解脂耶氏酵母底盘细胞的工程改造及应用

doi:10.12211/2096-8280.2022-060

摘要/Abstract

摘要：

基于性能卓越的微生物底盘细胞，开发高效的绿色生物制造技术，已经成为合成生物学领域的研究前沿。解脂耶氏酵母作为一种非常规产油酵母，由于其独特的生理生化特征，正迅速成为面向绿色生物制造的合成生物学研究领域的热门底盘细胞之一。近年来，围绕解脂耶氏酵母底盘细胞工程改造的研究与应用日益增多，促进了解脂耶氏酵母底盘细胞的进一步升级。本文总结了针对解脂耶氏酵母底盘细胞的工程改造策略及其在生物制造中的应用，从遗传改造技术及工具开发，基因的表达与调控策略等方面介绍各类合成生物学工具及技术在解脂耶氏酵母中的研究进展，并从底盘细胞合成高附加值产品的研究进展方面介绍了其工程改造效果。最后，对解脂耶氏酵母底盘细胞的应用前景和未来发展方向进行了展望。

关键词: 解脂耶氏酵母, 底盘细胞, 产油酵母, 细胞工厂, 合成生物学, 生物制造

Abstract:

Engineering microbial chassis cells to efficiently synthesize high value-added products has received increasing attention. This biomanufacturing mode based on excellent performance microbial chassis cells has become the research frontier in the field of synthetic biology. Yarrowia lipolytica, an unconventional oleaginous yeast, is emerging as one of the popular microbial chassis cells in the field of advanced and green biomanufacturing. This is due to its unique physiological and biochemical characteristics, such as the inherent mevalonate pathway, adequate acetyl-CoA supply, broad substrate spectrum, and high tolerance to multiple extreme environments. These characteristics make Y. lipolytica a superior chassis candidate for the advanced and green biomanufacturing. In recent years, the researches and applications on the rewiring of Y. lipolytica chassis cell for biomanufacturing have gradually increased, which promoted the further upgrading of Y. lipolytica chassis cells. This review firstly describes the development of the genetic elements for rewiring Y. lipolytica chassis cell, including promoters, terminators, and selecting markers. Then, this review summarizes the expression modes and integration methods for endogenous and heterogenous genes, including gene expression based on episomal plasmid, genomic integration based on homologous recombination (HR) and non-homologous end joining (NHEJ). This review further summarizes the research progress of various synthetic biology tools developed for Y. lipolytica, including various gene overexpression methods, biosensor-based dynamic regulation strategies, CRISPR/Cas-based gene expression regulation methods, and the emerging strategies such as genome-scale metabolic modelling, genome-wide mutational screening, etc. This review also introduces the achievements of rewiring Y. lipolytica chassis cell for the synthesis of different high value-added products, including proteins, organic acids, terpenes, functional sugars and sugar alcohols, fatty acids and their derivatives, flavonoids and polyketides, and amino acid derivatives. In addition, the prospects of Y. lipolytica chassis cell-based biomanufacturing are discussed in light of the current progresses, challenges, and trends in this field. Finally, guidelines for building next-generation Y. lipolytica chassis cell for production of the aforementioned products are also emphasized. {L-End}

Key words: Yarrowia lipolytica, chassis cell, oleaginous yeast, cell factory, synthetic biology, biomanufacturing

中图分类号:

Q819

孙美莉, 王凯峰, 陆然, 纪晓俊. 解脂耶氏酵母底盘细胞的工程改造及应用[J]. 合成生物学, 2023, 4(4): 779-807.

Meili SUN, Kaifeng WANG, Ran LU, Xiaojun JI. Rewiring and application of Yarrowia lipolytica chassis cell[J]. Synthetic Biology Journal, 2023, 4(4): 779-807.

图/表 6

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表征的启动子强度排序	表征方法	参考文献
pEXP1> pTEF> pGPD > pGPAT> pYAT1 >pXPR2 > pFBA1	报告蛋白：绿色荧光蛋白分析：流式细胞仪	[25]
pCTR1 > pTEF > pCTR2	报告蛋白：β-半乳糖苷酶分析：紫外-可见分光光度计	[22]
pTEFin>php4d > pTEF	报告蛋白：β-半乳糖苷酶分析：分光光度计	[26]
pTEF> pGAP > pACL2 > pICL > pIDH2 > pFAS1 > pDGA1 > pFAS2 > pZWF1 > pPOX4 > pACC1 > pIDP2	报告蛋白: 荧光素酶分析：酶标仪	[20]
pFBAin> pFBA> pTDH1 > pGPM1= pTEF	报告蛋白：β-葡萄糖醛酸酶分析：荧光微孔板读数仪	[23]

表征的启动子强度排序	表征方法	参考文献
pEXP1> pTEF> pGPD > pGPAT> pYAT1 >pXPR2 > pFBA1	报告蛋白：绿色荧光蛋白分析：流式细胞仪	[25]
pCTR1 > pTEF > pCTR2	报告蛋白：β-半乳糖苷酶分析：紫外-可见分光光度计	[22]
pTEFin>php4d > pTEF	报告蛋白：β-半乳糖苷酶分析：分光光度计	[26]
pTEF> pGAP > pACL2 > pICL > pIDH2 > pFAS1 > pDGA1 > pFAS2 > pZWF1 > pPOX4 > pACC1 > pIDP2	报告蛋白: 荧光素酶分析：酶标仪	[20]
pFBAin> pFBA> pTDH1 > pGPM1= pTEF	报告蛋白：β-葡萄糖醛酸酶分析：荧光微孔板读数仪	[23]

