Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

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Di-D-fructofuranosyl 2,6':2',6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.