5 results on '"Yang, Zhiliang"'
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2. Metabolic and Process Engineering of Pichia Pastoris for the Production of Value-added Products
- Author
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Yang, Zhiliang
- Subjects
Pichia pastoris ,Process engineering ,Metabolic engineering - Abstract
Motivated by the surging demand of recombinant proteins and biofuels derived from renewable substrates, increasing attention has been paid to the development of novel strains via metabolic engineering strategies. Pichia pastoris is a eukaryotic platform suitable for protein expression and potentially for biofuel production due to its advantageous traits over Escherichia coli or Saccharomyces cerevisiae. In this thesis, we constructed a xylanase-producing P. pastoris strain. The fungal xylanase Xyn11A was successfully overexpressed under the constitutive GAP promoter. Biochemical characterization of the xylanase revealed that Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase was stable over a wide range of pH ranging from pH 2 to pH 11. Excellent thermal stability was observed at temperature 60 °C. Enhanced production of Xyn11A was achieved by investigating the effect of carbon source and feeding strategies. The highest xylanase activity was detected at 15000 U/mL using high cell density cultivation. Production of optically pure (2R, 3R)-2, 3-BD was achieved by engineering P. pastoris with a heterologous pathway. The pathway genes consisting of Bacillus subtilis alsS, alsD and S. cerevisiae BDH1 were assembled and transformed into P. pastoris. Cultivation conditions were optimized and the highest titer of 2, 3-BD obtained using YPD media was 45 g/L in fed-batch cultivation. To enhance the economic viability of 2, 3-BD production in P. pastoris, statistical medium optimization was performed. It was found that 75 g/L of 2, 3-BD was produced using optimized media in fed-batch cultivation.
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- 2017
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3. Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review.
- Author
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Yang, Zhiliang and Zhang, Zisheng
- Subjects
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PROTEIN expression , *PICHIA pastoris , *PROMOTERS (Genetics) , *ALCOHOL oxidase , *FERMENTATION , *BIOLOGICAL products - Abstract
Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies. [ABSTRACT FROM AUTHOR]
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- 2018
- Full Text
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4. Codon-optimized expression and characterization of a pH stable fungal xylanase in Pichia pastoris.
- Author
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Yang, Zhiliang and Zhang, Zisheng
- Subjects
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XYLANASES , *PICHIA pastoris , *YEAST fungi , *PROTEIN expression , *POLYACRYLAMIDE gel electrophoresis , *FUNGAL reproduction - Abstract
Novel xylanase (EC 3.2.1.8) is in great demand due to its industrial significance. In this study, we have developed and characterized a novel xylanase-producing yeast strain. This mature xylanase gene xyn 11A consists of 870 base pairs and belongs to GH11 family. The gene sequence was optimized and synthesized, and was then cloned into yeast vector pGAPZαA under the control of the constitutive GAP promoter. SDS-PAGE analysis indicates that Xyn11A is extracellularly expressed as a glycosylated protein in P. pastoris . Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase retained more than 90% of its activity after incubation at 50 °C and 60 °C for up to 1 h. Xyn11A is also stable over a wide range of pH (2.0–11.0). Most metal ions tested such as copper (Cu 2+ ) and lead (Pb 2+ ) have little inhibitory effects on Xyn11A. It is also resistant to pepsin and proteinase K digestion, retaining 80% and 90% of its activity after digestion at 37 °C for 1 h, respectively. Those superior properties make Xyn11A a robust xylanase with great potential for industrial use. To the best of our knowledge, this is the first report of xylanase from the fungus Corynascus thermophilus . [ABSTRACT FROM AUTHOR]
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- 2017
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5. Production of (2R, 3R)-2,3-butanediol using engineered <italic>Pichia pastoris</italic>: strain construction, characterization and fermentation.
- Author
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Yang, Zhiliang and Zhang, Zisheng
- Subjects
AIRCRAFT fuels ,ISOMERS ,KLEBSIELLA pneumoniae ,PLASMIDS ,PICHIA pastoris - Abstract
Background: 2,3-butanediol (2,3-BD) is a bulk platform chemical with various potential applications such as aviation fuel. 2,3-BD has three optical isomers: (2R, 3R)-, (2S, 3S)- and meso-2,3-BD. Optically pure 2,3-BD is a crucial precursor for the chiral synthesis and it can also be used as anti-freeze agent due to its low freezing point. 2,3-BD has been produced in both native and non-native hosts. Several pathogenic bacteria were reported to produce 2,3-BD in mixture of its optical isomers including
Klebsiella pneumoniae andKlebsiella oxytoca . Engineered hosts based on episomal plasmid expression such asEscherichia coli ,Saccharomyces cerevisiae andBacillus subtilis are not ideal for industrial fermentation due to plasmid instability. Results:Pichia pastoris is generally regarded as safe and a well-established host for high-level heterologous protein production. To produce pure (2R, 3R)-2,3-BD enantiomer, we developed aP. pastoris strain by introducing a synthetic pathway. Theals S andals D genes fromB. subtilis were codon-optimized and synthesized. The BDH1 gene fromS. cerevisiae was cloned. These three pathway genes were integrated into the genome ofP. pastoris and expressed under the control of GAP promoter. Production of (2R, 3R)-2,3-BD was achieved using glucose as feedstock. The optical purity of (2R, 3R)-2,3-BD was more than 99%. The titer of (2R, 3R)-2,3-BD reached 12 g/L with 40 g/L glucose as carbon source in shake flask fermentation. The fermentation conditions including pH, agitation speeds and aeration rates were optimized in batch cultivations. The highest titer of (2R, 3R)-2,3-BD achieved in fed-batch fermentation using YPD media was 45 g/L. The titer of 2,3-BD was enhanced to 74.5 g/L through statistical medium optimization. Conclusions: The potential of engineeringP. pastoris into a microbial cell factory for biofuel production was evaluated in this work using (2R, 3R)-2,3-BD as an example. EngineeredP. pastoris could be a promising workhorse for the production of optically pure (2R, 3R)-2,3-BD. [ABSTRACT FROM AUTHOR]- Published
- 2018
- Full Text
- View/download PDF
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