合成生物学改造酵母驱动丁二酸绿色生物制造

doi:10.11949/0438-1157.20231395

摘要/Abstract

摘要：

丁二酸是一种重要的C4平台化合物，采用微生物以可再生生物质资源为原料制造丁二酸具有显著的优势，是当前国内外的研究热点。合成丁二酸的微生物主要包括细菌和酵母两大类，与细菌相比，虽然酵母的丁二酸生产得率低于细菌，但酵母对酸性等逆境的抗性更强，因此，酵母可以在低pH条件下合成丁二酸，避免了中和剂的添加，从而使丁二酸的下游纯化过程更简单、成本更低。本文综述了近年来，利用酵母类微生物合成丁二酸的研究进展，重点介绍了以酿酒酵母、解脂耶氏酵母和东方伊萨酵母为代表的酵母类生产菌株的合成生物学改造策略；在此基础上，展望了通过构建酵母细胞工厂绿色生物制造丁二酸的未来发展方向。

关键词: 酵母, 丁二酸, 细胞工厂, 代谢工程, 合成生物学, 生物技术

Abstract:

Succinic acid is an important C4 platform chemical, and its industrial biomanufacturing route has significant advantages, which is a research hotspot. The microorganisms for succinic acid biosynthesis mainly include bacteria and yeasts. Compared with bacteria, although the product yield of yeasts is lower, the yeasts generally have high acid tolerance, therefore, they can synthesize succinic acid under low pH value conditions, thus avoiding adding additional neutralization agent to maintain the neutral pH conditions, therefore, the downstream process for yeast-based succinic acid purification is simpler and costs lower. In this paper, the research progress of biomanufacturing succinic acid using yeasts in recent years is summarized, and the progress of engineering the representative yeasts including Saccharomyces cerevisiae, Yarrowia lipolytica, and Issatchenkia orientalis using the metabolic engineering and the emerging synthetic biology tools is then reviewed. Finally, the future directions for green biomanufacturing succinic acid by constructing yeast cell factories are proposed.

Key words: yeast, succinic acid, microbial cell factory, metabolic engineering, synthetic biology, biotechnology

中图分类号:

Q 815

孙涛, 孙美莉, 陆然, 余一梓, 王凯峰, 纪晓俊. 合成生物学改造酵母驱动丁二酸绿色生物制造[J]. 化工学报, DOI: 10.11949/0438-1157.20231395.

Tao SUN, Meili SUN, Ran LU, Yizi YU, Kaifeng WANG, Xiaojun JI. Synthetic biology of yeasts drives the green biomanufacturing of succinic acid[J]. CIESC Journal, DOI: 10.11949/0438-1157.20231395.

图/表 5

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宿主菌株	酵母	细菌
耐酸性	高，但需要ATP维持	低
CO₂排放	释放CO₂	固定CO₂
限制因素	底物得率低	生产成本高
碱中和剂	不添加	需要且易染菌
下游处理	步骤相对少	需要电渗析或酸化
提取副产物	无	硫酸铵（硫酸钙）
设备和能源	相对较少	需要中和及处理设备

宿主菌株	酵母	细菌
耐酸性	高，但需要ATP维持	低
CO₂排放	释放CO₂	固定CO₂
限制因素	底物得率低	生产成本高
碱中和剂	不添加	需要且易染菌
下游处理	步骤相对少	需要电渗析或酸化
提取副产物	无	硫酸铵（硫酸钙）
设备和能源	相对较少	需要中和及处理设备

