聚羟基脂肪酸酯的低碳生物制造：基于碳转化率的分析与应用

doi:10.12211/2096-8280.2021-088

摘要/Abstract

摘要：

随着环境污染日益加剧以及全球范围禁/限塑令出台，以聚羟基脂肪酸酯（PHA）为代表的生物基可降解塑料的生物制造看到了曙光。然而如何实现低成本、绿色低碳可持续的PHA生产仍然面临巨大挑战。采用合成生物学方法，创建合成PHA的微生物细胞工厂，将廉价碳源以及可再生原料高效转化为种类繁多和性能多样的PHA是解决所面临问题的重要途径。本文通过总结各种PHA单体的生物合成途径并分析了各途径的理论碳转化率，提出了提高共聚物单体产量的重要策略，即优先选择高碳转化率PHA单体的合成。同时本文汇总了当前在创建低碳生物合成途径及利用一碳化合物合成PHA的研究进展，为可降解塑料的低碳生物合成提供有效方法。最后，对合成生物学在PHA低碳生物制造领域的发展趋势进行了总结和展望。在未来，随着合成生物学及新技术的融合发展，绿色生物制造可以生产更多低成本高附加值的PHA，促进生物基塑料的产业化发展，从而更好地服务于全球绿色低碳文明。

关键词: 聚羟基脂肪酸酯, PHA, 低碳生物制造, 碳转化率

Abstract:

With the increasing environmental pollution and the policy introduction of global ban on free plastic bags, the bio-manufacturing of bio-based degradable plastics represented by polyhydroxyalkanoates (PHAs) is one of promising ways to respond this challenge. PHAs are polyesters of hydroxyalkanoates synthesized by many bacteria and haloarchaea as carbon and energy storage materials. There are more than 150 types of polyhydroxyalkanoate monomers reported, resulting in a variety of PHAs with diverse properties. However, how to ensure low-cost, green, low-carbon and sustainable production of PHAs is still facing huge challenges. By using synthetic biology approaches, it is an important solution to construct microbial cell factories that synthesize PHAs and efficiently convert cheap carbon sources and renewable raw materials into various types of PHAs with excellent mechanical properties. This paper summarizes the biosynthetic pathways of various PHA monomers and analyzes the theoretical carbon conversion rate of various monomer synthesis pathways. It is proposed that the synthesis of PHA monomers with high carbon conversion rate is preferred to increase the yield of copolymer monomers. At the same time, it summarizes the current research progress in the creation of low-carbon biosynthesis pathways and the use of one-carbon compounds as carbon sources to synthesize PHAs. These are all effective ways to achieve low-carbon biosynthesis of degradable plastics. Finally, we summarized and prospected the development trend of synthetic biology in the field of low-carbon biomanufacturing of PHAs. In the future, with the integration and development of synthetic biology and new technologies, more low-cost and high-value-added PHAs can be obtained through green bio-manufacturing, thereby promoting the industrialization of bio-based plastics and better serving the global green and low-carbon society.

Key words: polyhydroxyalkanoates, PHA, low-carbon bio-manufacturing, carbon conversion rate

中图分类号:

王倩, 祁庆生. 聚羟基脂肪酸酯的低碳生物制造：基于碳转化率的分析与应用[J]. 合成生物学, 2022, 3(4): 748-762.

Qian WANG, Qingsheng QI. Low-carbon biomanufacturing of polyhydroxyalkanoates: analysis and application based on carbon conversion rate[J]. Synthetic Biology Journal, 2022, 3(4): 748-762.

图/表 4

图1 PHA的分子式及分类

Fig. 1 Molecular structure of PHA and its classification

图2 微生物合成PHA的代谢途径［其中包括天然PHA单体（3HB、4HB、3HV、3HA、3HP单体）和代表性的非天然PHA（LA、GA、2HB单体）；3HP—3-羟基丙酸；3HP-CoA—3-羟基丙酰辅酶A；3HB-CoA—3-羟基丁酰辅酶A； 3-HA-CoA—3-羟基酯酰辅酶A；3HV-CoA—3-羟基戊酰辅酶A；SSA—琥珀酸半醛； 4HB—4-羟基丁酸； 4HB-CoA—4-羟基丁酰辅酶A；2HB—2-羟基丁酸；2HB-CoA—2-羟基丁酰辅酶A］

