DETAILED ACTION
Objections and rejections stated in prior Office Actions are withdrawn unless restated below.
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 .
Claim Interpretation
GPD and GPP as recited in claim 1 are understood as being abbreviations and not recitation of any specific gene from any specific source. As such, a glycerol-3-phosphate dehydrogenase identified, for example, as DAR1 or GDP1, are embodiments of a glycerol-3-phosphate dehydrogenase as recited.
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.
Claim(s) 1-8, 10-12 and 17-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Cervin et al. (U.S. 2008/0176302 A1) further in view of Zhang et al. (Production of C2–C4 diols from renewable bioresources, Biotechnol. Biofuels 10, 2017, 299), Eliot et al. (U.S. 2011/0144377 A1) and Lee (Production of 1,3-Propanediol from Glucose by Recombinant Escherichia coli BL21(DE3), Biotechnol. Bioprocess Eng. 23, 2018, 250-58).
Cervin, abstract, discloses or teaches:
The present invention provides a microorganism useful for biologically producing 1,3-propanediol from a fermentable carbon source at higher yield than was previously known. The complexity of the cofactor requirements necessitates the use of a whole cell catalyst for an industrial process that utilizes this reaction sequence to produce 1,3-propanediol. The invention provides a microorganism with disruptions in specified genes and alterations in the expression levels of specified genes that is useful in a higher yielding process to produce 1,3-propanediol.
Cerin, in the claims, states:
1. A method for the bioproduction of 1,3-propanediol comprising contacting with a suitable carbon substrate under suitable conditions a E. coli strain comprising:
a) a disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system comprising one or more of:
i) a genetically disrupted endogenous ptsH gene preventing expression of active phosphocarrier protein;
ii) a genetically disrupted endogenous ptsl gene preventing expression of active phosphoenolpyruvate-protein phosphotransferase; and
iii) a genetically disrupted endogenous crr gene preventing expression of active glucose-specific IIA component;
b) a genetically up regulated endogenous galP gene encoding active galactose-proton symporter, said up regulation resulting in an increased galactose-proton symporter activity;
c) a genetically up regulated endogenous glk gene encoding active glucokinase, said up regulation resulting in an increased glucokinase activity; and
d) a genetically down regulated endogenous gapA gene encoding active glyceraldehyde 3-phosphate dehydrogenase, said down regulation resulting in a reduced glyceraldehyde 3-phosphate dehydrogenase activity;
whereby said E. coli strain is capable of bioconverting a suitable carbon source to 1,3-propanediol.
2. The method of claim 1, wherein the E. coli strain comprises a genetically disrupted endogenous arcA gene preventing expression of active aerobic respiration control protein.
3. The method of claim 1, wherein the E. coli strain further comprises:
(i) glycerol-3-phosphate dehydrogenase;
(ii) glycerol-3-phosphatase;
(iii) dehydratase [i.e. glycerol dehydratase]; and
(iv) dehydratase reactivation factor.
4. The method of claim 2, wherein the E. coli strain further comprises:
(v) glycerol-3-phosphate dehydrogenase;
(vi) glycerol-3-phosphatase;
(vii) dehydratase; and
(viii) dehydratase reactivation factor.
Cervin further states:
Applicants have provided an E. coli strain comprising:
[0019]
a) a disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system preventing expression of active PEP-glucose phosphotransferase system proteins;
b) an up regulated endogenous galP gene encoding active galactose-proton symporter [i.e. galactose permease];
c) an up regulated endogenous glk gene encoding active glucokinase; and
d) a down regulated endogenous gapA gene encoding active glycerolaldehyde 3-phosphate dehydrogenase.
[0024]
Applicants have also provided an E. coli strain described above wherein the disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system comprises one or more of
a1) a disrupted endogenous ptsH gene preventing expression of active phosphocarrier protein proteins;
a2) a disrupted endogenous ptsl gene preventing expression of active phosphoenolpyruvate-protein phosphotransferase; and
a3) a disrupted endogenous crr gene preventing expression of active glucose-specific IIA component.
[0028]
The E. coli embodiments described above can further comprise one or more of
e) a disrupted endogenous arcA gene preventing expression of active aerobic respiration control protein;
f) an up regulated endogenous ppc gene encoding active phosphoenolpyruvate carboxylase;
g) an up regulated endogenous btuR gene encoding active cob(I)alamin adenosyltransferase; and
h) an up regulated yqhD gene encoding active alcohol dehydrogenase.
