巴斯德毕赤酵母是一种优良的外源蛋白生产平台,近年来也被用于发酵生产高附加值化学品。为了使产品生产适应发酵工业的需要,常需要对毕赤酵母进行代谢改造,同时表达多个基因。该文设计并建立了一种毕赤酵母多基因组装系统,利用golden gate克隆实现多元件整合,并通过Cre-lox系统去除抗性标签。该系统可实现同时游离或整合表达多个基因,插入位点、启动子、终止子均可灵活调整。利用这一系统对毕赤酵母进行改造,用于发酵合成高价值化学品2-苯乙醇。通过游离表达比较不同来源的关键基因,并整合表达2-酮-3-脱氧-D-阿拉伯庚酮-7-磷酸合酶ARO3、ARO4K229L和ARO5,分支酸变位酶ARO7G141S和预苯酸脱水酶PHA2基因,最终重组菌株PE-3合成了408.4 mg/L 2-苯乙醇,是出发菌株的10倍以上。该研究建立多基因组装的系统可用于毕赤酵母代谢工程,构建适用于发酵生产蛋白或其他代谢产物的重组菌株。
Komagataella phaffii, or Pichia pastoris, is widely applied for the production of heterologous proteins. In recent years, it has also been used for the production of high value-added chemicals including organic acids, terpenoids, and polyketides. Metabolic engineering was normally performed in fermentation industries, and more than one gene were required to be overexpressed. In this study, a system for multigene assembly system (MGAS) of K. phaffi was established. Golden gate cloning was used for multi-elements assembly and the resistance maker was rescued by Cre-lox system. Positive colonies could be easily screened by the replacement of sfGFP instead of colonies PCR. With the system we constructed, multi genes can be expressed at the same time by integrated into the genome or on episomal plasmids in K. phaffii, and the integration sites, promoters and terminators can be adjusted flexibly. White colonies (positive transformants) of seven elements assembly were over 90%, proving that this system was intuitive and efficient. After that, K. phaffii GS115 was modified for the production of a high-value added chemical 2-phenylethanol with the application of this system. By comparing key genes from different sources on episomal plasmids and integrating 2-keto-3-deoxy-D-arabinoheptanone-7-phosphate (DAHP) synthase genes ARO3, ARO4K229L and ARO5, chorismate mutase gene ARO7G141S and prephenate dehydratase gene PHA2, the recombinant strain PE-3 produced 408.4 mg/L 2-phenylethanol, which was more than 10 times that of the original strain. The study established an efficient system for industrial applications of K. phaffii to produce protein or other metabolites.
[1] PEÑA D A.Metabolic engineering of Pichia pastoris[J].Metabolic Engineering, 2018, 50:2-15.
[2] GUO F, DAI Z X, PENG W F, et al.Metabolic engineering of Pichia pastoris for malic acid production from methanol[J].Biotechnology and Bioengineering, 2021, 118(1):357-371.
[3] CAI P, DUAN X P, WU X Y, et al.Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris[J].Nucleic Acids Research, 2021, 49(13):7 791-7 805.
[4] SANTOS-MORENO J, SCHAERLI Y.A framework for the modular and combinatorial assembly of synthetic gene circuits[J].Acs Synthetic Biology, 2019, 8(7):1 691-1 697.
[5] WEBER E, ENGLER C, GRUETZNER R, et al.A modular cloning system for standardized assembly of multigene constructs[J].PLoS One, 2011, 6(2):e16765.
[6] ENGLER C, GRUETZNER R, KANDZIA R, et al.Golden gate shuffling:A one-pot DNA shuffling method based on type IIs restriction enzymes[J].Plos One, 2009, 4(5):e5553.
[7] PRIELHOFER R, BARRERO J J, STEUER S, et al.GoldenPiCS:A Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris[J].BMC Systems Biology, 2017, 11(1):123.
[8] LEE M E, DELOACHE W C, CERVANTES B, et al.A highly characterized yeast toolkit for modular, multipart assembly[J].Acs Synthetic Biology, 2015, 4(9):975-986.
[9] 丁东栋, 崔志峰, 徐翔, 等.生物转化法合成2-苯乙醇的研究进展[J].工业微生物, 2017, 47(2):53-60.
DING D D, CUI Z F, XU X, et al.Research progress in biotransformation production of 2-phenylethanol[J].Industrial Microbiology, 2017, 47(2):53-60.
[10] GU Y, MA J B, ZHU Y L, et al.Engineering Yarrowia lipolytica as a chassis for de novo synthesis of five aromatic-derived natural products and chemicals[J].ACS Synthetic Biology, 2020, 9(8):2 096-2 106.
[11] LI D, ZHANG B, LI S T, et al.A novel vector for construction of markerless multicopy overexpression transformants in Pichia pastoris[J].Frontiers in Microbiology, 2017, 8:1 698.
[12] HUA D L, XU P.Recent advances in biotechnological production of 2-phenylethanol[J].Biotechnology Advances, 2011, 29(6):654-660.
[13] WANG Y, ZHANG H, LU X, et al.Advances in 2-phenylethanol production from engineered microorganisms[J].Biotechnology Advances, 2019, 37(3):403-409.
[14] HASSING E J, DE GROOT P A, MARQUENIE V R, et al.Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae[J].Metabolic Engineering, 2019, 56:165-180.
[15] KONG S, PAN H, LIU X, et al.De novo biosynthesis of 2-phenylethanol in engineered Pichia pastoris[J].Enzyme and Microbial Technology, 2020, 133:109459.
[16] AGMON N, MITCHELL L A, CAI Y Z, et al.Yeast golden gate (YGG) for the efficient assembly of S.cerevisiae transcription units[J].ACS Synthetic Biology, 2015, 4(7):853-859.
[17] CELINSKA E, LEDESMA-AMARO R, LARROUDE M, et al.Golden gate assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica[J].Microbial Biotechnology, 2017, 10(2):450-455.
[18] KIRIYA K, TSUYUZAKI H, SATO M.Module-based systematic construction of plasmids for episomal gene expression in fission yeast[J].Gene, 2017, 637:14-24.
