高灵敏度和严格的胆碱诱导基因表达控制器的定向进化。

IF 0.8 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal of General and Applied Microbiology Pub Date : 2024-11-06 Epub Date: 2024-06-17 DOI:10.2323/jgam.2024.05.004
Yuki Yanai, Takayuki Hoshino, Yuki Kimura, Shigeko Kawai-Noma, Daisuke Umeno
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引用次数: 0

摘要

基因表达控制器是微生物生产重组蛋白质和有价值的生物基化学品的有用工具。尽管基因表达控制器非常有用,但由于缺乏符合诱导剂分子低成本、安全和严格控制这三个要求的系统,它们很少被应用到实际的工业生物工艺中。此前,我们已经开发出了由安全廉价的诱导剂胆碱控制的高规格基因诱导系统。然而,该系统需要相对高浓度(约 100 mM)的胆碱才能完全诱导所控制的基因。在这项工作中,我们试图大幅提高这种诱导系统的灵敏度,以进一步降低诱导成本。为此,我们设计了一个简单的电路,将基因诱导系统与胆碱导入蛋白 BetT 的正反馈回路(P-loop)结合起来。在对 BetT 的翻译水平(P-loop 的强度)和内源 betI(噪声源)进行调节和删除后,只需减少约 100 倍的诱导剂分子量,就能实现对基因表达的高活性和严格控制。本研究开发的胆碱诱导系统与现有的最高启动子(如 PT5 系统)相比,具有最低的基础表达量、激活所需的最低胆碱量和最高的诱导幅度。有了这个系统,人们就可以用可忽略不计的诱导剂分子成本来严格控制相关基因的表达水平,而诱导剂分子成本一直是应用于大规模工业过程的瓶颈。
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Directed evolution of highly sensitive and stringent choline-induced gene expression controllers.

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couples gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as PT5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

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来源期刊
Journal of General and Applied Microbiology
Journal of General and Applied Microbiology 生物-生物工程与应用微生物
CiteScore
2.40
自引率
0.00%
发文量
42
审稿时长
6-12 weeks
期刊介绍: JGAM is going to publish scientific reports containing novel and significant microbiological findings, which are mainly devoted to the following categories: Antibiotics and Secondary Metabolites; Biotechnology and Metabolic Engineering; Developmental Microbiology; Environmental Microbiology and Bioremediation; Enzymology; Eukaryotic Microbiology; Evolution and Phylogenetics; Genome Integrity and Plasticity; Microalgae and Photosynthesis; Microbiology for Food; Molecular Genetics; Physiology and Cell Surface; Synthetic and Systems Microbiology.
期刊最新文献
Marine bacteria have multiple polyamide 4-degrading enzymes. Structure of the SigF1-dependent pilA1 gene promoter and characterization of the light-activated response in the cyanobacterium Synechococcus elongatus PCC 7942. The chromosome level whole genome sequence and the seconary matabolism gene cluster prediction of Fusarium meridionale, the pathogen causing maize ear rot. Directed evolution of highly sensitive and stringent choline-induced gene expression controllers. Rational Design of a Yeast-derived 3',5'-bisphosphate Nucleotidase with Improved Substrate Specificity.
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