AUTHOR=Yang Shuling , Pan Xuewei , Wang Qiang , Lv Qinglan , Zhang Xian , Zhang Rongzhen , Rao Zhiming 

TITLE=Enhancing erythritol production from crude glycerol in a wild-type Yarrowia lipolytica by metabolic engineering

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.1054243

DOI=10.3389/fmicb.2022.1054243

ISSN=1664-302X

ABSTRACT=<p><bold>Background:</bold> Erythritol is a zero-calorie sweetener that is widely used in the food, pharmaceutical, and medical industries. Crude glycerol is the main by-product of biodiesel, and the effective utilization of crude glycerol will help to improve biodiesel viability. Previous studies on the production of erythritol from <italic>Y. lipolytica</italic> using crude glycerol as a carbon source have focused on optimizing the fermentation process of the mutant <italic>Y. lipolytica</italic> Wratislavia K1, while metabolic engineering has not been successfully applied.</p><p><bold>Results:</bold> To this end, we engineered the yeast <italic>Y. lipolytica</italic> to increase the productivity of this strain. Wild strains tolerant to high concentrations of crude glycerol were screened and identified. A series of rational metabolic approaches were employed to improve erythritol production. Among them, the engineered strain Y-04, obtained by tandem overexpression of <italic>GUT1</italic> and <italic>GUT2</italic>, significantly increased glycerol assimilation by 33.3%, which was consistent with the results of RT-qPCR analysis. The effects of tandem overexpression of <italic>GUT1</italic>, <italic>GUT2</italic>, <italic>TKL1,</italic> and <italic>TAL1</italic> on erythritol synthesis were also evaluated. The best results were obtained using a mutant that overexpressed <italic>GUT1</italic>, <italic>GUT2,</italic> and <italic>TKL1</italic> and knocked out <italic>EYD1</italic>. The final Y-11 strain produced 150 g/l erythritol in a 5-L bioreactor with a yield and productivity of 0.62 g/g and 1.25 g/l/h, respectively. To the best of our knowledge, this is the highest erythritol yield and productivity from crude glycerol ever reported in <italic>Y. lipolytica</italic>.</p><p><bold>Conclusion:</bold> This work demonstrated that overexpression of <italic>GUT1</italic>, <italic>GUT2,</italic> and <italic>TKL1</italic> and knockdown of <italic>EYD1</italic> could be used to improve crude glycerol utilization and erythritol synthesis in <italic>Y. lipolytica</italic>. The process parameters such as erythritol yield and productivity were significantly elevated, which is valuable for industrial applications. Crude glycerol, as a carbon source, could efficiently restrict the synthesis of by-products while enhancing the generation of erythritol, compared to glucose. This indicates considerable potential for synthesizing value-added products from crude glycerol by <italic>Y. lipolytica</italic>.</p>