Fig. 2 Metabolic pathways of microbial biosynthesis of PHA, including natural PHA monomers (3HB, 4HB, 3HV, 3HA, 3HP) and representative non-natural PHA monomers (LA, GA, 2HB)

表1 各类PHA单体的代表性合成途径及生产

Tab. 1 Representative biosynthesis pathways and production of various PHA monomers

单体	合成途径	理论碳摩尔得率	宿主	途径基因	碳源	单体比例（摩尔分数）/%	文献
3HB	乙酰辅酶A	0.66	E. coli	—	葡萄糖	100	[20-21]
3HV	苏氨酸途径	0.83	E. coli	E. colithrABC, C.glutamicumilvA	葡萄糖	17.5	[22]
			C.glutamicum WM001	—	葡萄糖	72.5	[23]
	柠苹酸途径	0.56	E. coli	M.jannaschiicimA, E. colileuBCD	葡萄糖	11.5	[24]
	甲基丙二酰 CoA途径	0.83	S.typhimurium	E. colisbm, ygfG	葡萄糖	30	[25]
4HB	琥珀酰CoA途径	0.66～1.33	E. coli	C.kluyveri4hbD, sucD, orfZ	葡萄糖	12.5	[26]
			H.bluephagenesis	C.kluyverior fZ (genomeintg)	葡萄糖	25	[27]
3HP	甘油途径	1.00	E. coli	C.butyricumdhaB1, S.entericapduP	甘油	100	[16]
	丙二酰CoA途径	1.00	E. coli	C.aurantiacusmcr, E. coliAccABCD, prpE	葡萄糖	100	[28]
LA	乳酸合成途径	1.00	E. coli	E. colildhA, C.propionicumpct	葡萄糖	30～60	[29]
2HB	柠苹酸途径	0.66	E. coli	E. colithrABC, C.glutamicumilvAL.lactispanE	葡萄糖	10～60	[30]
GA	木糖Dahms途径	0.40	E. coli	C.crescentusxylBC	木糖	29.5	[31]
	乙醛酸途径	0.66	E. coli	E. coliycdW, aceAK	葡萄糖	39	[32]
3HHx	丁酰辅酶A途径	0.66	E. coli	C.necatorbktBandphaB1, A.caviaephaJ, T.denticolater,E. colifadBandfadA	葡萄糖	13.2	[33]
			C.necator	C.necatorbktB, crthad,M.extorquensccr	葡萄糖	12.1	[34]
3HA	脂肪酸从头合成	0.66	E. coli	P.putidaphaG, PP_0763	葡萄糖	100	[35]
	反向β氧化	0.66	E. coli	E. colifadBA、T.denticolater、P.aeruginosaPhaJ1	葡萄糖	78.8	[36]

