High-yield secretory production of stable, active trypsin through engineering of the N-terminal peptide and self-degradation sites in Pichia pastoris

Zhang, Yunfeng; Huang, Hao; Yao, Xinhui; Du, Guocheng; Chen, Jian; Kang, Zhen

doi:10.1016/j.biortech.2017.08.006

Cited by 32 publications

(31 citation statements)

References 46 publications

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“…The SGT contains 16 potential autolysis residues (Arg, Lys). In previous study, the stability and production of SGT were improved with tbcf (K101A) mutant [13]. For further investigating the autolysis of tbcf (K101A), its' hydrolysate was analyzed by MALDI-TOF-MS. Based on the autolysis fragments of tbcf (K101A), five autolysis residues were identified as R21, R32, K122, R153, and R201 ( Fig.…”

Section: Results and Discussion Identification Of The Autolysis Sitesmentioning

confidence: 99%

“…For scale-up production of the trypsin, the strain GS115-tbcf (K101A, R201V)_SU was cultured in 3-L bio-reactor, according to the published method [13]. After glycerol fed-batch cultivation, a higher density of cells (68.02 ± 1.5 g/L, DCW) was achieved with glycerol feeding and high agitation speed (850 r•min − 1 ) ( Fig.…”

Section: Application Of High-yield Trypsin To Processing Insulin Precmentioning

confidence: 99%

“…For NeEMO prediction, the pH and temperature were set at 6.0 and 30 ºC. And R32A, K122A, and R153A mutation was individually intraduced into tbcf (K101A), for the increased activity in previous study [13].…”

Section: Prediction Of the Point Mutationsmentioning

confidence: 99%

“…Thus, high-yield production of trypsin might be hindered by autolysis of active trypsin. The mutant tbcf (K101A), with R145I and R201V mutations, afforded the higher stability against autolysis [13]. However, the autolysis residues of SGT have not been investigated in detail comparing with BT [14,15].…”

Section: Introductionmentioning

confidence: 99%

“…Over the past decade, SGT has been investigated in different microbial hosts, such as Escherichia coli [16], Bacillus subtilis [17], S. griseus [18,19] and Pichia pastoris [20,21]. Notably, by engineering the N-terminal peptide, the members of Kang group achieved high-yield production of trypsin (227.65 U•mL − 1 ) in P. pastoris [13,22]. However, the expression of trypsin caused the unfolding protein response (UPR), and trigged endoplasmic reticulum-associated degradation (ERAD) in P. pastoris.…”

Section: Introductionmentioning

confidence: 99%

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Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

Zhang

Liang

Zhang³

et al. 2019

Preprint

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Background: Trypsin has a plenty application in food and pharmaceutical manufacture. While, the commercial trypsin is usually extracted from pork pancreas, which has the risk of infectious and immunogenicity. Therefore, the microbial Streptomyces griseus trypsin (SGT) is a prior alternation because it processes efficient hydrolysis activity without the aforementioned risk. The remarkable hydrolysis efficiency of SGT caused its autolysis, and five autolysis sites R21, R32, K122, R153, and R201 were identified from its' autolysate. Results: The tbcf (K101A, R201V) mutant was screened by directed selection approach for improved activity in flask culture (60.85 ± 3.42 U·mL -1 , increased 1.5-fold). From the molecular dynamics simulation, the K101A/R201V mutation shortened the distant between catalytical residues D102 and H57 from 6.5 Å vs 7.0 Å, which afforded the improved specific activity 1527.96 ± 62.81 U·mg -1 . Further, the production of trypsin was increased 302.8% (689.47 ± 6.78 U·mL −1 ) in 3-L bio-reactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. Conclusions: The SGT protein could be an adequate trypsin for insulin production. When working with hydrolysates analysis and direction selection, the tbcf (K101A, R201V) mutant increased 1.5-fold activity. Further, the production of trypsin was improved 3-fold by overexpressing chaperone protein in Pichia pastoris . The future study should be emphasized on the application of SGT in insulin manufacture and pharmaceutical.

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Section: Results and Discussion Identification Of The Autolysis Sitesmentioning

confidence: 99%

Section: Application Of High-yield Trypsin To Processing Insulin Precmentioning

confidence: 99%

Section: Prediction Of the Point Mutationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

Zhang

Liang

Zhang³

et al. 2019

Preprint

Self Cite

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Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion

Zhou,

Zhang,

Qian

2023

Biotech & Bioengineering

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Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD‐hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild‐type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7‐fold higher kcat/KM. Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high‐producing strain, 8GM1, containing eight copies of AOX1 promoter‐GAS1 signal peptide‐MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co‐expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2‐fold. In the 5‐L bioreactor, conventional fed‐batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3‐fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high‐producing strains for other industrial enzymes.

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Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

Keikha

Rezaee

Farsiani

2020

Journal Cellular Physiology

333

220

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One of the most important branches of genetic engineering is the expression of recombinant proteins using biological expression systems. Nowadays, different expression systems are used for the production of recombinant proteins including bacteria, yeasts, molds, mammals, plants, and insects. Yeast expression systems such as Saccharomyces cerevisiae (S. cerevisiae) and Pichia pastoris (P. pastoris) are more popular. P. pastoris expression system is one of the most popular and standard tools for the production of recombinant protein in molecular biology. Overall, the benefits of protein production by P. pastoris system include appropriate folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins to the external environment of the cell. Moreover, in the P. pastoris expression system due to its limited production of endogenous secretory proteins, the purification of recombinant protein is easy. It is also considered a unique host for the expression of subunit vaccines which could significantly affect the growing market of medical biotechnology. Although P. pastoris expression systems are impressive and easy to use with well-defined process protocols, some degree of process optimization is required to achieve maximum production of the target proteins. Methanol and sorbitol concentration, Mut forms, temperature and incubation time have to be adjusted to obtain optimal conditions, which might vary among different strains and externally expressed protein. Eventually, optimal conditions for the production of a recombinant protein in P. pastoris expression system differ according to the target protein.

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High-yield secretory production of stable, active trypsin through engineering of the N-terminal peptide and self-degradation sites in Pichia pastoris

Cited by 32 publications

References 46 publications

Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion

Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins

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