Direct fermentation of 2‐keto‐l‐gulonic acid in recombinant Gluconobacter oxydans

Abstract

We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from D-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of L-sorbosone and 2-KLGA, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine resulted in improvement of the production level.

Mutation of Gluconobacter oxydans and Bacillus megaterium in a two-step process of l-ascorbic acid manufacture by ion beam

“…The general approaches for the improvement of 2‐KLG production is to generate recombinant strains using genetic engineering methods, but the yield of 2‐KLG is still insufficient for industry application (Shinjoh et al. 1995; Saito et al. 1998).…”

Contributions of Microorganisms to Industrial Biology

“…A recombinant strain of Gluconobacter oxydans containing genes encoding L-sorbose dehydrogenase and L-sorbosone dehydrogenase from G. oxydans T-100 is an improved producer of 2-KGA [63]. Cloning of the gene encoding Dsorbitol dehydrogenase from Gluconobacter suboxydans G24 into Pseudomonas putida IFO 3738, along with the above two genes and other genetic manipulations led to production of 2-KGA from D-sorbitol [64] A genetically engineered strain of Serratia herbicola produces 120 g/l of 2-KGA and another G. oxydans strain makes 130 g/l [65].…”

Gluconobacter in biosensors: applications of whole cells and enzymes isolated from gluconobacter and acetobacter to biosensor construction

“…Regioselective oxidations by Gluconobacter have been used to produce a number of chiral compounds (Kelliang and Dongzhi 2006), but yet no biosensors. Also, reports on preparing recombinant strains (Saito et al 1998) or mutants deficient in an enzymatic activity (Gupta et al 1999), both aimed to improve fermentation production, have appeared repeatedly. The knowledge of the genome sequence (Prust et al 2005) will likely lead to novel applications of Gluconobacter in biotransformations and biosensors.…”