DETAILED ACTION
All objections and rejections raised in prior office actions are withdrawn unless restated below.
The Terminal Disclaimer over Application No. 18005369 filed 03/24/2026 is acknowledged.
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03/24/2026 has been entered.
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 10 and 11 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipation by Kim et al. (High‐level production of 3‐hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli, Biotechnol. Bioeng. 117, Jan. 2020, 2139-52).
Kim, abstract, states:
As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L−1·h−1, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.
The above meets the features of claim 10 being a cell strain producing 3-HP at concentration of 60 g/L or more in a production medium. Regarding recitation of “wherein proliferation of the cells does not occur,” the broadest reasonable interpretation of the same is a culture containing 1) a strain (cells) producing 3-HP, 2) 60 g/L or more of 3-HP, and 3) the strain cells are not underdoing active proliferation. Kim, Fig. 3(e) shows the following (annotations added):
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That is, as shown in Fig. 3(e), after about 35 hours, a culture exits wherein ) a strain (cells) producing 3-HP, 2) 60 g/L or more of 3-HP in the culture (closed diamonds), and 3) the strain cells are not underdoing active proliferation (cell density shown by OD600 by closed squares). It is noted that any population of cells must have undergone prior proliferation as to become a population. The claims do not recite a method. Any culture not undergoing active proliferation meets the claim limitations “wherein proliferation of the cells does not occur” regardless of any method through which such culture is produced. As such, Kim anticipates the features of claim 10 for these reasons.
Regarding claim 11, , Fig. 3(e) of Kim presents the following embodiment wherein open triangle is glycerol. In the annotated Fig. 3(e) below, the open triangle for glycerol is straddling the 0 g/L line (highlighted by annotated arrow) such that it is clear that the ending glycerol concentration is less than 0.3% w/v (3 g/L). It is noted that glycerol is added during fermentation such that glycerol concentration fluctuates during fermentation; nevertheless, glycerol concentration is near zero at conclusion of fermentation with 3-HP concentration is above 60 g/L, which meets the features of claim 10 as well as discussed above.
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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, 4, 5 and 10-11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (High‐level production of 3‐hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli, Biotechnol. Bioeng. 117, Jan. 2020, 2139-52) further in view of Niu et al. (3-Hydroxypropionic acid production by recombinant Escherichia coli ZJU-3HP01 using glycerol–glucose dual-substrate fermentative strategy, Biotechnol. Appl. Biochem., 2017, 572-78).
The anticipation rejection of claims 10 and 11 over Kim are incorporated herein by reference.
As an initial matter, the broadest reasonable interpretation of the claims will be discussed.
Claim 1 after the preamble recites the open transitional phrase “comprising” such that claim 1 is open to the performance of unrecited method steps. The method requires two steps:
Inoculating cells with 3-HP production ability into a production medium; and
Producing 3-HP by culturing the inoculated cells.
The producing/culturing step is qualified adding glucose when dissolved oxygen in the medium is 0.1% or more and wherein in step (2) of producing 3-HP proliferation of cells does not occur.
The claims do not positively exclude the performance of a step wherein cells are proliferated. Since the claims recite the open transitional phrase “comprising,” the following is an embodiment of claim 1:
1. (Currently amended) A method of producing 3-hydroxypropionic acid (3-HP), comprising
(1) inoculating cells having 3-hydroxypropionic acid (3-HP) production ability into a production medium; [[and]]
(1a) proliferating the cells having 3-HP production ability in the production medium; and
(2) producing 3-HP by culturing the inoculated cells, wherein the production medium comprises glycerol as a carbon source, and the culturing is performed either under a condition of adding glucose when the dissolved oxygen in the medium is 0.1% or more, or a condition of adding glucose when the dissolved oxygen in the medium is 0.1% or more, and maintaining dissolved oxygen (DO) in the production medium at a level of 50% or less,
wherein in step (2) of producing 3-HP, proliferation of cells does not occur.
