I 1111111111111111 1111111111 1111111111 11111 1111111111 111111111111111 IIII IIII USO 10920251B2 c12) United States Patent (IO) Patent No.: US 10,920,251 B2 San et al. (45) Date of Patent: Feb.16,2021 (54) MICROBIAL PRODUCTION OF FATS (56) References Cited (71) Applicant: William Marsh Rice University, U.S. PATENT DOCUMENTS Houston, TX (US) 4,762,784 A 8/1988 Keith et al. 7,262,046 B2 8/2007 Ka-Yiu et al. (72) Inventors: Ka-Yiu San, Houston, TX (US); Zhilin 7,326,557 B2 2/2008 San et al. Li, Houston, TX (US); Xian Zhang, 7,569,380 B2 8/2009 San et al. Houston, TX (US) 7,901,924 B2 3/2011 San et al. 8,129,157 B2 3/2012 Gonzalez (73) Assignee: WILLIAM MARSH RICE 8,486,686 B2 7/2013 Segueilha et al. 8,691,552 B2 4/2014 Gonzalez et al. UNIVERSITY, Houston, TX (US) 8,795,991 B2 8/2014 San et al. 8,962,272 B2 2/2015 San et al. ( *) Notice: Subject to any disclaimer, the term ofthis 2010/0317086 Al* 12/2010 Segueilha ................. Cl2P 7/04 patent is extended or adjusted under 35 435/252.33 U.S.C. 154(b) by 103 days. 2014/0093921 Al 4/2014 San et al. 2014/0193867 Al 7/2014 Sanetal. 2014/0212935 Al 7/2014 Sanetal. (21) Appl. No.: 16/098,914 2014/0273114 Al 9/2014 San et al. 2015/0037853 Al 2/2015 Fischer et al. (22) PCT Filed: May 5, 2017 2015/0259712 Al 9/2015 San et al. (86) PCT No.: PCT/US2017 /03117 4 FOREIGN PATENT DOCUMENTS § 371 (c)(l), WO 2015054138 4/2015 (2) Date: Nov. 5, 2018 (87) PCT Pub. No.: WO2017/192925 OTHER PUBLICATIONS PCT Pub. Date: Nov. 9, 2017 Olsson, J., & Andrews, J.F., "The dissolved oxygen profile-A valuable tool for control of the activated sludge process," Water (65) Prior Publication Data Research, vol. 12, Issue 11, pp. 985-1004 (Mar. 3, 1978). Sun, Z., et al., "Amino acid substitutions at glutamate-354 in US 2019/0144898 Al May 16, 2019 dihydrolipoamide dehydrogenase of Escherichia coli lower the sensitivity of pyruvate dehydrogenase to NADH," Microbiology, vol. 158, pp. 1350-1358 (May 2012). Related U.S. Application Data Wu, H., et al., "Metabolic transistor strategy for controlling electron transfer chain activity in Escherichia coli," Metabolic Engineering, (60) Provisional application No. 62/332,308, filed on May vol. 28, pp. 159-168 (Mar. 2015). 5, 2016. Zhang, X., et al., "Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases," Metabolic Engineering, (51) Int. Cl. vol. 13, Issue 6, pp. 713-722, Nov. 2011. C12N 1120 (2006.01) Tatusova, T.A., and Madden, T.T., "Blast 2 Sequences, a new tool C12P 7164 (2006.01) for comparing protein and nucleotide sequences," FEMS Microbi­ ology Letters, vol. 174, pp. 247-250 (1998). C12N 9102 (2006.01) International Search Report & Written Opinion of the International C12N 15152 (2006.01) Searching Authority received in Application No. PCT/US2017/ C12N 9/16 (2006.01) 031174, dated Jul. 20, 2017. C12N 9/88 (2006.01) C12N 9/04 (2006.01) * cited by examiner C12N 15174 (2006.01) (52) U.S. Cl. Primary Examiner - Tekchand Saidha CPC .............. C12P 716409 (2013.01); C12N 1120 (74) Attorney, Agent, or Firm - Boulware & Valoir (2013.01); C12N 9/0006 (2013.01); C12N 9/0008 (2013.01); C12N 9/16 (2013.01); (57) ABSTRACT C12N 9/88 (2013.01); C12N 15152 (2013.01); This invention describes a method of using microbial to C12N 15174 (2013.01); C12Y 102/01051 produce fats, such as fatty acids and their derivatives, or (2013.01); C12Y 102/07001 (2013.01) products derived from the fatty acid synthesis cycle, such as (58) Field of Classification Search hydroxyfatty acids, methyl ketones, and the like. CPC ..................................................... C12P 7/6409 See application file for complete search history. 9 Claims, 4 Drawing Sheets U.S. Patent Feb.16,2021 Sheet 1 of 4 US 10,920,251 B2 "' 14 0 18 0 ell 1,0 -Qj 15 12 ... ~ -...~ 0 10 ~ "'C 12 ~ =~ 8 -"'C 9 -Glucose ~ =~ - 6 Qj ell -+-QD600 ...~~ -Qj 6 ... - rll 4 Qj - ~ ---+E-Fatty acid 0 ~ 3 ~ = 2 -e' -Acetate -~ ... ~ 0 0 ~ .........Lactate 0 12 24 36 48 60 72 ...~ ~ Time (h) ~ ML103(pXZ18Z) FIGURE 1 Gas inlet ---,"-""-......................... h Gas outlet ...... ,..·' ...... '-....: () Gas-bubble •'"\ ....• C"·""'· ............., \...-----.) () ........~)...........................i).......()4·~~-~----- Sparger/aerator FIGURE2 U.S. Patent Feb.16,2021 Sheet 2 of 4 US 10,920,251 B2 Gas outlet Heads pace FIGURE3 U.S. Patent Feb.16,2021 Sheet 3 of 4 US 10,920,251 B2 18 5 -~ 0 ell -Qj 0 15 1,0 4 ... -~ Q ...u 0 12 ~ "'C 3 -"'C =~ 9 =~ -Glucose ~ ...Qj - ~ ell 2 ... ~OD600 -Qj Qj rll 6 u - ~ -.-Fatty acid 0 u "' 1 ...."'C -e-Acetate = 3 u -~ ~ ...>. ...._Lactate 0 0 ~ ~ 0 12 24 36 48 60 Time (h) A-1 ML103(pXZ18Z) FIGURE4A 450 100 8.5 I 90 400 \ \ 80 8.0 350 ;, ,.,-.:·;.---, 70 \ ..:, \ 300 I I I _.·, ' 7.5 I I •; ' 60 •. I I ;'- ' a 250 "J ': ' - ( \ 0.. _,.... I ' 50 ~_, 7.0 ' I ::c: .-=:: 200 ' 40 N 0.. 00 •.. .• •• . ......·,1 --Agit '~ 0 < "O 150 I --- -dO2 30 6.5 : 1·•· ....... ..... pH • I 20 100 : I I : I I 6.0 • I I 10 50 I I ·.__: ,_ --L-------- _1 0 0 5.5 0 12 24 36 48 60 Time (h) A-2 l\1L103(p:XZ18Z) FIGURE4B U.S. Patent Feb.16,2021 Sheet 4 of 4 US 10,920,251 B2 18 C 5 ~ 0 - 0 ell 1,0 15 -Qj 4 ~ ... -~ 0 ... ~ "'C 12 ~ 3 =~ -"'C 9 -Glucose ~ =~ - Qj ell 2 ... ~OD600 -Qj ~ 6 rll ... - Qj -+-Fatty acid 0 ~ ~ 1 ~ = 3 -e' -+-Acetate -~ ... ~ ~ -+-Lactate 0 0 >. 0 12 24 36 48 60 ... ~ Time (h) ~ B-1 ZL302(pXZ18Z) FIGURE SA 450 100 8.5 90 400 ' - , -- -- ... ~------:--•.>· \ ''.,' .... :,; 80 8.0 350 \ 70 300 I ... 7.5 ' : •: 60 •: • i 50 ~ _.·. '._/ 7.0::g_ , I --Agit 4o 8 "O 150 .-. -·- ----dO2 30 6.5 .,:•· .. -·· ...... pH 100 ;: 20 : ' ~~ 6.0 50 . : ' lO .. •-1----------·-----~ 0 0 -+--"""T"""-"""T"""----r-----T"-----.-----.------,--,---..-------1 5.5 0 12 24 36 48 60 Time (h) B-2 ZL302(pXZ18Z) FIGURE SB US 10,920,251 B2 1 2 MICROBIAL PRODUCTION OF FATS What is needed in the art is a novel culture method that allows high yield and rates of production of compounds, PRIOR RELATED APPLICATIONS such as fatty acids and their derivatives, yet is amenable to scale up and is cost effective. This application claims priority to U.S. Ser. No. 62/332, 5 308, IMPROVED MICROBIAL PRODUCTION OF FATS, SUMMARY OF THE INVENTION filed May 5, 2016, and PCT/US2017/031174, filed May 5, 2017, each of which is incorporated by reference