Ex Parte Chao et alDownload PDFPatent Trial and Appeal BoardMay 19, 201713656654 (P.T.A.B. May. 19, 2017) Copy Citation United States Patent and Trademark Office UNITED STATES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONER FOR PATENTS P.O.Box 1450 Alexandria, Virginia 22313-1450 www.uspto.gov APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO. 13/656,654 10/19/2012 YUN-PENG CHAO KS-00011 2557 88174 7590 05/19/2017 Flasih Intellectual Prnnertv Tnr EXAMINER Attn. Cheng-Ju Chiang P.O. Box 766 EPSTEIN, TODD MATTHEW Chino, CA 91708 ART UNIT PAPER NUMBER 1652 MAIL DATE DELIVERY MODE 05/19/2017 PAPER Please find below and/or attached an Office communication concerning this application or proceeding. The time period for reply, if any, is set in the attached communication. PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte YUN-PENG CHAO, CHUNG-JEN CHIANG, HONG-MIN LEE, ZEI-WEN WANG, and PO-TING CHEN1 Appeal 2016-000365 Application 13/656,654 Technology Center 1600 Before ERICA A. FRANKLIN, ULRIKE W. JENKS, and JOHN G. NEW, Administrative Patent Judges. JENKS, Administrative Patent Judge. DECISION ON APPEAL This is an appeal under 35 U.S.C. § 134(a) involving claims directed to a method of modifying E. coli to ferment pentose and hexose simultaneously. The Examiner rejects the claims as obvious. We have jurisdiction under 35 U.S.C. § 6(b). We AFFIRM. 1 According to Appellants, the real party in interest is Feng Chia University. Appeal Br. 4. Appeal 2016-000365 Application 13/656,654 STATEMENT OF THE CASE The Specification explains that ordinarily “A. coli metabolizes glucose first. After glucose is totally consumed, other monosaccharides are utilized. Therefore, E. coli is unable to metabolize different monosaccharides at the same time in the presence of glucose.” Spec. |3. To overcome this obstacle a “gene sequence encoding a glucose permease in the phosphotransferase system is deleted to reduce the catabolite repression.” Id. |9. Claims 1, 5, 7—12, 16, 18, 22—26, and 31—33 are on appeal, and can be found in the Claims Appendix of the Appeal Brief. Claim 1 is representative of the claims on appeal, and reads as follows: 1. A method enabling Escherichia coli to ferment pentose and hexose simultaneously, which method comprises steps of: (a) deleting a ptsG gene sequence in Escherichia coli; (b) introducing a gif gene sequence of Zymomonas mobilis into Escherichia coli', (c) introducing at least one copy of the gene sequence in the pentose phosphate pathway of Escherichia coli, wherein upstream of the at least one copy of the gene sequence in the pentose phosphate pathway is introduced with at least one promoter; and (d) deleting a set of gene sequences responsible for the synthesis of organic acids in Escherichia consisting of an IdhA, a pta, apoxB, and an frdA gene sequences. 2 Appeal 2016-000365 Application 13/656,654 Appellants request review of the following rejections: I. Claims 1, 5, 8—12, 16, 18, 22—26, and 31—33 under 35 U.S.C. § 103(a) as unpatentable over Trinh2 in view of Nichols,3 Snoep,4 Wang,5 and Menart.6 II. Claims 1, 5, 7—12, 16, 18, 22—26, and 31—33 under 35 U.S.C. § 103(a) as unpatentable over Trinh in view of Nichols, Snoep, Wang, Menart, and Chiang.7 As Appellants do not argue the claims separately, we focus our analysis on claim 1, and claims 5, 7—12, 16, 18, 22—26, and 31—33 stand or fall with that claim. 37 C.F.R. § 41.37 (c)(l)(iv). 2 Cong T. Trinh et al., Minimal Escherichia coli Cell for the most Efficient Production of Ethanol from Hexoses and Pentoses, 74:12 Applied & Environmental Microbiology 3634—43 (2008) (“Trinh”). 3 N. N. Nichols et al., Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol, Appl Microbiol Biotechnol 120—25 (2001) (“Nichols”). 4 Jacky L. Snoep et al., Reconstitution of Glucose Uptake and Phosphorylation in a Glucose-Negative Mutant of Escherichia coli by Using Zvmomonas mobilis Genes Encoding the Glucose Facilitator Protein and Glucokinase, 176:7 J. of Bacteriology 2133-35 (1994) (“Snoep”). 5 Zhijun Wang et al., Effects of the presence of ColEl plasmid DNA in Escherichia coli on the host cell metabolism, Microbial Cell Factories 1-18 (2006) (“Wang”). 