That is, the broadest reasonable interpretation of claim 1 is that there is a period of 3-HP producing wherein cell proliferation does not occur. Claim 1 does not exclude either 1) performance of a prior proliferation step, or 2) performance of prior proliferation step wherein 3-HP production also occurs.
Kim, abstract, states:
As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L−1·h−1, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.
Regarding claim 1, Kim, page 2141, left col., states: Another factor that needs to be considered for efficient 3-HP production is dissolved oxygen (DO) level during the cultivation because the presence of oxygen inactivates glycerol dehydratase and inhibits the biosynthesis of coenzyme B12, a cofactor of glycerol dehydratase. However, at the same time, oxygen is necessary for efficient regeneration of NAD+, which is required for the activity of aldehyde dehydrogenase. Surprisingly, not many studies have been performed on examining the effect of DO on 3-HP production, and the fermentation condition of fixed agitation speed at various aeration rates was applied in most studies. Thus, the fermentation strategy including the DO management and nutrient feed strategy should be carefully optimized since 3-HP production is sensitive to fermentation conditions.
Kim, sec 2.3 states:
To produce 3-HP with recombinant E. coli strains in flask, the MR medium was used. The MR medium (pH 7.0) containing 6.67 g/L of KH2PO4, 4 g/L of (NH4)2HPO4, 0.8 g/L of citric acid, and 5 ml/L of trace metal solution was supplemented with 0.8 g/L of MgSO4·7H2O, 4 μM vitamin B12, 1 g/L of yeast extract, and 15 g/L of glycerol.
Fed-batch fermentation was carried out using a 6.6-L jar bioreactor (Bioflo 3000; New Brunswick Scientific Co., Edison, NJ) containing 2 L of total working volume at 37°C and pH 7.0. The ammonia solution (28%, v/v) was automatically fed into the bioreactor to control the culture pH at 7.0. The vessel aspect ratio was 1.4:1 (height of 24 cm and internal diameter of 17 cm). The standard Rushton impeller (a size of 6 cm in a diameter) with six vertical blades (blade length of 1.9 cm, height of 1.5 cm, and width of 1 mm) was used and positioned at a distance of 24 cm below the headplate. A filtered air was provided at an aeration rate of 2 L/min into the bioreactor using a ring sparger. The same MR medium, as used in flask cultivations, was also used in bioreactor cultures, except that 40 g/L of glycerol or crude glycerol (thankfully given by Hanwha Chemical in Daejeon) was supplemented as an initial carbon source. As the DO probe calibration method, an electronic zero (0%) on the bioreactor was set first and the culture medium was saturated with air at least for 8 hr.
Cells grown in two flasks (200 ml total) were transferred into the bioreactor, making a 10% (v/v) inoculum size [i.e. inoculating cells having 3-hydroxypropionic acid (3-HP) production ability into a production medium]. A bioreactor feeding solution was consisted of 700 g/L of glycerol and 8 g/L of MgSO4·7H2O. For fedbatch fermentations with intermittent feeding, 50 ml of feeding solution (equivalent to 35 g of glycerol and 4 g of MgSO4·7H2O) was manually added into the bioreactor when residual glycerol concentration was below 10 g/L, to maintain the glycerol in excess.
Kim, page, 2146, right col., states:
As a next step, the DO level was controlled by automatically increasing the agitation from 500 to 1000 rpm to further enhance 3-HP production. In the abovementioned fermentations, the agitation speed was fixed at 500 rpm rather than controlling the DO level (eventually reaching the DO level of 0% of air saturation) since glycerol dehydratase is known to be sensitive to oxygen. However, it was hypothesized that increasing oxygen supply would increase 3-HP titer since it can regenerate NAD+, a cofactor for aldehyde dehydrogenase, through electron transport chain. Thus, it was reasoned that there must be an optimal DO level for the enhanced 3-HP production. Fed-batch fermentation was performed by controlling the DO level to 10% of air saturation. This DO control level was chosen based on our previous experience on the successful production of lactate-containing polyhydroxyalkanoates, which required the activity of oxygen-sensitive lactate dehydrogenase and also cell growth at the same time. By controlling the DO level at 10% of air saturation, 3-HP titer dramatically increased to 71.9 g/L with the yield and productivity of 0.466 g/g glycerol and 1.94 g·L−1·h−1, respectively (Figure 3e).