图3 PHA生产的低碳及固碳生物合成途径（低碳生物合成途径包括如NOG途径、EP-Bifido途径等；固碳生物合成途径包括卡尔文循环、丝氨酸途径、5-磷酸核酮糖单磷酸途径等。酶：Fxpk—F6P/Xu5P磷酸解酮酶； Tal—转醛醇酶； Tkt—转酮醇酶； Fbp—果糖-1，6-二磷酸；Fba—果糖-1，6-二磷酸醛缩酶； tpi—丙糖磷酸异构酶； Rpe—核酮糖-5-磷酸异构酶； Rpi—核糖-5-磷酸异构酶； CODH—一氧化碳脱氢酶；FDH—甲酸脱氢酶；FTS—甲酰基-四氢叶酸合酶；FTC—甲酰基-四氢叶酸环化水解酶； MTD—亚甲基-四氢叶酸脱氢酶； MTR—亚甲基-四氢叶酸还原酶；MTF—甲基转移酶； ACS—乙酰辅酶A合成酶； PRK—磷酸核酮糖激酶； GAPDH—3-磷酸甘油醛脱氢酶； PGK—磷酸甘油酸激酶； RuBisCO—核酮糖-1，5-二磷酸羧化酶； MMO—甲烷单氧化酶； MDH—甲醇脱氢酶； H4MPTP—四氢甲烷蝶呤途径； GlyA—丝氨酸羟甲基转移酶；MtkAB—苹果酸硫激酶； Sga—丝氨酸-乙醛酸氨基转移酶； Mcl—苹果酰辅酶A裂合酶； 6PGDH—6-磷酸葡萄糖酸脱氢酶； H6PS—己酮糖-6-磷酸合酶；H6PI—己酮糖-6-磷酸异构酶；PGI—磷酸葡萄糖异构酶； G6PDH—葡萄糖-6-磷酸脱氢酶。代谢物：F6P—果糖-6-磷酸； PEP—磷酸烯醇式丙酮酸；Pyr—丙酮酸； E4P—赤藓糖-4-磷酸； S7P—景天庚酮糖-7-磷酸； R5P—核糖-5-磷酸；Ru5P—核酮糖-5-磷酸； X5P—木酮糖-5-磷酸； GAP—3-磷酸甘油醛； DHAP—磷酸二羟丙酮； FBP—果糖-1，6-二磷酸； Ribulose 1，5-BP—核酮糖-1，5-二磷酸； 1，3-BP-glycerate—1，3-二磷酸甘油酸；6P-Gluconate—6-磷酸葡萄糖酸； Hexulose-6P—己酮糖-6-磷酸； AcP—乙酰磷酸； Ac-CoA—乙酰辅酶A； Formyl-THF—甲酰基-四氢叶酸； Methenyl-THF—亚甲基四氢叶酸； Methylene-THF—亚甲基四氢叶酸； Methyl-THF—甲基-四氢叶酸； Methyl-CoFeS-P—甲基类咕啉铁硫蛋白）

Fig. 3 Low carbon and carbon sequestration biosynthetic pathways for PHA production(Low carbon biosynthesis pathways include NOG pathway, EP-bifido pathway, etc. Carbon sequestration biosynthesis pathways include Calvin cycle, serine pathway, ribulose 5-phosphate monophosphate pathway, etc)

