毕赤酵母中外源蛋白表达量的提升策略

doi:10.13560/j.cnki.biotech.bull.1985.2023-0857

摘要/Abstract

摘要：

利用异源重组表达系统表达外源蛋白是基因工程研究的重点。巴斯德毕赤酵母（Pichia pastoris）是一种甲基营养型酵母，由于其易于遗传操作、高水平分泌外源蛋白、翻译后修饰等特点，已成为工业应用中蛋白质生产的重要菌株，常被用于酶制剂的生产。然而，部分外源蛋白在毕赤酵母系统中的表达水平较低，仍有待提升的空间。因此，进一步探索提升毕赤酵母中外源蛋白表达量的原理和方法，将对降低毕赤酵母表达系统工业化生产成本，提高经济效益，具有重要的意义。本文主要从基因水平、转录水平、翻译水平、折叠分泌水平、抗逆水平、发酵工艺六个方面归纳总结了近年来毕赤酵母提高外源蛋白表达的优化策略的研究进展，旨在为提高外源蛋白在毕赤酵母表达系统中的表达水平提供有益参考。

关键词: 毕赤酵母, 重组表达, 外源蛋白, 优化, 酶

Abstract:

The production of heterologous proteins via recombinant expression system is a focus of genetic engineering research. Because of its ease of genetic manipulation, high degree of heterologous protein secretion, and post-translational modification, Pichia pastoris, a methylotrophic yeast, has become an essential strain for protein production in industrial applications and is frequently employed in the manufacturing of enzymes. Although some heterologous proteins can be expressed in P. pastoris, the amount of expression is modest and has to be increased. Therefore, it is critical to investigate the principle and method of boosting the expression of heterologous proteins in P. Pastoris in order to lower industrial production costs and improve economic benefits. In this review, the optimizing strategies for improving heterologous protein expression in P. Pastoris in recent years are summarized from six perspectives: Gene level, transcription level, translation level, protein folding and secretion levels, cell resistance level, and fermentation technology, with the goal of demonstrating comprehensively how to improve the expression of heterologous protein in P. Pastoris expression system.

Key words: Pichia pastoris, recombinant expression, heterologous protein, optimization, enzyme

茹扎·也里扎提, 杨宇. 毕赤酵母中外源蛋白表达量的提升策略[J]. 生物技术通报, 2024, 40(3): 118-134.

RUZHA Yelizhati, YANG Yu. Strategies for Increasing Heterologous Protein Expression in Pichia pastoris[J]. Biotechnology Bulletin, 2024, 40(3): 118-134.

图/表 10

图1 毕赤酵母中外源蛋白表达的优化策略 Gene of interest（GOI）：目的基因；Posttransformational vector amplification（PTVA）：转化后载体扩增；Geneticin（G418）：遗传霉素；Protein disulfide isomerase（PDI）：二硫键异构酶；Endoplasmic reticulum oxidation 1（ERO1）：内质网蛋白折叠氧化还原辅助因子； Immunoglobulin heavy chain binding protein（BiP）：免疫球蛋白结合蛋白； Process analytical technology（PAT）：应用过程分析技术； Eukaryotic translation initiation factor 4F（eIF4F）：真核起始因子4F；43S preinitiation complex（43S PIC）：43S前起始复合物

Fig. 1 Strategies for increasing heterologous protein expression in Pichia pastoris

表1 外源蛋白在毕赤酵母中通过基因水平的优化提高表达策略

Table 1 Gene level-optimizing strategies for enhancing heterologous proteins expression in P. pastoris

优化策略Optimizing strategy	外源蛋白Heterologous protein	优化效果Optimizing effect	发酵规模Fermentation scale/L	参考文献Reference
密码子优化	果胶酶	酶活提升1.19倍	-	[6]
	α-淀粉酶	酶活提升2.31倍	5	[8]
	α-淀粉酶	酶活提升2.62倍	50	[8]
	木聚糖酶	酶活提升1.44倍	5	[16]
	豆血红蛋白	酶活提升4.5倍	10	[17]
高拷贝筛选	甘露聚糖酶	酶活提升2.2倍	-	[10]
	L-天冬酰胺酶	酶活提升1.4倍	-	[11]
	壳聚糖酶	酶活提升1.61倍	50	[18]
	明胶	蛋白表达量提高1.31倍	140	[19]

图2 转录水平上提高毕赤酵母中外源蛋白表达的示意图 Iron-responsive transcription factor（Aft1）：铁响应转录因子； forkhead-like transcription factor（Fhl1p）：叉头转录因子；Zn[II]2Cys6-transcription factor（SUT2）：Zn[II]2Cys6-转录因子；Saccharomyces cerevisiae GAL4-like transcription factor（ATT1）：酿酒酵母GAL4转录因子同源物；glucose transcriptional repressor（Mig）：葡萄糖转录抑制因子；transcriptional repressor for glucose and glycerol repression（Nrg1）：葡萄糖及甘油转录抑制因子

Fig. 2 A workflow diagram of enhancing heterologous proteins expression in P. pastoris at transcription levels

表2 外源蛋白在毕赤酵母中通过转录水平的优化提高表达策略

Table 2 Transcription level-optimizing strategies for enhancing heterologous proteins expression in P. pastoris

