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. CphA6308Δ1, which was truncated by one amino acid at the C terminus; point mutated CphA6308C595S; and the combined double-mutant CphA6308Δ1C595S 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 CphA6308 (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA6308Δ2) or three (CphA6308Δ3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA6308. In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA6308Δ1 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°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 CphA6308 and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA6308Δ1.
Recombinant Ganoderma lucidum Immunomodulatory Protein Improves the Treatment for Chemotherapy-Induced Neutropenia