[0033]
The E. coli embodiments described above can further comprise one or more of
i) a disrupted endogenous mgsA gene preventing the expression of active methylglyoxal synthase;
j) a disrupted endogenous ackA gene preventing the expression of active acetate kinase;
k) a disrupted endogenous pta gene preventing the expression of active phosphotrasacetylase;
l) a disrupted endogenous aldA gene preventing the expression of active aldehyde dehydrogenase A; and
m) a disrupted endogenous aldB gene preventing the expression of active aldehyde dehydrogenase B.
[0039]
The E. coli embodiments described above can further comprise one or more of:
n) a disrupted endogenous edd gene preventing expression of active phosphogluconate dehydratase;
o) a disrupted endogenous glpK gene preventing expression of active glycerol kinase; and
p) a disrupted endogenous gldA gene preventing expression of active NADH-dependent glycerol dehydrogenase.
[0043]
Additionally, 1,3-propanediol can be bioproduced by contacting an E. coli strain described herein with a suitable carbon substrate such as glucose under suitable conditions for fermentation. In addition, 1,3-propanediol can be bioproduced by contacting an E. coli strain described herein, the E. coli strain further comprising an active:
(i) glycerol-3-phosphate dehydrogenase;
(ii) glycerol-3-phosphatase;
(iii) dehydratase; and
(iv) dehydratase reactivating activity;
“Strain KLP23 was transformed with plasmids pAH48 and pDT29 or pKP32. Production of 1,3-propanediol (and glycerol) was determined in 14 L fermenters as described in General Methods.” Cervin, para. [0267].
Cervin, para. [0026], shows that strain KLP23 is in E. coli with deleted glpK (glycerol kinase) and gldA (glycerol dehydrogenase) genes.
“Duplicate shake flasks cultures were grown with strains Triple btuR 1.6 yqhD, pSYCO109, and TT pSYCO109..” Cervin, para. [0314].
The genotype for E. coli strain Triple btuR 1.6 yqhD is as follows from para. [0026] of Cervin:
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The genotype shows, inter alia, gene deletion of glpK (glycerol kinase) [see, Cervin, para. [0118], deletion of gldA (aldehyde dehydrogenase), deletion of ptsHIcrr [i.e. weakening/inactivation of a glucose- phosphotransferase system (PTS)], replacement of galP promoter (galactose permease) with a trc promoter that causes increased expression of galP, replacement of glk promoter (glucose kinase or glucokinase) with a trc promoter that causes its increased expression of glk, and enhanced activity of btuR (adenosyltransferase) by replacement of its endogenous promoter. The pSYCO109 plasmid is “Particularly useful in the present invention are the vectors pSYCO101, pSYCO103, pSYCO106, and pSYCO109. The essential elements are derived from the dha regulon isolated from Klebsiella pneumoniae and from Saccharomyces cerevisiae. Each contains the open reading frames dhaB1, dhaB2, dhaB3, dhaX, orfX, DAR1, and GPP2 arranged in three separate operons, nucleotide sequences of which are given in SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68, respectively.” Cervin, para. [0174]. “The gene encoding glycerol-3-phosphate dehydrogenase (DAR1, GPD1) has been cloned and sequenced from S. diastaticus.” Cervin, para. [0011]. That is DAR1 is GPD1. Genes dhaB1, dhaB2, dhaB3 are all subunits of glycerol dehydratase, and orfX is dehydratase reactivation factor for glycerol dehydratase (Cervin, para. [0006]).
The above is further supported in Cervin of:
“an up regulated endogenous btuR gene encoding active cob(I)alamin adenosyltransferase.” Cervin, para. [0032].
“an up regulated endogenous glk gene encoding active glucokinase.” Cervin, para. [0019].
“an up regulated endogenous galP gene encoding active galactose-proton symporter” (i.e, galactose permease). Cervin, para. [0019].
“a disrupted endogenous crr gene preventing expression of active glucose-specific IIA component” of the phosphotransferase PTS system. Cervin, para. [0024].
“a disrupted endogenous glpK gene preventing expression of active glycerol kinase.” Cervin, para. [0039].
As such, the above is a description or teaching of a modified E. coli (modified from a parent) having all of:
(1) enhancement of activity of adenosyltransferase (btuR); (2) enhancement of activity of glycerol-3-phosphate dehydrogenase (GPD) and glycerol-3-phosphate phosphatase (GPP); (3) enhancement of activity of glycerol dehydratase, and glycerol dehydratase reactivase (4) enhancement of activity of galactose permease and glucokinase; (5) weakening or inactivation of activity of the phosphotransferase system (PTS), and (6) deletion of glycerol kinase.
