巴斯德毕赤酵母底盘细胞的工程化改造及应用

doi:10.12211/2096-8280.2022-039

合成生物学 ›› 2022, Vol. 3 ›› Issue (6): 1150-1173.DOI: 10.12211/2096-8280.2022-039

巴斯德毕赤酵母底盘细胞的工程化改造及应用

刘启, 钱芷兰, 宋丽丽, 要超颖, 徐名强, 任燕娜, 蔡孟浩

华东理工大学生物工程学院，生物反应器工程国家重点实验室，上海 200237

收稿日期:2022-07-04 修回日期:2022-08-03 出版日期:2022-12-31 发布日期:2023-01-17
通讯作者: 蔡孟浩
作者简介:刘启（1991—），男，博士后。研究方向为酵母底盘的改造优化及合成遗传线路设计。E-mail：liuqi0496@ecust.edu.cn
蔡孟浩（1984—），男，博士，副教授，博士生导师。研究方向为合成生物学与发酵工程，通过遗传元件挖掘与工具开发、真核微生物底盘设计与改造、细胞代谢重构与发酵优化，实现药/食源功能分子的规模化发酵制备。E-mail：cmh022199@ecust.edu.cn
基金资助:
国家自然科学基金面上项目(31870073);国家重点研发计划(2018YFC1706202);上海市青年科技启明星项目(19QA1402700)

Rewiring and application of Pichia pastoris chassis cell

Qi LIU, Zhilan QIAN, Lili SONG, Chaoying YAO, Mingqiang XU, Yanna REN, Menghao CAI

State Key Laboratory of Bioreactor Engineering，School of Biotechnology，East China University of Science and Technology，Shanghai 200237，China

Received:2022-07-04 Revised:2022-08-03 Online:2022-12-31 Published:2023-01-17
Contact: Menghao CAI

摘要/Abstract

摘要：

优质的微生物底盘宿主是实现绿色、可持续生物制造的重要平台。巴斯德毕赤酵母底盘宿主因其在蛋白表达和发酵生产中的诸多优势受到了广泛的关注和应用。而作为一种工业甲基营养酵母，其可以有效地利用来源广泛的甲醇作为唯一碳源，使其成为碳一化合物潜在的生物转化平台。近年来，随着合成生物技术和生物制药技术的快速发展，围绕毕赤酵母底盘的工程化改造研究逐渐增多，并取得了卓有成效的进展，促进了毕赤酵母底盘的发展和升级。本文简述了毕赤酵母底盘细胞的发展和应用现状，从基因操纵技术、基因表达调控、代谢工程改造等方面介绍了毕赤酵母的工程化改造策略及应用效果，总结了毕赤酵母中合成生物技术、调控元器件、新型表达平台和生物转化体系的建立与开发情况。在此基础上，进一步强调了毕赤酵母中CRISPR介导的基因编辑及调控、转录系统的重构及人工设计，介绍了其在蛋白表达和化合物合成方面的应用，并分析了其在实际应用中的优势和问题。最后，对毕赤酵母在后续研究中的底盘升级方向和应用场景进行了展望。

关键词: 毕赤酵母, 底盘细胞, 甲基营养型酵母, 细胞工厂, 合成生物系统

Abstract:

Microbial chassis hosts are important platforms for green and sustainable biomanufacturing. Pichia pastoris has served as a preferred chassis for heterologous protein expression and fermentation production, which is attributed to its numerous advantages in expression capacity, post-translational modification, high cell density culture, and extracellular product purification. Moreover, as an industrial methylotrophic yeast, P. pastoris effectively utilizes cheap and widely sourced methanol as the sole carbon source, making it a potential biotransformation platform for C1 compounds. Recently, scientists have endowed this nonconventional yeast as an efficient microbial cell factory for biosynthesis of small molecule products beyond its traditional role of a protein expression workhorse. The growing of synthetic biology and biopharmaceutical technology has promoted the rapid development on the genetic rewiring of P. pastoris chassis host. A series of engineering strategies have been developed to break the restrictions and bottlenecks of P. pastoris in both academic and industrial applications. This allowed the updated chassis versions adapting to diversified application scenarios. In this review, we briefly introduce the advances and current status of P. pastoris. We describe the development and application of this chassis from the genetic manipulation technology, regulation of gene expression, and metabolic engineering. We summarize the establishment and characterization of synthetic biological techniques, regulatory parts and devices, novel expression platform, and bioconversion system in P. pastoris. We emphasize the CRISPR-mediated gene editing, transcription regulation, rewiring of natural transcription system, and the design of artificial biosystems. Then the production of glycoprotein and the synthesis of natural products based on alcohols are concisely summarized. Also, the advantages and limitations of this host in practical application are analyzed and discussed. Finally, we propose the research directions for further updating versions of P. pastoris and provide a perspective on their future application scenarios.

