Effects On 1,3-Dihydroxyacetone Production By Overexpression Of Glycerol Uptake Facilitator And Glycerol Dehydrogenase

Dihydroxyacetone(DHA)is the simplest ketose,has been widely used in food,chemical,cosmetics and other fields.Dihydroxyacetone was produced by glycerol through the incomplete oxidation of Gluconbacter oxydans.Gluconbacter oxydans is an obligate aerobic,gram-negative bacteria.In the process of DHA production,oxygen,substrate inhibition and product inhibition are important influencing factors.Therefore,in order to improve the production efficiency of dihydroxyacetone,we overexpressed glycerol uptake facilitator protein(glpF)and glycerol dehydrogenase(GDH)in Gluconbacter oxydans,the main contents are as follows:1 Overexpressed glpF in Gluconbacter oxydans and studied its effect on cell growth.To improve the tolerance of glycerol,we overexpress glpF controlled by four different promoters in strains.After RT-PCR,we found that the transcriptional level of glpF controlled by its own length promoter,tuf promoter,its own short promoter,gmr promoter are 10-fold,10-fold,5-fold and 3-fold than the original bacteria respectly.When concentration of glycerol was 100 g/L,its final biomass OD600 were 4.8?4.7?4.2?3.5,and the OD600 of original bacteria was 2.5.With the rise of glycerol concentration,the original bacteria growth was severely inhibited,while 250 g/L glycerol,original bacteria basically stopped growing,and engineered bacteria could survive,expression of the enzyme could effectively improve the tolerance of bacteria to glycerol.2 overexpressing of glycerol dehydrogenase and its effect on growth of bacterial cells and the production of DHAIn this experiment,overexpression was controlled by tufB promoter and promoter of glycerol dehydrogenase.It was found that expression of the glycerol dehydrogenase tuf B promoter and the promoter itself is substantially the same,as about 5 times to the original strain,the glycerol dehydrogenase activity is also increased from the 1.74 U/mg(original bacteria)to 2.25 U/mg and 2.14 U/mg.After overexpression of glycerol dehydrogenase,growth of recombinant bacteria in seed medium and 100 g/L glycerol medium bacteria is basically the same as the original.When at 100 g/L concentration of glycerol,it was found that improvement of DHA is about 8 g/L,about 12.5%.3 double overexpression of glycerol transport proteins and glycerol dehydrogenase in the cells and its impact of the production of DHATo improve the yield of DHA,we overexpressed glycerol dehydrogenase and glycerol transport proteins in bacterial cells,the study shows that while the single expression of a protein could improve the tolerance to glycerol and promote cell growth;single expression of dehydrogenation of glycerol enzyme could increase production of dihydroxyacetone,but production of dihydroxyacetone was not significantly improved with the over-expression of both membrane-bound enzymes.To be carried out with next experiment.