Metabolic engineering of Yarrowia lipolytica to produce fatty acids and their derivatives

doi:10.11949/0438-1157.20201043

Abstract

Abstract:

Microbial-derived fatty acids and their derivatives are widely used in the fields of energy, materials and nutritional chemicals, and can be used to produce aviation fuels, polymers, plasticizers, lubricants and food additives. Yarrowia lipolytica is one of the well-studied oleaginous yeasts, which has the potential of producing various fatty acids and their derivatives. In this paper, the development of genetic manipulation tools of Yarrowia lipolytica is firstly summarized, and the progress of engineering Yarrowia lipolytica to produce fatty acids and their derivatives using the metabolic engineering and the emerging synthetic biology tools is then reviewed. Finally, the trends of constructing Yarrowia lipolytica cell factory to produce these products are prospected.

Key words: Yarrowia lipolytica, fatty acid, cell factory, metabolic engineering, synthetic biology, biotechnology

摘要：

微生物来源的脂肪酸及其衍生物广泛应用于能源、材料和营养化学品等领域，可用于生产航空燃油、聚合物、增塑剂、润滑剂和食品添加剂等。解脂耶氏酵母是一种研究最为透彻的产油脂酵母，具有高产各种脂肪酸及其衍生物的潜力。本文综述了近年来解脂耶氏酵母遗传操作工具的发展，并介绍了通过代谢工程技术改造解脂耶氏酵母生产脂肪酸及其衍生物的进展，在此基础上，展望了通过构建解脂耶氏酵母细胞工厂合成特定脂肪酸及其衍生物的未来发展方向。

关键词: 解脂耶氏酵母, 脂肪酸, 细胞工厂, 代谢工程, 合成生物学, 生物技术

CLC Number:

Q 815

WANG Kaifeng, WANG Jinpeng, WEI Ping, JI Xiaojun. Metabolic engineering of Yarrowia lipolytica to produce fatty acids and their derivatives[J]. CIESC Journal, 2021, 72(1): 351-365.

王凯峰, 王金鹏, 韦萍, 纪晓俊. 代谢工程改造解脂耶氏酵母生产脂肪酸及其衍生物[J]. 化工学报, 2021, 72(1): 351-365.