Further, as directly recited in claims 1 and 6, Cervin, para. [0174]-[0184], describe expression of heterologous glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase genes that is considered to meet claim 6. Regarding features directly recited in claim 17 and requirement in the claims including claim 1 that a product is produced from glucose, Cervin, para. [0262], describes that glucose (i.e. a medium containing glucose) is used in cultures to produce 1,3-propanediol as disclosed by Cervin.
Regarding E. coli specifically producing 3-hydroxypropionic acid from glucose as recited in claim 1, and enhancement of activity of glycerol dehydratase, and glycerol dehydratase reactivase and aldehyde dehydrogenase as recited in claim 4, Cervin, para. [0199], describes:
The production of 1,3-propanediol from glucose can be accomplished by the following series of steps. This series is representative of a number of pathways known to those skilled in the art. Glucose is converted in a series of steps by enzymes of the glycolytic pathway to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, or reduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis. The hydrolysis step can be catalyzed by any number of cellular phosphatases, which are known to be non-specific with respect to their substrates, or the activity can be introduced into the host by recombination. The reduction step can be catalyzed by a NAD+ (or NADP+) linked host enzyme or the activity can be introduced into the host by recombination. It is notable that the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) that catalyzes the reversible reaction of Equation 3.
Glycerol→3-HPA+H2O (Equation 1)
3-HPA [3-hydroxypropionaldehyde] +NADH+H+→1,3-Propanediol+NAD+ (Equation 2)
Glycerol+NAD+→DHA+NADH+H+ (Equation 3)
For clearer comprehension, Zhang, Fig. 3, shows the same or very similar pathway for production of 1,3-propanediol including the following:
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As can be seen, the various modifications taught by Cervin and Zhang produce glycerol and then 3-hydroxypropionaldehye as the immediate precursor or intermediate to 1,3-propanediol. However, the prior art teaches that other useful products can be made by culture/fermentation of E. coli from a 3-hyroxypropionaldehyde intermediate.
Eliot, abstract, teaches:
The present invention provides a microorganism useful for biologically producing 3-hydroxypropionic acid from a fermentable carbon source. Further, the microorganism comprises disruptions in specified genes and alterations in the expression levels of specified genes that are useful in a higher yielding process to produce 3-hydroxypropionic acid, compositions comprising renewably sourced 3-hydroxypropionic acid provided by said microorganism, and industrial relevant products made using such renewably sourced 3-hydroxypropionic acid.
Eliot, in the claims, states:
1. An E. coli strain comprising:
a) an exogenous gene encoding a glycerol-3-phosphate dehydrogenase;
b) an exogenous gene encoding a glycerol 3-phosphatase;
c) exogenous genes encoding alpha, beta, and gamma subunits of glycerol dehydratase; and
d) an overexpression of a gene encoding an aldehyde dehydrogenase;
whereby said E. coli strain is capable of bioconverting a suitable carbon source to 3-hydroxypropionic acid.
3. The E. coli strain of claim 1 further comprising a deletion of an endogenous gene encoding a 1,3-propanediol dehydrogenase.
5. The E. coli strain of claim 1 further comprising:
e) a disrupted endogenous phosphoenolpyruvate-glucose phosphotransferase system comprising one or more of:
i) a genetically disrupted endogenous ptsH gene preventing expression of active phosphocarrier protein;
ii) a genetically disrupted endogenous ptsl gene preventing expression of active phosphoenolpyruvate-protein phosphotransferase; and
iii) a genetically disrupted endogenous crr gene preventing expression of active glucose-specific IIA component;
f) a genetically up regulated endogenous galP gene encoding active galactose-proton symporter, said up regulation resulting in an increased galactose-proton symporter activity; wherein the up regulation is produced by (a) by introducing additional copies of said gene into host cell followed by integration or (b) by replacing native regulatory sequence with strong non-native promoter or altered native promoter;
g) a genetically up regulated endogenous glk gene encoding active glucokinase, said up regulation resulting in an increased glucokinase activity; wherein the up regulation is produced by a) by introducing additional copies of said gene into host cell followed by integration or b) by replacing native regulatory sequence with strong non-native promoter or altered native promoter, and
h) a genetically down regulated endogenous gapA gene encoding active glyceraldehyde-3-phosphate dehydrogenase, said down regulation resulting in a reduced glyceraldehyde-3-phosphate dehydrogenase activity.
