合成类胡萝卜素细胞工厂的快速构建和定向进化

doi:10.16085/j.issn.1000-6613.2020-1944

摘要/Abstract

摘要：

类胡萝卜素具有丰富的颜色和强抗氧化活性，这赋予了该类产品较高的商业价值，加速了该类产品合成路径在异源底盘中的构建进程，使之成为发展非理性设计策略的有效研究对象。本文主要针对微生物合成类胡萝卜素细胞工厂构建中的瓶颈问题，从增强合成类胡萝卜素前体GGPP的供给利用以及缓解各类胡萝卜素产品对微生物底盘细胞的胁迫压力两个角度，系统综述非理性设计策略对元件优化、模块适配和多维调控等路径构建优化方法的丰富，以及对不同尺度的DNA变异和适应性进化等底盘进化手段的推动作用。并从扩展类胡萝卜素合成底盘物种的多样性和丰富非天然类胡萝卜素产品结构的可获得性的需求出发，对构建合成该类产品微生物细胞工厂自动化和智能化的发展趋势进行展望。

关键词: 合成生物学, 细胞工厂, 类胡萝卜素, 非理性设计, 路径构建, 底盘进化

Abstract:

Carotenoids have brilliant colors and strong antioxidant activity, which not only endow these compounds with high commercial value, but also accelerate the construction process for building efficient biosynthetic pathways in heterogeneous chassis. Carotenoids have become an ideal object to develop irrational design strategy. Here we critically review recent advances about carotenoid biosynthesis via irrational design strategies on pathway construction by parts optimization, modular compatibility, multidimensional regulation etc., as well as chassis evolution by DNA mutation and adaptive evolution. These strategies aims to break the bottleneck of carotenoid biosynthesis through improving the supply and utilization of precursor GGPP as well as relieving the stress from carotenoid accumulation on microbiological chassis. Based on the demand of employing unconventional chassis and expanding carotenoid kingdom, we also propose the development of the construction strategies for carotenoid microbiological cell factories towards the automation and intellectualization.

Key words: synthetic biology, cell factories, carotenoids, irrational design, pathway construction, chassis evolution

中图分类号:

Q819

王颖, 曲俊泽, 梁楠, 郝鹤, 元英进. 合成类胡萝卜素细胞工厂的快速构建和定向进化[J]. 化工进展, 2021, 40(3): 1187-1201.

WANG Ying, QU Junze, LIANG Nan, HAO He, YUAN Yingjin. Rapid construction and directed evolution of cell factories for carotenoid biosynthesis[J]. Chemical Industry and Engineering Progress, 2021, 40(3): 1187-1201.