The above is a disclosure of a method of producing 3-hydroxypropionic acid (3-HP), comprising (1) inoculating cells having 3-hydroxypropionic acid (3-HP) production ability into a production medium; and (2) producing 3-HP by culturing the inoculated cells, wherein the production medium comprises glycerol as a carbon source, and the culturing is performed under a condition of the dissolved oxygen (DO) in the medium is 0.1% or more (i.e. 10%).
Regarding recitation of “wherein in step (2) of producing 3-HP, proliferation of cells does not occur,” Fig. 3(e) with added annotation dashed box below shows within the region of the dashed box a step of producing 3-HP (filled diamonds) by culturing the inoculated cells wherein cell proliferation (filled squares) does not occur. Again, the claims within their broadest reasonable interpretation do not exclude a prior step of proliferation following inoculation due to recitation of the open transitional phrase “comprising.”
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However, Kim does not disclose a condition of adding glucose when the dissolved oxygen in the medium is 0.1% or more (i.e. 10%).
Regarding a condition of adding glucose when the dissolved oxygen in the medium is 0.1% or more (i.e. 10%) and the features of claim 5, “In this study, glycerol was chosen as a sole carbon source due to its large amount of availability at low price from biodiesel industry.” Kim, page 2150, left col. However, combined carbon sources of glycerol and glucose for production of 3-HP are known in the prior art, since while glycerol is inexpensive as taught, specific benefits are obtainable by inclusion of glucose.
Niu, abstract, teaches:
3-Hydroxypropionic acid (3-HP) is an important platform synthesis block for sets of chemicals, but the relatively low production of 3-HP from biological sources presented major barriers for its industrial applications. In this study, a dual-substrate fermentative strategy by glycerol and glucose was proposed, and the aim was to evaluate the effect of different substrate addition strategies on the fermentation process. The results indicated that the optimal cosubstrate was glucose (20 g/L), and the enzymatic activity of aldehyde dehydrogenase (AldH) could be improved 3.5-fold as compared with no glucose addition. Continuous fed-batch fermentation at a constant speed displayed better 3-HP production of 17.20 g/L and highest specific 3-HP productivity of 1.79 mmol/(g cell·H) than the other fed-batch mode. The addition of glucose could greatly reduce the imbalance of the activity between glycerol dehydratase and AldH and provide a feasible method for improving 3-HP production. These results would be helpful in developing the 3-HP fermentation process.
“It was reported that the metabolism of glycerol in E. coli required the presence of external electron acceptors; otherwise, the fermentation processes could be hampered due to its higher reduction state. Carbon sources affect the kinetics of 3-HP fermentation through a cellular response that redistributes the composition of the fermentation end-products to achieve an oxidation–reduction balance. Besides, glucose metabolism increases the available NAD+ for the synthesis of 3-HP from 3-HPA. Therefore, it was logical to investigate the effects of glycerol/glucose mixtures on cell growth and 3-HP production from glycerol by this recombinant strain.”
“To investigate the optimal concentration of glucose, 30 g/L glycerol was used as the substrate for 3-HP production, and 0–60 g/L glucose was added as the cosubstrate. The results in Fig. 1 indicate that the glucose concentration had little effect on cell growth but played an important role in 3-HP production.” “These results further indicated that during glycerol/glucose cofermentation, glucose was metabolized to produce some electron acceptors substrate to provide both oxidation–reduction balance and ATP for biomass formation as well as cofactor regeneration, while glycerol was mainly used as the substrate for conversion to 3-HP.”
