Establishment of a novel anabolism-based addiction system with an artificially introduced mevalonate pathway: Complete stabilization of plasmids as universal application in white biotechnology

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Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-L-arginylpoly-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 ⌬1, which was truncated by one amino acid at the C terminus; point mutated CphA 6308 C595S; and the combined double-mutant CphA 6308 ⌬1C595S 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 ⌬2) or three (CphA 6308 ⌬3) 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 ⌬1 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 CphA 6308 and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA 6308 ⌬1.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases

Steinle

Witthoff

Krause

et al. 2010

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“…In addition, the feeding solution contained 50 g/liter kanamycin to maintain the plasmid stability of the culture. In order to investigate the plasmid stability of the cultivated recombinant S. blattae strain, diluted samples of the fermentation broth were spread on LB agar plates containing kanamycin as well as on LB agar plates lacking kanamycin, and the plates were then incubated at 30°C for 24 h. Plasmid stability was determined by comparing the numbers of CFU on LB agar plates without kanamycin with those on LB agar plates containing kanamycin (32).…”

Section: Methodsmentioning

confidence: 99%

From Waste to Plastic: Synthesis of Poly(3-Hydroxypropionate) in Shimwellia blattae

Heinrich¹,

Andreeßen²,

Madkour

et al. 2013

bIn recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a byproduct of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ؎ 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fedbatch fermentation process.

“…Plasmid instability is mainly due to the segregational plasmid loss during cell division that was observed in the second, third, and fourth cultivations, which had longer incubation periods than the first cultivation. Strategies to improve the stability of recombinant plasmids to achieve high density of cells without losing the ability of product formation are being investigated in our laboratory (25).…”

Section: Vol 76 2010mentioning

confidence: 99%

Pilot-Scale Production of Fatty Acid Ethyl Esters by an Engineered Escherichia coli Strain Harboring the p(Microdiesel) Plasmid

Elbahloul

Steinbüchel

2010

Fatty acid ethyl esters (FAEEs) were produced in this study by the use of an engineered Escherichia coli p(Microdiesel) strain. Four fed-batch pilot scale cultivations were carried out by first using glycerol as sole carbon source for biomass production before glucose and oleic acid were added as carbon sources. Cultivations yielded a cell density of up to 61 ؎ 3.1 g of cell dry mass (CDM) per liter and a maximal FAEE content of 25.4% ؎ 1.1% (wt/wt) of CDM.