Key words: Pichia pastoris, chassis cell, methylotrophic yeast, cell factory, synthetic biological system

中图分类号:

刘启, 钱芷兰, 宋丽丽, 要超颖, 徐名强, 任燕娜, 蔡孟浩. 巴斯德毕赤酵母底盘细胞的工程化改造及应用[J]. 合成生物学, 2022, 3(6): 1150-1173.

Qi LIU, Zhilan QIAN, Lili SONG, Chaoying YAO, Mingqiang XU, Yanna REN, Menghao CAI. Rewiring and application of Pichia pastoris chassis cell[J]. Synthetic Biology Journal, 2022, 3(6): 1150-1173.

图/表 7

图1 毕赤酵母CRISPR介导的基因编辑过程及工程化改造策略［29-30， 41， 57］（标注蓝色字体代表该蛋白敲除后可以有效抑制NHEJ作用；标注红色字体代表该蛋白过表达可以提升HDR介导的修复效率）

Fig. 1 CRISPR mediated gene editing process and engineering strategy in Pichia pastoris[29-30, 41, 57](Blue font indicates that the knockout of target protein can inhibit NHEJ; red font indicates that the overexpression of target protein can enhance HDR efficiency)

表1 毕赤酵母中CRISPR介导的HDR效率及工程化改造策略

Tab. 1 CRISPR mediated HDR efficiency and engineering strategy in Pichia pastoris

菌株改造策略	Cas9/gRNA表达方式	游离质粒ARS	HDR效率		参考文献
菌株改造策略	Cas9/gRNA表达方式	游离质粒ARS	敲除	敲入	参考文献
Wild type	P _HTX1 -Cas9 P _HTX1 -HH-sgRNA-HDV	PARS1	100%（单基因）	—	[56]
敲除ku70	P _HTX1 -Cas9 P _HTX1 -HH-sgRNA-HDV	PARS1	100%（单基因）	—	[57]
敲除ku70	P _HTX1 -Cas9 P _HTX1 -HH-sgRNA-HDV	PARS1	—	75%～97.9%（单位点） 57.7%～70.0%（双位点） 12.5%～32.1%（三位点）	[65]
Wild type	P _GAP- Cas9 P _SER -sgRNA	panARS	80%（单基因）	—	[39]
敲除ku70	P _ENO1 -Cas9 P _tRNA1 -tRNA1-sgRNA	PARS1	—	40%（三位点）	[58]
敲除MPH1和过表达PpRAD52	P _HTX1 -Cas9 P _HTX1 -HH-sgRNA-HDV	panARS	90%（单基因）	43%～70%（单位点） 25%（三位点）	[29]
Wild type	P _GAP -Cas9（整合） P _SER -sgRNA	panARS	—	98.6%（单位点）	[60]
Wild type	P _GAP -Cas9（整合） P _SER -sgRNA	PARS1	—	33.3%～93.1%（双位点） 10%～75%（三位点）	[60]
敲除ku70	P _GAP -Cas9（整合） P _SER -sgRNA	PARS1	—	33.3%～57%（三位点）	[60]
过表达ScRAD52 ScRAD59 ScMRE11	P _GAP -Cas9（整合） P _SER -sgRNA	PARS1	—	100%（单位点） 95.7%～97.9%（双位点） 64.5%～80.9%（三位点）	[30]
Wild type	P _GAP -FnCpf1（整合） P _SER -crRNA	panARS	99%（单基因） 65%～80%（双基因） 30%（三基因）	—	[31]

图2 毕赤酵母中转录调控系统的改造及工程化设计［24-28， 40， 59， 78， 82， 88， 94-103， 108-115］URS—上游调控序列；CP—核心启动子；AD—激活结构域；RD—阻遏结构域；DBP—DNA结合蛋白；BS—结合位点；ER—雌二醇受体；βE—β-雌二醇

Fig. 2 Rewiring and engineering design of transcription regulation system in Pichia pastoris[24-28, 40, 59, 78, 82, 88, 94-103, 108-115]URS—upstream regulatory sequence; CP—core promoter; AD—activation domain; RD—repression domain; DBP—DNA binding protein; BS—binding site; ER—estradiol receptor; βE—β-estradiol

图3 毕赤酵母N糖基化过程及工程化改造［128-141］

Fig. 3 The N-glycosylation and N-glycan engineering in Pichia pastoris[128-141]

表2 分子伴侣共表达提高毕赤酵母蛋白表达量

Tab. 2 Co-expression of chaperone to enhance protein expression in Pichia pastoris