As discussed above, Kim teaches maintaining DO at 10% saturation for the entire course of culture, which meets the limitation of “the dissolved oxygen in the medium is 0.1% or more” wither periodic addition of glycerol. While, as discussed, Kim teaches glycerol as an only carbon source, Niu teaches that “glucose metabolism increases the available NAD+ for the synthesis of 3-HP from 3-HPA. Therefore, it was logical to investigate the effects of glycerol/glucose mixtures on cell growth and 3-HP production from glycerol” in an engineered E. coli otherwise suitable for production of 3-HP from glycerol. As such, in addition to provision of glycerol as a carbon source as taught by Kim, an ordinarily skilled artisan would have been motivated to provide a mixture of glucose and glycerol to E. coli for production of 3-HP in order to achieve the benefit of increased 3-HP production due to increased availability of NAD+ as taught by Niu. In the alternative, an ordinarily skilled artisan at the time of filing would have understood that some carbon source must be provided for production of 3-HP. As far as the prior art of Kim and Niu teach that glycerol and a mixture of glycerol and glucose, respectively, are both functional carbon sources for production of 3-HP for culturing E. coli producing 3-HP, an ordinarily skilled artisan at the time of filing would have been motivated to utilize either carbon source. Both Kim and Niu teach batch-fed techniques where a carbon source is periodically added during culture. “[G]lucose metabolism increases the available NAD+ for the synthesis of 3-HP from 3-HPA. Therefore, it was logical to investigate the effects of glycerol/glucose mixtures on cell growth and 3-HP production from glycerol.” Kim, sec. 2.3. “In the fed-batch I, glucose concentration was controlled at 3–8 g/L, and the initial glycerol concentration was 30 g/L. Figure 3 shows that cell growth and 3-HP production were all improved. Glucose feeding was stopped after 24 H.” Niu, sec. 3.4. Fig. 3 of Niu shows that after 10 hours of culture time, glucose is periodically added during the culture which meets the claim feature of “adding glucose,” wherein as discussed Kim teaches maintaining 10% DO in the medium throughout the culturing time, which is a level of dissolved oxygen at a level of 50% or less as recited in claim 4. That is, in following the teachings of Kim and Niu regarding fed-batch culturing methods, a carbon source is added during culturing wherein when a mixture of glucose and glycerol is used as a carbon source glucose and/or glycerol is added to a culture as to meet the claim limitation “a condition of adding glucose when the dissolved oxygen in the medium is 0.1% or more” (i.e. 10%) that is also 50% or less.
Regarding the range of 0.01 to 3 g/L glucose in the production medium in claim 5, “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. ‘[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.’" MPEP 2144.05(II)(A). Niu teaches “0–60 g/L glucose was added as the cosubstrate.” Niu, page 574, left col. Both Kim and Niu teach that glucose need not be added at all to successfully production 3-HP wherein glycerol is a sufficient sole carbon source. Nevertheless, Niu teaches that regeneration of NAD+ and 3-HP production may increase by including glucose along with glycerol wherein Niu in connection with Fig. 3 teaches maintaining a glucose range of 3-8 g/L as discussed, such range maintained by adding glucose periodically to the 3-HP production medium. However, in view of Kim and Niu teaching that glucose is not required for 3-HP production, it is not inventive for an ordinarily skilled artisan at time of filing to discover that maintaining a glucose concentration by batch-fed addition of glucose somewhere within the range of 0.01 to 3 g/L is workable for production of 3-HP where the prior art teaches the general condition that no glucose is required and suggests the broader range of 0-60 g/L glucose as workable. See, Niu, Fig. 1 wherein every concentration of glucose trialed including zero g/L produces 3-HP.
Regarding claim 3, as discussed, Kim does not utilize glucose at any point including in a production medium into which cells are inoculated. Further, Niu discusses conditions in which no glucose is present. “To investigate the optimal concentration of glucose, 30 g/L glycerol was used as the substrate for 3-HP production, and 0–60 g/L glucose was added as the cosubstrate.”
“[S]election of any order of performing process steps is prima facie obvious in the absence of new or unexpected results.” MPEP 2144.04(IV)(C). The features of claims 3 are met by forming a production medium without glucose, inoculation such medium, and if an election to include an amount of glucose is made in view of the teachings of Niu, then adding glucose after the inoculation. Such is the section of the order of performing process steps that is prima facie obvious in the absence of new or unexpected results.