图/表 7

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底盘物种	产品	工程化手段	产量/mg·L^-1	产率/mg·g DCW^-1	参考文献
大肠杆菌	番茄红素	过表达磷酸戊糖途径和TCA循环关键蛋白戊二酸脱氢酶SucAB、琥珀酸脱氢酶SdhABCD和转醛酶B TalB，提高底盘NADPH和ATP的供给基于RBS序列调节dxs、idi和crt基因簇的转录水平	3520	50.6	[16]
	β-胡萝卜素	将合成路径划分为MEP路径、β-胡萝卜素合成、ATP合成、磷酸戊糖途径、TCA循环5个模块，分别进行工程化优化改造调节MEP路径模块和β-胡萝卜素合成模块表达强度，过表达dxs和idi 过表达α-酮戊二酸脱氢酶SucAB和琥珀酸脱氢酶SdhABCD，增强TCA循环，提高能量和还原力NADPH供给过表达转醛酶TalB，增强磷酸戊糖途径，提高NADPH供给	2100	59.9	[23]
	β-胡萝卜素	敲除葡萄糖-6-磷酸脱氢酶编码基因zwf，减弱碳代谢流进入磷酸戊糖途径；以半乳糖渗透酶系统（galP）替代葡萄糖磷酸转移酶系统（ptsHIcrr）负责葡萄糖转运，减少磷酸烯醇式丙酮酸的消耗，提高MEP路径前体供给敲除醛还原酶编码基因yjgB，降低NADPH的消耗；过表达nadK，将NADH转化为NADPH，提高NADPH供给	2579	NP	[18]
	β-胡萝卜素	引入异源MVA路径上调dxs、fni、GPPS2，优化内源MEP路径发酵条件优化，以甘油为碳源	3200	NP	[11]
	玉米黄质	应用可调控基因间序列平衡异MVA路径蛋白表达使用IPP/FPP响应启动子动态调控异源MVA路径基因，避免有毒中间代谢物积累	722.5	23.2	[24]
	虾青素	组合筛选路径基因启动子，促进番茄红素向β-胡萝卜素转化 OmpF和TrxA标签分别与截短蛋白N端或C端融合，实现CrtW的稳定表达和质膜定位优化培养基碳源和发酵温度，降低IPTG诱导浓度基于代谢流分析选择过表达靶点ispD和ispF 引入毒素-抗毒素系统hok/sok，提高表达质粒稳定性	432.8	7.12	[25]
	藏红花酸	组合筛选CCD、ALD、UGT 体外酶活实验确定UGT功能	4.42	NP	[26]
酿酒酵母	番茄红素	CrtB序列的筛选 CrtYB与CrtE的定向进化调节Crt编码基因拷贝数构建非营养缺陷二倍体细胞	1610	24.41	[27]
	番茄红素	过表达乙醇脱氢酶ADH2、乙醛脱氢酶ALD6和来自沙门氏菌的乙酰-CoA合成酶ACS，提高前体乙酰-CoA的供给过表达tHMGR，增强MVA路径过表达NADH激酶POS5，促进NADH向NADPH的转化增加CrtE和CrtI的拷贝数敲除YPL062W和EXG1 引入乙酰-CoA羧化酶ACC1突变体S659A/S1157A，过表达磷脂酸磷酸酶PAH1和二酰基甘油酰基转移酶DGA1，增强甘油三酯的合成过表达脂肪酸脱氢酶OLE1，增加不饱和脂肪酸的含量敲除Seipin编码基因FLD1，增大脂质体体积	2370	73.3	[14]
	番茄红素	启动子筛选过表达tHMGR，增强MVA路径筛选CrtE、CrtB、CrtI来源，增加CrtE和CrtI的拷贝数过表达NADH激酶POS5，促进NADH向NADPH的转化过表达乙醇脱氢酶ADH2、乙醛脱氢酶ALD6和来自沙门氏菌的乙酰-CoA合成酶ACS，提高前体乙酰-CoA的供给 ΔGAL80与ΔGAL1,7,10诱导系统的比较	3280	NP	[17]
	番茄红素	敲除YPL062W提高乙酰-CoA供给组合筛选CrtE、CrtB、CrtI来源调节CrtI表达水平选择底盘交配型工程化非路径相关靶点，即敲除ROX1和过表达INO2 引入异源血红蛋白VHb 连续自循环发酵	5880	NP	[28]
	β-胡萝卜素	以GAL系统和HXT1启动子时序调控胡萝卜素和竞争性鲨烯的合成	1156	20.8	[29]
	虾青素	CrtZ和CrtW协同定向进化温敏调控系统控制关键基因表达	235.0	NP	[30]
	虾青素	等离子体诱变与适应性进化交替，促进菌株进化	404.8	NP	[31]
	藏红花酸	组合筛选CrtZ、CCD、ALD 发酵温度优化 CCD和ALD的过表达	6.278	NP	[32]
	藏红花酸	引入温敏调控系统控制藏红花酸合成，隔离细胞生长和生产调节CCD2和ALDH的拷贝数培养基碳源葡萄糖和半乳糖比例优化	NP	0.14	[33]
	藏红花酸	强化前体供应：敲除柠檬酸合酶CIT2、苹果酸合酶MLS1 CrtZ和CCD2的融合表达	12.43	NP	[34]
耶氏解脂酵母	番茄红素	引入异戊烯醇同化路径，增强前体IPP/DMAPP供给增强脂质积累增加IDI拷贝	4200	约170	[35]
	β-胡萝卜素	敲除KU70，增强同源重组效率敲除过氧化氢酶编码基因YALI0F30987、脂代谢相关基因LIP1、POX3-6，并作为后续表达盒整合位点过表达3个拷贝tHMGR、4个拷贝CrtYB和1个拷贝的CrtE、CrtI 过表达2个拷贝的MVA路径蛋白ERG10、ERG13、ERG12、ERG8、ERG19、IDI、ERG20 用受葡萄糖和甘油抑制的启动子ALK1p替换ERG9本源启动子，以实现ERG9下调氮源抑制发酵	3968	49.0	[13]
	β-胡萝卜素	敲除脂代谢基因POX2、MFE、POX3、LIP1，增强脂质积累过表达HMGR、ERG13，增强MVA路径增加3个拷贝的CrtYB、2个拷贝的CrtI和1个拷贝的CrtE 下调鲨烯合酶的表达	4500	57.5	[36]
	β-胡萝卜素	选择高度积累脂质的底盘路径基因启动子的组合适配发酵条件优化	6500	89.6	[37]
	虾青素	下调鲨烯合酶的表达同时表达不同来源的CrtE 筛选CrtZ/CrtW来源，调节表达量	285	6.0	[38]