10. The method of claim 9 wherein said suitable carbon substrate is glucose.
“In one embodiment, the present invention utilizes a preferred pathway for the production of 3-HP from a sugar substrate where the carbon flow moves from glucose to DHAP, G3P, glycerol, 3-HPA, and finally to 3-HP.” Eliot, para. [0152].
That is, the pathway of carbon frow from DHAP, G3P, glycerol, to 3-HPA as stated by Eliot is identical to that taught by Cervin (and Zhang). Cerin teaches expression of an alcohol dehydrogenase as a final step to produce 1,3-propanediol from 3-HPA. Eliot in the alternative teaches expression of an exogenous aldehyde dehydrogenase for “conversion of 3-hydroxypropionaldehdye [3-HPA] to 3-HP [3-hydroxypropionic acid].” Eliot, para. [0114].
As such, at the time of filing an ordinarily skilled artisan would have been motivated to replace expression of an alcohol dehydrogenase for 1,3-propanediol production with enhanced expression of a heterologous aldehyde dehydrogenase as taught by Eliot for production of 3HP from 3-HPA. An ordinarily skilled artisan would have been motivated to do this since Eliot teaches that 3HP is an advantageous product to be produced by fermentation of E. coli wherein the carbon flow from glucose to DHAP, G3P, glycerol, to 3-HPA is identical between Cervin and Eliot. Upon making such a modification, just as Cervin describes production of 3-hydroxypropionaldehdye and 1,3-hydroxypropanediol from glucose, produced 3-hydroxypropionic acid due to expression of a heterologous aldehyde dehydrogenase is from glucose in the microorganism so modified.
As such, at the time of filing an ordinarily skilled artisan would have been motivated to make this one gene substitution in embodiment E. coli of Cervin (expression of an alcohol dehydrogenase to an exogenous aldehyde dehydrogenase as taught by Eliot) in order to gain the benefit of 3HP production. Again, both Cervin and Eliot teach the same carbon flow from glucose to DHAP, G3P, glycerol, 3-HPA, and finally to 3-HP wherein Cervin has some additional teachings of modifications to improve such carbon such as increased expression btuR. That is, Cervin and Eliot teach that a cell with carbon flow from glucose to DHAP, G3P, glycerol, to 3-HPA can be used to produce either desirable product being 1,3-propanediol or 3-HP depending upon whether an alcohol dehydrogenase or an aldehyde dehydrogenase, respectively, is expressed for the final conversion step of 3-HPA, which directly motivates an ordinarily skilled artisan to express an aldehyde dehydrogenase (in replacement of alcohol dehydrogenase) in E. coli engineered as taught by Cervin in order to achieve the advantage of 3-HP production. Upon making such a modification to embodiments of Cervin, the features of enhancement of activity of glycerol dehydratase, glycerol dehydratase reactivase and aldehyde dehydrogenase as recited in claim 4 are reached. Further, an ordinarily skilled artisan would have been motivated to culture any E. coli in accordance with Cervin (with substitution of aldehyde dehydrogenase) on a glucose-containing media as taught by Cervin to produce 3HP as to meet the features of claims 9-11, wherein Eliot also teaches culturing or a recombinant E. coli on a medium containing glucose to produce 3-HP.
Regarding a requirement in claim 4 for weakening/inactivation of a glucose-specific transporter of a phosphotransferase system (PTS), as discussed Cervin directly teaches deletion of genes ptsHIccr that interrupts PTS system, which is further taught by Eliot, claim 5. Lee, abstract, also relates to production of 1,3-propanediol from glucose using a recombinant E. coli with many commonalities with Cervin and Eliot as shown in Fig. 1 of Lee including production of glycerol (GLY) and 3-hydroxypropinaldehye (3-HPA) by heterologous expressing of GPD and GPP. Lee, sec. 3.7, sets forth:
“In E. coli, there are five glucose transport systems. Among them, the phosphoenolpyruvate dependent phosphotransferase system (PTS system) plays a key role. The PTS system consists of EI, HPr, EII, etc., and the EII consists of the cytosolic IIAGlc protein (encoded by crr gene) and membranous IICBGlc protein (encoded by ptsG gene). In this PTS system, conversion of PEP to pyruvate is coupled with glucose transport and this coupling also contributes to the overflow metabolism. The ptsG gene was disrupted in EB6 strain, and the EB9 and EB9-1 strains were constructed. As expected, in the newly developed ptsG-deficient strains, the glucose consumption rate and cell growth decreased significantly.”
