ArticleApplied and environmental microbiology2010
Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases.
Article in Applied and environmental microbiology, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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8 citing papers in PubMed, 42 citations in OpenAlex.
- A cryptic third active site in cyanophycin synthetase creates primers for polymerization.Nature communications · 2022Article
- Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum.AMB Express · 2021Article
- Cyanophycin Modifications-Widening the Application Potential.Frontiers in bioengineering and biotechnology · 2021Review
- Active Expression of Human Tissue Plasminogen Activator (t-PA) c-DNA from Pulmonary Metastases in the Methylotrophic Yeast Pichia Pastoris KM71H StrainAsian Pacific journal of cancer prevention : APJCP · 2017Article
- Increased lysine content is the main characteristic of the soluble form of the polyamide cyanophycin synthesized by recombinant Escherichia coli.Applied and environmental microbiology · 2013Article
- Autotrophic production of stable-isotope-labeled arginine in Ralstonia eutropha strain H16.Applied and environmental microbiology · 2012Article
- Production of cyanophycin in Rhizopus oryzae through the expression of a cyanophycin synthetase encoding gene.Applied microbiology and biotechnology · 2012Article
- Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1.AMB Express · 2011Article
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4 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
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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