参考文献 90

1	ANDERSON A J, DAWES E A. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates[J]. Microbiological Reviews, 1990, 54(4): 450-472.
2	MADISON L L, HUISMAN G W. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic[J]. Microbiology and Molecular Biology Reviews, 1999, 63(1): 21-53.
3	祁庆生, 王倩, 梁泉峰. 生物合成可降解材料PHA的研究进展和产业化趋势[J]. 中国基础科学, 2009, 11(5): 96-99, 74.
	QI Q S, WANG Q, LIANG Q F. Research progress and industrialization trend of biodegradable materials PHA[J]. China Basic Science, 2009, 11(5): 96-99, 74.
4	STEINBÜCHEL A. Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example[J]. Macromolecular Bioscience, 2001, 1(1): 1-24.
5	SUDESH K, ABE H, Synthesis DOI Y., structure and properties of polyhydroxyalkanoates : biological polyesters[J]. Progress in Polymer Science, 2000, 25(10): 1503-1555.
6	LI Z B, YANG J, LOH X J. Polyhydroxyalkanoates: opening doors for a sustainable future[J]. NPG Asia Materials, 2016, 8(4): e265.
7	REHM B H A, STEINBÜCHEL A. Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis[J]. International Journal of Biological Macromolecules, 1999, 25(1/2/3): 3-19.
8	赵国强, 李亚丽, 武双, 等. 基于低成本碳源微生物合成聚羟基脂肪酸酯的研究进展[J]. 高分子通报, 2020(11): 22-30.
	ZHAO G Q, LI Y L, WU S, et al. Research progress in polyhydroxyalkanoates biosynthesis by microorganisms based on inexpensive carbon sources[J]. Polymer Bulletin, 2020(11): 22-30.
9	SANTIMANO M C, PRABHU N N, GARG S. PHA production using low-cost agro-industrial wastes by Bacillus sp. strain COL1/A6[J]. Research Journal of Microbiology, 2009, 4(3): 89-96.
10	KIM H S, OH Y H, JANG Y A, et al. Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution[J]. Microbial Cell Factories, 2016, 15: 95.
11	SHEU D S, CHEN W M, YANG J Y, et al. Thermophilic bacterium Caldimonas taiwanensis produces poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from starch and valerate as carbon sources[J]. Enzyme and Microbial Technology, 2009, 44(5): 289-294.
12	BUDDE C F, RIEDEL S L, WILLIS L B, et al. Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains[J]. Applied and Environmental Microbiology, 2011, 77(9): 2847-2854.
13	KOLLER M, HESSE P, BONA R, et al. Potential of various archae- and eubacterial strains as industrial polyhydroxyalkanoate producers from whey[J]. Macromolecular Bioscience, 2007, 7(2): 218-226.
14	PARK S J, PARK J P, LEE S Y. Production of poly(3-hydroxybutyrate) from whey by fed-batch culture of recombinant Escherichia coli in a pilot-scale fermenter[J]. Biotechnology Letters, 2002, 24(3):185-189.
15	OH Y H, LEE S H, JANG Y A, et al. Development of rice bran treatment process and its use for the synthesis of polyhydroxyalkanoates from rice bran hydrolysate solution[J]. Bioresource Technology, 2015, 181: 283-290.
16	ANDREESSEN B, LANGE A B, ROBENEK H, et al. Conversion of glycerol to poly(3-hydroxypropionate) in recombinant Escherichia coli [J]. Applied and Environmental Microbiology, 2010, 76(2): 622-626.
17	VERLINDEN R A, HILL D J, KENWARD M A, et al. Production of polyhydroxyalkanoates from waste frying oil by Cupriavidus necator [J]. AMB Express, 2011, 1(1): 11.
18	NEELAMEGAM A, AL-BATTASHI H, AL-BAHRY S, et al. Biorefinery production of poly-3-hydroxybutyrate using waste office paper hydrolysate as feedstock for microbial fermentation[J]. Journal of Biotechnology, 2018, 265: 25-30.
19	POVOLO S, TOFFANO P, BASAGLIA M, et al. Polyhydroxyalkanoates production by engineered Cupriavidus necator from waste material containing lactose[J]. Bioresource Technology, 2010, 101(20): 7902-7907.
20	CHOI J I, LEE S Y, HAN K. Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for enhanced production of poly(3-hydroxybutyrate) in Escherichia coli [J]. Applied and Environmental Microbiology, 1998, 64(12): 4897-4903.
21	ELHADI D, LÜ L, JIANG X R, et al. CRISPRi engineering E. coli for morphology diversification[J]. Metabolic Engineering, 2016, 38: 358-369.
22	CHEN Q, WANG Q, WEI G Q, et al. Production in Escherichia coli of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with differing monomer compositions from unrelated carbon sources[J]. Applied and Environmental Microbiology, 2011, 77(14): 4886-4893.
23	MA W J, WANG J L, LI Y, et al. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) co-produced with L-isoleucine in Corynebacterium glutamicum WM001[J]. Microbial Cell Factories, 2018, 17(1): 93.
