Improvement Of Dihydroxyacetone Production In Gluconobacter Oxydans By Gene Recombination

Dihydroxyacetone (DHA) is an important ketose sugar, which is extensively used in the cosmetic, chemical and pharmaceutical industries. Commercial synthesis of DHA has been realized via the incomplete oxidation of glycerol by G. oxydans, an obligate aerobic Gram-negative bacterium belonging to the family Acetobacteriaceae. In G. oxydans, the enzyme responsible for the oxidative reaction is a membrane-bound, PQQ-dependent glycerol dehydrogenase (GDH) which employs oxygen as the final electron acceptor without NADH involvement. So, the oxidation of glycerol to DHA is a high oxygen-consuming reaction and oxygen should be supplied enough for the production of DHA in a biocatalytic process. In addition, high concentration of substrate and product could also affect DHA production. In order to improve DHA production, Vitreoscilla Hemoglobin (VHb) was expressed to enhance the oxygen uptake of G. oxydans. Besides, the glycerol dehydrogenase was also homologously overexpressed in G. oxydans to facilitate the oxidation of glycerol to DHA.In order to expression of VHb in G. oxydans, the gene vgb coding for VHb was inserted to the broad host range vector pBBR1MCS-5, and then the promoters of tufB, bla, and tac were inserted into the upstream of VHb gene respectively. When the VHb gene was controlled by the constitutive promoter of tufB, the VHb-expressing strain displayed the highest VHb expression level, reaching 19.0nmol/g cell wet weight determined by analysis of CO-difference spectra.The expression of VHb improved the cell growth and DHA production in G. oxydans. Under low aeration conditions, the VHb+ strain displayed 19.27% and 22.96% more biomass compared to VHb- strain when cultured in sorbitol medium and glycerol medium in a 5-L fermentor. In a resting cell system, the DHA production was increased by 21.55%, from 49.65g/L to 60.35g/L, when 100g/L glycerol was supplied. In compared to the control strain, the VHb+ strain exhibited 35.39% more DHA production attributing to high oxygen uptake and more cell biomass in a fermentation process. Results also indicated that the expression of VHb could not increase the GDH expression level, and the enhancement of DHA production by VHb only resulted from the improvement of oxygen uptake rate. The Km and Vmax for glycerol of GDH were determined in whole cells of VHb+ and VHb- strains under both high and low aeration conditions. It was apparent that the presence of VHb increased the enzyme's Vmax by 39.92% to 62.46% and decreased the Km by 19.15% to 23.48%.When the GDH gene was overexpressed in the wild strain G. oxydans M5, the GDH activity was increased by 26% and the DHA production was improved by 20% in shake flask test. Unexpectedly, the activity of GDH was increased by 74.57% when the GDH gene was overexpressed in M5AM strain, in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH) was interrupted. Overproduction of GDH in M5AM strain led to a 2.28-fold increased DHA productivity of 2.39g/g CDW/h using a batch biotransformation process in a 5-L fermentor, yielding 96.74g/L DHA from 100g/L glycerol. When 140g/L glycerol was supplied, all the glycerol could be exhausted by the recombinant strain within 14h, and a final DHA concentration of 133.81g/L was accumulated. In repeated batch biotransformations, 385.32g/L DHA over a time period of 34h was achieved, with an average productivity of 2.27g/g CDW/h. However, when the initial glycerol concentration was increased to 180g/L, the DHA reached an approximately maximum concentration of about 152.55g/L after 18 h. Prolonged incubation for another 10h had little effect on DHA yield, so did increase in concentration of resting cells from 20g/L to 40g/L. High concentration of DHA could not result in cell lysis but probably lead to irreversible damage of GDH by interacting with the amine function localized on the enzymatic enzyme site. So, the glycerol at an initial concentration of 180g/L could not be oxidated completely and only a DHA yield of 87.4% was achieved after 18h bioconversion. In a word, this newly developed recombinant strain G. oxydans M5AM/GDH with high productivity and yield exhibits potential for the industrial production of DHA.Both the expression of VHb and GDH gene could improve the production of DHA, so the two genes were co-expressed in G.oxydans M5, generating strain G.oxydans M5/TVTG. Analysis by CO-difference spectra showed that the expression level of VHb in M5/TVTG strain was 17.01nmol/g cell wet weight. And this recombinant strain displayed a GDH activity of 2.16U/g protein, 15.51% more than that of M5/pBBR strain. When biotransformation was performanced in a fermentor under low aeration conditions, the M5/TVTG strain exhibited a rapid oxidation of glycerol rate and 63.38g/L DHA was accumulated after 48h, 27.65% more than that of the M5/pBBR strain.