底盘物种	产品	工程化手段	产量/mg·L^-1	产率/mg·g DCW^-1	参考文献
大肠杆菌	番茄红素	过表达磷酸戊糖途径和TCA循环关键蛋白戊二酸脱氢酶SucAB、琥珀酸脱氢酶SdhABCD和转醛酶B TalB，提高底盘NADPH和ATP的供给基于RBS序列调节dxs、idi和crt基因簇的转录水平	3520	50.6	[16]
	β-胡萝卜素	将合成路径划分为MEP路径、β-胡萝卜素合成、ATP合成、磷酸戊糖途径、TCA循环5个模块，分别进行工程化优化改造调节MEP路径模块和β-胡萝卜素合成模块表达强度，过表达dxs和idi 过表达α-酮戊二酸脱氢酶SucAB和琥珀酸脱氢酶SdhABCD，增强TCA循环，提高能量和还原力NADPH供给过表达转醛酶TalB，增强磷酸戊糖途径，提高NADPH供给	2100	59.9	[23]
	β-胡萝卜素	敲除葡萄糖-6-磷酸脱氢酶编码基因zwf，减弱碳代谢流进入磷酸戊糖途径；以半乳糖渗透酶系统（galP）替代葡萄糖磷酸转移酶系统（ptsHIcrr）负责葡萄糖转运，减少磷酸烯醇式丙酮酸的消耗，提高MEP路径前体供给敲除醛还原酶编码基因yjgB，降低NADPH的消耗；过表达nadK，将NADH转化为NADPH，提高NADPH供给	2579	NP	[18]
	β-胡萝卜素	引入异源MVA路径上调dxs、fni、GPPS2，优化内源MEP路径发酵条件优化，以甘油为碳源	3200	NP	[11]
	玉米黄质	应用可调控基因间序列平衡异MVA路径蛋白表达使用IPP/FPP响应启动子动态调控异源MVA路径基因，避免有毒中间代谢物积累	722.5	23.2	[24]
	虾青素	组合筛选路径基因启动子，促进番茄红素向β-胡萝卜素转化 OmpF和TrxA标签分别与截短蛋白N端或C端融合，实现CrtW的稳定表达和质膜定位优化培养基碳源和发酵温度，降低IPTG诱导浓度基于代谢流分析选择过表达靶点ispD和ispF 引入毒素-抗毒素系统hok/sok，提高表达质粒稳定性	432.8	7.12	[25]
	藏红花酸	组合筛选CCD、ALD、UGT 体外酶活实验确定UGT功能	4.42	NP	[26]
酿酒酵母	番茄红素	CrtB序列的筛选 CrtYB与CrtE的定向进化调节Crt编码基因拷贝数构建非营养缺陷二倍体细胞	1610	24.41	[27]
	番茄红素	过表达乙醇脱氢酶ADH2、乙醛脱氢酶ALD6和来自沙门氏菌的乙酰-CoA合成酶ACS，提高前体乙酰-CoA的供给过表达tHMGR，增强MVA路径过表达NADH激酶POS5，促进NADH向NADPH的转化增加CrtE和CrtI的拷贝数敲除YPL062W和EXG1 引入乙酰-CoA羧化酶ACC1突变体S659A/S1157A，过表达磷脂酸磷酸酶PAH1和二酰基甘油酰基转移酶DGA1，增强甘油三酯的合成过表达脂肪酸脱氢酶OLE1，增加不饱和脂肪酸的含量敲除Seipin编码基因FLD1，增大脂质体体积	2370	73.3	[14]
	番茄红素	启动子筛选过表达tHMGR，增强MVA路径筛选CrtE、CrtB、CrtI来源，增加CrtE和CrtI的拷贝数过表达NADH激酶POS5，促进NADH向NADPH的转化过表达乙醇脱氢酶ADH2、乙醛脱氢酶ALD6和来自沙门氏菌的乙酰-CoA合成酶ACS，提高前体乙酰-CoA的供给 ΔGAL80与ΔGAL1,7,10诱导系统的比较	3280	NP	[17]
	番茄红素	敲除YPL062W提高乙酰-CoA供给组合筛选CrtE、CrtB、CrtI来源调节CrtI表达水平选择底盘交配型工程化非路径相关靶点，即敲除ROX1和过表达INO2 引入异源血红蛋白VHb 连续自循环发酵	5880	NP	[28]
	β-胡萝卜素	以GAL系统和HXT1启动子时序调控胡萝卜素和竞争性鲨烯的合成	1156	20.8	[29]
	虾青素	CrtZ和CrtW协同定向进化温敏调控系统控制关键基因表达	235.0	NP	[30]
	虾青素	等离子体诱变与适应性进化交替，促进菌株进化	404.8	NP	[31]
	藏红花酸	组合筛选CrtZ、CCD、ALD 发酵温度优化 CCD和ALD的过表达	6.278	NP	[32]
	藏红花酸	引入温敏调控系统控制藏红花酸合成，隔离细胞生长和生产调节CCD2和ALDH的拷贝数培养基碳源葡萄糖和半乳糖比例优化	NP	0.14	[33]
	藏红花酸	强化前体供应：敲除柠檬酸合酶CIT2、苹果酸合酶MLS1 CrtZ和CCD2的融合表达	12.43	NP	[34]
耶氏解脂酵母	番茄红素	引入异戊烯醇同化路径，增强前体IPP/DMAPP供给增强脂质积累增加IDI拷贝	4200	约170	[35]
	β-胡萝卜素	敲除KU70，增强同源重组效率敲除过氧化氢酶编码基因YALI0F30987、脂代谢相关基因LIP1、POX3-6，并作为后续表达盒整合位点过表达3个拷贝tHMGR、4个拷贝CrtYB和1个拷贝的CrtE、CrtI 过表达2个拷贝的MVA路径蛋白ERG10、ERG13、ERG12、ERG8、ERG19、IDI、ERG20 用受葡萄糖和甘油抑制的启动子ALK1p替换ERG9本源启动子，以实现ERG9下调氮源抑制发酵	3968	49.0	[13]
	β-胡萝卜素	敲除脂代谢基因POX2、MFE、POX3、LIP1，增强脂质积累过表达HMGR、ERG13，增强MVA路径增加3个拷贝的CrtYB、2个拷贝的CrtI和1个拷贝的CrtE 下调鲨烯合酶的表达	4500	57.5	[36]
	β-胡萝卜素	选择高度积累脂质的底盘路径基因启动子的组合适配发酵条件优化	6500	89.6	[37]
	虾青素	下调鲨烯合酶的表达同时表达不同来源的CrtE 筛选CrtZ/CrtW来源，调节表达量	285	6.0	[38]