24	WANG Q, LIU X L, QI Q S. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose with elevated 3-hydroxyvalerate fraction via combined citramalate and threonine pathway in Escherichia coli [J]. Applied Microbiology and Biotechnology, 2014, 98(9): 3923-3931.
25	ALDOR I S, KIM S W, PRATHER K L, et al. Metabolic engineering of a novel propionate-independent pathway for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Salmonella enterica serovar typhimurium[J]. Applied and Environmental Microbiology, 2002, 68(8): 3848-3854.
26	LI Z J, SHI Z Y, JIAN J, et al. Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli [J]. Metabolic Engineering, 2010, 12(4): 352-359.
27	YE J W, HU D K, CHE X M, et al. Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose[J]. Metabolic Engineering, 2018, 47: 143-152.
28	WANG Q, LIU C S, XIAN M, et al. Biosynthetic pathway for poly(3-hydroxypropionate) in recombinant Escherichia coli [J]. Journal of Microbiology, 2012, 50(4): 693-697.
29	JUNG Y K, KIM T Y, PARK S J, et al. Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers[J]. Biotechnology and Bioengineering, 2010, 105(1): 161-171.
30	PARK S J, LEE T W, LIM S C, et al. Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered Escherichia coli [J]. Applied Microbiology and Biotechnology, 2012, 93(1): 273-283.
31	CHOI S Y, PARK S J, KIM W J, et al. One-step fermentative production of poly(lactate-co-glycolate) from carbohydrates in Escherichia coli [J]. Nature Biotechnology, 2016, 34(4): 435-440.
32	LI Z J, QIAO K J, SHI W C, et al. Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli [J]. Metabolic Engineering, 2016, 35: 1-8.
33	WANG Q, LUAN Y Q, CHENG X L, et al. Engineering of Escherichia coli for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from glucose[J]. Applied Microbiology and Biotechnology, 2015, 99(6): 2593-2602.
34	ZHANG M X, KURITA S, ORITA I, et al. Modification of acetoacetyl-CoA reduction step in Ralstonia eutropha for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from structurally unrelated compounds[J]. Microbial Cell Factories, 2019, 18(1): 147.
35	WANG Q, TAPPEL R C, ZHU C J, et al. Development of a new strategy for production of medium-chain-length polyhydroxyalkanoates by recombinant Escherichia coli via inexpensive non-fatty acid feedstocks[J]. Applied and Environmental Microbiology, 2012, 78(2): 519-527.
36	ZHUANG Q Q, WANG Q, LIANG Q F, et al. Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid β-oxidation cycle in Escherichia coli [J]. Metabolic Engineering, 2014, 24: 78-86.
37	CHOI J I, LEE S Y. High level production of supra molecular weight poly(3-hydroxybutyrate) by metabolically engineered Escherichia coli [J]. Biotechnology and Bioprocess Engineering, 2004, 9(3): 196-200.
38	MARANGONI C, FURIGO A, DE ARAGÃO G M F. Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstonia eutropha in whey and inverted sugar with propionic acid feeding[J]. Process Biochemistry, 2002, 38(2): 137-141.
39	KOLLER M, BONA R, CHIELLINI E, et al. Polyhydroxyalkanoate production from whey by Pseudomonas hydrogenovora [J]. Bioresource Technology, 2008, 99(11): 4854-4863.
40	STEINBÜCHEL A, HUSTEDE E, LIEBERGESELL M, et al. Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria[J]. FEMS Microbiology Letters, 1992, 103(2/3/4): 217-230.
41	HAYWOOD G W, ANDERSON A J, WILLIAMS D R, et al. Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126[J]. International Journal of Biological Macromolecules, 1991, 13(2): 83-88.
42	ESCHENLAUER A C, STOUP S K, SRIENC F, et al. Production of heteropolymeric polyhydroxyalkanoate in Escherichia coli from a single carbon source[J]. International Journal of Biological Macromolecules, 1996, 19(2): 121-130.
43	YANG J E, CHOI Y J, LEE S J, et al. Metabolic engineering of Escherichia coli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose[J]. Applied Microbiology and Biotechnology, 2014, 98(1): 95-104.
44	CHEN Y, CHEN X Y, DU H T, et al. Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV)[J]. Metabolic Engineering, 2019, 54: 69-82.
45	AKDOĞAN M, ÇELIK E. Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biopolymer by recombinant Bacillus megaterium in fed-batch bioreactors[J]. Bioprocess and Biosystems Engineering, 2021, 44(2): 403-416.