6 Viktor Menart et al., Constitutive Versus Thermoinducible Expression of Heterologous Proteins in Escherichia coli Based on Strong Pr,Pl Promoters From Phage Lambda, 83:2 Biotechnology and Bioengineering 181-90 (2003) (“Menart”). 7 Chung-Jen Chiang et al., Replicon-Free and Markerless Methods for Genomic Insertion of DNAs in Phage Attachment Sites and Controlled Expression of Chromosomal Genes in Escherichia coli, 101:5 Biotechnology and Bioengineering 985-95 (2008) (“Chiang”). 3 Appeal 2016-000365 Application 13/656,654 Obviousness over Trinh, Nichols, Snoep, Wang, and Menart The Examiner finds that Trinh “teach[es] a CT1101 strain having deletions to the ptsG, glk, manX, poxB, pta, IdhA and frdA genes to generate a strain specific for growth on pentoses also further transformed with the pl01297 plasmid (pUC18-based) containing genes for pdc (pyruvate decarboxylase) and adhB (i.e., adhll, alcohol dehydrogenase) from Z. mobilise Ans. 2. The Examiner acknowledges that Trinh “do[es] not teach introducing a gif gene sequence of Z. mobilis into the cell.” Ans. 3. The Examiner finds that Snoep “directly exemplif[ies] the construction of such strains lacking the ptsG gene [function] and expressing the g// gene from Z. mobilis in replacement thereof to achieve the benefits of improving glucose utilization in strains lacking the ptsG gene.” Ans. 5. “[T]he g//gene from Z. mobilis ‘functioned well in E. coli [to] provide an alternative to the native glucose-PTS system’ in E. coli strains where the PTS system is not functional.” Ans. 5. The Examiner concludes that “it would have been obvious for the skilled artisan to have constructed a strain having deletion of ptsG [such as in the CT1101 strain of Trinh] and expression of gif and glk from Z. mobilis ... to achieve the benefits of improving glucose utilization in strains lacking the ptsG gene.” Ans. 5. The Examiner acknowledges that Trinh does not teach “introducing at least one copy of a gene sequence in the pentose phosphate pathway with at least one least one promoter that is a k-PRPL promoter.” Ans. 3. The Examiner finds that Wang teach that the pentose phosphate (PP) pathway [(pentose phosphate pathway)] is the only metabolic pathway used by E. coli to metabolize xylose, where the PP pathway is comprised of a limited number of enzymes encoded by their respective 4 Appeal 2016-000365 Application 13/656,654 genes. As such, at the time of the invention, one having ordinary skill in the art would have been motivated [to try] to introduce additional copies of one or more of the genes of the PP pathway to increase the capacity of E. coli TCS083 to metabolize and to ferment xylose into ethanol. Ans. 6. Appellants contend: (1) that the Examiner has not provided “any objective reason to reduce the size of gene knockouts taught by Trinh et al. (i.e., the zwf ndh, sfcA, maeB, ldhA,frdA,poxB, andpta genes) to a subset recited in the independent claims” (Appeal Br. 11), (2) that the prior art combination should not alter the principle of operation of the reference (Appeal Br. 12), and (3) that the Examiner failed to articulate the rationale “to explain what in the prior art would have prompted a person having ordinary skill in the art to include Wang et al. into the E. coli strain taught by Trinh.” Appeal Br. 16. The issue is: Does the preponderance of evidence of record support the Examiner’s conclusion that the combined references teach the method as claimed? Findings of Fact We adopt the Examiner’s findings of fact and reasoning regarding the scope and content of the prior art. See Final Action (mailed Nov. 13, 2014) and Answer. For emphasis only we highlight the following: FF1. Trinh teaches the production of E. coli with gene knockouts for producing strains that can efficiently ferment ethanol from pentoses and hexoses. See Trinh, Title, Abstract. Trinh teaches the production of the E. coli strain CT1101 that contains the following gene deletions: Azwf Andh, AsfcA, AmaeB, AldhA, AfrdA, ApoxB, Apia, 5 Appeal 2016-000365 Application 13/656,654 AptsG, Aglk, AmanX::Kan. Trinh, Table 1. “In the