文献类型：期刊文献

英文题名：Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism

作者：Xu, Qin[1];Bai, Chenxiao[1];Liu, Yiqi[1];Song, Lili[1];Tian, Lin[1];Yan, Yunfeng[1];Zhou, Jinfeng[1];Zhou, Xiangshan[1];Zhang, Yuanxing[1,2];Cai, Menghao[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg, Shanghai 200237, Peoples R China

年份：2019

卷号：12

期号：1

外文期刊名：BIOTECHNOLOGY FOR BIOFUELS

收录：;EI(收录号：20191306701465);WOS:【SCI-EXPANDED(收录号:WOS:000462447500001)】；

基金：This work was supported by the National Key R&D Program of China (2018YFC1706200), National Natural Science Foundation of China (31870073), Fundamental Research Funds for the Shanghai Science and Technology Innovation Action Plan (17JC1402400), Shanghai Rising-Star Program (19QA1402700), the 111 Project (B18022), Fundamental Research Funds for the Central Universities (22221818014), Research Program of State Key Laboratory of Bioreactor Engineering and Talent Program of School of Biotechnology in East China University of Science and Technology.

语种：英文

外文关键词：Komagataella phaffii; Acetate utilization; Acetyl-CoA; Kinase screening; Metabolic engineering

摘要：BackgroundAcetate, an economical industrial chemical, which is also the precursor of acetyl-CoA, could serve as an alternative substrate for biomanufacturing. This nontraditional substrate can be widely produced from syngas via hydrolysis or pyrolysis of the cellulosic biomass, chemical or microbial catalysis, anaerobic fermentation in treated wastewater, etc. However, the toxicity of acetate to microorganisms has held back its utilization, especially for the eukaryotes that are good hosts for production of complicated pharmaceuticals or chemicals. This study seeks to improve acetate utilization in a widely used yeast host, Komagataella phaffii (previously Pichia pastoris), by metabolic engineering of acetate tolerance, transport, and metabolism.ResultsA kinase-deficient library of K. phaffii was firstly used to screen acetate-resistant kinases. The HRK1 knockout strain was sensitive to acetate and overexpression of this gene improved acetate tolerance and cell growth of the strain. Also, overexpression of HRK1 caused a 55% productivity improvement of acetyl-CoA-dependent 6-methylsalicylic acid (6-MSA). However, activation of Hrk1 on membrane H(+)-ATPase Pma1 seemed not to work in the engineered strain. Acetate transporter gene ScFPS1* was further overexpressed, despite of not improving 6-MSA biosynthesis. To enhance acetate metabolism, acetyl-CoA synthesizing related genes, yeast PpACS1, ScACS1*, and E. coli ackA/pta were overexpressed separately. Introduction of PpACS1 and ScACS1* each increased biosynthesis of 6-MSA by approximately 20% on 20mM acetate. Finally, co-overexpression of HRK1 and ScACS1* improved 6-MSA productivity by 51% on 20mM acetate, despite that a low expression level of HRK1 happened when genes were expressed under the same promoter.ConclusionsHRK1 screened by K. phaffii kinase-deficient library played an important role in acetate tolerance and was proved to profit the biosynthesis of acetyl-CoA-derived chemicals. It could be a potential target for metabolic engineering of acetate utilization in other eukaryotic hosts as well. A combined strategy of introducing genes for acetate tolerance and metabolism further improved biosynthesis of acetyl-CoA derived reporter compound in K. phaffii. This makes it a good choice for acetyl-CoA-derived chemicals with acetate as substrate or precursor in K. phaffii, which would also extend the use of this chassis host.