46	DOI Y, KANESAWA Y, KUNIOKA M, et al. Biodegradation of microbial copolyesters: poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate)[J]. Macromolecules, 1990, 23(1): 26-31.
47	VIGNESWARI S, VIJAYA S, MAJID M I A, et al. Enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer with manipulated variables and its properties[J]. Journal of Industrial Microbiology and Biotechnology, 2009, 36(4): 547-556.
48	SÖHLING B, GOTTSCHALK G. Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri [J]. Journal of Bacteriology, 1996, 178(3): 871-880.
49	VALENTIN H E, DENNIS D. Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose[J]. Journal of Biotechnology, 1997, 58(1): 33-38.
50	NAKAMURA S, KUNIOKA M, DOI Y. Biosynthesis and characterization of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)[J]. Journal of Macromolecular Science: Part A- Chemistry, 1991, 28(sup1): 15-24.
51	VALENTIN H E, MITSKY T A, MAHADEO D A, et al. Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli [J]. Applied and Environmental Microbiology, 2000, 66(12): 5253-5258.
52	FUKUI T, SUZUKI M, TSUGE T, et al. Microbial synthesis of poly[(R)-3-hydroxybutyrate-co-3-hydroxypropionate] from unrelated carbon sources by engineered Cupriavidus necator [J]. Biomacromolecules, 2009, 10(4): 700-706.
53	WANG Q, YANG P, XIAN M, et al. Biosynthesis of poly(3-hydroxypropionate-co-3-hydroxybutyrate) with fully controllable structures from glycerol[J]. Bioresource Technology, 2013, 142: 741-744.
54	GAO Y Q, LIU C S, DING Y M, et al. Development of genetically stable Escherichia coli strains for poly(3-hydroxypropionate) production[J]. PLoS One, 2014, 9(5): e97845.
55	DEB P K, KOKAZ S F, ABED S N, et al. Pharmaceutical and biomedical applications of polymers[M]// Basic fundamentals of drug delivery. Amsterdam: Elsevier, 2019: 203-267.
56	GIAMMONA G, CRAPARO E F. Biomedical applications of polylactide (PLA) and its copolymers[J]. Molecules, 2018, 23(4): 980.
57	YANG T H, KIM T W, KANG H O, et al. Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase[J]. Biotechnology and Bioengineering, 2010, 105(1): 150-160.
58	YANG T H, JUNG Y K, KANG H O, et al. Tailor-made type II Pseudomonas PHA synthases and their use for the biosynthesis of polylactic acid and its copolymer in recombinant Escherichia coli [J]. Applied Microbiology and Biotechnology, 2011, 90(2): 603-614.
59	REN Y L, MENG D C, WU L P, et al. Microbial synthesis of a novel terpolyester P(LA-co-3HB-co-3HP) from low-cost substrates[J]. Microbial Biotechnology, 2017, 10(2): 371-380.
60	PARK S J, KIM T W, KIM M K, et al. Advanced bacterial polyhydroxyalkanoates: towards a versatile and sustainable platform for unnatural tailor-made polyesters[J]. Biotechnology Advances, 2012, 30(6): 1196-1206.
61	DAVID Y, JOO J C, YANG J E, et al. Biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates by employing butyryl-CoA transferases in metabolically engineered Escherichia coli [J]. Biotechnology Journal, 2017, 12(11): 1700116.
62	CHOI S Y, KIM W J, YU S J, et al. Engineering the xylose-catabolizing Dahms pathway for production of poly(D-lactate-co-glycolate) and poly(D-lactate-co-glycolate-co-D-2-hydroxybutyrate) in Escherichia coli [J]. Microbial Biotechnology, 2017, 10(6): 1353-1364.
63	LI Z J, QIAO K J, CHE X M, et al. Metabolic engineering of Escherichia coli for the synthesis of the quadripolymer poly(glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose[J]. Metabolic Engineering, 2017, 44: 38-44.
64	NODA I, GREEN P R, SATKOWSKI M M, et al. Preparation and properties of a novel class of polyhydroxyalkanoate copolymers[J]. Biomacromolecules, 2005, 6(2): 580-586.
65	YU B Y, CHEN P Y, SUN Y M, et al. Response of human mesenchymal stem cells (hMSCs) to the topographic variation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHH _x ) films[J]. Journal of Biomaterials Science Polymer Edition, 2012, 23(1/2/3/4): 1-26.
66	INSOMPHUN C, XIE H, MIFUNE J, et al. Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C₆-monomer composition from fructose in Ralstonia eutropha [J]. Metabolic Engineering, 2015, 27: 38-45.
67	FUKUI T, DOI Y. Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae [J]. Journal of Bacteriology, 1997, 179(15): 4821-4830.