As far as claim 4 may require the deletion of a specific transporter gene being ptsG as set forth in para. [46] of the specification, all of Cervin, Eliot and Lee teach interruption of PTS system as beneficial for supporting carbon flow from glucose to glycerol to 3-HPA and then to 1,3-propanediol or 3-HP. While Cervin and Eliot teach interruption of PTS by deletion of ptsHIccr genes, Lee teach that deletion of ptsG gene is also a technical solution for interruption of PTS. More specifically, Cervin, para. [0019], teaches that the endogenous PTS system should be disrupted to prevent expression of active PEP-glucose phosphotransferase system proteins and it not limited to specific proteins to be disrupted. Substitution of known elements is obvious upon a finding of:
(1) a finding that the prior art contained a device (method, product, etc.) which differed from the claimed device by the substitution of some components (step, element, etc.) with other components;
(2) a finding that the substituted components and their functions were known in the art;
(3) a finding that one of ordinary skill in the art could have substituted one known element for another, and the results of the substitution would have been predictable; and
(4) whatever additional findings based on the Graham factual inquiries may be necessary, in view of the facts of the case under consideration, to explain a conclusion of obviousness. (MPEP 2143(I)(B)).
Here, as set forth above, Lee teach that deletion of ptsG gene is also a technical solution for interruption of PTS such that deletion of ptsG can substitute for deletion of ptsHIccr as taught by Cervin in a manner consistent with para. [0019] of Cervin with a predictable result of interrupting the PTS system (see Cervin, para. [0019]) such that an ordinarily skilled artisan at time of filing would have been motivated to do the same in view of MPEP 2143(I)(B).
Regarding claim 8, as discussed, Cervin teaches culturing of E. coli engineered to produce 1,3-propanediol from glucose, wherein glucose is the only carbon source provided such that glucose supports both cell growth and reproduction as well as propanediol production. Upon expression of an aldehyde dehydrogenase to produce 3-hydroxypropionic acid, glucose similarly supports both cell growth and reproduction as well as propanediol production as well as 3-hydroxypropionic acid production. An ordinarily skilled artisan at the time of filing would have recognized that when any E. coli cell is engineered to produce significant quantities of product by fermentation, then glucose that would be used for cell growth is diverted to production of that product such that cell growth will be inhibited due to the metabolic burden of production of such product (i.e. 3-hydroxypropionic acid). “The fact that appellant has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious." MPEP 2145(II). While inhibited cell growth may not be an “advantage,” the same is described in para. [112] of the specification as a direct result of transcription of GDP and GPP genes that diverts carbon flow from glucose to 3-hdyroxypropionate production.
Regarding claim 12, Cervin, para. [0258], states pH is adjusted to 6.8. As such, the prior art expressly teaches that a pH of 6.8 falling within the range as recited in claim 12 is appropriate for culturing E. coli to make a product.
Regarding claim 18, Cervin, para. [0314], states: Final 1,3-propanediol concentration was 135.3 g/L and the mass yield was 46.1%. An ordinarily skilled artisan at time of filing would have fully expected that alternate expression of an aldehyde dehydrogenase for 3HP production will produce a corresponding amount of 3HP meeting the features of claim 18. As discussed above, glycerol produced to 3-HPA and other further downstream products such that its concentration would be expected to low including below 10 g/L. Table 2.2 of Cerin shows that at several points during fermentation (e.g. 30 hours) the product 1,3-PD is between 60-200 g/L and glycerol less than 10 g/L.
Claim(s) 1-8, 10-13 and 17-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Cervin et al. (U.S. 2008/0176302 A1), Zhang et al. (Production of C2–C4 diols from renewable bioresources, Biotechnol. Biofuels 10, 2017, 299), Eliot et al. (U.S. 2011/0144377 A1) and Lee (Production of 1,3-Propanediol from Glucose by Recombinant Escherichia coli BL21(DE3), Biotechnol. Bioprocess Eng. 23, 2018, 250-58) as applied to claims 1-8, 10-12 and 17-18 above, and further in view of Sanil et al. (In situ pH management for microbial culture in shake flasks and its application to increase plasmid yield, J. Ind. Microbiol. Biotechnol. 41, 2014: 647-55).
