An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibri
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RESEARCH ARTICLE
Open Access
An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris) Zhiqun Che1, Xiaoyan Cao2, Guiguang Chen1 and Zhiqun Liang1*
Abstract Background: As a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii). Results: After codon optimization, the fibase from a marine Bacillus subtilis was expressed and secreted in K. phaffii GS115. Recombinant strains harboring different copies of the fib gene (fib-nc) were successfully obtained via Geneticin (0.25–4 mg/ml) screening on minimal dextrose selection plates and assessment via real-time quantitative PCR. The respective levels of fibase produced by strains expressing fib-5.4c, fib-6c, fib-8c, fib-9c, and fib-12c were 4428, 5781, 7323, 7930, and 2472 U/ml. Levels increased as the copy number increased from 4 to 9, but decreased dramatically at copy number 12. After high cell density fermentation optimization, the highest fibase activity of the strain expressing fib-9c was 7930 U/ml in a shake flask and increased to 12,690 U/ml after 3 days of continuous culture in a 5-L fermenter, which is one of the highest levels of production reported. The recombinant fibase was maximally active at pH 9.0 and 45 °C, and was remarkably stable at pH levels ranging from 5 to 10 and temperatures up to 50 °C. As a metal-dependent serine protease, fibase did not cause hemolysis in vitro and preferentially degraded fibrin directly. Conclusions: The combination of codon optimization, gene dosage, and process optimization described herein could be used for the expression of other therapeutic proteins difficult to express. The characteristics of the recombinant fibase suggest that it has potential applications for thrombosis prevention and therapy. Keywords: Fibrinolytic enzyme, Recombinant engineered strain, Real-time quantification PCR, Fermentation optimization, Characteristics
* Correspondence: [email protected]; [email protected] 1 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Microorganism and Enyme Research Center of Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning 530004, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes wer
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