通过酵母人工染色体转移二硫键形成模块可促进马氏假丝酵母中异源蛋白的表达。

IF 4.5 Q1 MICROBIOLOGY
mLife Pub Date : 2024-03-22 eCollection Date: 2024-03-01 DOI:10.1002/mlf2.12115
Pingping Wu, Wenjuan Mo, Tian Tian, Kunfeng Song, Yilin Lyu, Haiyan Ren, Jungang Zhou, Yao Yu, Hong Lu
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引用次数: 0

摘要

马氏酵母(Kluyveromyces marxianus)是一种食品安全酵母,在生产异源蛋白方面具有巨大潜力。提高 K. marxianus 的产量仍然是一个挑战,而整合大规模功能模块则是工程中的一个技术障碍。为了解决这些问题,我们构建了马钱子酵母的线性和环状酵母人工染色体(KmYACs),并加载了来自 Pichia pastoris 或马钱子酵母的二硫键形成模块。这些模块最多包含 7 个基因,最大大小为 15 kb。KmYACs 带有来自 K. marxianus 或四膜虫的端粒。无论端粒的类型和 KmYACs 的配置如何,KmYACs 都能成功转移到 K. marxianus 中,并在不影响宿主正常生长的情况下稳定繁殖。KmYACs 提高了二硫键形成基因的整体表达水平,并显著提高了各种异源蛋白的产量。在高密度发酵中,使用 KmYACs 可使葡萄糖淀粉酶产量达到 16.8 克/升,这是迄今为止所报道的 K. marxianus 的最高水平。对含有 KmYACs 的细胞进行的转录组和代谢组分析表明,黄素腺嘌呤二核苷酸生物合成增加、进入三羧酸循环的通量增加,以及对赖氨酸和精氨酸的优先需求是过表达异源蛋白细胞的特征。同样,补充赖氨酸或精氨酸可进一步提高产量。因此,KmYAC 为操作大型模块提供了一个强大的平台,在工业应用和基础研究方面具有巨大潜力。事实证明,通过YACs转移二硫键形成模块是提高异源蛋白产量的有效策略,这一策略可用于优化其他微生物细胞工厂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

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