非常规酵母细胞工厂合成天然产物

doi:10.13560/j.cnki.biotech.bull.1985.2021-0815

摘要/Abstract

摘要：

天然产物在医药、美容、食品等行业具有重要的应用,随着人们需求的增加,迫切需要绿色可持续的生产过程。近年来,微生物细胞工厂合成天然产物发展迅速,目前常用的细胞工厂宿主包括大肠杆菌和酿酒酵母。与此同时,非常规酵母因其性状独特,如高密度有氧发酵、耐受温度和pH范围广,底物谱广泛（长链糖、脂肪酸、甲醇等）,逐渐成为极具潜力的细胞工厂宿主。本文综述了近年来非常规酵母在天然产物合成方面的研究进展,主要集中在萜类和黄酮类化合物的生物合成,并对常用的改造手段和策略进行了介绍。在此基础上,系统讨论了新型非常规酵母细胞工厂存在的优势和不足之处,并总结了非常规酵母细胞工厂合成天然产物的挑战和机遇。

关键词: 非常规酵母, 萜类化合物, 黄酮类化合物, 微生物细胞工厂, 合成生物学

Abstract:

Natural products have been widely applied in medical,cosmetic and food industries. With the increasing of people’s demand,the green sustainable producing process is urgently needed. Recently,microbial cell factories for synthesizing natural products have been rapidly developing,and the current common-used cell factory hosts include Saccharomyces cerevisiae and Escherichia coli. Non-conventional yeasts have becoming very potential cell factory hosts due to their specific advantages,such as high-cell-density aerobic fermentation,tolerance to a wide range of temperature and pH,and wide substrate spectrum（long-chain hydrocarbons,fatty acids and methanol）. In this review,we summarized recent progresses on engineering non-conventional yeasts for the synthesis of natural products mainly terpenoids and flavonoids,and introduced common engineering methods and strategies. On this basis,we systematically discussed their strengths and weaknesses of novel non-conventional yeasts cell factories,and finally listed the challenges and opportunities while using non-conventional yeasts cell factories for the synthesis of natural products.

Key words: non-conventional yeasts, terpenoids, flavonoids, microbial cell factories, synthetic biology

叶敏, 高教琪, 周雍进. 非常规酵母细胞工厂合成天然产物[J]. 生物技术通报, 2021, 37(8): 12-24.

YE Min, GAO Jiao-qi, ZHOU Yong-jin. Engineering Non-conventional Yeast Cell Factory for the Biosynthesis of Natural Products[J]. Biotechnology Bulletin, 2021, 37(8): 12-24.

图/表 8

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产物 Products	菌株 Strains	产量 Productions	发酵条件 Conditions	培养基 Culture medium	参考文献 Reference
柠檬烯	酿酒酵母	2.6 g/L	摇瓶,半连续发酵	YNB+糖	[33]
	大肠杆菌	3.6 g/L	3.1 L发酵罐	基本培养基/甘油	[34]
	解脂耶氏酵母	165 mg/L	1.5 L发酵罐	YP+甘油+柠檬酸	[35]
芳樟醇	大肠杆菌	1.0 g/L	1.3 L发酵罐	基本培养基	[36]
芳樟醇	解脂耶氏酵母	7 mg/L	摇瓶	YPD	[28]
α-法尼烯	酿酒酵母	130 g/L	工业发酵罐	基本培养基	[13]
α-法尼烯	解脂耶氏酵母	25.6 g/L	1.5L发酵罐	YPD	[18]
	毕赤酵母	2.56 g/L	摇瓶	丰富培养基+复合碳源	[37]
α-檀香烯	解脂耶氏酵母	27.9 mg/L	5 L发酵罐	YPD	[38]
紫穗槐二烯	酿酒酵母	41 g/L	3.1 L发酵罐	基本培养基	[12]
	大肠杆菌	30 g/L	摇瓶,半连续发酵	丰富培养基+甘油	[39]
	解脂耶氏酵母	171 mg/L	摇瓶	YPD	[26]
诺卡酮	酿酒酵母	59.78 ug/L	摇瓶	YPD	[40]
	毕赤酵母	208 mg/L	1 L发酵罐	基本培养基	[30]
	解脂耶氏酵母	978 μg/L	摇瓶	YPD	[41]
番茄红素	酿酒酵母	3.3 g/L	7 L发酵罐	基本培养基	[42]
	毕赤酵母	0.7 g/L	3 L发酵罐	基本培养基+甘油	[43]
	解脂耶氏酵母	4.2 g/L	3 L发酵罐	丰富培养基+异戊烯醇	[15]
	假丝酵母	7.8 mg/g DCW	摇瓶	YPD	[32]
β-胡萝卜素	大肠杆菌	2.6 g/L	5 L发酵罐	丰富培养基	[44]
β-胡萝卜素	解脂耶氏酵母	6.5 g/L	5 L发酵罐	YPD	[27]
β-紫罗兰酮	酿酒酵母	33 mg/L	摇瓶	YPD	[45]
β-紫罗兰酮	解脂耶氏酵母	0.98 g/L	3 L发酵罐	YPD	[46]
虾青素	酿酒酵母	217.9 mg/L	5 L发酵罐	YPD	[47]
	大肠杆菌	1.2 g/L	5 L发酵罐	酵母提取物+甘油+柠檬酸复合培养基	[48]
	解脂耶氏酵母	285 mg/L	1 L发酵罐	YPD	[49]
	克鲁维酵母	10 mg/g DCW	5 L发酵罐	YP+半乳糖	[31]
	红发夫酵母	27.8 mg/L	7.5 L发酵罐	基本培养基	[50]

