Codon optimisation improves the expression of Trichoderma viride sp. endochitinase in Pichia pastoris.
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ABSTRACT:
SUBMITTER: Yu P
PROVIDER: S-EPMC3807108 | biostudies-other | 2013 Oct
REPOSITORIES: biostudies-other
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Project description:The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZ?A-kerAwt, pPICZ?A-kerAopti1 and pPICZ?A-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both ?-keratin (keratin azure) and ?-keratin (chicken feather meal). These properties make the P. pastoris pPICZ?A-kerAopti1 a suitable candidate for industrial production of keratinases.
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Project description:BackgroundPichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.ResultsPotential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter.DiscussionThe microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.
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Project description:?-Amylase as an important industrial enzyme has been widely used in starch processing, detergent, and paper industries. To improve expression efficiency of recombinant ?-amylase from Bacillus licheniformis (B. licheniformis), the ?-amylase gene from B. licheniformis was optimized according to the codon usage of Pichia pastoris (P. pastoris) and expressed in P. pastoris. Totally, the codons encoding 305 amino acids were optimized in which a total of 328 nucleotides were changed and the G+C content was increased from 47.6 to 49.2%. The recombinants were cultured in 96-deep-well microplates and screened by a new plate assay method. Compared with the wild-type gene, the optimized gene is expressed at a significantly higher level in P. pastoris after methanol induction for 168 h in 5- and 50-L bioreactor with the maximum activity of 8100 and 11000 U/mL, which was 2.31- and 2.62-fold higher than that by wild-type gene. The improved expression level makes the enzyme a good candidate for ?-amylase production in industrial use.
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Project description:BACKGROUND: Sustainable utilization of plant biomass as renewable source for fuels and chemical building blocks requires a complex mixture of diverse enzymes, including hydrolases which comprise the largest class of lignocellulolytic enzymes. These enzymes need to be available in large amounts at a low price to allow sustainable and economic biotechnological processes.Over the past years Pichia pastoris has become an attractive host for the cost-efficient production and engineering of heterologous (eukaryotic) proteins due to several advantages. RESULTS: In this paper codon optimized genes and synthetic alcohol oxidase 1 promoter variants were used to generate Pichia pastoris strains which individually expressed cellobiohydrolase 1, cellobiohydrolase 2 and beta-mannanase from Trichoderma reesei and xylanase A from Thermomyces lanuginosus. For three of these enzymes we could develop strains capable of secreting gram quantities of enzyme per liter in fed-batch cultivations. Additionally, we compared our achieved yields of secreted enzymes and the corresponding activities to literature data. CONCLUSION: In our experiments we could clearly show the importance of gene optimization and strain characterization for successfully improving secretion levels. We also present a basic guideline how to correctly interpret the interplay of promoter strength and gene dosage for a successful improvement of the secretory production of lignocellulolytic enzymes in Pichia pastoris.
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Project description:Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P. pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), Km (0.041 mM), and Vmax (2.008 µmol x mg protein(-1) x min(-1)) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg x L(-1) vs 36 mg x L(-1)) and total enzyme activity increased about 5 folds (1900 IU x L(-1) vs 367 IU x L(-1)) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.
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Project description:The growth of the mould Trichoderma viride on a defined medium containing either potassium d-glucose 6-O-sulphate or potassium d-galactose 6-O-sulphate as sole sources of both carbon and sulphur is marked by the production of an enzyme system capable of liberating inorganic SO(4) (2-) ions from either of the sulphate esters. The enzyme is not produced when the organism is grown with glucose (or galactose) and potassium sulphate or with glucose and methionine as sole sources of carbon and sulphur. Experimental conditions are described whereby inorganic SO(4) (2-) ions liberated from potassium glucose 6-O-sulphate by the growing mould appear in the culture medium after a constant lag period of 21-24hr. The enzyme has been shown to be a simple glycosulphatase that is active towards the 6-O-sulphate esters of d-glucose and d-galactose but not towards potassium glucose 3-O-sulphate. The properties of the crude glycosulphatase show the enzyme to be appreciably different from analogous molluscan enzymes that can degrade monosaccharide sulphate esters.
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Project description:BACKGROUND: In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast Pichia pastoris can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes. RESULTS: The Xyn2 gene, which encodes the main Trichoderma reesei Rut C-30 endo-beta-1, 4-xylanase was cloned into the pPICZalphaA vector and expressed in Pichia pastoris. The selected P. pastoris strains produced as 4,350 nkat/ml beta-xylanase under the control of the methanol inducible alcohol oxidase 1 (AOX1) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60 degrees C and it was active over a broad range of pH (3.0-8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50 degrees C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent Km and kcat values were 2.1 mg/ml and 219.2 S-1, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity. CONCLUSION: The P. pastoris expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, P pastoris is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.
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Project description:D-lactic acid is a three-carbon organic acid with a chiral structure and can improve the thermostability of polylactic acid. Microorganisms such as the methylotrophic yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of D-lactic acid, have been engineered to produce it in high titers. However, tolerance to D-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to D-lactic acid and leads to increased D-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high D-lactic acid concentrations. Furthermore, integrating a D-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that can produce D-lactic acid at a titer of 5.12 0.35 g/L in 48 hours , a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to D-lactic acid including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of D-lactic acid by manipulating yeast flocculation.
2023-04-01 | GSE224837 | GEO