Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis

Abstract

Background Lacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation. Results In this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in Bacillus subtilis, resulting in an LNnT titer of 0.61 g/L. Then, by fine-tuning the expression level of LgtB, the growth inhibition was reduced and the LNnT titer was increased to 1.31 g/L. In addition, by modular pathway engineering, the positive-acting enzymes of the UDP-GlcNAc and UDP-Gal pathways were strengthened to balance the two key precursors supply, and the LNnT titer was improved to 1.95 g/L. Finally, the LNnT titer reached 4.52 g/L in a 3-L bioreactor with an optimal glucose and lactose feeding strategy. Conclusions In general, this study showed that the LNnT biosynthesis could be significantly increased by optimizing enzymes expression levels and modular pathway engineering for balancing the precursors supply in B. subtilis.

Metabolic engineering of Escherichia coli for the production of Lacto-N-neotetraose (LNnT)

“…For SDS-PAGE analysis, the cell was diluted to OD 600 of 5, and then the cells were boiled at 100 °C for 10 min. The concentrations of lactose, LNTII and LNnT in the fermentation were measured with high-performance anion exchange chromatography-pulsed amperometric detection (HPEAC-PAD) [22,36]. The column was Car-boPac PA10 (4 × 250 mm) and the column temperature was 30 °C.…”

“…The flow phase was 36 mM NaOH at a flow rate of 1.00 mL/min. Before the detection, the samples were preprocessed under the extracting with a solvent of chloroform/n-butanol (4:1 v/v) in a 1 mL tube for five times [22]. All experiments were repeated at least three times independently.…”

“…However, the lack of metabolic regulation results a low conversion rate of carbon [14,21]. Recent research has constructed the pathway of LNnT in Bacillus subtilis [22,23]. Engineering of modular pathway of role precursors has improved the titer of LNnT in some degree, but the metabolic regulation and the utilization of substrate still have difficulties.…”

“…UDP-glucose (UDP-Gal) and UDP-N-acetyglucosamine (UDP-GlcNAc) are considered the principal precursors among the biosynthesis pathway of LNnT in E. coli. In the metabolic pathway, glucose-6-phosphate and fructose-6-phosphate are converted to UDP-GlcNAc through Embden-Meyerhof-Parnas pathway (EMP) [22,24]. In this study, Escherichia coli K12 MG1655, which is the most common biosynthesis host, was chosen as the original strain.…”

Recent advances and challenges in microbial production of human milk oligosaccharides

“…Bacillus subtilis was also reconstructed to produce LNnT. Dong et al constructed and optimized the LNnT biosynthesis by modular pathway engineering and CRISPRi-guided multiplexed fine-tuning of metabolic flux in B. subtilis 168 [59,60]. Apart from strategies mentioned in the reconstructed E. coli, the synthesis of cofactors UDP-galactose and UDP-GlcNac was improved by overexpressing heterologous pathway genes lgtA and lgtB (Fig.…”