产物 Products	菌株 Strains	产量 Productions	发酵条件 Conditions	培养基 Culture medium	参考文献 Reference
柠檬烯	酿酒酵母	2.6 g/L	摇瓶,半连续发酵	YNB+糖	[33]
	大肠杆菌	3.6 g/L	3.1 L发酵罐	基本培养基/甘油	[34]
	解脂耶氏酵母	165 mg/L	1.5 L发酵罐	YP+甘油+柠檬酸	[35]
芳樟醇	大肠杆菌	1.0 g/L	1.3 L发酵罐	基本培养基	[36]
芳樟醇	解脂耶氏酵母	7 mg/L	摇瓶	YPD	[28]
α-法尼烯	酿酒酵母	130 g/L	工业发酵罐	基本培养基	[13]
α-法尼烯	解脂耶氏酵母	25.6 g/L	1.5L发酵罐	YPD	[18]
	毕赤酵母	2.56 g/L	摇瓶	丰富培养基+复合碳源	[37]
α-檀香烯	解脂耶氏酵母	27.9 mg/L	5 L发酵罐	YPD	[38]
紫穗槐二烯	酿酒酵母	41 g/L	3.1 L发酵罐	基本培养基	[12]
	大肠杆菌	30 g/L	摇瓶,半连续发酵	丰富培养基+甘油	[39]
	解脂耶氏酵母	171 mg/L	摇瓶	YPD	[26]
诺卡酮	酿酒酵母	59.78 ug/L	摇瓶	YPD	[40]
	毕赤酵母	208 mg/L	1 L发酵罐	基本培养基	[30]
	解脂耶氏酵母	978 μg/L	摇瓶	YPD	[41]
番茄红素	酿酒酵母	3.3 g/L	7 L发酵罐	基本培养基	[42]
	毕赤酵母	0.7 g/L	3 L发酵罐	基本培养基+甘油	[43]
	解脂耶氏酵母	4.2 g/L	3 L发酵罐	丰富培养基+异戊烯醇	[15]
	假丝酵母	7.8 mg/g DCW	摇瓶	YPD	[32]
β-胡萝卜素	大肠杆菌	2.6 g/L	5 L发酵罐	丰富培养基	[44]
β-胡萝卜素	解脂耶氏酵母	6.5 g/L	5 L发酵罐	YPD	[27]
β-紫罗兰酮	酿酒酵母	33 mg/L	摇瓶	YPD	[45]
β-紫罗兰酮	解脂耶氏酵母	0.98 g/L	3 L发酵罐	YPD	[46]
虾青素	酿酒酵母	217.9 mg/L	5 L发酵罐	YPD	[47]
	大肠杆菌	1.2 g/L	5 L发酵罐	酵母提取物+甘油+柠檬酸复合培养基	[48]
	解脂耶氏酵母	285 mg/L	1 L发酵罐	YPD	[49]
	克鲁维酵母	10 mg/g DCW	5 L发酵罐	YP+半乳糖	[31]
	红发夫酵母	27.8 mg/L	7.5 L发酵罐	基本培养基	[50]