酵母种类	出发菌株	代谢工程改造策略	底物	是否添加中和剂	产量、得率、生产强度	参考文献
酿酒酵母	酿酒酵母XU-1	敲除SDH1-2	葡萄糖	否	产量409±68 mg/L、提升1.9倍	[30]
	酿酒酵母AH22ura3	敲除SDH1-2、IDH1、IDP1	葡萄糖	否	产量3.62 g/L、提升4.8倍，得率0.11 mol/mol	[31]
	酿酒酵母TAM	表达ScPYC2、ScMDH3、ScFRDS1、EcFUMC，敲除ScFUM1、GPD1	葡萄糖	否，pH 3.8	产量12.97±0.42 g/L，得率0.21 mol/mol	[34]
	酿酒酵母UBR2_CBS-DHA	表达ScMDH3、RoFUM、TbFRDg、AnDCT-02	甘油	是，pH 5.0	产量10.7 g/L，得率0.22±0.01 g/g	[35]
	酿酒酵母CEN.PK113-1A	表达CjFPS1、OpGDH、ScDAK1、ScMDH3、RoFUM、TbFRDg、AnDCT-02、ScPYC2，敲除GUT1	甘油	是，pH 5.0-6.0	产量35 g/L，得率0.6 g/g、理论得率的47.1%	[36]
	酿酒酵母BY4739	敲除SDH1-2、ADH1-5，表达SpMAE1	葡萄糖	否	得率2.36±0.06 mol/mol	[37]
	酿酒酵母CEN.PK113-5D	敲除SDH3、SER3、SER33，适应性进化，表达ScICL1	葡萄糖	是，pH 5.0	产量0.9 g/L、提高了30倍，得率0.05 g/g	[39]
	酿酒酵母BY4741	敲除DIC1	葡萄糖	是，pH 5.0	得率0.02 mol/mol。	[40]
解脂耶氏酵母	解脂耶氏酵母Po1f	敲除SDH2，化学诱变	甘油	否，pH 3.2	产量17.4 g/L	[43]
	解脂耶氏酵母Po1f	敲除SDH5	粗甘油	是，pH 6	产量160.2 g/L，得率0.40 g/g、理论得率的62.4%，生产强度0.40 g/(L·h)	[45]
	解脂耶氏酵母H222	SDH2启动子替换为诱导型启动子	甘油	是，pH 5	产量25 g/L，得率0.26 g/g，生产强度0.152 g/(L·h)	[47]
	解脂耶氏酵母ST6512	截短SDH1启动子，表达AsPCK、YlICL、YlMLS、YlMDH、YlSCS2、YlKGDH，适应性进化	葡萄糖	是，pH 5	产量35.3 g/L，得率0.26 g/g，生产强度0.60 g/(L·h)	[17]
	解脂耶氏酵母PGC01003	敲除ACH1，表达ScPCK、YlSCS2	甘油	否，pH 3.4	产量110.7 g/L，得率0.53 g/g、理论得率的81.8%	[48]
	解脂耶氏酵母PGC91	表达TbFRD×2、EcFUM、YlMDH1、YlPYC、YlMDH2、YlFUM、SpMAE1，适应性进化	葡萄糖	否，pH 2.49	产量111.9 g/L，得率0.79 g/g，生产强度1.79 g/(L·h)	[49]
	解脂耶氏酵母PGC62	表达SpMAE1、TbFRD、YlSCS2、YlICL、YlMLS、YlYHM2	葡萄糖	是，pH 5.5	产量101.4 g/L，得率0.37 g/g，生产强度0.70 g/(L·h)	[50]
	解脂耶氏酵母 PGC01003	-	甘油	是，pH 6.0	产量198.2 g/L，生产强度0.84 g/(L·h)	[52]
	解脂耶氏酵母 PGC01003	-	粗甘油	是，pH 6.0	产量209.7 g/L，生产强度0.65 g/(L·h)	[53]
	解脂耶氏酵母 PSA02004	代谢进化	葡萄糖	否	产量76.8 g/L	[55]
东方伊萨酵母	东方伊萨酵母SD108	表达IoPYC、IoMDH、IoFUM、TbFRD	葡萄糖	否	产量11.63 g/L，得率0.12 g/g，生产强度0.11 g/(L·h)	[69]
东方伊萨酵母	东方伊萨酵母SA	表达SpMAE1、PaGDH、IoDAK，敲除ADH、GPD、g3473、g3837	葡萄糖和甘油	否，pH 3.0	产量109.5 g/L，得率0.65 g/g，生产强度0.54 g/(L·h)	[70]

