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Efficient production of 2'-fucosyllactose in unconventional yeast Yarrowia lipolytica.

ABSTRACT: 2'-Fucosyllactose (2'-FL) has great application value as a nutritional component and the whole cell biosynthesis of 2'-FL has become the focus of current research. Yarrowia lipolytica has great potential in oligosaccharide synthesis and large-scale fermentation. In this study, systematic engineering of Y. lipolytica for efficient 2'-FL production was performed. By fusing different protein tags, the synthesis of 2'-FL was optimized and the ubiquitin tag was demonstrated to be the best choice to increase the 2'-FL production. By iterative integration of the related genes, increasing the precursor supply, and promoting NADPH regeneration, the 2'-FL synthesis was further improved. The final 2'-FL titer, 41.10 g/L, was obtained in the strain F5-1. Our work reports the highest 2'-FL production in Y. lipolytica, and demonstrates that Y. lipolytica is an efficient microbial chassis for the synthesis of oligosaccharides.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC10694633 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Efficient production of 2'-fucosyllactose in unconventional yeast Yarrowia lipolytica.

Zhang Yan Y Zhang Xuejing X Liu Haiyan H Hou Jin J Liu Mengmeng M Qi Qingsheng Q

Synthetic and systems biotechnology 20231115 4

2'-Fucosyllactose (2'-FL) has great application value as a nutritional component and the whole cell biosynthesis of 2'-FL has become the focus of current research. Yarrowia lipolytica has great potential in oligosaccharide synthesis and large-scale fermentation. In this study, systematic engineering of Y. lipolytica for efficient 2'-FL production was performed. By fusing different protein tags, the synthesis of 2'-FL was optimized and the ubiquitin tag was demonstrated to be the be ...[more]

PMID: 38053583

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Project description:BackgroundMycosporine-like amino acids (MAAs), including shinorine and porphyra-334, are gaining attention as safe natural sunscreens. The production of MAAs has been achieved in diverse microbial hosts, including Saccharomyces cerevisiae. While S. cerevisiae is the most extensively studied model yeast, the oleaginous yeast Yarrowia lipolytica has emerged as a promising candidate for the synthesis of valuable products. In this study, we explored the potential of Y. lipolytica as a host for producing MAAs, utilizing its advantages such as a robust pentose phosphate pathway flux and versatile carbon source utilization.ResultsWe produced MAAs in Y. lipolytica by introducing the MAA biosynthetic genes from cyanobacteria Nostoc punctiforme and Anabaena variabilis. These genes include mysA, mysB, and mysC responsible for producing mycosporine-glycine (MG) from sedoheptulose 7-phosphate (S7P). The two strains utilize different enzymes, D-Ala-D-Ala ligase homologue (MysD) in N. punctiforme and NRPS-like enzyme (MysE) in A. variabilis, for amino acid conjugation to MG. MysE specifically generated shinorine, a serine conjugate of MG, while MysD exhibited substrate promiscuity, yielding both shinorine and a small amount of porphyra-334, a threonine conjugate of MG. We enhanced MAAs production by selecting mysA, mysB, and mysC from A. variabilis and mysD from N. punctiforme based on their activities. We further improved production by strengthening promoters, increasing gene copies, and introducing the xylose utilization pathway. Co-utilization of xylose with glucose or glycerol increased MAAs production by boosting the S7P pool through the pentose phosphate pathway. Overexpressing GND1 and ZWF1, key genes in the pentose phosphate pathway, further enhanced MAAs production. The highest achieved MAAs level was 249.0 mg/L (207.4 mg/L shinorine and 41.6 mg/L of porphyra-334) in YP medium containing 10 g/L glucose and 10 g/L xylose.ConclusionsY. lipolytica was successfully engineered to produce MAAs, primarily shinorine. This achievement involved the introduction of MAA biosynthetic genes from cyanobacteria, establishing xylose utilizing pathway, and overexpressing the pentose phosphate pathway genes. These results highlight the potential of Y. lipolytica as a promising yeast chassis strain for MAAs production, notably attributed to its proficient expression of MysE enzyme, which remains non-functional in S. cerevisiae, and versatile utilization of carbon sources like glycerol.

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Efficient production of 2'-fucosyllactose in unconventional yeast Yarrowia lipolytica.

Publications

Efficient production of 2'-fucosyllactose in unconventional yeast <i>Yarrowia lipolytica</i>.

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