产物类型 Type	改造策略 Strategy	产物 Product	菌株 Strain	改造基因 Gene	效果 Effect	参考文献 Reference
萜类化合物	MVA途径优化	柠檬烯	解脂耶氏酵母	过表达HMGR,ERG12	产量提升112倍	[51]
	MVA途径优化	诺卡酮	解脂耶氏酵母	过表达tHMGR,ERG20	产量提升20.5倍	[41]
	加强前体和辅因子供给	番茄红素	解脂耶氏酵母	抑制TCA循环,过表达ACL1/ACL2/AMPD	产量提升2-3倍	[25]
	加强前体和辅因子供给	角鲨烯	解脂耶氏酵母	过表达甘露醇脱氢酶MnMDH2	产量提升11%	[52]
	弱化副反应	芳樟醇	解脂耶氏酵母	过表达ERG20 ^F88W-N119W突变体	产量提升4倍	[28]
	弱化副反应	虾青素	解脂耶氏酵母	ERG9启动子截短	产量提升2.5倍	[53]
	区室化策略	番茄红素	毕赤酵母	番茄红素合成基因定位至过氧化物酶体	比定位至细胞质产量略高	[43]
	区室化策略	角鲨烯	解脂耶氏酵母	过表达脂质体合成关键基因 DGA1	产量提升2.1倍	[54]
黄酮类化合物	莽草酸途径优化	柚皮素	解脂耶氏酵母	过表达ARO1	产量提升51%	[55]
	莽草酸途径优化	白藜芦醇	解脂耶氏酵母	过表达突变体ARO4^K221L,ARO7^G141S解除反馈抑制	产量提升2.2倍	[56]
	加强前体供给	柚皮素	解脂耶氏酵母	过表达ACC1	产量提升1.6倍	[55]
	加强前体供给	柚皮素	解脂耶氏酵母	过表达PEX10促进β-氧化	产量略有提升	[57]
	外源基因表达水平优化	柚皮素	解脂耶氏酵母	优化外源基因表达配比	产量波动1-3.8倍	[55]
		白藜芦醇	解脂耶氏酵母	增加外源基因拷贝数	产量提高4.8倍	[56]
		白藜芦醇	解脂耶氏酵母	启动子强度调节	产量波动较大	[56]

产物类型 Type	改造策略 Strategy	产物 Product	菌株 Strain	改造基因 Gene	效果 Effect	参考文献 Reference
萜类化合物	MVA途径优化	柠檬烯	解脂耶氏酵母	过表达HMGR,ERG12	产量提升112倍	[51]
	MVA途径优化	诺卡酮	解脂耶氏酵母	过表达tHMGR,ERG20	产量提升20.5倍	[41]
	加强前体和辅因子供给	番茄红素	解脂耶氏酵母	抑制TCA循环,过表达ACL1/ACL2/AMPD	产量提升2-3倍	[25]
	加强前体和辅因子供给	角鲨烯	解脂耶氏酵母	过表达甘露醇脱氢酶MnMDH2	产量提升11%	[52]
	弱化副反应	芳樟醇	解脂耶氏酵母	过表达ERG20 ^F88W-N119W突变体	产量提升4倍	[28]
	弱化副反应	虾青素	解脂耶氏酵母	ERG9启动子截短	产量提升2.5倍	[53]
	区室化策略	番茄红素	毕赤酵母	番茄红素合成基因定位至过氧化物酶体	比定位至细胞质产量略高	[43]
	区室化策略	角鲨烯	解脂耶氏酵母	过表达脂质体合成关键基因 DGA1	产量提升2.1倍	[54]
黄酮类化合物	莽草酸途径优化	柚皮素	解脂耶氏酵母	过表达ARO1	产量提升51%	[55]
	莽草酸途径优化	白藜芦醇	解脂耶氏酵母	过表达突变体ARO4^K221L,ARO7^G141S解除反馈抑制	产量提升2.2倍	[56]
	加强前体供给	柚皮素	解脂耶氏酵母	过表达ACC1	产量提升1.6倍	[55]
	加强前体供给	柚皮素	解脂耶氏酵母	过表达PEX10促进β-氧化	产量略有提升	[57]
	外源基因表达水平优化	柚皮素	解脂耶氏酵母	优化外源基因表达配比	产量波动1-3.8倍	[55]
		白藜芦醇	解脂耶氏酵母	增加外源基因拷贝数	产量提高4.8倍	[56]
		白藜芦醇	解脂耶氏酵母	启动子强度调节	产量波动较大	[56]

产物 Product	菌株 Strain	产量 Production	发酵条件 Condition	培养基 Culture medium	参考文献 Reference
白藜芦醇	酿酒酵母	800 mg/L	摇瓶	基本培养基	[68]
	大肠杆菌	2.3 g/L	摇瓶	丰富培养基+对香豆酸	[69]
	解脂耶氏酵母	12.4 g/L	1 L发酵罐	基本培养基	[56]
柚皮素	酿酒酵母	1.1 g/L	5 L发酵罐	YPD	[70]
	大肠杆菌	484 mg/L	摇瓶	基本培养基+酪氨酸	[71]
	解脂耶氏酵母	898 mg/L	3 L发酵罐	基本培养基	[55]
黄杉素	酿酒酵母	3.5 g/L	5 L发酵罐	基本培养基+柚皮素	[72]
黄杉素	解脂耶氏酵母	134 mg/L	3 L发酵罐	基本培养基	[55]
圣草酚	酿酒酵母	3.3 g/L	5 L发酵罐	YPD+5 g/L柚皮素	[73]
	大肠杆菌	107 mg/L	摇瓶	基本培养基+酪氨酸	[74]
	解脂耶氏酵母	110 mg/L	3 L发酵罐	基本培养基	[55]