Regarding claim 4, Kim, page, 2146, right col., as discussed, sets froth that 10% DO was maintained by controlling agitation (i.e. stirring speed), which appears to meet the features of claim 4 requiring production medium DO to be at a level of 50% or less by adjusting stirring speed.
Further regarding claim 11, the features of claim 11 are anticipated by Kim. However, in the event that it is argued that Kim does not teach a glycerol concentration of 0.3%(w/v) or less, the following is noted.
“Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. ‘[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.’" MPEP 2144.05(II)(A).
As discussed, Fig. 3(e) of Kim shows depletion of glycerol after each addition of glycerol wherein at the end of culture the open triangle representing glycerol is on the zero line. Since glycerol is depleted during culture to a level near zero, it is not inventive to discover culture conditions and times that will be less than 0.3%(w/v) or 3 g/L. Further, Fig. 4(d) of Kim shows metabolite production from several strains for 3-HP with acetate shown by black bar which are well below 2 g/L (0.2% w/v) for all strains shown. As such, at the time of filing an ordinarily skilled artisan would have had an expectation that the strains taught by Kim including when cultured to produce a 3-HP concentration of 60 g/L or more (as shown in Fig. 3(e)) can also produce less than 0.2% w/v of residual acetate in view of Kim disclosing multiple strain producing less than 2 g/L (0.2% w/v).
Claim(s) 1 and 3-11 (all pending claims) is/are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. (High‐level production of 3‐hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli, Biotechnol. Bioeng. 117, Jan. 2020, 2139-52) and Niu et al. (3-Hydroxypropionic acid production by recombinant Escherichia coli ZJU-3HP01 using glycerol–glucose dual-substrate fermentative strategy, Biotechnol. Appl. Biochem., 2017, 572-78) as applied to claims 1, 4, 5 and 10-11 above, and further in view of Takayama et al. (Enhancing 3‑hydroxypropionic acid production in combination with sugar supply engineering by cell surface‑display and metabolic engineering of Schizosaccharomyces pombe, Microb. Cell. Fact. 17, 2018, 176) and MacWilliams et al. (Luria Broth (LB) and Luria Agar (LA) media and their uses protocol, Am. Soc. Microbiol., 2006).
Regarding claims 6 and 9, “Two identical seed cultures were prepared by transferring cells (1 ml) cultured in 10‐ml LB test tube for 12 hr into 250‐ml Erlenmeyer flasks containing 100 ml LB medium [i.e. growth medium], and cultivating for 6 hr at 37°C in a 200 rpm rotary shaker. Cells grown in two flasks (200 ml total) were transferred [i.e. inoculated] into the bioreactor, making a 10% (v/v) inoculum size.” Kim, sec. 2.3. The bioreactor contains a production medium (MR medium as described in sec. 2.3 of Kim).
Kim is silent regarding OD600 or other cell density measurement with respect to the described seed cultures used for inoculation. Regardless, “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. ‘[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.’" MPEP 2144.05(II)(A).
Here, Kim in Fig. 3(f) shows culturing of cells during production of 3-HP up to an OD600 of near 70. Cell density as measured by OD600 is a concentration. Since it is known in the prior art as demonstrated by Kim that E. coli cells for 3-HP production can be cultured to densities of OD600 of 50 or more, it is not inventive to discover that it is workable to produce a seed culture (i.e. a high concentration culture in LB growth medium) having an OD600 of greater than 50 wherein “differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical.”
