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Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

ABSTRACT: BACKGROUND: Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. RESULTS: In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. CONCLUSIONS: We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.

SUBMITTER: Varnai A

PROVIDER: S-EPMC4021498 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

Várnai Anikó A Tang Campbell C Bengtsson Oskar O Atterton Andrew A Mathiesen Geir G Eijsink Vincent G H VG

Microbial cell factories 20140418 1

<h4>Background</h4>Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone ...[more]

PMID: 24742273

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Project description:The infections caused by antibiotic multidrug-resistant bacteria seriously threaten human health. To prevent and cure the infections caused by multidrug-resistant bacteria, new antimicrobial agents are required. Antimicrobial peptides are ideal therapy candidates for antibiotic-resistant pathogens. However, due to high production costs, novel methods of large-scale production are urgently needed.The novel plectasin-derived antimicrobial peptide-MP1102 gene was constitutively expressed under the control of the GAP promoter. The optimum carbon source and concentration were determined, and 4% glucose (w/v) was initially selected as the best carbon source. Six media were assayed for the improved yield of recombinant MP1102 (rMP1102). The total protein and rMP1102 yield was 100.06 mg/l and 42.83 mg/l, which was accomplished via the use of medium number 1. The peptone and yeast extract from Hongrun Baoshun (HRBS, crude industrial grade, Beijing, China) more effectively improved the total protein and the yield of rMP1102 to 280.41 mg/l and 120.57 mg/l compared to 190.26 mg/l and 78.01 mg/l that resulted from Oxoid (used in the research). Furthermore, we observed that the total protein, antimicrobial activity and rMP1102 yield from the fermentation supernatant increased from 807.42 mg/l, 384,000 AU/ml, and 367.59 mg/l, respectively, in pH5.0 to 1213.64 mg/l, 153,600 AU/ml and 538.17 mg/ml, respectively in pH 6.5 in a 5-l fermenter. Accordingly, the productivity increased from 104464 AU/mg rMP1102 in pH 5.0 to a maximum of 285412 AU/mg rMP1102 in pH 6.5. Finally, the recombinant MP1102 was purified with a cation-exchange column with a yield of 376.89 mg/l, 96.8% purity, and a molecular weight of 4382.9 Da, which was consistent with its theoretical value of 4383 Da.It's the highest level of antimicrobial peptides expressed in Pichia pastoris using GAP promoter so far. These results provide an economical method for the high-level production of rMP1102 under the control of the GAP promoter.

Project description:Pichia pastoris (P. pastoris) is one of the most popular cell factories for expressing exogenous proteins and producing useful chemicals. The alcohol oxidase 1 promoter (PAOX1) is the most commonly used strong promoter in P. pastoris and has the characteristic of biphasic expression. However, the inducer for PAOX1, methanol, has toxicity and poses risks in industrial settings. In the present study, analyzing transcriptomic data of cells collected at different stages of growth found that the formate dehydrogenase (FDH) gene ranked 4960th in relative expression among 5032 genes during the early logarithmic growth phase but rose to the 10th and 1st during the middle and late logarithmic growth phases, respectively, displaying a strict biphasic expression characteristic. The unique transcriptional regulatory profile of the FDH gene prompted us to investigate the properties of its promoter (PFDH800). Under single-copy conditions, when a green fluorescent protein variant was used as the expression target, the PFDH800 achieved 119% and 69% of the activity of the glyceraldehyde-3-phosphate dehydrogenase promoter and PAOX1, respectively. After increasing the copy number of the expression cassette in the strain to approximately four copies, the expression level of GFPuv driven by PFDH800 increased to approximately 2.5 times that of the strain containing GFPuv driven by a single copy of PAOX1. Our PFDH800-based expression system exhibited precise biphasic expression, ease of construction, minimal impact on normal cellular metabolism, and high strength. Therefore, it has the potential to serve as a new expression system to replace the PAOX1 promoter.IMPORTANCEThe alcohol oxidase 1 promoter (PAOX1) expression system has the characteristics of biphasic expression and high expression levels, making it the most widely used promoter in the yeast Pichia pastoris. However, PAOX1 requires methanol induction, which can be toxic and poses a fire hazard in large quantities. Our research has found that the activity of PFDH800 is closely related to the growth state of cells and can achieve biphasic expression without the need for an inducer. Compared to other reported non-methanol-induced biphasic expression systems, the system based on the PFDH800 offers several advantages, including high expression levels, simple construction, minimal impact on cellular metabolism, no need for an inducer, and the ability to fine-tune expression.

Dataset Information

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

Publications

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

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