Production Of1,3-dihydroxyacetone From Glycerol By Gluconobacter Oxydans WD

1,3-Dihydroxyacetone (DHA) is an important organic widely used in pharmaceuticalsindustry and mainly produced from glycerol by genus Gluconobacter and Acetobacter.In this study, a DHA-producing strain WD stored in our lab was identified to be Glu-conobacter by multiphase classification. The cultural and fermentation conditions of strainWD were optimized. In addition, the sorbitol dehydrogenase of strain WD was also investi-gated. The results of this thesis were summarized as follow:1. The morphology, physiology of the strain WD were analyzed and the phylogenetictree was established according to16S rDNA sequence alignment. Based on these results, theDHA-producing strain WD was identified as Gluconobacter oxydans WD.2. The cultural and fermentation conditions of G. oxydans WD were optimized in shake-flask. The largest biomass (OD6006.0) was harvested when sorbitol was used as carbon source,and20g L-1yeast extraction as nitrogen source. Moreover,95g L-1DHA was obtained underthe condition of100g L-1glycerol,30g L-1yeast extraction and5.0g L-1CaCO3with5%in-oculation volume. The yield of DHA was improved in fermenter.120g L-1DHA was obtainedin batch fermentation while140g L-1DHA was obtained in fed-batch fermentation.3. According to the published sequences of sorbitol dehydrogenase, primers were de-signed and used to amplify the two coding genes sldA and sldB in the strain G. oxydans WD.The target genes were cloned to T-vector and sequenced, respectively. The sequences of thesetwo sorbitol dehydrogenase genes of G. oxydans WD were highly consistent with G. oxydans621H. Then, the two coding genes of sorbitol dehydrogenase of G. oxydans WD were ex-pressed in Escherichia coli C43. The recombinants carrying the sldB and sldA were proved tohave the ability to produce DHA from glycerol. In addition, the SD sequence of sldA, whichlocated in the end of sldB could play a equal role as the SD sequence of pET28a during trans-lation. We also found that the interval sequence downstream of SD sequence could affect thetranslation of Enhanced Green Fluorescent Protein (EGFP).