AUTHOR=Wang Yanhong , Liu Wei , Chen Jingyao , Li Zhijiang , Hu Yijia , Fan Zixiang , Yan Liangyuan , Liu Jiahui , Zhou Yuao , Jiang Wei , Rui Haiying , Dai Lingyan 

TITLE=Overexpression of the FBA and TPI genes promotes high production of HDMF in Zygosaccharomyces rouxii

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1366021

DOI=10.3389/fmicb.2024.1366021

ISSN=1664-302X

ABSTRACT=<p>4-Hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) is widely used in the food industry as a spice and flavoring agent with high market demand. In this study, fructose-1,6-bisphosphate aldolase (FBA) and triose phosphate isomerase (TPI) were overexpressed in <italic>Zygosaccharomyces rouxii</italic> in the form of single and double genes, respectively, via electroporation. High-yield HDMF-engineered yeast strains were constructed by combining the analysis of gene expression levels obtained by real-time fluorescence quantitative PCR technology and HDMF production measured by HPLC. The results showed that there was a significant positive correlation between the production of HDMF and the expression levels of the <italic>FBA</italic> and <italic>TPI</italic> genes in yeast; the expression levels of the <italic>FBA</italic> and <italic>TPI</italic> genes were also positively correlated (<italic>p</italic> &lt; 0.05). Compared with the wild type (WT), the engineered strains F10-D, T17-D, and TF15-A showed marked increases in HDMF production and <italic>FBA</italic> and <italic>TPI</italic> gene expression (<italic>p</italic> &lt; 0.05) and exhibited great genetic stability with no obvious differences in biomass or colony morphology. In addition, the exogenous addition of <sc>d</sc>-fructose promoted the growth of <italic>Z. rouxii</italic>. Among the engineered strains, when fermented in YPD media supplemented with <sc>d</sc>-fructose for 5 days, TF15-A (overexpressing the <italic>FBA</italic> and <italic>TPI</italic> genes) generated the highest HDMF production of 13.39 mg/L, which is 1.91 times greater than that of the wild-type strain. The results above indicated that FBA and TPI, which are key enzymes involved in the process of HDMF biosynthesis by <italic>Z. rouxii</italic>, positively regulate the synthesis of HDMF at the transcriptional level. <sc>d</sc>-fructose can be used as a precursor for the biosynthesis of HDMF by engineered yeast in industrial production.</p>