دانلود رایگان مقاله ساخت ژنتیکی کلرورافیس نوترکیب سودوموناس

Abstract

This study is to use genetic engineering to improve the glycerol metabolic capability of Pseudomonas chlororaphis which is capable of producing commercially valuable biodegradable poly(hydroxyalkanoate) (PHA) biopolymers and biosurfactant rhamnolipids (RLs). In the study, the glycerol uptake facilitator or aquaglyceroporin gene (glpF) and the glycerol kinase (glpK) gene were PCR-cloned from E. coli, inserted into a shuttle vector pBS29P2-gfp, and expressed in P. chlororaphis by a Pseudomonas promoter P2. The P. chlororaphis recombinants were then tested for cell growth and glycerol metabolism in chemically defined medium containing 0.5% and 1.0% (v/v) glycerol. The simultaneous expression of glpF and glpK resulted in a shorter lag time for cell growth and a more immediate glycerol consumption by P. chlororaphis. In conclusion, the recombinant P. chlororaphis that grows more efficiently in glycerol is expected to improve the technoeconomics of PHA and RL production using the surplus bioglycerol byproduct stream from biodiesel production.

3. Results and discussion

Table 1 shows the PCR primers used to amplify glpF (glycerol uptake facilitator or aquaglyceroporin), glpK (glycerol kinase), glpD (glycerol 3-phosphate dehydrogenase), and the contiguous glpFK genes from E. coli K12. These amplicons were spliced using an In while the growth of [-gfp] Control and [-glpF] was only beginning with an A600 nm of ca. 1. The glycerol consumption curves (Fig. 3B) show that P. chlororaphis [pBS29P2-glpFK] started to consume glycerol at a much earlier time than the other two strains, nearly exhausting the glycerol at ca. 30 h while the other two cultures still contained ca. 40% of the substrate. From these data, it is apparent that P. chlororaphis [pBS29P2-glpFK] has a cell growth and glycerol utilization advantage compared to the other two strains. We next assessed the advantageous properties of P. chlororaphis [pBS29P2-glpFK] in MSM þ1.0% (w/v) glycerol (MSMþG1.0). The results showed that the glpFK-expressing cells started to grow at a much earlier time-point (ca. 11 h) than the other two strains (ca. 24–27 h), reaching a stationary phase at ca. 37 h as opposed to ca. 49 h for the control and the glpF-expressing strains (Fig. 3C). Fig. 3D shows that at 22 h, half of the initial glycerol had been consumed by P. chlororaphis [pBS29P2-glpFK]. In contrast,o20% of the glycerol was consumed by P. chlororaphis [pBS29P2-gfp] or [pBS29P2-glpF] at 22 h. As expected, MSMþG1.0 with more substrate afforded a higher cell mass at final A600 nm values of 6 (Fig. 3C), while the final A600 nm's in MSMþG0.5 were only 3 (Fig. 3A).