模型名称	基因数	代谢物数	反应数	代谢区室数	准确性	年份	参考文献
iNL895	895	1847	1989	16	0.65	2012	[109]
iYL619_PCP	619	849	1142	2	0.83	2012	[110]
iMK735	735	1111	1336	8	0.80	2015	[111]
iYali4	901	1683	1985	16	Not determined	2016	[112]
iYL_2.0	645	1083	1471	4	0.97	2017	[115]
iYLI647	647	1119	1347	8	0.80	2018	[116]
iYli21	1058	1868	2285	16	0.86	2022	[121]

模型名称	基因数	代谢物数	反应数	代谢区室数	准确性	年份	参考文献
iNL895	895	1847	1989	16	0.65	2012	[109]
iYL619_PCP	619	849	1142	2	0.83	2012	[110]
iMK735	735	1111	1336	8	0.80	2015	[111]
iYali4	901	1683	1985	16	Not determined	2016	[112]
iYL_2.0	645	1083	1471	4	0.97	2017	[115]
iYLI647	647	1119	1347	8	0.80	2018	[116]
iYli21	1058	1868	2285	16	0.86	2022	[121]

种类	产品	生产规模	生产水平	参考文献
有机酸	柠檬酸	1L 生物反应器	97.1 g/L	[138]
	异柠檬酸	1L 生物反应器	136.7 g/L	[139]
	琥珀酸	1L 生物反应器	101.4 g/L	[140]
	衣康酸	5L 生物反应器	22.03 g/L	[141]
	α-酮戊二酸	3L 生物反应器	67.4 g/L	[144]
	丙酮酸	3L 生物反应器	39.1 g/L	[144]
萜烯类	柠檬烯	摇瓶	35.9 mg/L	[150]
	α-法尼烯	1L 生物反应器	25.55 g/L	[56]
	β-法尼烯	摇瓶	955 mg/L	[150]
	α-葎草烯	5L 生物反应器	3.2 g/L	[151]
	(-)-α-红没药醇	5L 生物反应器	4.4 g/L	[57]
	脱落酸	摇瓶	263.5 mg/L	[163]
	赤霉素3	摇瓶	17.29 mg/L	[153]
	赤霉素4	摇瓶	12.81 mg/L	[153]
	角鲨烯	摇瓶	731.18 mg/L	[154]
	桦木酸	摇瓶	51.87 mg/L	[155]
	人参皂苷CK	5L 生物反应器	161.8 mg/L	[164]
	番茄红素	3L 生物反应器	17.6 g/L	[156]
	β-胡萝卜素	5L 生物反应器	6.5 g/L	[157]
	虾青素	摇瓶	858 mg/L	[158]
功能糖及糖醇类	赤藓糖醇	3L生物反应器	148 g/L	[102]
	D-苏糖醇	摇瓶	112 g/L	[168]
	木糖醇	摇瓶	53.2 g/L	[170]
	2′-岩藻糖基乳糖	2L 生物反应器	24 g/L	[172]
	异麦芽酮糖	10L 生物反应器	572.1 g/L	[173]
	海藻糖	3L 生物反应器	219 g/L	[174]
脂肪酸及衍生物类	奇数链脂肪酸(C₁₅～C₁₉)	摇瓶	1.87 g/L	[215]
	(10E,12Z)十八碳二烯酸	5L 生物反应器	4 g/L	[216]
	油酸	5L 生物反应器	46.23 g/L	[78]
	α-亚麻酸	2L 生物反应器	1.42 g/L	[183]
	γ-亚麻酸	摇瓶	71.6 mg/L	[186]
	花生四烯酸	摇瓶	118.1 mg/L	[189]
	二十碳五烯酸	摇瓶	占总脂肪酸的56.6%	[184]
	二十二碳六烯酸	1L 生物反应器	350 mg/L	[185]
	环丙烷脂肪酸(C₁₇、C₁₉)	5L 生物反应器	7.49 g/L	[217]
	10-甲基支链脂肪酸	1L 生物反应器	1.2 g/L	[218]
	蓖麻油酸	摇瓶	12 g/L	[219]
	甲基酮	0.5L 生物反应器	314.8 mg/L	[180]
	聚羟基脂肪酸酯	摇瓶	占细胞干重7.3%	[181]
	γ-癸内酯	300L 生物反应器	12.3 g/L	[220]
	δ-癸内酯	摇瓶	1.9 g/L	[221]
	γ-十二内酯	1L 生物反应器	282 mg/L	[222]
	烷（烯）烃	摇瓶	1.47 g/L	[223]
	烯烃	摇瓶	554.4 mg/L	[224]
聚酮类和黄酮类	三乙酸内酯	3L 生物反应器	(35.9±3.9) g/L	[195]
	白藜芦醇	5L 生物反应器	430 mg/L	[200]
	柚皮素	3L 生物反应器	898 mg/L	[199]
	圣草酚	摇瓶	134.2 mg/L	[96]
氨基酸衍生物类	2-苯乙醇	摇瓶	2669.54 mg/L	[209]
	对香豆酸	摇瓶	(593.53 ± 28.75) mg/L	[210]
	紫杆菌素	摇瓶	(366.30 ± 28.99) mg/L	[210]
	犬尿酸	5L 生物反应器	培养液：68 mg/L 生物量：542 mg/kg	[213]
	麦角硫因	1L 生物反应器	1.63 g/L	[214]