68	LEE S H, OH D H, AHN W S, et al. Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by high-cell-density cultivation of Aeromonas hydrophila[J]. Biotechnology and Bioengineering, 2000, 67(2): 240-244.
69	TIMM A, STEINBÜCHEL A. Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads[J]. Applied and Environmental Microbiology, 1990, 56(11): 3360-3367.
70	REHM B H, MITSKY T A, STEINBÜCHEL A. Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli [J]. Applied and Environmental Microbiology, 2001, 67(7): 3102-3109.
71	PARK S J, CHOI J I, LEE S Y. Engineering of Escherichia coli fatty acid metabolism for the production of polyhydroxyalkanoates[J]. Enzyme and Microbial Technology, 2005, 36(4): 579-588.
72	HOKAMURA A, WAKIDA I, MIYAHARA Y, et al. Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant Escherichia coli from glucose[J]. Journal of Bioscience and Bioengineering, 2015, 120(3): 305-310.
73	TSAY J T, OH W, LARSON T J, et al. Isolation and characterization of the beta-ketoacyl-acyl carrier protein synthase III gene (fabH) from Escherichia coli K-12[J]. The Journal of Biological Chemistry, 1992, 267(10): 6807-6814.
74	NOMURA C T, TANAKA T, GAN Z H, et al. Effective enhancement of short-chain-length-medium-chain-length polyhydroxyalkanoate copolymer production by coexpression of genetically engineered 3-ketoacyl-acyl-carrier-protein synthase III (fabH) and polyhydroxyalkanoate synthesis genes[J]. Biomacromolecules, 2004, 5(4): 1457-1464.
75	BOGORAD I W, LIN T S, LIAO J C. Synthetic non-oxidative glycolysis enables complete carbon conservation[J]. Nature, 2013, 502(7473): 693-697.
76	WANG Q, XU J S, SUN Z J, et al. Engineering an in vivo EP-bifido pathway in Escherichia coli for high-yield acetyl-CoA generation with low CO₂ emission[J]. Metabolic Engineering, 2019, 51: 79-87.
77	OPGENORTH P H, KORMAN T P, BOWIE J U. A synthetic biochemistry module for production of bio-based chemicals from glucose [J]. Nat Chem Biol 2016, 12 (6):393-395.
78	DUDLEY Q M, KARIM A S, JEWETT M C. Cell-free metabolic engineering: Biomanufacturing beyond the cell[J]. Biotechnology Journal, 2015, 10(1): 69-82.
79	DÜRRE P, EIKMANNS B J. C₁-carbon sources for chemical and fuel production by microbial gas fermentation[J]. Current Opinion in Biotechnology, 2015, 35: 63-72.
80	BRIGHAM C. Perspectives for the biotechnological production of biofuels from CO₂ and H₂ using Ralstonia eutropha and other 'Knallgas' bacteria[J]. Applied Microbiology and Biotechnology, 2019, 103(5): 2113-2120.
81	ISHIZAKI A, TANAKA K, TAGA N. Microbial production of poly-D-3-hydroxybutyrate from CO₂ [J]. Applied Microbiology and Biotechnology, 2001, 57(1/2): 6-12.
82	VOLOVA T G, KALACHEVA G S, STEINBÜCHEL A. Biosynthesis of multi-component polyhydroxyalkanoates by the Bacterium Wautersia eutropha [J]. Macromolecular Symposia, 2008, 269(1): 1-7.
83	DE SOUZA PINTO LEMGRUBER R, VALGEPEA K, TAPPEL R, et al. Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)[J]. Metabolic Engineering, 2019, 53: 14-23.
84	WU G F, WU Q Y, SHEN Z Y. Accumulation of poly-β-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803[J]. Bioresource Technology, 2001, 76(2): 85-90.
85	CARPINE R, DU W, OLIVIERI G, et al. Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction[J]. Algal Research, 2017, 25: 117-127.
86	WENDLANDT K D, JECHOREK M, HELM J, et al. Producing poly-3-hydroxybutyrate with a high molecular mass from methane[J]. Journal of Biotechnology, 2001, 86(2): 127-133.
87	BOURQUE D, POMERLEAU Y, GROLEAU D. High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB[J]. Applied Microbiology and Biotechnology, 1995, 44(3/4): 367-376.
88	ORITA I, NISHIKAWA K, NAKAMURA S, et al. Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions[J]. Applied Microbiology and Biotechnology, 2014, 98(8): 3715-3725.
89	WANG J, TAN H, LI K, et al. Two-stage fermentation optimization for poly-3-hydroxybutyrate production from methanol by a new Methylobacterium isolate from oil fields[J]. Journal of Applied Microbiology, 2020, 128(1): 171-181.
90	王颖, 陈国强. 合成生物学技术在聚羟基脂肪酸酯PHA生产中的应用[J]. 中国科学: 生命科学, 2015, 45(10): 1003-1014.
	WANG Y, CHEN G Q. Application of synthetic biology for the production of polyhydroxyalkanoates[J]. Scientia Sinica (Vitae), 2015, 45(10): 1003-1014.