Abstract
l-Sorbose is an essential intermediate for the industrial production of vitamin C (l-ascorbic acid). However, the formation of fructose and some unknown by-products significantly reduces the conversion ratio of D-sorbitol to l-sorbose. This study aimed to identify the key D-sorbitol dehydrogenases in Gluconobacter oxydans WSH-003 by gene knockout. Then, a total of 38 dehydrogenases were knocked out in G. oxydans WSH-003, and 23 dehydrogenase-deficient strains could increase l-sorbose production. G. oxydans-30, wherein a pyrroloquinoline quinone-dependent glucose dehydrogenase was deleted, showed a significant reduction of a by-product with the extension of fermentation time. In addition, the highest conversion ratio of 99.60% was achieved in G. oxydans MD-16, in which 16 different types of dehydrogenases were inactivated consecutively. Finally, the gene vhb encoding hemoglobin was introduced into the strain. The titer of l-sorbose was 298.61 g/L in a 5-L bioreactor. The results showed that the systematic engineering of dehydrogenase could significantly enhance the production of l-sorbose.