Figures/Tables 3

References 112

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品类	脂肪酸及其衍生物	代谢工程改造策略	有益效果	文献
能源化学品	中链脂肪酸	大肠杆菌来源的硫酯酶(EcTesA′)和加州月桂来源的硫酯酶(UcBTE)替换FASI中的MPT	C14和C12脂肪酸分别占总脂肪酸的29.2%和7.5%	[39]
	中链脂肪酸	用芳香族残基代替酮酰基合酶(KS)中异亮氨酸残基I1220	C14脂肪酸占总脂肪酸11.6%	[48]
	中链脂肪酸	过表达油棕来源的对中链脂肪酸有特异性的甘油三酯酰转移酶	中链脂肪酸占总脂肪酸45%	[49]
	脂肪酸乙酯	将异源的蜡酯合酶基因(AbAtfA)靶向内质网和过氧化物酶体	脂肪酸乙酯分别为136.5和110.9 mg/L	[39]
	脂肪酸乙酯	过表达异源的蜡酯合酶基因(MhAtfA)，过表达柠檬酸裂解酶、乙酰辅酶A羧化酶、酿酒酵母来源的乙酰辅酶A合成酶，敲除dga1、pex10	外源添加5%(体积)乙醇，脂肪酸乙酯达1.18 g/L	[54]
	脂肪酸乙酯	表达来自酿酒酵母来源的丙酮酸脱羧酶PDC1和醇脱氢酶ADH1，表达异源的蜡酯合酶基因(MhAtfA)并将解脂耶氏酵母和酿酒酵母共培养	脂肪酸乙酯产量达500.4 mg /L	[55]
	烷烃	在高产亚油酸解脂耶氏酵母菌株中引入大豆脂氧合酶I (Gmlox1)，敲除β-氧化第二步的多功能酶MFE1	戊烷产量达1.56 mg/L	[58]
	烷烃	表达海洋分枝杆菌来源的羧酸还原酶(MmCAR)、枯草芽孢杆菌来源的磷酰转移酶(BsuSfp)和海洋原绿球藻来源的醛脱甲氧合酶(PmADO)	烷烃产量达23.3 mg/L	[39]
材料化学品	蓖麻油酸	敲除pox1-6、Ylfad2、dga1、dga2，过表达lro1和异源Δ12油酸羟化酶(CpFAH12)	蓖麻油酸占总脂肪酸的43%	[68]
	蓖麻油酸	敲除pox1-6、Ylfad2、dga1、dga2, 表达2拷贝lro1和3拷贝的异源Δ12油酸羟化酶(CpFAH12)	蓖麻油酸占总脂肪酸的60%，产量达12 g/L	[68]
	长链二元酸	敲除不同pox基因并添加烷烃和烷烃降解中间产物作为底物	十二烷二酸产量达5~20 mg/ml	[69]
	长链二元酸	敲除pox1-6基因之后，过表达编码NADPH细胞色素P450还原酶和细胞色素 P450单加氧酶基因	增加第一步羟基化活性，长链二元酸产量显著提高	[70]
	长链二元酸	敲除pox1-6基因，并过表达新的醇氧化酶(FAO)	十二烷二酸产量达11 g/L	[72]
	长链二元酸	基于基因组规模代谢网络模型筛选出能提高十二烷二酸产量的三种酶并过表达	十二烷二酸产量提高48%	[73]
	聚羟基脂肪酸酯	敲除油脂合成途径并过表达MFE蛋白的2-烯脂酰辅酶A水合酶结构	PHA占细胞干重7％	[76]
	聚羟基脂肪酸酯	表达Ralstonia eutropha来源的编码β-酮硫解酶基因(phaA)，乙酰乙酰辅酶A还原酶基因(phaB)和PHA合酶基因(phaC)	乙酸盐作为唯一碳源，PHB占细胞干重的10.2%，达7.35 g/L	[77]
	聚羟基脂肪酸酯	多拷贝表达密码子优化后铜绿假单胞菌 PHA合酶 (PhaC1)	PHA占细胞干重0.205％	[79]
	聚羟基脂肪酸酯	表达PHA合酶(PhaC)，油酸诱导型启动子POX2过表达MFE蛋白	mclPHA占细胞干重27%	[78]