底盘物种	优势	产品	产量 /mg·L^-1	产率 /mg·g DCW^-1	参考文献
恶臭假单胞菌（Pseudomonas putida）	化学品、有机溶剂耐受性高 NADPH再生快	玉米黄质	239^①	NP	[105]
谷氨酸棒杆菌（Corynebacterium glutamicum）	可利用多种碳源天然合成C₅₀类胡萝卜素	虾青素	NP	1.6	[51]
毕赤酵母（Pichia pastoris）	蛋白表达的优势宿主，翻译后修饰更接近天然蛋白	番茄红素	714	9.319	[116]
克鲁维酵母（Kluyveromyces marxianus）	耐热性好，可在25~52℃的温度下发酵单位碳源生物量转化率高	虾青素	NP	9.972	[114]
集胞藻（Synechocystis sp.）	光自养型微生物；单位碳源生物量转化率高	虾青素	NP	29.6^①	[121]

底盘物种	优势	产品	产量 /mg·L^-1	产率 /mg·g DCW^-1	参考文献
恶臭假单胞菌（Pseudomonas putida）	化学品、有机溶剂耐受性高 NADPH再生快	玉米黄质	239^①	NP	[105]
谷氨酸棒杆菌（Corynebacterium glutamicum）	可利用多种碳源天然合成C₅₀类胡萝卜素	虾青素	NP	1.6	[51]
毕赤酵母（Pichia pastoris）	蛋白表达的优势宿主，翻译后修饰更接近天然蛋白	番茄红素	714	9.319	[116]
克鲁维酵母（Kluyveromyces marxianus）	耐热性好，可在25~52℃的温度下发酵单位碳源生物量转化率高	虾青素	NP	9.972	[114]
集胞藻（Synechocystis sp.）	光自养型微生物；单位碳源生物量转化率高	虾青素	NP	29.6^①	[121]

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[2]	郭峰, 张尚杰, 蒋羽佳, 姜万奎, 信丰学, 章文明, 姜岷. 一碳资源在酵母中的利用与转化[J]. 化工进展, 2023, 42(1): 30-39.
[3]	陶雨萱, 张尚杰, 景艺文, 信丰学, 董维亮, 周杰, 蒋羽佳, 章文明, 姜岷. 甲基营养型大肠杆菌构建策略的研究进展[J]. 化工进展, 2021, 40(7): 3932-3941.
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