wild type, the glucose was completely exhausted before consumption of xylose could be observed, while the constructed mutant TCS083 [containing ptsG, glk, manX genes] consumed both sugars simultaneously and strain CT1101 used only xylose even when glucose was present.” Trinh 3641. FF2. Trinh teaches: The pyruvate decarboxylase [reaction] does not exist in E. coli but was introduced into E. coli through the plasmid pLOI297 (ATCC 68239) (3). The introduction of pyruvate decarboxylase into the strain contributed to the use of the precursor pyruvate for the ethanol production pathway and mimicked the ethanol-producing pathway of native ethanologenic strains such as Zymomonas mobilis and Saccharomyces cerevisiae. Trinh 3635. FF3. “Xylose and arabinose are the dominant pentoses found in biomass.” Trinh 3636. Trinh teaches that when the E. coli strain CT1101 containing the plasmid pL01297 is grown “in a mixture of xylose and glucose. This strain evidently has all catabolite repression removed and can grow on pentoses even in the presence of glucose.” Trinh 3641. FF4. Nicholas teaches that ptsG mutants “grew on glucose at rates approximately equal to the parent strain and co-metabolized pentoses with glucose.” Nicholas 124. “Due to inactivation of the glucose phosphotransferase system ptsG gene, the strains lacked the molecular machinery required for catabolite repression.” Nicholas 124. 6 Appeal 2016-000365 Application 13/656,654 FF5. Snoep teaches strain ZSC113 an E. coli that “is a glucose-negative strain which contains mutations [rather than deletions] in glucose- specific and mannose-specific phosphotransferase genes and in GLK [glucokinase].” Snoep 2133—2134. FF6. Snoep teaches that “glucose uptake (gif) and phosphorylation genes (glk) from Z. mobilis functioned well in E. coli and provide an alternative to the native glucose-PTS system” of E. coli. Snoep 2134. FF7. Wang teaches that “[t]he PP pathway has fewer enzymes than glycolysis, however, this pathway is more complicated ... [it is, however,] the only pathway that allows E. coli to use some sugars, such as D-xylose, D-ribose, or L-arabinose.” Wang 13. FF8. Wang teaches that “[p]lasmids, as non-essential DNA molecules, require additional intracellular materials and energy to exist in host cell, thus usually reducing the growth rate of a bacterial culture.” Wang 10. Wang teaches results of “DNA microarray experiments. In the carbon catabolism, there were number of genes, which were up- regulated significantly, suggesting that these groups of genes were helpful for maintaining plasmid DNA in E. coli.'” Wang. 9, 11 (Table 7). FF9. Wang teaches that “modification of the PP pathway by enhancement of the rpiA gene expression can significantly increase ColEl-like plasmid DNA copy number in E. coli.” Wang 13. Principle of Law “The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results.” KSRInt’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007). 7 Appeal 2016-000365 Application 13/656,654 Analysis Trinh teaches the creation of minimal E. coli strains for ethanol production from pentose sugars. CT1101 is strain of E. coli that contains the following gene deletion: AIdhA, AfrdA, ApoxB, Apia, and AptsG in addition to other genes. FF1. This CT1101 strain can only use xylose for ethanol production, but also does not show catabolite repression in the presence of glucose. FF1 andFF3. Nichols explains that catabolite repression is linked to the ptsG gene. FF4. Ethanol production from glucose in the CT1101 strain was restored by introducing a plasmid containing pyruvate decarboxylase from Z. mobilis. FF2. Snoep teaches “glucose uptake (gif) and phosphorylation genes (glk) from Z. mobilis [also] functioned well in E. coli and provide an alternative to the native glucose-PTS system.” FF5 and FF6. Wang teaches that in E. coli the PP pathway is the only pathway that can utilize certain sugars such as D-xylose, D-ribose, or L-arabinose. FF7. Wang also teaches that expression of the PP pathway gene rpiA helps maintain plasmid copy number in E. coli. FF7—FF9. Applying the KSR standard of obviousness to the findings of fact we agree