Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids)

Abstract

A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 x 10(5) CFUs/microgram plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans.

Prediction and optimization of gene transfection and drug delivery by electroporation

“…Gene Therapy determined experimentally that long, low-voltage pulses give the greatest transfection, 43 in agreement with our prediction. In contrast, clinical electrochemotherapy protocols have used multiple, short pulses at moderate field strengths.…”

“…Based on comparison with five studies, correlation predictions of peak transfection conditions in vivo [39][40][41][42][43] were of similar accuracy as for cell suspensions (MAPE = 51 ± 53%), which suggests that this correlation may be of importance in developing clinical applications. Predictions of peak transfection conditions for bacteria and yeast 22, [44][45][46][47][48] (MAPE = 48 ± 62%) were not as good as for mammalian cells, but predictions of 50% viability conditions were similarly accurate (MAPE = 21 ± 8%) (eg Figure 2d).…”

Biodegradation of the xenobiotic organic disulphide 4,4′-dithiodibutyric acid by Rhodococcus erythropolis strain MI2 and comparison with the microbial utilization of 3,3′-dithiodipropionic acid and 3,3′-thiodipropionic acid

“…Electroporation of Rhodococcus erythropolis strain MI2 was accomplished based on the methods described by Desomer et al (1990) and Kalscheuer et al (1999).…”

Metabolic engineering of microorganisms to produce omega-3 very long-chain polyunsaturated fatty acids

“…the average yield of 2.5% of total seed fatty acids) [24]. 142 Subsequently, the same authors also described the successful 143 reconstruction of the EPA and DHA biosynthetic pathway in the 144 seeds of an oilseed crop, C. sativa. They successfully engineered this 145 species to accumulate high levels of x3-VLCPUFAs in its seed oils 146 and yielded two iterations, in which one accumulated EPA up to 147 31% of the seed oils and the other accumulated up to 12% EPA 148 and 14% DHA in the seed oils [25].…”