黑曲霉作为分泌蛋白细胞工厂的研究进展

doi:10.11949/0438-1157.20190591

摘要/Abstract

摘要：

黑曲霉具有极强的分泌蛋白的能力，其分泌的木质纤维素降解酶、淀粉酶、蛋白酶、脂肪酶等广泛应用于食品、饲料和生物技术等相关工业中。本文围绕黑曲霉生产同源和异源分泌蛋白，阐述黑曲霉作为分泌蛋白细胞工厂的巨大潜力。首先，通过总结黑曲霉表达系统的优越性，确定了黑曲霉作为分泌蛋白细胞工厂的开发前景。随后，介绍了初步构建黑曲霉分泌蛋白细胞工厂的一般流程。接着，总结了近十年来黑曲霉作为同源分泌酶细胞工厂的研究进展，提出在黑曲霉中定向表达分泌酶是深入研究黑曲霉自身分泌酶性质、功能和结构的理想策略。最后，总结了近年来黑曲霉作为异源分泌蛋白细胞工厂的研究进展，并从异源蛋白表达中遇到的难点出发介绍了完善黑曲霉细胞工厂来提高异源蛋白产量的对策。

关键词: 生物工程, 黑曲霉, 酶, 细胞工厂, 定向表达, 同源分泌酶, 异源分泌蛋白

Abstract:

Aspergillus niger has a strong ability to secrete proteins, and its secreted lignocellulose degrading enzymes, amylases, proteases, and lipases are widely used in related industries such as food, feed, and biotechnology. We describe its great potential as cell factory for secretory proteins based on the production of homologous and heterologous secretory proteins by A. niger. Firstly, the prospect of A. niger as cell factory for secretory proteins is ascertained based on the superiority of its expression system. Then the standard procedure for construction of A. niger cell factory for secretory proteins is introduced. Subsequently, the advances on A. niger as cell factory for homologous secretases in the past decade are summarized. It is proposed that the directed expression of inherent secretases in A. niger is an ideal strategy to study their properties, functions and structures. Finally, the advances on A. niger as cell factory for heterologous secretory proteins in recent years are summarized, and based on the difficulties encountered in the expression of heterologous proteins, the countermeasures to improve the production of heterologous proteins by further improvement of A. niger cell factory are introduced.

Key words: biochemical engineering, Aspergillus niger, enzyme, cell factory, directed expression, homologous secretase, heterologous secretory protein

中图分类号:

TQ 028.8

姚善泾, 蔡礼年, 林东强. 黑曲霉作为分泌蛋白细胞工厂的研究进展[J]. 化工学报, 2019, 70(10): 3690-3703.

Shanjing YAO, Linian CAI, Dongqiang LIN. Progress in Aspergillus niger as cell factory for secretory proteins[J]. CIESC Journal, 2019, 70(10): 3690-3703.

图/表 7

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参考依据	大肠杆菌	黑曲霉	酵母	昆虫细胞	哺乳动物细胞	植物细胞
转化难易	简单	较难	一般	较难	较难	较难
培养周期	1天以内	1周左右	1周左右	1周左右	1周左右	1月左右
培养成本	低	低	低	高	高	较高
分泌性能	低	高	较高	高	高	高
蛋白折叠	低	较高	较高	高	高	高
N-糖基化	无	哺乳动物型的糖核	高比例甘露聚糖	与哺乳动物细胞类似	哺乳动物细胞类型	与哺乳动物细胞类似
O-糖基化	无	有	有	有	有	有

参考依据	大肠杆菌	黑曲霉	酵母	昆虫细胞	哺乳动物细胞	植物细胞
转化难易	简单	较难	一般	较难	较难	较难
培养周期	1天以内	1周左右	1周左右	1周左右	1周左右	1月左右
培养成本	低	低	低	高	高	较高
分泌性能	低	高	较高	高	高	高
蛋白折叠	低	较高	较高	高	高	高
N-糖基化	无	哺乳动物型的糖核	高比例甘露聚糖	与哺乳动物细胞类似	哺乳动物细胞类型	与哺乳动物细胞类似
O-糖基化	无	有	有	有	有	有

