Functional Identification Of Subunit ? Of Membrane-bound Alcohol Dehydrogenase And Enhancement Of The Hydroxy Acid Production Capacity Of Gluconobacter Oxydans

A large amount of membrane-bound alcohol dehydrogenase (mADH) is located on the peripalsmic side of the cytoplasmic membrane in Gluconobacter oxydans, and could incompletely oxidize a wide range of primary alcohols and diols to the corresponding acids or hydroxy acids. Based on earlier studies, mADH might be related to the cyanide-resistant bypass respiratory chain of G. oxydans, and mediates the electron transfer from another primary membrane-bound D-glucose dehydrogenase through the subunit ? of mADH to ubiquinone. In our previous study, we have purified and characterized the subunit I and subunit ? of mADH from G. oxydans DSM 2003, which are encoded by adhA and adhB, respectively. This study aimed to identify the subunit ? of mADH in G oxydans DSM 2003, investigate its function and rationally enhance the hydroxyl acid production capacity.Firstly, identification and functional analysis of the subunit ? of mADH. We found the putative adhS from the genome of G. oxydans DSM 2003 through sequence alignment, and constructed three differential overexpression strains G. oxydans-adhS, G oxydans-adhABS, and G. oxydans-adhAB. By comparison of the transcription levels, the expression levels, and the enzyme activities of mADH, we found that adhS overexpression could promote the subunit ? which serves as the primary dehydrogenase transfer from the periplasmic space to the periplasmic surface of the membrane, increase the amount of active mADH and thus enhance the mADH activity. For mADH activity, the subunit ? might be a limiting component in quantitySecondly, effects of mADH overexpression on the hydroxyl acid production capacity. For the conversion of 1,2-propanediol in shake flasks, G. oxydans-adhABS could increase D-lactic acid production by 53.8%, and the space-time yield of D-lactic acid increased from 0.65 g/(1·h) to 1.0 g/(1·h). For the conversion of ethylene glycol in shake flasks, G oxydans-adhABS could increase glycolic acid production by 164%, and the space-time yield of glycolic acid increased from 0.3 g/(1·h) to 0.78 g/(1·h). Therefore, overexpression of mADH could greatly enhance the hydroxyl acid production capacity of G. oxydans. Fed-batch bioconversion of ethylene glycol was performed in a 7 L bioreactor. The pH was controlled automatically by computer, and dissolve oxygen was controlled by increasing agitation speed, airflow and bioreactor pressure. The concentration of ethylene glycol was kept by controlling the feeding rate. After 45 h of bioconversion, G. oxydans-adhABS could produce 73.3 g/1 glycolic acid with a molar yield of 93.5%, and the space-time yield was 1.63 g/(l·h). Fed-batch fermentation of ethylene glycol for glycolic acid production was also carried out in a 7 L bioreactor. After 45 h of fermentation,113.8 g/1 glycolic acid was obtained with a molar yield of 92.9% in G. oxydans-adhABS, and the space-time yield of glycolic acid increased to 2.53 g/(l·h), which is the highest reported glycolic acid yield to date.Thirdly, effects of mADH overexpression on the respiratory chain and other membrane-bound dehydrogenases. The transcription level of respiratory chain components except NADH was significantly up-regulated in the overexpression strain G. oxydans-adhABS, especially the two terminal oxidases (bo3 and bd). When cultivated in a 7 L bioreactor, G. oxydans-adhABS showed a higher OUR in the exponential phase, and the biomass was increased by 26%-33% after 36 h of culture. Moreover, the transcription levels and enzyme activities of other major membrane-bound dehydrogenases in G. oxydans-adhABS were also increased to some extent, especially membrane-bound aldehyde dehydrogenase (mALDH), which was 26% higher. It might be beneficial to produce organic acid.Fourthly, effects of the differential expression of mADH on the product selectivity during glycerol oxidation. It has been reported that mADH is essential for the conversion of glycerol to glyceraldehyde. Based on the different mADH activity, only approximately 30% of glycerol was catalyzed by G. oxydans-adhS and G. oxydans-adhABS, but the flux of glycerol to glyceric acid in the two strains was obviously increased 7.6- and 7.9-fold, respectively. On the contrary, up to 94% of glycerol was catalyzed by G. oxydans-adhAB, and the flux of glycerol to dihydroxyacetone was increased to 92.6%. It was demonstrated that the differential expression of the three mADH subunits could significantly alter product selectivity during glycerol oxidation.