营养化学品	共轭亚油酸	多拷贝表达经过密码子优化的痤疮丙酸杆菌来源的亚油酸异构酶基因	反-10,顺-12共轭亚油酸占总脂肪酸5.6%	[83]
	共轭亚油酸	用hp16d强启动子共表达高山被孢酶来源的Δ12去饱和酶和密码子优化的痤疮丙酸杆菌来源的亚油酸异构酶基因	以葡萄糖和大豆油发酵，反-10，顺-12共轭亚油酸分别占总脂肪酸10%和44%	[84]
	共轭亚油酸	敲除pox1-6、dga1、dga2、are1和lro1，过表达内源Δ12去饱和酶和2拷贝的痤疮丙酸杆菌来源的亚油酸异构酶	反-10,顺-12共轭亚油酸占总脂肪酸6.5%，共轭亚油酸的降解速率明显降低	[85]
	α-亚麻酸	过表达双功能镰刀菌来源的Δ15去饱和酶	α-亚麻酸占总脂肪酸28%	[88]
	α-亚麻酸	在高产油酸菌株中表达3个拷贝的Rhodosporidium kratochvilovae来源的Δ15去饱和酶编码基因	α-亚麻酸占总脂肪酸8.1%；低温发酵后占17.0%，产量达1.4g/L	[89]
	二十碳五烯酸	过表达胆碱酯酶(CPT)，敲除pex10，导入Δ6和Δ9需氧去饱延长酶途径所需的延长酶和去饱和酶，并优化基因拷贝数	EPA占总脂肪酸56.6%，菌株实现工业化应用	[92-93]
	二十二碳六烯酸	过表达高山被孢霉来源的Δ4，Δ5，Δ6，Δ7去饱和酶、C18~C20延长酶，以及Thraustochytrium aureum来源的C20~C22延长酶	DHA占总脂肪酸5.6%	[94]
	二十二碳六烯酸	表达经过密码子优化后的4′-磷酸泛乙烯基转移酶和长链多不饱和脂肪酸合成酶基因簇	DHA占总脂肪酸10.5%	[95]
	γ-亚麻酸	表达密码子优化高山孢霉来源的Δ6去饱和酶	γ-亚麻酸占总脂肪酸6.1%	[97]
	花生四烯酸	表达密码子优化高山被饱霉来源的Δ6去饱和酶、Δ6延长酶和Δ5去饱和酶	花生四烯酸产占总脂肪酸0.4%	[98]
	花生四烯酸	融合表达Δ9延长酶和Δ8去饱和酶，并过表达Δ5去饱和酶	花生四烯酸产量达118.1mg/L	[100]
其他化学品	奇数链脂肪酸	敲除2-甲基柠檬酸脱水酶(PHD1)，葡萄糖与丙酸盐共培养	奇数链脂肪酸产量达0.75 g/L	[107]
	奇数链脂肪酸	敲除POX1-6、脂肪酶(TGL4)、过表达二酰甘油酰基转移酶(DGA2)和3-磷酸甘油脱氢酶(GPD1)，过表达苏氨酸的生物合成途径相关的7个基因，葡萄糖为唯一碳源	奇数链脂肪酸占总脂肪酸5.64%	[108]
	脂肪醇	过表达Synechococcus elongatus来源的脂酰ACP还原酶(SeFAR)和大肠杆菌来源的醛还原酶(EcAHR)	脂肪醇产量达2.5 mg/L	[39]
	脂肪醇	过表达Marinobacter aquaeolei来源的脂酰辅酶A还原酶(MaQu2220)和大肠杆菌来源的脂酰辅酶A合成酶(EcFadD)	脂肪醇产量达205.4 mg/L	[39]
	脂肪醇	敲除脂肪醇氧化酶(FAO1)、DGA1、过表达5个拷贝猫头鹰来源的脂酰辅酶A还原酶(TaFAR)基因	细胞内十六醇产量达636.89 mg/L；细胞外十六醇产量达53.32 mg/L	[109]
	脂肪醇	过表达脂酰辅酶A还原酶和去饱和酶(OLE1)，敲除与脂肪醇竞争的13个基因	脂肪醇产量达5.75 g/L	[110]
	脂肪醇	过表达拟南芥来源的脂酰辅酶A还原酶，敲除PEX10	1-癸醇产量达500 mg/L	[111]
	脂肪醇	过表达异源的脂酰辅酶A还原酶(MhFAR)	脂肪醇产量达5.8 g/L，在高产α-亚麻酸菌株中亚麻油醇产量达2 mg/L	[112]

