKETOLASE_2; 1.
PE 3: Inferred from homology;
KW Isoprene biosynthesis; Magnesium; Metal-binding; Reference proteome;
KW Thiamine biosynthesis; Thiamine pyrophosphate; Transferase.
FT CHAIN 1..633
FT /note="1-deoxy-D-xylulose-5-phosphate synthase"
FT /id="PRO_0000189086"
FT BINDING 72
FT /ligand="thiamine diphosphate"
FT /ligand_id="ChEBI:CHEBI:58937"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
FT BINDING 113..115
FT /ligand="thiamine diphosphate"
FT /ligand_id="ChEBI:CHEBI:58937"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
FT BINDING 144
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
FT BINDING 145..146
FT /ligand="thiamine diphosphate"
FT /ligand_id="ChEBI:CHEBI:58937"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
FT BINDING 173
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
FT BINDING 173
FT /ligand="thiamine diphosphate"
FT /ligand_id="ChEBI:CHEBI:58937"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
FT BINDING 284
FT /ligand="thiamine diphosphate"
FT /ligand_id="ChEBI:CHEBI:58937"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
FT BINDING 367
FT /ligand="thiamine diphosphate"
FT /ligand_id="ChEBI:CHEBI:58937"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_00315"
SQ SEQUENCE 633 AA; 69559 MW; 333B65355F0CEF8F CRC64;
Query Match 100.0%; Score 3282; Length 633;
Best Local Similarity 100.0%;
Matches 633; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MDLLSIQDPSFLKNMSIDELEKLSDEIRQFLITSLSASGGHIGPNLGVVELTVALHKEFN 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MDLLSIQDPSFLKNMSIDELEKLSDEIRQFLITSLSASGGHIGPNLGVVELTVALHKEFN 60
Qy 61 SPKDKFLWDVGHQSYVHKLLTGRGKEFATLRQYKGLCGFPKRSESEHDVWETGHSSTSLS 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 SPKDKFLWDVGHQSYVHKLLTGRGKEFATLRQYKGLCGFPKRSESEHDVWETGHSSTSLS 120
Qy 121 GAMGMAAARDIKGTDEYIIPIIGDGALTGGMALEALNHIGDEKKDMIVILNDNEMSIAPN 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 GAMGMAAARDIKGTDEYIIPIIGDGALTGGMALEALNHIGDEKKDMIVILNDNEMSIAPN 180
Qy 181 VGAIHSMLGRLRTAGKYQWVKDELEYLFKKIPAVGGKLAATAERVKDSLKYMLVSGMFFE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VGAIHSMLGRLRTAGKYQWVKDELEYLFKKIPAVGGKLAATAERVKDSLKYMLVSGMFFE 240
Qy 241 ELGFTYLGPVDGHSYHELIENLQYAKKTKGPVLLHVITKKGKGYKPAETDTIGTWHGTGP 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 ELGFTYLGPVDGHSYHELIENLQYAKKTKGPVLLHVITKKGKGYKPAETDTIGTWHGTGP 300
Qy 301 YKINTGDFVKPKAAAPSWSGLVSGTVQRMAREDGRIVAITPAMPVGSKLEGFAKEFPDRM 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 YKINTGDFVKPKAAAPSWSGLVSGTVQRMAREDGRIVAITPAMPVGSKLEGFAKEFPDRM 360
Qy 361 FDVGIAEQHAATMAAAMAMQGMKPFLAIYSTFLQRAYDQVVHDICRQNANVFIGIDRAGL 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 FDVGIAEQHAATMAAAMAMQGMKPFLAIYSTFLQRAYDQVVHDICRQNANVFIGIDRAGL 420
Qy 421 VGADGETHQGVFDIAFMRHIPNMVLMMPKDENEGQHMVHTALSYDEGPIAMRFPRGNGLG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 VGADGETHQGVFDIAFMRHIPNMVLMMPKDENEGQHMVHTALSYDEGPIAMRFPRGNGLG 480
Qy 481 VKMDEQLKTIPIGTWEVLRPGNDAVILTFGTTIEMAIEAAEELQKEGLSVRVVNARFIKP 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 VKMDEQLKTIPIGTWEVLRPGNDAVILTFGTTIEMAIEAAEELQKEGLSVRVVNARFIKP 540
Qy 541 IDEKMMKSILKEGLPILTIEEAVLEGGFGSSILEFAHDQGEYHTPIDRMGIPDRFIEHGS 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 IDEKMMKSILKEGLPILTIEEAVLEGGFGSSILEFAHDQGEYHTPIDRMGIPDRFIEHGS 600
Qy 601 VTALLEEIGLTKQQVANRIRLLMPPKTHKGIGS 633
|||||||||||||||||||||||||||||||||
Db 601 VTALLEEIGLTKQQVANRIRLLMPPKTHKGIGS 633
Prosecution Insights