| Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases. | |
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MedLine Citation:
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PMID: 20038708 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-L-arginyl-poly-L-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA(6308)) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA(6308) and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA(6308)Delta1, which was truncated by one amino acid at the C terminus; point mutated CphA(6308)C595S; and the combined double-mutant CphA(6308)Delta1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA(6308) (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA(6308)Delta2) or three (CphA(6308)Delta3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA(6308). In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA(6308)Delta1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26 degrees C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308) and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308)Delta1. |
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Authors:
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Anna Steinle; Sabrina Witthoff; Jens P Krause; Alexander Steinbüchel |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2009-12-28 |
Journal Detail:
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Title: Applied and environmental microbiology Volume: 76 ISSN: 1098-5336 ISO Abbreviation: Appl. Environ. Microbiol. Publication Date: 2010 Feb |
Date Detail:
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Created Date: 2010-02-08 Completed Date: 2010-04-02 Revised Date: 2010-09-28 |
Medline Journal Info:
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Nlm Unique ID: 7605801 Medline TA: Appl Environ Microbiol Country: United States |
Other Details:
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Languages: eng Pagination: 1062-70 Citation Subset: IM |
Affiliation:
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Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Bacterial Proteins
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chemistry,
genetics*,
metabolism* Base Sequence Cloning, Molecular DNA, Recombinant / genetics Escherichia coli / enzymology, genetics Fermentation Genes, Bacterial Hydrogen-Ion Concentration Kinetics Mutagenesis, Site-Directed Peptide Synthases / chemistry, genetics*, metabolism* Pichia / genetics*, growth & development, metabolism* Plant Proteins / biosynthesis* Protein Engineering Recombinant Proteins / chemistry, genetics, metabolism Synechocystis / enzymology, genetics Temperature |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Proteins; 0/DNA, Recombinant; 0/Plant Proteins; 0/Recombinant Proteins; 0/cyanophycin; EC 6.3.2.-/Peptide Synthases; EC 6.3.2.-/cyanophycin synthase, bacteria |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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