Claim 6 is interpreted as reciting that the cells are “isolated” prior to inoculation into a production medium (i.e. medium containing glycerol for 3-HP production as discussed above), which implies some degree of separation between the cells and a high concentration culture. Takayama relates to 3-hydroxypropionic acid production in yeast cells (S. pombe). “SPO-07 was pre-cultured in 5 mL YM medium for 1 day; the preculture then was inoculated into 100 mL YM medium in a 1-L baffled flask and cultured for approximately 24 h. Cells were collected by centrifugation at 900g for 10 min and used to inoculate 400 mL EMM medium containing cellobiose as a carbon source in a 1-L jar fermenter.” Takayama, page 5, left col. While Takayama relates to yeast cells, Takayama nevertheless teaches that it is a known inoculation method in the art applicable to microorganisms in general to grow cells in a first culture (i.e. a seed culture or high concentration culture), isolate or collect such cells by centrifugation, and employing the isolated/collected cells to inoculate a second culture for production of 3-hydroxypropionic acid or any other product. Since isolating cells by centrifugation is routine in the prior art for inoculation of a further culture as evidenced by Takayama, at the time of filing an ordinarily skilled artisan would have been motivated to isolate cells from a seed culture (i.e. high density culture) as taught by Kim followed by inoculation of isolated cells into a medium present in a bioreactor.
Regarding claim 7, “E. coli K‐12 W3110 ΔlacI (WL) strain was used as a base strain to constitutively express the genes under trc or tac promoter without using IPTG; this is because the use of IPTG is undesirable for the production of a bulk chemical like 3‐HP in large‐scale. To enable conversion of glycerol to 3‐HPA, pTac15kBAB was constructed to overexpress the dhaB1234 genes from K. pneumoniae encoding glycerol dehydratase and the K. pneumoniae gdrAB genes encoding glycerol reactivase, which reactivates damaged vitamin B12, a cofactor necessary for the activity of glycerol dehydratase.” Kim, sec. 3.1.
Regarding claim 8, LB media (i.e. growth medium) is well-known to not contain glycerol. While the precise composition of LB media may vary, LB media is a combination of tryptone, yeast extract and NaCl as evidenced by page 2 of MacWilliams and does not contain glycerol or other carbon sources outside of tryptone. Regardless, in view of Kim not providing an exact composition for LB media as taught, at the time of filing an ordinarily skilled artisan would have been motivated to utilize any standard composition for LB medium including the composition taught on page 2 of MacWilliams that does not contain glycerol.
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 and 3-11 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 7-19 of copending Application No. 18/028,005 in view of Kim et al. (High‐level production of 3‐hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli, Biotechnol. Bioeng. 117, Jan. 2020, 2139-52), Takayama et al. (Enhancing 3‑hydroxypropionic acid production in combination with sugar supply engineering by cell surface‑display and metabolic engineering of Schizosaccharomyces pombe, Microb. Cell. Fact. 17, 2018, 176), MacWilliams et al. (Luria Broth (LB) and Luria Agar (LA) media and their uses protocol, Am. Soc. Microbiol., 2006) and Niu et al. (3-Hydroxypropionic acid production by recombinant Escherichia coli ZJU-3HP01 using glycerol–glucose dual-substrate fermentative strategy, Biotechnol. Appl. Biochem., 2017, 572-78).
Rejections of claims 1 and 3-13 under 35 U.S.C. 102 and/or 103 stated above are incorporated herein by reference. The copending claims recite:
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As discussed above: Kim, page 2141, left col., states: Another factor that needs to be considered for efficient 3-HP production is dissolved oxygen (DO) level during the cultivation because the presence of oxygen inactivates glycerol dehydratase and inhibits the biosynthesis of coenzyme B12, a cofactor of glycerol dehydratase. However, at the same time, oxygen is necessary for efficient regeneration of NAD+, which is required for the activity of aldehyde dehydrogenase. The microorganism of the copending claims, on the face of the claims, employs glycerol dehydratase and aldehyde dehydrogenase on its face to produce 3-HP. Since Kim expressly teaches that its methods (culturing at 10% DO) is desirable for maximizing effect of these enzymes for producing 3-HP, an ordinarily skilled artisan at the time of filing would have been motivated to culture the microorganism of the copending claims (i.e. modify embodiments of the copending claims) in the precise same manner as taught by Kim since Kim teaches that culturing under such conditions is beneficial for 3-HP production, as to reach the features of claims 1, 3, 4 and 10-13 taught by Kim. While it is recognized that the microorganisms of the copending claims may be modified to only require glucose, as taught by Kim and indicated in the copending claims, 3-HP is made from glycerol by the action of glycerol dehydrogenase and an aldehyde dehydrogenase such that the microorganism of the copending claims would be expected to have capacity to utilize glycerol in producing 3-HP just as the recombinant E. coli taught by Kim have such capacity.
