Dataset Information

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris).

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.

SUBMITTER: Che Z

PROVIDER: S-EPMC7716587 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris).

Che Zhiqun Z Cao Xiaoyan X Chen Guiguang G Liang Zhiqun Z

BMC biotechnology 20201204 1

<h4>Background</h4>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).<h4>Results</h4>After codon optimization, the fibase from a marine Bacillus sub ...[more]

PMID: 33276774

Similar Datasets

Project description:Pichia pastoris (Komagataella phaffii) is a methylotrophic yeast that is widely used in industry as a host system for heterologous protein expression. Heterologous gene expression is typically facilitated by strongly inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters. However, protein production is usually accomplished by a fed-batch induction process, which is known to negatively affect cell physiology, resulting in limited protein yields and quality. To assess how yields of exogenous proteins can be increased and to further understand the physiological response of P. pastoris to the carbon conversion of glycerol and methanol, as well as the continuous induction of methanol, we analyzed recombinant protein production in a 10,000-L fed-batch culture. Furthermore, we investigated gene expression during the yeast cell culture phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth and may regulate the alcohol oxidase1 (AOX1) promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris heterologous protein expression, regulation, and secretory mechanisms.

Dataset Information

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris).

Background

Results

Conclusions

Publications

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris).

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