文献类型：期刊文献

英文题名：CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris

作者：Liu, Qi[1];Shi, Xiaona[1];Song, Lili[1];Liu, Haifeng[2];Zhou, Xiangshan[1,2];Wang, Qiyao[1];Zhang, Yuanxing[1,3];Cai, Menghao[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]Chinare Resources Angde Biotech Pharmaceut Co Ltd, 78 E Jiao St, Liaocheng, Shandong, Peoples R China;[3]Shanghai Collaborat Innovat Ctr Biomfg, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2019

卷号：18

期号：1

外文期刊名：MICROBIAL CELL FACTORIES

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000483292300001)】；

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, China (19QA1402700), the 111 Project, China (B18022), Fundamental Research Funds for the Central Universities, China (22221818014), Research Program of State Key Laboratory of Bioreactor Engineering.

语种：英文

外文关键词：Pichia pastoris; CRISPR-Cas9; Homology directed repair; Multiloci integration; Multistep enzymatic pathway

摘要：BackgroundPichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris.ResultsA non-homologous-end-joining defective strain (Delta ku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (PAOX1UP-g2, PTEF1UP-g1, and PFLD1UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR-Cas9-mediated gene co-integration method in P. pastoris.ConclusionsA CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.