Construction of Halomonas bluephagenesis capable of high cell density growth for efficient PHA production.

Abstract

High-cell-density cultivation is an effective way to improve the productivity of microbial fermentations and in turn reduce the cost of the final products, especially in the case of intracellular products. Halomonas bluephagenesis TD01 is a halophilic platform bacterium for the next generation of industrial biotechnology with a native PHA synthetic pathway, able to grow under non-sterile continuous fermentation conditions. A selection strategy for mutant strains that can grow to a high cell density was developed. Based on an error-prone DNA polymerase III ε subunit, a genome-wide random mutagenesis system was established and used in conjunction with an artificial high cell density culture environment during the selection process. A high-cell-density H. bluephagenesis TDHCD-R3 obtained after 3 rounds of selection showed an obvious enhancement of resistance to toxic metabolites including acetate, formate, lactate and ethanol compared to wild-type. H. bluephagenesis TDHCD-R3-8-3 constructed from H. bluephagenesis TDHCD-R3 by overexpressing an optimized phaCAB operon was able to grow to 15g/L cell dry weight (CDW) containing 94% PHA in shake flask studies. H. bluephagenesis TDHCD-R3-8-3 was grown to more than 90g/L CDW containing 79% PHA compared with only 81g/L with 70% PHA by the wild type when incubated in a 7-L fermentor under the same conditions.