with the Examiner’s conclusion that based on the combination of references it would have been obvious to introduce a plasmid carrying the Z. mobilis glucose uptake (gif) and phosphorylation genes (glk) into the E. coli strain CT1101 of Trinh in order to restore glucose utilization in this strain. We also agree with the conclusion that one of ordinary skill in the art would have been motivate to express additional PP pathway genes to help increase expression of PP pathway proteins to utilize xylose, ribose, or arabinose energy sources, and additionally Wang suggests that expression of rpiA gene helps maintain plasmid copy numbers in the transformed E. coli strains. 8 Appeal 2016-000365 Application 13/656,654 After considering the evidence and the arguments, we conclude that the weight of the evidence favors the Examiner’s conclusion of obviousness. Accordingly, we adopt the Examiner’s reasoning (see Grounds of Rejection, Ans. 2—8), and agree that the Examiner properly found Appellants’ arguments unpersuasive (see Response to Argument, Ans. 8—15). We provide the following points for emphasis. Appellants contend that Trinh contains more gene deletions than are set out in the claim and “[t]he Examiner has not provided any objective reason to reduce the size of gene knockouts taught by Trinh et al. (i.e., the zwf ndh, sfcA, maeB, ldhA,frdA, poxB, andpta genes) to a subset recited in the independent claims 1,16, and 33.” Appeal Br. 11. Appellants contend that “the E. coli strain with only a set of gene sequence [deletions] consisting of IdhA, pta, poxB and frdA is never being reduced to practice during the construction of TCS083.” Reply Br. 6. We are not persuaded. We begin with claim interpretation, so that its scope may properly be compared to the prior art. Claim 1 uses the transitional phrase a “method comprises steps of’ followed by the listing of several steps including “(a) deleting a ptsG gene sequence in Escherichia coir and “(d) “deleting a set of gene sequences responsible for the synthesis of organic acids in Escherichia consisting of an IdhA, a pta, a poxB, and an frdA gene sequences.” “‘Comprising’ is a term of art used in claim language which means that the named elements are essential, but other elements may be added and still form a construct within the scope of the claim.” Genentech, Inc. v. Chiron Corp., 112 F.3d 495, 501 (Fed. Cir. 1997). Therefore, the broadest reasonable interpretation of the transitional phrase “method comprises steps 9 Appeal 2016-000365 Application 13/656,654 of’ is that the method contains the recited steps but can include additional steps including additional gene deletions and still fall within the scope of the claim. The specifically recited steps of “(a) deleting a ptsG gene sequence in Escherichia coif’ and “(d) deleting a set of gene sequences responsible for the synthesis of organic acids in Escherichia consisting of an IdhA, a pta, a poxB, and an frdA gene sequences” does not narrow this interpretation because the method may contain additional steps that lead to additional gene deletions. The recitation of step “(d) deleting a set of gene sequences responsible for the synthesis of organic acids in Escherichia consisting of an IdhA, a pta, a poxB, and an frdA gene sequences.” This limitation is reasonably interpreted to require that an IdhA, a pta, a poxB, and frdA gene sequences are removed from the E. coli organism without placing constraints on how the deletion process is ultimately carried out. See Ans. 12. Our reviewing court has found that, “[t]he reasonable interpretation of the claims containing both of the terms ‘comprising’ and ‘consists’ is that the term ‘consists’ limits the ‘said portion’ language to the subsequently recited numbered nucleotides, but the earlier term ‘comprising’ means that the claim can include that portion plus other nucleotides.” In re Crish, 393 F.3d 1253, 1257 (Fed. Cir. 2004). In other words, step (d) as recited in claim 1 requires that the recited genes are deleted from an E. coli strain but does not place any limitations on the starting material that is used to arrive at the final construct