外源蛋白	分子伴侣	效价提升倍数	参考文献
狂犬病病毒糖蛋白	PDI1 ERO1 GPX1	9.6倍 3倍 8.2倍	[159]
白介素-2-人血清白蛋白融合蛋白	PDI1 KAR2 ERO1	2.2倍 1.9倍 2.3倍	[161]
猪肽聚糖识别蛋白	PDI1	5倍（高拷贝）	[162]
疏水蛋白HFBI	KAR2	14倍（单拷贝） 9.8倍（双拷贝） 22倍（三拷贝）	[163]
疏水蛋白HFBI	PDI ERO1	7.8倍（三拷贝） 30倍（三拷贝）	[163]
人溶菌酶	HAC1	1.2倍	[156]
铜绿假单胞菌弹性蛋白酶	HAC1	1.8～3.9倍	[155]
大肠杆菌植酸酶	HAC1 PDI1	1.36倍 1.40倍	[158]
家蚕乙酰胆碱酯酶	PDI1	5倍	[160]
几丁质酶	HAC1	1.3倍	[157]

图4 毕赤酵母中不同碳源底物的代谢途径及各类天然产物的合成［33-34， 60， 82， 171-184， 199-203］DHA—二羟丙酮；GAP—3-磷酸甘油醛；Xu5P—5-磷酸木酮糖；DHAP—磷酸二羟丙酮；XuMP cycle—单磷酸木酮糖循环；Aox—醇氧化酶；Cat—过氧化氢酶；Das—二羟丙酮合成酶；Dak—二羟丙酮激酶；Tpi—磷酸丙糖异构酶；Fld—甲醛脱氢酶；Fgh—S-甲酰基谷胱甘肽水解酶；Fdh—甲酸脱氢酶；Pdh—丙酮酸脱氢酶；Pdc—丙酮酸脱羧酶；Adh—乙醇脱氢酶；Ald—乙醛脱氢酶；Acs—乙酰辅酶A合成酶；Acc—乙酰辅酶A羧化酶；Fas—脂肪酸合成酶；表示酶催化反应；表示代谢物在细胞内外或不同细胞器间的穿梭；表示多步代谢途径

Fig. 4 Metabolic pathways of different carbon sources and synthesis of various natural products based on in Pichia pastoris[33-34, 60, 82, 171-184, 199- 203]DHA—dihydroxyacetone; GAP—glyceraldehyde-3-phosphate; Xu5P—xylulose 5-phosphate; DHAP—dihydroxyacetone phosphate; XuMP cycle—xylulose monophosphate cycle; Aox—alcohol oxidase; Cat—catalase; Das—dihydroxyacetone synthase; Dak—dihydroxyacetone kinase; Tpi—triosephosphate isomerase; Fld—formaldehyde dehydrogenase; Fgh—S-formylglutathione hydrolase; Fdh—formate dehydrogenase; Pdh—Pyruvate dehydrogenase; Pdc—pyruvate decarboxylase; Adh—alcohol dehydrogenase; Ald—acetaldehyde dehydrogenase; Acs—acetyl-CoA synthetase; Acc—acetyl-CoA carboxylase; Fas—fatty acid synthetase; indicate enzyme catalyzed reaction; indicate the shuttle of metabolites between extracellular and intracellular or between different organelles; indicate a multistep metabolic pathway.

表3 基于毕赤酵母底盘细胞的天然产物合成

Tab. 3 Production of natural products in Pichia pastoris chassis cell

种类	化合物	底物及培养方式	产量	参考文献
四碳有机酸	苹果酸	甲醇（摇瓶）	2.79 g/L	[172]
		葡萄糖（摇瓶）	8.55 g/L
		甲醇（反应器）	42.28 g/L	[171]
	富马酸	甲醇（反应器）	0.76 g/L
	琥珀酸	甲醇（反应器）	9.42 g/L
脂肪酸衍生物	蓖麻油酸	甲醇（摇瓶）	171.44 mg/L	[175]
	长链α-烯烃	甲醇（摇瓶）	1.6 mg/L	[174]
	脂肪酸	甲醇（反应器）	23.4 g/L	[173]
	脂肪醇	甲醇（反应器）	2.0 g/L
萜类化合物	番茄红素	葡萄糖（摇瓶）	1.141 μg/g DCW	[202]
		甲醇（反应器）	714 mg/L	[180]
	β-胡萝卜素	葡萄糖（摇瓶）	339 μg/g DCW	[202]
	虾青素	葡萄糖（摇瓶）	3.7 μg/g DCW	[203]
	诺卡酮	甲醇（反应器）	208 mg/L	[178]
	达玛烯二醇	甲醇（摇瓶）	1.073 mg/g DCW	[179]
聚酮类化合物	6-甲基水杨酸	甲醇（反应器）	2.2 g/L	[181]
	橘霉素	甲醇（摇瓶）	0.6 mg/L	[182]
	洛伐他汀	甲醇（反应器）	250.8 mg/L	[184]
		甲醇（反应器）	419.0 mg/L	[201]
	莫纳可林J	甲醇（反应器）	593.9 mg/L	[184]
		乙醇（反应器）	3.2 g/L	[33]
黄酮类化合物	黄芩素	乙醇（摇瓶）	401.9 mg/L	[34]
	千层纸素	乙醇（摇瓶）	339.5 mg/L

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巴斯德毕赤酵母底盘细胞的工程化改造及应用

Rewiring and application of Pichia pastoris chassis cell

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