生物合成聚乳酸研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2024-0982

摘要/Abstract

摘要：

聚乳酸（polylactic acid, PLA）是以乳酸为原料聚合而成的非天然生物可降解塑料，具有优良的生物降解性，是碳达峰、碳中和背景下传统石油基塑料的重要替代品之一。作为典型的碳中和材料，PLA正逐步成为国民经济和社会发展所需的基础性大宗原材料。目前，PLA主要通过生物发酵与化学聚合相结合的工艺生产，生产过程复杂，成本较高，且存在毒性物质残留的隐患。因此，更加绿色便捷的生产方法开发成为PLA合成领域的关注热点。随着合成生物学、蛋白质工程和代谢工程的快速发展，PLA全生物合成关键酶被逐渐挖掘和改造，PLA全生物合成路径被设计和组装，以具有工业化属性的微生物为底盘构建一步合成PLA的细胞工厂成为现实，为PLA的绿色合成提供了新的解决方案。然而，生物合成法面临PLA产量低、产品性能差等问题，难以满足工业化生产的要求。因此，提高PLA生物合成效率和改善产品性能成为PLA生物合成技术开发的重点。本文首先对PLA及其合成方法进行了介绍，并系统分析了化学合成法与生物合成法各自的优势和不足；随后，总结了PLA生物合成的途径和关键酶，着重从蛋白质工程和代谢工程两个方面归纳了PLA生物合成的调控策略；最后，对PLA生物合成技术升级发展中面临的关键挑战和未来研究趋势进行了系统的分析和展望，旨在为更高效、更绿色的PLA生物合成系统的设计和开发提供有益参考。

关键词: 聚乳酸, 生物合成, 关键酶, 蛋白质工程, 代谢工程, 辅酶A转移酶, PHA合成酶

Abstract:

Polylactic acid (PLA) is a non-natural biodegradable plastic polymerized from lactic acid. It demonstrates remarkable biodegradability and serves as a significant alternative to traditional petroleum-based plastics, particularly in the background of achieving carbon peak and carbon neutrality. As a typical carbon-neutral material, PLA is increasingly recognized as a crucial raw material for fostering national economic and social development. Currently, PLA is primarily produced through a combination of biological fermentation and chemical polymerization. However, this systhesis method is fraught with complexities, high costs, and potential risks of toxic residue accumulation. Consequently, the exploration of more environmentally sustainable and efficient production methods has become a central focus in the field of PLA synthesis. With the rapid advancements in synthetic biology, protein engineering and metabolic engineering, the key enzymes of PLA biosynthesis have been gradually identified and modified, the PLA biosynthesis pathways have been designed and assembled, leading to the establishment of cell factories for one-step synthesis of PLA utilizing microorganisms with industrial properties, thereby offering a new solution for the green synthesis of PLA. Nonetheless, the biosynthesis method is confronted with challenges, including low PLA yield and suboptimal product performance, which hinder its alignment with industrial standard. Therefore, enhancing the efficiency of PLA biosynthesis and improving product performance have emerged as critical objectives in the development of PLA biosynthesis technologies. In this paper, PLA and its synthesis methods were introduced briefly, and the advantages and disadvantages of chemical synthesis and biosynthesis were systematically analyzed. Subsequently, the pathways and key enzymes of PLA biosynthesis were summarized, and the regulatory strategies of PLA biosynthesis were summarized from the aspects of protein engineering and metabolic engineering. Finally, the key challenges and future research trends in the upgrading and development of PLA biosynthesis technology were systematically analyzed and prospected, aiming to provide useful reference for the design and development of more efficient and greener PLA biosynthesis system.

Key words: polylactic acid, biosynthesis, key enzyme, protein engineering, metabolic engineering, CoA transferase, PHA synthase

鲁天怡, 李爱朋, 费强. 生物合成聚乳酸研究进展[J]. 生物技术通报, 2025, 41(4): 47-60.

LU Tian-yi, LI Ai-peng, FEI Qiang. Research Progress in the Biosynthesis of Polylactic Acid[J]. Biotechnology Bulletin, 2025, 41(4): 47-60.