also there is no limitation with regard to additional gene deletions. The recitation of separate steps “(a) deleting a ptsG gene sequence in Escherichia coif and “(d) “deleting a set of gene sequences responsible for the synthesis of organic acids in Escherichia consisting of an IdhA, a pta, a 10 Appeal 2016-000365 Application 13/656,654 poxB, and an frdA gene sequences” does not place any limitations in what order those steps are carried out. It has been held that the selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results. In re Hampel, 162 F.2d 483, 485—86 (CCPA 1947) (“There is nothing in the instant record which indicates that the particular order of steps produces results differing in any way from those which would be brought about if another order of steps were followed.”). We find no evidence in the record, and Appellants do not direct us to such evidence, that would indicate the order of deletion of the various genes in claim 1 has any effect on the pentose and hexose utilization in the E. coli organism constructed to with such deletions. The broadest reasonable interpretation of claim 1 is that a ptsG gene is deleted from Escherichia coli and that another process step results in the deletion of IdhA, a pta, a poxB, and frdA gene sequences. The claim language, however, does not limit the gene deletion to only those deletions recited in the claim, and there is no limitation as to whether the ptsG gene is deleted from the organism before or after the deletion of the other recited genes. See also Ans. 12 (the claims “therefore encompass any number and quantity of additional unrecited method steps including deletion of additional genes not directly recited in the claims. That is, claims 1,16 and 33 are not limited to deletion of only the specific subset of genes IdhA, pta, poxB and frdA at the exclusion of deletion of additional genes”). In light of our claim interpretation above, we are not persuaded by Appellants’ contention that the claims require the production of an E. coli strain containing only the recited gene deletions. See Appeal Br. 11; Reply Br. 6. The Examiner finds, and Appellants do not contest, that “TCS083 11 Appeal 2016-000365 Application 13/656,654 E. coli strain having deletions to the poxB, pta, IdhA and frdA genes” in addition to other deletions. Ans. 2; Reply Br. 5—6. This strain is created from a TCS062 that already contains deletions of the IdhA and frdA from the wild type strain. Trinh 3635 (Table 1); FF1. However, the deletion of poxB and pta gene from the TCS062 strain to create TCS083 strain meets the limitation claim 1, step (d) because this deletion step results in a strain that has all four recited genes deleted. Accordingly, we find no error with the Examiner’s reliance on Trinh for teaching the production of an E. coli deletion mutant that shows glucose utilization is restored by the addition of a plasmid containing genes for pyruvate decarboxylase from Z. mobilis. See FF1-FF3. Appellants contend that the prior art combination should not alter the principle of operation of the reference. Appeal Br. 12. “[T]he key element underlying the CT 1101 strain is to prevent glucose transportations in order to achieve the intended purpose.” Appeal Br. 13. We are not persuaded by Appellants’ contention. We agree with the Examiner that while Trinh et al. envisions that the “E. coli strain CT1101 can be combined with a hexose -utilizing organism to arbitrarily adjust the individual sugar consumption rates in a mixed culture,” Table 5 of the same reference shows that the CT1101/ plO 12997 stain can function without a hexose-utilizing organism present to produce ethanol from a mixed sugar source of glucose and xylose. ... As such, the intended purpose and explicitly demonstrated function of the CT1101 strain is not limited only to adjusting pentose consumption rates in a mixed culture. Ans. 13. Thus, improving glucose utilization is consistent with the teaching a Trinh. 