Production of mannitol from molasses and Jerusalem artichoke tubers at elevated temperatures using engineered <i>Kluyveromyces marxianus</i> through consolidated bioprocessing.

Abstract

In this study, an engineered <i>Kluyveromyces marxianus</i> 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 (<i>CmMDH</i>) from <i>Candida magnoliae</i> identified as the most effective enzyme. Mannitol production was further enhanced by increasing the copy number of <i>CmMDH.</i> Among the four tested NADPH synthesis-related genes, overexpression of <i>KmUTR1</i> 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.