Regarding claim 13, Cervin, para. [0258], states pH is adjusted to 6.8; however, Cervin does not state how pH is adjusted. Sanil discussed the use of magnesium-hydroxide hydrogels to manage the pH of E. coli culture. While Cervin is not explicit regarding the reagent used to adjust pH, as evidenced by Sanil it is known in the prior art to employ magnesium hydroxide to adjust the pH of an E. coli culture such that an ordinarily skilled artisan at the time of filing would have been motivated to add magnesium hydroxide in a solution or otherwise to adjust the pH of an E. coli culture as needed. It is noted that Sanil, page 648, right col., describes addition of 150 Mg(OH)2 to a solution such that an ordinarily skilled artisan at the time of filing would have understood that solutions of magnesium hydroxide are to be used in adjusting pH of a culture as opposed to addition of solid or neat magnesium hydroxide.
Claim(s) 1-8, 10-12, 14-15 and 17-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Cervin et al. (U.S. 2008/0176302 A1), Zhang et al. (Production of C2–C4 diols from renewable bioresources, Biotechnol. Biofuels 10, 2017, 299), Eliot et al. (U.S. 2011/0144377 A1) and Lee (Production of 1,3-Propanediol from Glucose by Recombinant Escherichia coli BL21(DE3), Biotechnol. Bioprocess Eng. 23, 2018, 250-58) as applied to claims 1-8, 10-12 and 17-18 above, and further in view of Nakamura et a. (U.S. 6,013,494 A) and Overton (Recombinant Protein production in bacterial hosts, Drug Discovery Today 19, 2014, 590-601).
Regarding claims 14-15, “Initiation control regions, or promoters, which are useful to drive expression of the G3PDH and G3P phosphatase genes (DAR1 and GPP2, respectively) in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, λPL, λPR, T7, tac, and trc (useful for expression in E. coli).” Cervin, para. [0171]. As discussed above, G3PDH and G3P are the two enzymes directly responsible for glycerol production.
As discussed above, at least Cervin an Eliot teach that E. coli can be engineered to produce glycerol and 3-hydroxypropionaldehdye intermediates, which can be reduced to produce 1,3-propanediol or oxidized to produce 3-hyroxypropionate depending upon the desired product. In either case, the pathway for production is identical except for the last step of reduction or oxidation.
Nakamura similarly teaches production of glycerol from glucose in E. coli by expression of glycerol-3-phosphate phosphatase (GPP2) and glycerol-3-phosphate dehydrogenase (DAR1). “The pAH19 contains the GPP2 gene in the correct orientation for expression from the lac promoter.” Nakamura, col. 24, ln. 35-38. “Plasmid pAH40 contains the new RBS and DAR1 gene in the correct orientation for expression from the trc promoter of Trc99A (Pharmacia).” Nakamura, col. 25, ln. 1-2. Cervin, para. [0184], further indicates that in plasmid pSYCO109 indicates that GPP2 and DAR1 are expressed under control of a trc promoter. “The expression of the DAR1 and GPP2 genes may be enhanced by the addition of IPTG (0.2-2.0 mM) to the growth medium.” Nakamura, col. 30, ln, 41-42. Nakamura, Example 2, demonstrates production of 1,3-propanediol (through a glucose intermediate as discussed) by addition of 0.2 mM IPTG (an inducer) after 6 hours of culture with analysis for production of glycerol and 1,3-propanediol after addition of IPTG for up to 24 hours of culture time, which indicates that glucose was present and not exhausted at time of IPTG induction. The trc and lac promoters are known to be inducible by addition of IPTG provided that the lac repressor (LacI) protein is expressed. See Overton, Table 1 and related text for background information.
Cervin is silent regarding the application of IPTG or any other inducing agent. However, the prior art including Nakamura teaches that it is known to employ IPTG production for induced expression of glycerol-3-phosphate dehydrogenase and/or gelycerol-3-phosphate phosphatase for production of glycerol. While Nakamura exemplifies production of 1,3-propanediol, as discussed Eliot and other cited art teach that E. coli for production 1,3-propanediol can be converted to producing 3-hydroxypropionic acid by expression of an aldehyde dehydrogenase. As such, at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of Cervin (including after modification to produce 3-hydroxypropionic acid) to have induction by IPTG of glycerol-3-phosphate dehydrogenase and/or glycerol-3-phosphate phosphatase genes as shown by Nakamura, since Nakamura teach that the same is a suitable technological solution for overexpression of glycerol-3-phosphate dehydrogenase and/or gelycerol-3-phosphate phosphatase for production of glycerol and other products downstream from glycerol, wherein Nakamura teaches that IPTG inducer is added prior to consumption of all glucose in a culture.
