Abstract

In this study, an engineered Kluyveromyces marxianus strain with enhanced mannitol biosynthesis capacity was developed, enabling efficient conversion of molasses and Jerusalem artichoke tubers into mannitol via consolidated bioprocessing. An efficient mannitol-producing strain was constructed by screening of five mannitol synthesis-related genes, with mannitol dehydrogenase ( CmMDH ) from Candida magnoliae identified as the most effective enzyme. Mannitol production was further enhanced by increasing the copy number of CmMDH. Among the four tested NADPH synthesis-related genes, overexpression of KmUTR1 led to an additional increase in mannitol production. For the final engineered strain Y2CmMDH-KmUTR1, aeration enhancement and pH control substantially increased mannitol production. When employing fructose and glycerol as carbon sources, the maximum mannitol titer reached 175.43 ± 2.02 g/L in single-feed batch fermentation, with peak productivity of 5.26 ± 0.04 g/(L·h) achieved in two-feed batch fermentation. The Y2CmMDH-KmUTR1 strain produced 47.96 ± 0.65 g/L mannitol from non-sterilized molasses at 42 °C, which is higher than that reported in other studies, and produced 48.30 ± 1.18 g/L mannitol from non-sterilized Jerusalem artichoke tubers (JAT) at 42 °C through consolidated bioprocessing. To the best of our knowledge, this is the first successful demonstration of mannitol production at 42 °C and producing mannitol from JAT through consolidated bioprocessing. The developed strain exhibits significant industrial potential, particularly for valorizing agricultural waste molasses and Jerusalem artichoke tubers.