Metabolic Engineering Study on the Direct Fermentation of 2-Keto-_L-Gulonic Acid, a Key Intermediate of _L-Ascorbic Acid in Pseudomonas putida IFO3738

概要

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We have achieved production of 2-keto-_L-gulonic acid (2-KLGA) in recombinant Pseudomonas putida IFO3738. Firstly, the genes for sorbose dehydrogenase (SDH)/sorbosone dehydrogenase (SNDH) were introduced into P.putida. The recombinant P.putida/pBBR-SDH produced 0.7 mg/ml of 2-KLGA in a culture broth containing 5% _L-sorbose. Replacement of the native SNDH promoter by the Escherichia coli tufB promoter (pBBR-SDH-tufB) improved the productivity of 2-KLGA up to 11.4 mg/ml. Secondly, the sorbitol dehydrogenase (SLDH) gene was also introduced into P.putida. The recombinant P.putida/pUCP19-3DH carrying the genes for SDH, SNDH and SLDH had the ability to produce 2-KLGA (7.5mg/ml) in a 5% _D-sorbitol broth. The productivity of 2-KLGA was improved up to 9.8 mg/ml by changing to an expression system with two plasmids, pBBR-SDH-tufB (for SDH/SNDH) and pUCP19-SLDH(for SLDH), respectively. Moreover, the raplacement of the native SLDH promoter by the E.coli tufB promoter (pUCP19-SLDH-tufB) improved the 2-KLGA productivity up to 11.6 mg/ml. Optimization of cultivation conditions increased the conversion yield of 2-KLGA to 32% and that of _L-idonate, a metabolite of 2-KLGA, to 40%. These results indicate P.putida IFO3738 is one of the candidate strains for direct fermentation of 2-KLGA.
社団法人日本生物工学会の論文
2000-08-25