High-Yield Synthesis of Lacto-N-Neotetraose from Glycerol and Glucose in Engineered Escherichia coli

Abstract

Lacto-N-neotetraose (LNnT) is a neutral human milk oligosaccharide with important biological functions. However, the low LNnT productivity and the incomplete conversion of the intermediate lacto-N-tetraose II (LNT II) currently limited the sustainable biosynthesis of LNnT. First, the LNnT biosynthetic module was integrated in Escherichia coli. Next, the LNnT export system was optimized to alleviate the inhibition of intracellular LNnT synthesis. Furthermore, by utilizing rate-limiting enzyme diagnosis, the expressions of LNnT synthesis pathway genes were finely regulated to further enhance the production yield of LNnT. Subsequently, a strategy of cofermentation using a glucose/glycerol (4:6, g/g) mixed feed was employed to regulate carbon flux distribution. Finally, by overexpressing key transferases, LNnT and LNT II titers reached 112.47 and 7.42 g/L, respectively, in a 5 L fermenter, and 107.4 and 2.08 g/L, respectively, in a 1000 L fermenter. These are the highest reported titers of LNnT to date, indicating its significant potential for industrial production.

Engineering Escherichia coli for Highly Efficient Biosynthesis of Lacto-N-difucohexaose II through De Novo GDP-l-fucose Pathway

“…Recently, Liao constructed a plasmid-free engineered E. coli to produce LNnT with a titer of 112.47 g/L by fed-batch cultivation . But the previously reported plasmid-dependent strains only produced LNnT with titers of approximately 20 g/L. , Thus, genomic integration for plasmid-free strains may be another research direction for LNDFH II biosynthesis.…”

Combinatorial Optimization Strategies for 3-Fucosyllactose Hyperproduction in Escherichia coli

“…Biosynthesis of HMOs in microbial strains has increasingly focused on optimizing fermentation conditions during large-scale production. For example, Liao et al performed a glucose/glycerol cofermentation strategy for lacto- N -neotetraose (LNnT) production; the carbon-source ratio (glucose/glycerol) in the supplement was optimized, and the highest titer of LNnT (94.08 g/L) was obtained when the ratio was 4:6 . To decrease the accumulation of the byproduct acetic acid in fed-batch fermentation.…”

De Novo Biosynthesis of Difucosyllactose by Artificial Pathway Construction and α1,3/4-Fucosyltransferase Rational Design in Escherichia coli

“…At present, the microbial synthesis of 3–6 HMOs has become a mainstream approach. − In contrast to 2′-FL and 3-FL, which have been extensively studied for efficient synthesis through the continuous development of microbial cell factories for over 10 years, very few studies have reported the microbial synthesis of DFL. Typically, to synthesize DFL, the initial step involves monofucosylation of lactose by an α-1,2-fucosyltransferase or α-1,3-fucosyltransferase.…”