A strategy for successful dual-species protein expression of genes with non-optimal codon usage destined for bacterial and yeast cell factories.

IF 2.5 3区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Biotechnology Progress Pub Date : 2024-05-17 DOI:10.1002/btpr.3482
Marcus Wäneskog, Trine Bertram Rasmussen, Emil D Jensen
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Abstract

Recombinant protein expression on an industrial scale traditionally utilizes one of two microbial workhorses: Escherichia coli or Saccharomyces cerevisiae. Additionally, random protein engineering of enzymes and proteins aimed for expression in S. cerevisiae are often mutagenized and pre-screened in E. coli before expression in yeast. This introduces artificial bottlenecks as the bacterial expression vector needs to be substituted for a yeast expression vector via sub-cloning, and the new library re-evaluated before a final screening in yeast. Here, we put forward a protein expression and engineering strategy that involves the use of a dual-host shuttle vector (pYB-Dual) designed with both a strong inducible yeast promoter (pGAL1), and a strong inducible bacterial promoter (pT7-RNAP), which allows for inducible protein expression in both species. Additionally, we demonstrate that by transforming the pYB-Dual vector into the E. coli strain Rosetta 2, which has elevated levels of 7 rare tRNAs, we can achieve high-level protein expression in both yeast and bacteria, even when using a mNeonGreen gene codon optimized for yeast. This dual expression vector is expected to remove bottlenecks during protein engineering of commercially important enzymes destined for high-titer expression in yeast.

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成功表达细菌和酵母细胞工厂非最佳密码子使用基因的双物种蛋白质的策略。
传统上,工业规模的重组蛋白表达使用两种微生物中的一种:大肠杆菌或酿酒酵母。此外,用于在酿酒酵母中表达的酶和蛋白质的随机蛋白质工程通常先在大肠杆菌中进行诱变和预筛选,然后再在酵母中表达。这就造成了人为的瓶颈,因为细菌表达载体需要通过亚克隆来替代酵母表达载体,并在酵母中进行最终筛选之前对新文库进行重新评估。在这里,我们提出了一种蛋白质表达和工程策略,即使用双宿主穿梭载体(pYB-Dual),该载体同时设计有强诱导性酵母启动子(pGAL1)和强诱导性细菌启动子(pT7-RNAP),从而可以在两种物种中进行诱导性蛋白质表达。此外,我们还证明,通过将 pYB-Dual 载体转化到 7 种稀有 tRNA 水平较高的大肠杆菌菌株 Rosetta 2 中,即使使用针对酵母优化密码子的 mNeonGreen 基因,我们也能在酵母和细菌中实现高水平的蛋白质表达。这种双重表达载体有望消除在酵母中进行高滴度表达的重要商业酶的蛋白质工程瓶颈。
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来源期刊
Biotechnology Progress
Biotechnology Progress 工程技术-生物工程与应用微生物
CiteScore
6.50
自引率
3.40%
发文量
83
审稿时长
4 months
期刊介绍: Biotechnology Progress , an official, bimonthly publication of the American Institute of Chemical Engineers and its technological community, the Society for Biological Engineering, features peer-reviewed research articles, reviews, and descriptions of emerging techniques for the development and design of new processes, products, and devices for the biotechnology, biopharmaceutical and bioprocess industries. Widespread interest includes application of biological and engineering principles in fields such as applied cellular physiology and metabolic engineering, biocatalysis and bioreactor design, bioseparations and downstream processing, cell culture and tissue engineering, biosensors and process control, bioinformatics and systems biology, biomaterials and artificial organs, stem cell biology and genetics, and plant biology and food science. Manuscripts concerning the design of related processes, products, or devices are also encouraged. Four types of manuscripts are printed in the Journal: Research Papers, Topical or Review Papers, Letters to the Editor, and R & D Notes.
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Non-thermal plasma decontamination of microbes: a state of the art. Mechanistic model of minute virus of mice elution behavior in anion exchange chromatography purification. Comparing in silico flowsheet optimization strategies in biopharmaceutical downstream processes. General strategies for IgG-like bispecific antibody purification. Issue Information
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