Regarding claim 15, MPEP 2144.04(IV)(C) provides: “Selection of any order of mixing ingredients is prima facie obvious” in the absence of any particular unexpected result. As discussed, Nakamura teaches addition of an IPTG inducer during the culture to produce a product from glucose via a glycerol intermediate. The selection of any point during the culture (including at the beginning) is prima facie obvious outside of any particularly unexpected or technical effect.
Claim(s) 1-8, 10-12, and 14-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Cervin et al. (U.S. 2008/0176302 A1), Zhang et al. (Production of C2–C4 diols from renewable bioresources, Biotechnol. Biofuels 10, 2017, 299), Eliot et al. (U.S. 2011/0144377 A1), Lee (Production of 1,3-Propanediol from Glucose by Recombinant Escherichia coli BL21(DE3), Biotechnol. Bioprocess Eng. 23, 2018, 250-58), Nakamura et a. (U.S. 6,013,494 A) and Overton (Recombinant Protein production in bacterial hosts, Drug Discovery Today 19, 2014, 590-601) as applied to claims 1-8, 10-12, 14-15 and 17-18 above, and further in view of Hanko et al. (Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator, Sci. Reports 7, 2017, 1724).
Regarding claim 16, Hanko, abstract, teaches the following:
3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. . . . In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/PmmsA and PpHpdR/PhpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). . . . These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.”
As such, Hanko teaches promoters that are inducible with 3-HP and are intended for employment in producing 3-hydroxypropionic acid: “Although, relatively high titers of 3-HP have been reported in E. coli and K. pneumoniae, the challenge remains to develop a sustainable biotechnological production of this carboxylic acid.” Hanko, page 1. As such, while induction with IPTG is discussed above, at the time of filing an ordinarily skilled artisan would have been motivated to replace trc promoters for expressing glycerol-3-phosphate dehydrogenase and/or gelycerol-3-phosphate phosphatase genes as discussed above with the promoters taught by Hanko inducible with 3-hydroxypriopionic acid. An ordinarily skilled artisan at the time of filing would have been motivated to do this since Hanko teaches that such promoters are preferred for synthetic biology applications of producing 3-HP.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1-8 and 10-18 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-20 of copending Application No. 18/696,177 in view of Cervin et al. (U.S. 2008/0176302 A1), Zhang et al. (Production of C2–C4 diols from renewable bioresources, Biotechnol. Biofuels 10, 2017, 299), Eliot et al. (U.S. 2011/0144377 A1), Lee (Production of 1,3-Propanediol from Glucose by Recombinant Escherichia coli BL21(DE3), Biotechnol. Bioprocess Eng. 23, 2018, 250-58), Nakamura et a. (U.S. 6,013,494 A), Overton (Recombinant Protein production in bacterial hosts, Drug Discovery Today 19, 2014, 590-601), Sanil et al. (In situ pH management for microbial culture in shake flasks and its application to increase plasmid yield, J. Ind. Microbiol. Biotechnol. 41, 2014: 647-55) and Hanko et al. (Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator, Sci. Reports 7, 2017, 1724).
The rejections under 35 U.S.C. 103 stated above are incorporated herein by reference.
The copending claims include:
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The copending claims, particularly claim 1, are particularly broad as to include an E. coli bacterium with foreign genes for 3-HP production. Cervin, as discussed above, teach E. coli engineered to produce 1,3-propanediol that after modification to express an aldehyde dehydrogenase for 3-HP production as taught by Eliot, as discussed above, are embodiments of copending claims 1-4, and also meet the features of present claims as discussed above. For these reasons, at the time of filing, an ordinarily skilled artisan would have been motivated to form embodiments of the copending claims meeting the features of the rejected claims, and to apply the same to the methods of the rejected claims, for the same reasons stated under 35 U.S.C. 103 stated above. Specifically, practice of the copending claims requires specific E. coli embodiments wherein the copending claims do not recite all features necessary for 3-HP production. Since the prior art cited teaches and suggests E. coli having all needed structure for 3-HP, an ordinarily skilled artisan would have been motivated to form embodiments of the copending having the features taught by at least Cervin, Zhang, Eliot, Lee and other cited prior art, as discussed above, as advantageous for producing 3-HP from glucose, which further meet the features of the rejected claims.