酵母种类	出发菌株	代谢工程改造策略	底物	是否添加中和剂	产量、得率、生产强度	参考文献
酿酒酵母	酿酒酵母XU-1	敲除SDH1-2	葡萄糖	否	产量409±68 mg/L、提升1.9倍	[30]
	酿酒酵母AH22ura3	敲除SDH1-2、IDH1、IDP1	葡萄糖	否	产量3.62 g/L、提升4.8倍，得率0.11 mol/mol	[31]
	酿酒酵母TAM	表达ScPYC2、ScMDH3、ScFRDS1、EcFUMC，敲除ScFUM1、GPD1	葡萄糖	否，pH 3.8	产量12.97±0.42 g/L，得率0.21 mol/mol	[34]
	酿酒酵母UBR2_CBS-DHA	表达ScMDH3、RoFUM、TbFRDg、AnDCT-02	甘油	是，pH 5.0	产量10.7 g/L，得率0.22±0.01 g/g	[35]
	酿酒酵母CEN.PK113-1A	表达CjFPS1、OpGDH、ScDAK1、ScMDH3、RoFUM、TbFRDg、AnDCT-02、ScPYC2，敲除GUT1	甘油	是，pH 5.0-6.0	产量35 g/L，得率0.6 g/g、理论得率的47.1%	[36]
	酿酒酵母BY4739	敲除SDH1-2、ADH1-5，表达SpMAE1	葡萄糖	否	得率2.36±0.06 mol/mol	[37]
	酿酒酵母CEN.PK113-5D	敲除SDH3、SER3、SER33，适应性进化，表达ScICL1	葡萄糖	是，pH 5.0	产量0.9 g/L、提高了30倍，得率0.05 g/g	[39]
	酿酒酵母BY4741	敲除DIC1	葡萄糖	是，pH 5.0	得率0.02 mol/mol。	[40]
解脂耶氏酵母	解脂耶氏酵母Po1f	敲除SDH2，化学诱变	甘油	否，pH 3.2	产量17.4 g/L	[43]
	解脂耶氏酵母Po1f	敲除SDH5	粗甘油	是，pH 6	产量160.2 g/L，得率0.40 g/g、理论得率的62.4%，生产强度0.40 g/(L·h)	[45]
	解脂耶氏酵母H222	SDH2启动子替换为诱导型启动子	甘油	是，pH 5	产量25 g/L，得率0.26 g/g，生产强度0.152 g/(L·h)	[47]
	解脂耶氏酵母ST6512	截短SDH1启动子，表达AsPCK、YlICL、YlMLS、YlMDH、YlSCS2、YlKGDH，适应性进化	葡萄糖	是，pH 5	产量35.3 g/L，得率0.26 g/g，生产强度0.60 g/(L·h)	[17]
	解脂耶氏酵母PGC01003	敲除ACH1，表达ScPCK、YlSCS2	甘油	否，pH 3.4	产量110.7 g/L，得率0.53 g/g、理论得率的81.8%	[48]
	解脂耶氏酵母PGC91	表达TbFRD×2、EcFUM、YlMDH1、YlPYC、YlMDH2、YlFUM、SpMAE1，适应性进化	葡萄糖	否，pH 2.49	产量111.9 g/L，得率0.79 g/g，生产强度1.79 g/(L·h)	[49]
	解脂耶氏酵母PGC62	表达SpMAE1、TbFRD、YlSCS2、YlICL、YlMLS、YlYHM2	葡萄糖	是，pH 5.5	产量101.4 g/L，得率0.37 g/g，生产强度0.70 g/(L·h)	[50]
	解脂耶氏酵母 PGC01003	-	甘油	是，pH 6.0	产量198.2 g/L，生产强度0.84 g/(L·h)	[52]
	解脂耶氏酵母 PGC01003	-	粗甘油	是，pH 6.0	产量209.7 g/L，生产强度0.65 g/(L·h)	[53]
	解脂耶氏酵母 PSA02004	代谢进化	葡萄糖	否	产量76.8 g/L	[55]
东方伊萨酵母	东方伊萨酵母SD108	表达IoPYC、IoMDH、IoFUM、TbFRD	葡萄糖	否	产量11.63 g/L，得率0.12 g/g，生产强度0.11 g/(L·h)	[69]
东方伊萨酵母	东方伊萨酵母SA	表达SpMAE1、PaGDH、IoDAK，敲除ADH、GPD、g3473、g3837	葡萄糖和甘油	否，pH 3.0	产量109.5 g/L，得率0.65 g/g，生产强度0.54 g/(L·h)	[70]

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