期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 69, 期 5, 页码 1567-1577出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.0c07224
关键词
2 '-fucosyllactose; metabolic engineering; Escherichia coli; batch fermentation
资金
- National Natural Science Foundation of China [31922073]
- Key-Area Research and Development Program of Guangdong Province [2020B020226007]
- Zhangjiagang International Cooperation Project [ZKH1905]
In this study, the de novo and salvage pathways for GDP-fucose synthesis were engineered and optimized in E. coli, leading to increased 2'-FL production. By fine-tuning the expression of related genes, along with the introduction of fucT2 gene, the final engineered strains produced 2.62 and 14.1 g/L of 2'-FL in shake-flask and fed-batch cultivations, respectively.
2'-Fucosyllactose (2'-FL), one of the most valuable oligosaccharides in human milk, is used as an emerging food ingredient in the nutraceutical and food industries due to its numerous health benefits. Herein, the de novo and salvage pathways for GDP-fucose synthesis were engineered and optimized in Escherichia coli BL21 (DE3) to improve the production of 2'-FL. The de novo pathway genes encoding phosphomannomutase (ManB), mannose-1-phosphate guanyltransferase (ManC), GDP-D-mannose-4,6-dehydratase (Gmd), and GDP-L-fucose synthase (WcaG) combined with the gene from the salvage pathway encoding fucose kinase/fucose-1-phosphate guanylyltransferase (Fkp) were reconstructed in two vectors to evaluate the GDP-fucose biosynthesis. Then, the fucT2 gene, encoding alpha 1,2-fucosyltransferase, was introduced into the GDP-fucose-overproducing strains to realize 2'-FL biosynthesis. Furthermore, the genes in bypass pathways, including lacZ, fucI, fucK, and wcaJ, were inactivated to improve 2'-FL production. In addition, the two GDP-fucose synthesis pathways, along with fucT2, were transcriptionally fine-tuned to efficiently increase 2'-FL production. The final metabolically engineered E. coli produced 2.62 and 14.1 g/L in shake-flask and fed-batch cultivations, respectively.
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