Integration And Enhancement Of 2-keto-L-Gulonic Acid Synthesis Pathway In Gluconobacter Oxydans

2-keto-L-gulonic acid(2-KLG) is an important precursor of vitamin C. The one-step fermentation route using D-sorbitol as substrate could dramatically decrease product ion costs and is considered as a major technology improvement in vitamin C industry. In order to avoid the issues associated with the use of plasmids, such as the plasmid instability and the heavy reliance on antibiotics feeding in medium, the sorbose dehydrogenase gene(sdh) and sorbosone dehydrogenase gene(sndh) from Ketogulonigenium vulgare WSH-001 were integrated into the genome of Gluconobacter oxydans WSH-003. To further increase the production of 2-KLG, the gene clusters of pyrroloquinoline quinone(PQQ), which is a cofactor of SDH and SNDH, were expressed. The main results were listed as follows:(1) The recombinant strain G. oxydans-ss was constructed to produce 2-KLG. The recombinant gene fragment sr1-Ptuf-sdh-GS-sndh-km-sr2(ss) was obtained by ligating the gene fragments of sr1, Ptuf, sdh-GS-sndh, km and sr2. Then ss was introduced into G. oxydans WSH-003 to construct the recombinant strain G. oxydans-ss by electroporation. The recombinant strain G. oxydans-ss exhibited a 2.3-fold(22.1 g?L-1) and 2.2-fold increase(34.1 g?L-1) in the production of 2-KLG after 168 h fermentation in 500 m L flasks and 1 L fermentor respectively, compared to wild type G. oxydans WSH-003.(2) PQQ was enhanced in the recombinant strain G. oxydans-ss. The gene clusters of pqq A, pqq ABCDE and pqq ABCDEN from K. vulgare WSH-001 were expressed by the plasmid p BBR1MCS-5 under four promoters Ptuf, Ptuf-621, Psod and Paldh, which were derived from G. oxydans. Then the recombinant plasmids were introduced into G. oxydans-ss separately. Expression of pqq genes enhanced the concentration of PQQ and increased the production of 2-KLG. The recombinant strain G. oxydans-ss/Ptuf-pqq A exhibited 52.5% increase(33.7 g?L-1) in the production of 2-KLG after 168 h fermentation in 500 m L flasks, compared to G. oxydans-ss.(3) G. oxydans-ss/Ptuf-pqq A was cultivated to produce 2-KLG in 1 L fermentor. The highest production of 2-KLG was 40.5 g?L-1, which was 7.7% higher than the one produced by G. oxydans-ss after 84 h fermentation. Then the production was declined to 36.7 g?L-1 after 168 h fermentation. During the late stage fermentation, much L-sorbosone was remained in the medium. This may be related with the activity of SNDH. During that time, the cofactor supply was sufficient as the concentration of PQQ was high; the value of p H in the medium was decreased under 4.0. Maybe the acid environment affected the activity of SNDH.