herein in Fatty acids are aliphatic acids fundamental to energy its entirety for all purposes. production and storage, cellular structure and as intermedi- 10 ates in the biosynthesis of hormones and other biologically FEDERALLY SPONSORED RESEARCH important molecules. Fatty acids in E. coli, for example, are synthesized by a series of decarboxylative Claisen conden­ STATEMENT sation reactions using acetyl-CoA to add two carbon units to a growing fat. Following each round of elongation the beta This invention was made with govermnent support under 15 keto group is reduced to the fully saturated carbon chain by Grant No: 2012-10008-20263 awarded by the USDA. The the sequential action of a ketoreductase, a dehydratase, and govermnent has certain rights in the invention. an enol reductase. The growing fatty acid chain is carried between these FIELD OF THE DISCLOSURE active sites while attached covalently to the phosphoanteth- 20 eine prosthetic group of an acyl carrier protein (ACP), and This invention relates generally to the microbial produc­ is released from the ACP by the action ofa thioesterase (TE) tion of fats, such as fatty acids and their derivatives, or upon reaching a carbon chain length ofe.g., 16, although this products derived from the fatty acid synthesis cycle, such as can be varied by adding different TE enzymes to the cell. hydroxyfatty acids, methyl ketones, and the like. This invention relates to a technology to improve micro- 25 bial production of fatty acids, fatty acid derivatives and/or BACKGROUND OF THE DISCLOSURE products derived from the fatty acid synthesis cycle through the use of a new micro-aerobic/anaerobic cultivation Anaerobic fermentation and aerobic respiration have been method. The traditional fatty acid production was performed the two metabolic modes of interest for the industrial under fully aerobic conditions, because fatty acid production bioproduction of chemicals, such as fats. Oxygen rich res­ 30 dropped significantly under anaerobic conditions. piration offers very efficient cell growth (growth rate and There are two main reasons for the absolute requirement yield) and converts a high percentage of the carbon source of oxygen for high free fatty acid production. One reason is into carbon dioxide and cell mass ( see Table 1), but typically due to the limitation of ATP availability. Only 2 ATP per has low product yields. Anaerobic fermentation, on the other glucose will be generated in the glycolysis pathway under hand, has high product formation, but poor cell growth and 35 anaerobic conditions and 1 ATP is required for each fatty the synthesis of several competing fermentation products at acid elongation cycle. high yields (e.g. lactate, formate, ethanol, acetate, succinate, The second reason is due to limited reducing equivalent etc.), thus, diverting carbons away from the desired product. availability and is more problematic. Under normal aerobic conditions, the conversion of pyruvate to acetyl-CoA, a TABLE 1 40 precursor for fatty acid synthesis, and NAD+ to NADH is carried out by the enzyme pyruvate dehydrogenase (PDH).