优化策略Optimizing strategy	外源蛋白Heterologous protein	优化效果Optimizing effect	发酵规模Fermentation scale/L	参考文献Reference
启动子的选择和使用	几丁质酶	蛋白表达量提高1.5倍	1.5	[30]
	葡萄糖醛酸酯酶	酶活提升3.5倍	14	[53]
	南极假丝酵母脂肪酶B	蛋白表达量提高3倍	-	[42]
终止子的选择和使用	羧基酯酶	蛋白表达量提高2.5倍	1	[44]
转录因子的过表达	水蛭透明质酸酶	酶活提升1.47倍	3	[45]
	植酸酶	酶活提升20%	-	[46]
	果胶酶	酶活提升35%	-	[46]
转录因子的去表达	人表皮生长因子受体-2单抗	蛋白表达量提高1.5倍	1	[48]
	-	解除甘油环境对P_AOX1的抑制	-	[50]
	-	挽救过氧化物酶体相关缺陷	-	[52]

表3 外源蛋白在毕赤酵母中通过优化翻译水平提高表达策略

Table 3 Translation level-optimizing strategies for enhancing heterologous proteins expression in P. pastoris

优化策略Optimizing strategy	外源蛋白Heterologous protein	优化效果Optimizing effect	发酵规模Fermentation scale	参考文献Reference
翻译起始因子的过表达	纳米抗体vHH	蛋白表达量提高3倍	1 L	[55]
核糖体生物合成因子的过表达	增强型绿色荧光蛋白	蛋白表达和细胞生物量均提高20%	250 mL	[58]
核糖体生物合成因子的过表达	植酸酶	酶活提升26%	250 mL	[58]
其他翻译相关元件的过表达	β-半乳糖苷酶	酶活提升2倍	-	[59]

图3 分泌折叠水平上提高毕赤酵母中外源蛋白表达的示意图 Protein disulfide isomerase（PDI）：二硫键异构酶；Endoplasmic reticulum oxidation 1（ERO1）：内质网蛋白折叠氧化还原辅助因子； immunoglobulin heavy chain binding protein（BiP）：免疫球蛋白结合蛋白；Saccharomyces cerevisiae Heat shock protein（Ssa4）：热休克蛋白；Transcriptional activator（HAC1）：转录激活剂； Endoplasmic reticulum stress（ER stress）：内质网应激；unfolded protein response（UPR）：未折叠蛋白响应；ER-associated protein degradation（ERAD）：内质网相关蛋白降解；disulfide bond（S-S）：二硫键

Fig. 3 A schematic diagram of improving secretion and folding level of heterologous proteins in P. pastoris

表4 外源蛋白在毕赤酵母中通过优化分泌折叠水平提高表达策略

Table 4 Protein secretion and folding levels-optimizing strategies for enhancing heterologous proteins expression in P. pastoris

优化策略Optimizing strategy	外源蛋白Heterologous protein	优化效果Optimizing effect	发酵规模Fermentation scale/L	参考文献Reference
分子伴侣共表达帮助蛋白正确折叠	巴西甜蛋白	蛋白表达量提高2.59倍	5	[66]
	人溶菌酶	蛋白表达量提高1.54倍	5	[82]
	磷脂酶C	酶活提升4.2倍	5	[83]
分子伴侣共表达帮助蛋白易位	抗体片段Fabs、scFvs	蛋白表达量提高5倍	1	[75]
信号肽优化	果胶裂解酶	酶活提升1.14倍	50	[84]
信号肽优化	抗菌肽	蛋白表达量提高2倍	5	[85]

图4 毕赤酵母中提高细胞抗逆水平的示意图 Gene of interest（GOI）：目的基因；Htreoscilla Hemoglobin（VHb）：透明颤菌血红蛋白基因；small heat shock protein gene 17.2（CsHSP17.2）：小热休克蛋白热诱导基因；antioxidant genes（CAT、SOD1、GLR1）：抗氧化基因；reactive oxygen species（ROS）：活性氧

Fig. 4 A Schematic diagram of improving cell resistance level in P. pastoris

表5 外源蛋白在毕赤酵母中通过优化抗逆水平提高表达策略

Table 5 Cell resistance level-optimizing strategies for enhancing heterologous proteins expression in P. pastoris

优化策略Optimizing strategy	外源蛋白Heterologous protein	优化效果Optimizing effect	参考文献Reference
改善溶解氧限制	脂肪酶Lip2	蛋白表达量提高83%	[88]
	脂肪酶Lip2	酶活提升1.88倍	[89]
	β-甘露聚糖酶	蛋白表达量提高90%	[90]
提高耐热性	-	菌株生长率提高	[91]
提高耐热性	过氧化氢酶	酶活提升2.5倍	[92]
提高抗氧化性	脂肪酶r27RCL	蛋白表达量提高1.6倍	[94]

表6 外源蛋白在毕赤酵母中通过优化发酵水平提高表达策略

Table 6 Fermentation level-optimizing strategies for enhancing heterologous proteins expression in P. pastoris

优化策略Optimizing strategy	外源蛋白Heterologous protein	优化效果Optimizing effect	发酵规模Fermentation scale/L	参考文献Reference
培养基优化	人血清蛋白	蛋白表达量提高1.97倍	5	[99]
溶解氧比例优化	MAS1脂肪酶	细胞生长率提高	5	[93]
溶解氧比例优化	人类溶菌酶	细胞密度提高	5	[102]
分批补料策略优化	人生长激素	蛋白表达量提高1.16倍	2	[105]
	马铃薯糖蛋白	蛋白表达量提高6.9倍	5	[106]
	漆酶	蛋白表达量提高1.5倍	7.5	[107]

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