图/表 7

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关键酶 Key enzyme		来源菌株 Source strain	宿主菌株 Host strain	突变方式 Mutation method	酶改造效果 Effect of enzyme modification	参考文献 Reference
丙酰辅酶A转移酶 propionyl-CoA transferase		Clostridium ropionicum	E. coli XL1-Blue	V193A和4个沉默突变T78C、T669C、A1125G、T1158C（Pct540 _Cp ）	减缓酶表达对菌株的生长抑制，增强LA-CoA的合成，与野生型相比聚合物产量提高约5倍，聚合物中乳酸分数提高约8倍，酶体外特异性活性降低约39%	［22］
丙酰辅酶A转移酶 propionyl-CoA transferase				A243T和1个沉默突变A1200G（Pct532 _Cp ）	减缓酶表达对菌株的生长抑制，增强LA-CoA的合成，与野生型相比聚合物产量提高约4.5倍，聚合物中乳酸分数提高约9.5倍，酶体外特异性活性降低约52%
PHA合成酶 PHA synthetase	Ⅰ型 Type Ⅰ	Ralstonia eutropha	E. coli LS5218	A510S	赋予酶对LA-CoA的聚合能力，共聚物中乳酸分数最高可达26 mol%，且共聚物似为嵌段共聚物	［40］
	Ⅱ型 Type Ⅱ	Pseudomonas sp. 61-3	E. coli JM109	S325T/Q481K	赋予酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至6 mol%	［20］
			E. coli JW0885	S325T/Q481K	赋予酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至26 mol%	［41］
				S325T/Q481K/F392S	F392S的额外突变提高酶对LA-CoA的聚合能力，共聚物中乳酸分数提高约73%，聚合物含量提高约40.9%	［41］
		Pseudomonas sp. MBEL 6-19	E. coli XL1-blue	E130D/Q481K	赋予酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至35.3 mol%	［22］
				E130D/S477F/Q481K	首次实现PLA均聚物（0.5 wt%）的合成，提高共聚物中乳酸分数至36.2 mol%
				E130D/S325T/Q481K	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至39.1 mol%
				E130D/S325T/S477R/Q481M	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至41.2 mol%
				E130D/S325T/S477F/Q481K	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至46 mol%
				E130D/S325T/S477G/Q481K	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至49 mol%
		Pseudomonas resinovorans	E. coli XL1-Blue	E130D/Q481K	提高酶对LA-CoA的聚合能力，PLA产量提高至1.2 wt%	［7］
		Pseudomonas resinovorans	E. coli XL1-Blue	E130D/S325T/S477G/Q481K	提高酶对LA-CoA的聚合能力，PLA产量提高至7.3 wt%
	嵌合酶 Chimerase	N端：Aeromonas cavia（Ⅱ型）； C端：Ralstonia eutropha（Ⅰ型）	E. coli JM109	N149D/F314H	提高酶对LA-CoA的聚合能力，共聚物中乳酸分数提高9.5倍	［42］

关键酶 Key enzyme		来源菌株 Source strain	宿主菌株 Host strain	突变方式 Mutation method	酶改造效果 Effect of enzyme modification	参考文献 Reference
丙酰辅酶A转移酶 propionyl-CoA transferase		Clostridium ropionicum	E. coli XL1-Blue	V193A和4个沉默突变T78C、T669C、A1125G、T1158C（Pct540 _Cp ）	减缓酶表达对菌株的生长抑制，增强LA-CoA的合成，与野生型相比聚合物产量提高约5倍，聚合物中乳酸分数提高约8倍，酶体外特异性活性降低约39%	［22］
丙酰辅酶A转移酶 propionyl-CoA transferase				A243T和1个沉默突变A1200G（Pct532 _Cp ）	减缓酶表达对菌株的生长抑制，增强LA-CoA的合成，与野生型相比聚合物产量提高约4.5倍，聚合物中乳酸分数提高约9.5倍，酶体外特异性活性降低约52%
PHA合成酶 PHA synthetase	Ⅰ型 Type Ⅰ	Ralstonia eutropha	E. coli LS5218	A510S	赋予酶对LA-CoA的聚合能力，共聚物中乳酸分数最高可达26 mol%，且共聚物似为嵌段共聚物	［40］
	Ⅱ型 Type Ⅱ	Pseudomonas sp. 61-3	E. coli JM109	S325T/Q481K	赋予酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至6 mol%	［20］
			E. coli JW0885	S325T/Q481K	赋予酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至26 mol%	［41］
				S325T/Q481K/F392S	F392S的额外突变提高酶对LA-CoA的聚合能力，共聚物中乳酸分数提高约73%，聚合物含量提高约40.9%	［41］
		Pseudomonas sp. MBEL 6-19	E. coli XL1-blue	E130D/Q481K	赋予酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至35.3 mol%	［22］
				E130D/S477F/Q481K	首次实现PLA均聚物（0.5 wt%）的合成，提高共聚物中乳酸分数至36.2 mol%
				E130D/S325T/Q481K	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至39.1 mol%
				E130D/S325T/S477R/Q481M	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至41.2 mol%
				E130D/S325T/S477F/Q481K	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至46 mol%
				E130D/S325T/S477G/Q481K	提高酶对LA-CoA的聚合能力，提高共聚物中乳酸分数至49 mol%
		Pseudomonas resinovorans	E. coli XL1-Blue	E130D/Q481K	提高酶对LA-CoA的聚合能力，PLA产量提高至1.2 wt%	［7］
		Pseudomonas resinovorans	E. coli XL1-Blue	E130D/S325T/S477G/Q481K	提高酶对LA-CoA的聚合能力，PLA产量提高至7.3 wt%
	嵌合酶 Chimerase	N端：Aeromonas cavia（Ⅱ型）； C端：Ralstonia eutropha（Ⅰ型）	E. coli JM109	N149D/F314H	提高酶对LA-CoA的聚合能力，共聚物中乳酸分数提高9.5倍	［42］