12 Appeal 2016-000365 Application 13/656,654 Appellants contend that the Examiner failed to articulate the rationale “to explain what in the prior art would have prompted a person having ordinary skill in the art to include Wang et al. into the E. coli strain taught byTrinh.” Appeal Br. 16. We are not persuaded. The Examiner’s rationale is that “one having ordinary skill in the art would have been motivated to introduce additional copies of one or more of the genes of the PP pathway to increase the capacity of E. coli TCS083 to metabolize and to ferment xylose into ethanol.” Ans. 6. The Examiner explains that Wang “teach[es] that the pentose phosphate (PP) pathway is the only metabolic pathway used by E. coli to metabolize xylose, where the PP pathway is comprised of a limited number of enzymes encoded by their respective genes.” Ans. 6; see FF7— FF9. Thus, the Examiner reasoned that “expression of PP pathway genes, which are endogenous and present in the CT1101 strain taught by Trinh et al., is necessary for any metabolism and utilization of xylose.” Ans. 14. “The rejection does not rely upon an increase in the PP pathway as necessary or required for xylose utilization only that an increase in PP pathway may assist in xylose utilization.” Ans. 15. Here, the rationale provided by the Examiner is that expression of PP pathway genes could assist in xylose utilization by E. coli strains that are deficient in glucose utilization. We find that rationale to be reasonable and supported by the evidence. Appellants have not established otherwise. We additionally note that Wang specifically teaches that rpiA gene expression can significantly increase plasmid DNA copy number in E. coli. FF9. Increasing plasmid DNA is a desirable feature in E. coli strains in 13 Appeal 2016-000365 Application 13/656,654 which glucose utilization is restored using plasmids containing genes for glucose utilization. We conclude that the evidence cited by the Examiner supports a prima facie case of obviousness with respect to claim 1, and Appellants have not provided sufficient rebuttal evidence, or evidence of secondary considerations that outweighs the evidence supporting the prima facie case. As Appellants do not argue the claims separately, claims 5, 8—12, 16, 18, 22-26, and 31-33 fall with claim 1. 37 C.F.R. § 41.37 (c)(l)(iv). II. Obviousness over Trinh, Nichols, Snoep, Wang, Menart, and Chiang The Examiner has rejected claims 1, 5, 7—12, 16, 18, 22—26, and 31— 33 as obvious based the additional teaching of Chiang. Ans. 7—8. Specifically, the Examiner found that Chiang “teach[es] that the technical difficulties associated with maintaining plasmids with antibiotic selection, such as antibiotic-resistance, and physiological stress from redundant copies of DNA ‘can be circumvented by chromosome engineering of the strain based on the recombinase system.’” Ans. 7. Appellants’ only argument with respect to this rejection is that chromosomal insertion of homologous or heterologous gene insertion into E. coli is “irrelevant to the arguments discussed” with respect to Trinh, Nichols, Snoep, Wang, and Menart. Appeal Br. 17. This argument is not persuasive because, as discussed above (/.), we conclude that the method of producing an E. coli strain to ferment pentose and hexose simultaneously would have been obvious based on the teachings of Trinh, Nichols, Snoep, Wang, and Menart and the process of inserting the sequence into the gif gene into E. coli would be an obvious process based on the teachings of Chiang. We affirm the rejection of claims 14 Appeal 2016-000365 Application 13/656,654 1, 5, 7—12, 16, 18, 22—26, and 31—33 as obvious for the reasons given by the Examiner. SUMMARY We affirm the rejection of claim 1 under 35 U.S.C. § 103(a) over Trinh, Nichols, Snoep, Wang, and Menart. Claims 5, 8—12, 16, 18, 22—26, and 31—33 were not argued separately and fall with claim 1. We affirm the rejection of claim 1 under 35 U.S.C. § 103(a) over Trinh, Nichols, Snoep, Wang, Menart, and Chiang. Claims 5, 7—12, 16, 18, 22—26, and 31—33 were not argued separately and fall with claim 1. TIME PERIOD FOR RESPONSE No time period for taking any subsequent action in connection with this appeal may be extended under 37 C.F.R. § 1.136(a). AFFIRMED 15 Copy with citationCopy as parenthetical citation