Improving glycerol utilization during high-temperature xylitol production with Kluyveromyces marxianus using a transient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system

Glycerol is an ideal co-substrate for xylitol production with Kluyveromyces marxianus. This study demonstrated that K. marxianus catabolizes glycerol through the Gut1-Gut2 pathway instead of the previously speculated NADPH-dependent Gcy1-Dak1 pathway using the transient clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system. Additionally, Utr1p was demonstrated to mediate NADPH generation through NADH phosphorylation. YZB392, which was constructed by integrating Utr1 into the Ypr1 site in the strain overexpressing NcXyl1 and CiGxf1 and harboring disrupted Xyl2, exhibited enhanced glycerol utilization for xylitol production (from 2.50- to 3.30- g/L after consuming 1 g/L glycerol). Fed-batch fermentation at 42 °C with YZB392 yielded 322.07 g/L xylitol, which is the highest known xylitol titer obtained via biological method. Feeding crude glycerol, xylose mother liquor, and corn steep liquor powder into a bioreactor resulted in the production of 235.69 g/L xylitol. This study developed a platform for xylitol production from industrial by-products.