Search

Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida , an essential strategy to develop stable industrial strains producing heterologous bioproducts, requires development of a more efficient method. Current methods rely on time-consuming homologous recombination techniques and transposon-mediated random insertions. Here we report a RecET recombineering system for markerless integration of heterologous genes into the P. putida chromosome. The efficiency and capacity of the recombineering system were first demonstrated by knocking out various genetic loci on the P. putida chromosome with knockout lengths widely spanning 0.6–101.7 kb. The RecET recombineering system developed here allowed successful integration of biosynthetic gene clusters for four proof-of-concept bioproducts, including protein, polyketide, isoprenoid, and amino acid derivative, into the target genetic locus of P. putida chromosome. The markerless recombineering system was completed by combining Cre/ lox system and developing efficient plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida , a bacterial host strain of increasing academic and industrial interest. [ABSTRACT FROM AUTHOR]

Abstract: The yeast species Saccharomyces cerevisiae was engineered to produce 1,2-propanediol (1,2-PD) using the δ/UB sequential gene integration method. To study the effects of increased copy number, 2 genes (mgs and gldA from Escherichia coli) were sequentially integrated into the chromosomes of S. cerevisiae strains of opposite mating type. The resulting strains (containing 0–3 copies of either mgs or gldA) were mated to create all possible combinations of the 2 genes introduced for 1,2-PD production. Enzyme activities were generally correlated with copy number, although there was greater variation in GldA activity in the diploid cells. The integrated genes were confirmed by Southern blot and 1,2-PD production was analyzed by HPLC. The strain containing 3 copies of mgs and gldA showed the highest level of 1,2-PD; however, 1,2-PD concentration was not clearly related to gene copy number. 1,2-PD production did correlate with Mgs specific activity, and high GldA specific activity was found to be inhibitory. [Copyright &y& Elsevier]