Regarding features of the remaining claims, the above rejections under 35 U.S.C. 102 and/or 103 set forth why an ordinarily skilled artisan at the time of filing would have performed the method of claim 1 with the additional features of claims depending from claim 1 including wherein in step (2) of producing 3-HP proliferation of cells does not occur. As far as one having ordinarily skill in the art would have been motivated to modify embodiments of the copending claims to have all of the features of claim 1, an ordinarily skilled artisan would have been motivated further to perform embodiments of the copending claims having the features of claim 1 with the any additional features of claims 3-13 for the same reasons set forth above including addition of glucose as recited in claims 1 and 5 in order to increase availability of NAD+ as beneficial to 3-HP production as discussed in detail above.
This is a provisional nonstatutory double patenting rejection.
Claims 1 and 3-11 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6, 9, and 21-28 of copending Application No. 18/696,177 in view of Kim et al. (High‐level production of 3‐hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli, Biotechnol. Bioeng. 117, Jan. 2020, 2139-52), Takayama et al. (Enhancing 3‑hydroxypropionic acid production in combination with sugar supply engineering by cell surface‑display and metabolic engineering of Schizosaccharomyces pombe, Microb. Cell. Fact. 17, 2018, 176), MacWilliams et al. (Luria Broth (LB) and Luria Agar (LA) media and their uses protocol, Am. Soc. Microbiol., 2006) and Niu et al. (3-Hydroxypropionic acid production by recombinant Escherichia coli ZJU-3HP01 using glycerol–glucose dual-substrate fermentative strategy, Biotechnol. Appl. Biochem., 2017, 572-78).
Rejections of claims 1 and 3-13 under 35 U.S.C. 102 and/or 103 stated above are incorporated herein by reference. The copending claims recite:
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That is, the copending claims recite a generic E. coli with ability to produce 3-HP including those with a glycerol dehydratase and aldehyde dehydrogenase for production of 3-HP. Since Kim expressly teaches that its methods (culturing at 10% DO) is desirable for maximizing effect of these enzymes for producing 3-HP, an ordinarily skilled artisan at the time of filing would have been motivated to culture the microorganism of the copending claims (i.e. modify embodiments of the copending claims) in the precise same manner as taught by Kim since Kim teaches that culturing under such conditions is beneficial for 3-HP production, as to reach the features of claims 1, 3, 4 and 10-11 taught by Kim, except for the further addition of glucose as recited in claims 1 and 5.
Regarding features of the remaining claims, the above rejections under 35 U.S.C. 102 and/or 103 set forth why an ordinarily skilled artisan at the time of filing would have performed the method of claim 1 with the additional features of claims depending from claim 1 including wherein in step (2) of producing 3-HP proliferation of cells does not occur. As far as one having ordinarily skill in the art would have been motivated to modify embodiments of the copending claims to have all of the features of claim 1, an ordinarily skilled artisan would have been motivated further to perform embodiments of the copending claims having the features of claim 1 with the any additional features of claims 3-13 for the same reasons set forth above including addition of glucose as recited in claim 5 in order to increase availability of NAD+ as beneficial to 3-HP production as discussed in detail above.
This is a provisional nonstatutory double patenting rejection.
Response to arguments
As reviewed above, the broadest reasonable interpretation of at least claim 1 has been reevaluated, which applies to both rejections under 35 U.S.C. 103 and for double patenting. It is noted that the current grounds of rejection under 35 U.S.C. 103 may potentially be addressed by amending the “inoculating” step of claim 1 to require the inoculation to form a high cell concentration culture that does not undergo cell proliferation, and producing 3-HP with such high cell concentration culture.
Applicant states that claim 10 has been amended to include the limitation of previously pending claim 2. Previously pending claim 2 recited a method. Claim 10 recites a product. As such, claim 10 has not been amended to include the limitations of previously pending claim 2 as asserted.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652
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