品类	脂肪酸及其衍生物	代谢工程改造策略	有益效果	文献
能源化学品	中链脂肪酸	大肠杆菌来源的硫酯酶(EcTesA′)和加州月桂来源的硫酯酶(UcBTE)替换FASI中的MPT	C14和C12脂肪酸分别占总脂肪酸的29.2%和7.5%	[39]
	中链脂肪酸	用芳香族残基代替酮酰基合酶(KS)中异亮氨酸残基I1220	C14脂肪酸占总脂肪酸11.6%	[48]
	中链脂肪酸	过表达油棕来源的对中链脂肪酸有特异性的甘油三酯酰转移酶	中链脂肪酸占总脂肪酸45%	[49]
	脂肪酸乙酯	将异源的蜡酯合酶基因(AbAtfA)靶向内质网和过氧化物酶体	脂肪酸乙酯分别为136.5和110.9 mg/L	[39]
	脂肪酸乙酯	过表达异源的蜡酯合酶基因(MhAtfA)，过表达柠檬酸裂解酶、乙酰辅酶A羧化酶、酿酒酵母来源的乙酰辅酶A合成酶，敲除dga1、pex10	外源添加5%(体积)乙醇，脂肪酸乙酯达1.18 g/L	[54]
	脂肪酸乙酯	表达来自酿酒酵母来源的丙酮酸脱羧酶PDC1和醇脱氢酶ADH1，表达异源的蜡酯合酶基因(MhAtfA)并将解脂耶氏酵母和酿酒酵母共培养	脂肪酸乙酯产量达500.4 mg /L	[55]
	烷烃	在高产亚油酸解脂耶氏酵母菌株中引入大豆脂氧合酶I (Gmlox1)，敲除β-氧化第二步的多功能酶MFE1	戊烷产量达1.56 mg/L	[58]
	烷烃	表达海洋分枝杆菌来源的羧酸还原酶(MmCAR)、枯草芽孢杆菌来源的磷酰转移酶(BsuSfp)和海洋原绿球藻来源的醛脱甲氧合酶(PmADO)	烷烃产量达23.3 mg/L	[39]
材料化学品	蓖麻油酸	敲除pox1-6、Ylfad2、dga1、dga2，过表达lro1和异源Δ12油酸羟化酶(CpFAH12)	蓖麻油酸占总脂肪酸的43%	[68]
	蓖麻油酸	敲除pox1-6、Ylfad2、dga1、dga2, 表达2拷贝lro1和3拷贝的异源Δ12油酸羟化酶(CpFAH12)	蓖麻油酸占总脂肪酸的60%，产量达12 g/L	[68]
	长链二元酸	敲除不同pox基因并添加烷烃和烷烃降解中间产物作为底物	十二烷二酸产量达5~20 mg/ml	[69]
	长链二元酸	敲除pox1-6基因之后，过表达编码NADPH细胞色素P450还原酶和细胞色素 P450单加氧酶基因	增加第一步羟基化活性，长链二元酸产量显著提高	[70]
	长链二元酸	敲除pox1-6基因，并过表达新的醇氧化酶(FAO)	十二烷二酸产量达11 g/L	[72]
	长链二元酸	基于基因组规模代谢网络模型筛选出能提高十二烷二酸产量的三种酶并过表达	十二烷二酸产量提高48%	[73]
	聚羟基脂肪酸酯	敲除油脂合成途径并过表达MFE蛋白的2-烯脂酰辅酶A水合酶结构	PHA占细胞干重7％	[76]
	聚羟基脂肪酸酯	表达Ralstonia eutropha来源的编码β-酮硫解酶基因(phaA)，乙酰乙酰辅酶A还原酶基因(phaB)和PHA合酶基因(phaC)	乙酸盐作为唯一碳源，PHB占细胞干重的10.2%，达7.35 g/L	[77]
	聚羟基脂肪酸酯	多拷贝表达密码子优化后铜绿假单胞菌 PHA合酶 (PhaC1)	PHA占细胞干重0.205％	[79]
	聚羟基脂肪酸酯	表达PHA合酶(PhaC)，油酸诱导型启动子POX2过表达MFE蛋白	mclPHA占细胞干重27%	[78]
营养化学品	共轭亚油酸	多拷贝表达经过密码子优化的痤疮丙酸杆菌来源的亚油酸异构酶基因	反-10,顺-12共轭亚油酸占总脂肪酸5.6%	[83]
