Enhanced bioproduction of fucosylated oligosaccharide 3-fucosyllactose in engineered Escherichia coli with an improved de novo pathway.

3-fucosyllactose (3-FL) and 2'-fucosyllactose (2'-FL), are two important fucosylated oligosaccharides in human milk. Extensive studies on 2'-FL enabled its official approval for use in infant formula. However, development of 3-FL has been somewhat sluggish due to its low content in human milk and poor yield in enlarged production. Here, an α-1,3-fucosyltransferase mutant was introduced into an engineered Escherichia coli (E. coli) capable of producing GDP-L-fucose, leading to a promising 3-FL titer in a 5.0-L bioreactor. To increase the availability of cofactors (NADPH and GTP) for optimized 3-FL production, zwf, pntAB, and gsk genes were successively overexpressed, finally resulting in a higher 3-FL level with a titer of 35.72 g/L and a yield of 0.82 mol 3-FL/mol lactose. Unexpectedly, the deletion of pfkA gene led to a much lower performance of 3-FL production than the control strain. Still, our strategy achieved the highest 3-FL level in E. coli to date.

Multi-Enzymatic Cascade One-Pot Biosynthesis of 3'-Sialyllactose Using Engineered Escherichia coli.

Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3'-sialyllactose (3'-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3'-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that ß-galactosidase (lacZ) affects the yield of 3'-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3'-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3'-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3'-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3'-SL.

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

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.

Effect of Co-overexpression of Nisin Key Genes on Nisin Production Improvement in Lactococcus lactis LS01.

Nisin is a small antimicrobial peptide produced by several subset strains of Lactococcus lactis. To improve nisin yield in the producer L. lactis LS01, we proposed a successive fusion of nisA with nisRK and nisFEG into a single shuttle expression vector pMG36e under the control of the native strong constitutive promoter p32. Subsequently, the recombinant vectors were transplanted into the producer cell through electroporation. Nisin productivity was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and bioactivity assays. Expression of nisin peptide was detected by agar diffusion bioassay, and the transcriptional levels of the target genes involved in nisin biosynthesis were investigated via semi-quantitative reverse transcription PCR expression analysis using 16S ribosomal RNA (rRNA) as an internal control. Results suggested that the introduction of empty plasmid did not affect nisin production of L. lactis LS01, whereas by our rational construction and screening, the engineered strain co-overexpressing nisA, nisRK, and nisFEG achieved a maximum increment in bioactive nisin production with a yield of 2470 IU/ml in shake flasks and 4857 IU/ml in 1.0-l fermenters, which increased by approximately 66.3 and 52.6% (P < 0.05), respectively, compared with that of the original strain under the given fermentation conditions. Meanwhile, the transcriptional analysis revealed that the expression of most of these multicopy genes except nisE at transcriptional level were upregulated in the two recombinant strains (LS01/pAR and LS01/pARF), possibly contributing to the improved nisin production. Therefore, this study would provide a potential strategy to improve the economic benefits of nisin manufacture for large-scale industrial production.