酶分类	酶种类	获取方式^①
纤维素酶	内切葡聚糖酶	D^[39,40,41]，P^[42]
	外切葡聚糖酶	D^[43,44]，P^[45]
	β-葡萄糖苷酶	D^{[46,47,48,49]}，P^[50,51]，A^[38]
	裂解多糖单氧化酶	P^[52,53]
半纤维素酶	木聚糖酶	D^[54,55,56]，P^{[57,58,59,60]}，E^[61,62]
	木糖苷酶	D^[46,63]，P^[64,65]，A^[66]
	甘露聚糖酶	P^[67,68,69]
	甘露糖苷酶	P^[70,71,72]
	乙酰木聚糖酯酶	P^[73]
果胶酶	果胶甲酯酶	P^[74,75]
	果胶酶	D^[76]
	聚半乳糖醛酸酶	D^[77]，S^[78]
	果胶裂解酶	D^[79]
淀粉酶	葡糖淀粉酶	D^[80,81,82]
淀粉酶	淀粉酶	D^[83]，P^[84]
蛋白酶	肽酶	D^[85]
	角质蛋白酶	P^[86]，E^[87]
	酸性蛋白酶	D^[88,89]，P^[90]
	氨肽酶	P^[91]
其他酶	脂肪酶	D^[92,93]，P^[94]，E^[95]，A^[96]
	α-葡萄糖苷酶	D^[97]，P^[98]
	植酸酶	D^{[99,100,101,102]}，P^[103]，A^[104]
	芸香苷酶	P^[105]
	阿魏酸酯酶	P^[106]，A^[107]
	鞣酸酶	D^[108]，A^[109]
	菊粉酶	E^[110]
	天冬酰胺酶单宁酶	D^[111] A^[112]

酶分类	酶种类	获取方式^①
纤维素酶	内切葡聚糖酶	D^[39,40,41]，P^[42]
	外切葡聚糖酶	D^[43,44]，P^[45]
	β-葡萄糖苷酶	D^{[46,47,48,49]}，P^[50,51]，A^[38]
	裂解多糖单氧化酶	P^[52,53]
半纤维素酶	木聚糖酶	D^[54,55,56]，P^{[57,58,59,60]}，E^[61,62]
	木糖苷酶	D^[46,63]，P^[64,65]，A^[66]
	甘露聚糖酶	P^[67,68,69]
	甘露糖苷酶	P^[70,71,72]
	乙酰木聚糖酯酶	P^[73]
果胶酶	果胶甲酯酶	P^[74,75]
	果胶酶	D^[76]
	聚半乳糖醛酸酶	D^[77]，S^[78]
	果胶裂解酶	D^[79]
淀粉酶	葡糖淀粉酶	D^[80,81,82]
淀粉酶	淀粉酶	D^[83]，P^[84]
蛋白酶	肽酶	D^[85]
	角质蛋白酶	P^[86]，E^[87]
	酸性蛋白酶	D^[88,89]，P^[90]
	氨肽酶	P^[91]
其他酶	脂肪酶	D^[92,93]，P^[94]，E^[95]，A^[96]
	α-葡萄糖苷酶	D^[97]，P^[98]
	植酸酶	D^{[99,100,101,102]}，P^[103]，A^[104]
	芸香苷酶	P^[105]
	阿魏酸酯酶	P^[106]，A^[107]
	鞣酸酶	D^[108]，A^[109]
	菊粉酶	E^[110]
	天冬酰胺酶单宁酶	D^[111] A^[112]

蛋白来源	蛋白种类	表达水平^①	参考文献
人	α₁蛋白酶抑制剂	12 mg/L	[113]
人	白细胞介素-6	15 mg/L	[23]
人	溶菌酶	40 mg/L	[114]
马	溶菌酶	150 mg/L	[114]
牛	肠激酶	5 mg/L	[115]
利什曼虫	抗原蛋白	54 mg/L	[116]
福寿螺	纤维素酶	SDS-PAGE	[117]
杏鲍菇	过氧物酶	466 U/L	[118]
云芝	漆酶	2700 U/L	[119]
黄孢原毛平革菌	锰过氧物酶	100 mg/L	[120]
黄孢原毛平革菌	木质素过氧物酶	Western blot	[121]
里氏木霉	木聚糖酶，内切葡聚糖酶	SDS-PAGE	[122]
嗜热子囊菌，里氏木霉	29个纤维素酶	SDS-PAGE	[123]
白曲霉	酸性蛋白酶	9972 U/ml	[124]
米黑根毛霉	脂肪酶	15 U/ml	[125]
梨囊鞭菌属R	纤维素酶	SDS-PAGE	[126]
朱红密孔菌	漆酶	SDS-PAGE	[127]
Caldariomyces fumago	氯化物过氧物酶	SDS-PAGE	[128]
海栖热袍菌	木聚糖酶	500 mg/L	[129]
解淀粉芽孢杆菌	纤维素酶	SDS-PAGE	[130]