	共轭亚油酸	用hp16d强启动子共表达高山被孢酶来源的Δ12去饱和酶和密码子优化的痤疮丙酸杆菌来源的亚油酸异构酶基因	以葡萄糖和大豆油发酵，反-10，顺-12共轭亚油酸分别占总脂肪酸10%和44%	[84]
	共轭亚油酸	敲除pox1-6、dga1、dga2、are1和lro1，过表达内源Δ12去饱和酶和2拷贝的痤疮丙酸杆菌来源的亚油酸异构酶	反-10,顺-12共轭亚油酸占总脂肪酸6.5%，共轭亚油酸的降解速率明显降低	[85]
	α-亚麻酸	过表达双功能镰刀菌来源的Δ15去饱和酶	α-亚麻酸占总脂肪酸28%	[88]
	α-亚麻酸	在高产油酸菌株中表达3个拷贝的Rhodosporidium kratochvilovae来源的Δ15去饱和酶编码基因	α-亚麻酸占总脂肪酸8.1%；低温发酵后占17.0%，产量达1.4g/L	[89]
	二十碳五烯酸	过表达胆碱酯酶(CPT)，敲除pex10，导入Δ6和Δ9需氧去饱延长酶途径所需的延长酶和去饱和酶，并优化基因拷贝数	EPA占总脂肪酸56.6%，菌株实现工业化应用	[92-93]
	二十二碳六烯酸	过表达高山被孢霉来源的Δ4，Δ5，Δ6，Δ7去饱和酶、C18~C20延长酶，以及Thraustochytrium aureum来源的C20~C22延长酶	DHA占总脂肪酸5.6%	[94]
	二十二碳六烯酸	表达经过密码子优化后的4′-磷酸泛乙烯基转移酶和长链多不饱和脂肪酸合成酶基因簇	DHA占总脂肪酸10.5%	[95]
	γ-亚麻酸	表达密码子优化高山孢霉来源的Δ6去饱和酶	γ-亚麻酸占总脂肪酸6.1%	[97]
	花生四烯酸	表达密码子优化高山被饱霉来源的Δ6去饱和酶、Δ6延长酶和Δ5去饱和酶	花生四烯酸产占总脂肪酸0.4%	[98]
	花生四烯酸	融合表达Δ9延长酶和Δ8去饱和酶，并过表达Δ5去饱和酶	花生四烯酸产量达118.1mg/L	[100]
其他化学品	奇数链脂肪酸	敲除2-甲基柠檬酸脱水酶(PHD1)，葡萄糖与丙酸盐共培养	奇数链脂肪酸产量达0.75 g/L	[107]
	奇数链脂肪酸	敲除POX1-6、脂肪酶(TGL4)、过表达二酰甘油酰基转移酶(DGA2)和3-磷酸甘油脱氢酶(GPD1)，过表达苏氨酸的生物合成途径相关的7个基因，葡萄糖为唯一碳源	奇数链脂肪酸占总脂肪酸5.64%	[108]
	脂肪醇	过表达Synechococcus elongatus来源的脂酰ACP还原酶(SeFAR)和大肠杆菌来源的醛还原酶(EcAHR)	脂肪醇产量达2.5 mg/L	[39]
	脂肪醇	过表达Marinobacter aquaeolei来源的脂酰辅酶A还原酶(MaQu2220)和大肠杆菌来源的脂酰辅酶A合成酶(EcFadD)	脂肪醇产量达205.4 mg/L	[39]
	脂肪醇	敲除脂肪醇氧化酶(FAO1)、DGA1、过表达5个拷贝猫头鹰来源的脂酰辅酶A还原酶(TaFAR)基因	细胞内十六醇产量达636.89 mg/L；细胞外十六醇产量达53.32 mg/L	[109]
	脂肪醇	过表达脂酰辅酶A还原酶和去饱和酶(OLE1)，敲除与脂肪醇竞争的13个基因	脂肪醇产量达5.75 g/L	[110]
	脂肪醇	过表达拟南芥来源的脂酰辅酶A还原酶，敲除PEX10	1-癸醇产量达500 mg/L	[111]
	脂肪醇	过表达异源的脂酰辅酶A还原酶(MhFAR)	脂肪醇产量达5.8 g/L，在高产α-亚麻酸菌株中亚麻油醇产量达2 mg/L	[112]

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