碳源 Carbon source	宿主菌株 Host strain	使用的关键酶 Key enzyme	代谢工程调控策略 Metabolic engineering regulation strategies	产量 Yield	分子量 Molecular weight	参考文献 Reference
葡萄糖 Glucose	E. coli XL1-blue	Pct540 _Cp PhaC _Ps_6-19^{E130D/S325T/S477R/Q481M}	1）乳酸供应强化：过表达ldhA；敲除ppc 2）乙酰辅酶A供应强化：敲除ackA、adhE；过表达acs	11 wt%	N.D.	［46］
	E. coli XL1-blue	Pct540 _Cp PhaC _Ps_6-19^{E130D/S477F/Q481K}	1）启动子改造：敲除lacI 2）乳酸供应强化：替换ldhA的天然启动子为强启动子trc 3）乙酰辅酶A供应强化：过表达acs 4）副产物阻断：敲除pflB、frdABCD和adhE	4.2 wt%	21 000 Da	［47］
	E. coli BL21（DE3）	Pct540 _Cp PhaC _Cs	形态工程：过表达sulA	2.23 wt% （955 mg/L）	21 000 Da	［44］
	Corynebacterium glutamicum ATCC13803	Pct _Me PhaC1 _Ps^STQK	聚合途径强化：过表达PhaC1 _Ps^STQK	1.4 wt%	Mw： 5.7 kD； Mn： 4.3 kD	［48］
	Yarrowia lipolytica	Pct540 _Cp PhaC _Pa^{E130D/S325T/S477R/Q481M}	1）乳酸供应强化：敲除YlDLD1 2）聚合途径强化：过表达Pct540 _Cp 、PhaC _Pa^{E130D/S325T/S477R/Q481M} 3）区室化工程：细胞质表达Pct540 _Cp，过氧化物酶体表达PhaC _Pa^{E130D/S325T/S477R/Q481M}	26 mg/g DCW	50.5 kD	［49］
二氧化碳 Carbon dioxide	Synechococcus elongatus PCC7942	Pct540 _Cp PhaC _Ps_6-19	1）聚合途径强化：过表达Pct540 _Cp 、PhaC _Ps_6-19 2）乙酰辅酶A供给强化：下调ackA，过表达acs 3）脂肪酸途径弱化：下调accABCD、fabH、fabF 4）高密度发酵和培养条件优化	23 mg/g DCW （108 mg/L）	Mw： 62.5 kD；Mn： 32.8 kD	［50］

碳源 Carbon source	宿主菌株 Host strain	使用的关键酶 Key enzyme	代谢工程调控策略 Metabolic engineering regulation strategies	产量 Yield	分子量 Molecular weight	参考文献 Reference
葡萄糖 Glucose	E. coli XL1-blue	Pct540 _Cp PhaC _Ps_6-19^{E130D/S325T/S477R/Q481M}	1）乳酸供应强化：过表达ldhA；敲除ppc 2）乙酰辅酶A供应强化：敲除ackA、adhE；过表达acs	11 wt%	N.D.	［46］
	E. coli XL1-blue	Pct540 _Cp PhaC _Ps_6-19^{E130D/S477F/Q481K}	1）启动子改造：敲除lacI 2）乳酸供应强化：替换ldhA的天然启动子为强启动子trc 3）乙酰辅酶A供应强化：过表达acs 4）副产物阻断：敲除pflB、frdABCD和adhE	4.2 wt%	21 000 Da	［47］
	E. coli BL21（DE3）	Pct540 _Cp PhaC _Cs	形态工程：过表达sulA	2.23 wt% （955 mg/L）	21 000 Da	［44］
	Corynebacterium glutamicum ATCC13803	Pct _Me PhaC1 _Ps^STQK	聚合途径强化：过表达PhaC1 _Ps^STQK	1.4 wt%	Mw： 5.7 kD； Mn： 4.3 kD	［48］
	Yarrowia lipolytica	Pct540 _Cp PhaC _Pa^{E130D/S325T/S477R/Q481M}	1）乳酸供应强化：敲除YlDLD1 2）聚合途径强化：过表达Pct540 _Cp 、PhaC _Pa^{E130D/S325T/S477R/Q481M} 3）区室化工程：细胞质表达Pct540 _Cp，过氧化物酶体表达PhaC _Pa^{E130D/S325T/S477R/Q481M}	26 mg/g DCW	50.5 kD	［49］
二氧化碳 Carbon dioxide	Synechococcus elongatus PCC7942	Pct540 _Cp PhaC _Ps_6-19	1）聚合途径强化：过表达Pct540 _Cp 、PhaC _Ps_6-19 2）乙酰辅酶A供给强化：下调ackA，过表达acs 3）脂肪酸途径弱化：下调accABCD、fabH、fabF 4）高密度发酵和培养条件优化	23 mg/g DCW （108 mg/L）	Mw： 62.5 kD；Mn： 32.8 kD	［50］

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