Characterization and optimization of the LAC4 upstream region for low‐leakage expression in Kluyveromyces marxianus
Characterization and optimization of the LAC4 upstream region for low‐leakage expression in Kluyveromyces marxianus
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DOI：
10.1002/yea.3682
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发表时间：
2021-11
期刊：
影响因子：
2.6
通讯作者：
Benxin Liu;Pingping Wu;Jungang Zhou;Anqi Yin;Yao Yu;Hong Lu
中科院分区：
文献类型：
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作者：
Benxin Liu;Pingping Wu;Jungang Zhou;Anqi Yin;Yao Yu;Hong Lu
马氏克卢维酵母是生产异源蛋白、化学物质和生物乙醇的有前途的宿主。该物种的一个优越特征是其吸收乳糖的能力，这是由编码乳糖渗透酶和β -半乳糖苷酶的LAC12-LAC4基因对实现的。目前对LAC4在马氏夜蛾中的调控作用知之甚少。在本研究中，我们发现在对LAC4的调控中存在微弱的葡萄糖抑制，这可能是导致LAC4在葡萄糖培养基中漏表达的原因。在1000 bp LAC4上游区域的突变筛选中，一个名为H1的突变区域在葡萄糖培养基中驱动URA3报告基因的低泄漏表达。5 ' UTR的聚腺苷延伸段（poly(a)）内的两个突变是导致H1低渗漏表型的主要原因。H1引导GFP在质粒上的低泄漏表达和LAC4在葡萄糖培养基中的原位表达，这不是由于mRNA水平的降低。同时，H1不影响乳糖对GFP和LAC4的诱导。与野生型LAC4上游区域或强INU1启动子表达的Cre重组酶相比，H1表达的Cre重组酶在抑制条件下毒性较低，诱导后产量更高。我们的研究表明，5 ' UTR内的poly(A)在抑制条件下调节LAC4的表达。同时，为建立严格的工业蛋白，特别是毒性蛋白的诱导表达体系奠定了基础。
Kluyveromyces marxianus is a promising host for the production of heterologous proteins, chemicals, and bioethanol. One superior feature of this species is its capacity to assimilate lactose, which is rendered by the LAC12–LAC4 gene pair encoding a lactose permease and a β‐galactosidase enzyme. Little is known about the regulation of LAC4 in K. marxianus. In this study, we showed the presence of weak glucose repression in the regulation of LAC4 and that might contribute to the leaky expression of LAC4 in the glucose medium. In a mutagenesis screen of 1000‐bp LAC4 upstream region, one mutant region, named H1, drove low‐leakage expression of a URA3 reporter gene in glucose medium. Two mutations inside a polyadenosine stretch (poly(A)) of 5′ UTR were major contributors to the low‐leakage phenotype of H1. H1 directed low‐leakage expression of GFP on a plasmid and that of LAC4 in situ in the glucose medium, which was not due to the reduction of mRNA levels. Meanwhile, H1 did not affect the induction of GFP or LAC4 by lactose. Cre recombinase expressed by H1 caused lower toxicity in the repressive condition and achieved higher yield after induction, compared with that expressed by a wild‐type LAC4 upstream region or a strong INU1 promoter. Our study suggested that poly(A) inside 5′ UTR played a role in regulating the expression of LAC4 in the repressive condition. Meanwhile, H1 provided a base for the development of a strict inducible system for expressing industrial proteins, especially toxic proteins.