Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative

Abstract

2′-fucosyllactose (2′-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2′-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2′-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2′-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2′-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2′-FL production, ultimately a highest titer of 2′-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2′-FL was achieved with a yield of 0.89 mol 2′-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2′-FL. As a proof of concept, our preliminary 2′-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

“…Overexpression of rcsA and/or rcsB generally led to an obvious enhancement of 2′-FL in previous studies. 14,16,29 Drouillard et al developed a method for 2′-FL production by high-celldensity fermentation of an engineered E. coli strain only overexpressing rcsA and H. pylori f utC genes and inactivating the wcaJ gene. The maximum 2′-FL titer reached 3 g/L intracellularly and 11 g/L extracellularly from the initially added lactose at 15 g/L after fermentation for 45 h. 29 The 2′-FL concentration was greatly improved to 2.12 g/L in the engineered strain expressing rcsA gene, representing a 58.2% increase in comparison with that in the control strain.…”

“…In a previous study, the single expression of rcsA and coexpression of rcsA and rcsB both promoted GDP-Lfucose synthesis obviously in E. coli and the coexpression of rcsA and rcsB exhibited a better promotion effect than single expression of rcsA; in addition, the enhanced GDP-L-fucose supply favored to improve 2′-FL biosynthesis. 16 Integration of wbgL in recA Locus of the Engineered Host. Considering the supply of the GDP-L-fucose pathway via the overexpression of genes manC, manB, gmd, wcaG, rcsA, and rcsB, we speculated that a great increase in 2′-FL production might be obtained by further strengthening the expression of gene wbgL.…”

Designing a Highly Efficient Biosynthetic Route for Lacto-N-Neotetraose Production in Escherichia coli

“…It was indicated that the expression of LgtA on low-copy plasmid pACYCDuet-1 was beneficial for LNnT synthesis. Taken together, combinatorial optimization strategy via changing different plasmids has been widely used as a practical strategy for in vivo highly efficient production of other HMOs, such as 2′-fucosyllactose (2′-FL), , 3-fucosyllactose (3-FL), and LNTri II …”

Engineered Microbial Routes for Human Milk Oligosaccharides Synthesis

“…This method deleted the lacZ , gcd , glK , ptsG , and pfKA genes to overexpress the de novo UDP-D-galactose and GDP- l -fucose pathways, and used sugar efflux transporters to promote the export of 2′-FL. They increased the production of 2′-FL to 60 g/L after 48 h. The use of glycerol as a carbon source by Ni et al can also ensured reduced costs. They deleted genes involved in the degradation of precursors GDP- l -fucose and GDP-mannose, and further expanded production by introducing two heterologous positive regulators.…”