This is a provisional nonstatutory double patenting rejection.
Response to arguments
Applicant argues:
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In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e.,1) higher yield than previously obtained, and 2) yield greater than 35%) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Further, the prior art teaches that 3-HPA and any downstream product including 3-hydroxypropionic acid is produced from glucose.
Applicant argues:
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Zhang is cited for teaching the same pathway as described in Cervin, para. [0199].
Applicant argues:
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The burden is on applicant to establish that results are unexpected and significant. MPEP 716.02(b). "[A]ppellants have the burden of explaining the data in any declaration they proffer as evidence of non-obviousness." Ex parte Ishizaka, 24 USPQ2d 1621, 1624 (Bd. Pat. App. & Inter. 1992); MPEP 716.02(b). “An affidavit or declaration under 37 CFR 1.132 [or data from the specification] must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness.” MPEP 716.02(e). "Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof." In re Gershon, 372 F.2d 535, 538, 152 USPQ 602, 604 (CCPA 1967); MPEP 716.02(c)(II). “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.’" MPEP 716.02(d).
While Eliot does not disclose production date for 3-hydroxypropionic acid, Eliot discloses specific embodiments, which represents the closest prior art. Applicant has not made any comparison to the closest prior art of Eliot, as such unexpected results have not been established. Applicant has the burden of establishing unexpected results.
It is noted that Eliot, para. [0177] embodiments wherein “A deletion of the yqhD gene (given as SEQ ID NO:76), which encodes a nonspecific alcohol dehydrogenase, is made in E. coli strain TT/pSYCO109 (described in U.S. Pat. No. 7,371,558, Example 14) by P1 transduction.” U.S. Pat. No. 7,371,558, Example 14, describes “A P1 phage lysate was prepared and used to pass the mutation to strain Triple 1.6btuR 1.6yqhD to form strain Triple 1.6btuR 1.6yqhD ΔackA-pta::Cm.” U.S. Pat. No. 7,371,558, col. 4,ln. 60-61. The pSYCO109 plasmid further expresses GPD1 (Dar1), GPP, dhaB1, dhaB2, dhaB3 and dhaX wherein no comparison is made to an equivalent strain as disclosed by Cervin by reference to U.S. Pat. No. 7,371,558, Example 14. See U.S. Pat. No. 7,371,558, col. 22, ln. 1-10. Gene btuR encodes adenosyltransferase that is the closes prior art includes heterologous expression of adenosyltransferase and other heterologous genes. An affidavit or declaration under 37 CFR 1.132 [or data from the specification] must compare the claimed subject matter with the closest prior art [being the specific embodiment example E. coli in para. [0177] of Eliot] to be effective to rebut a prima facie case of obviousness.” MPEP 716.02(e).
Applicant argues:
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In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Cervin is cited for teaching adenosyltransferase. As stated in the rejection, the relationship between adenosyltransferase and 3-HP and propanediol production is that the immediate precursor to both is 3-HPA whose production is increased by expression of adenosyltarnsferase that is then converted to either 3-HP by oxidation or propanediol by reduction. Further, Eliot directly teaches that an E.coli pre-engineered for production of propanediol is an appropriate starting point for making an E. coli for 3-HP production. That is the E. coli strain TT/pSYCO109 (described in U.S. Pat. No. 7,371,558, Example 14) cited in Eliot, para. [0177] is a strain for 1,3-PDO production.
Applicant argues:
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The Examiner is bound by the requirements of the MPEP. “A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim.” MPEP 804(II)(B). In this instance, the rejected claims are obvious over the reference claims for the reasons stated. “It is important to note that the "obviousness" analysis for "obviousness-type" double-patenting is "similar to, but not necessarily the same as, that undertaken under 35 U.S.C. 103.” MPEP 804.
Regarding applicants arguments that a species is rarely render obvious by a genus, it is noted that claim 1 recites a genus (as evidenced by the presence of narrower depending claims), which is obviousness over the cited prior art for the reasons set forth above under 35 U.S.C. 103. Applicant’s arguments that the genus of claim 1 is not obvious over the prior art is addressed above.
Conclusion
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 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 TODD M EPSTEIN whose telephone number is (571)272-5141. The examiner can normally be reached Mon-Fri 9:00a-5:30p.
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652