Metabolic Engineering of Escherichia coli for Lacto-N-triose II Production with High Productivity

Abstract

Lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides (HMOs), has attracted substantial attention for its nutraceutical potentials and applications in the production of complex HMOs. In this study, Escherichia coli was metabolically engineered to efficiently produce LNT II using glycerol as a carbon source and lactose as a substrate. The UDP-Nacetylglucosamine (UDP-GlcNAc) biosynthesis pathway was strengthened, and β-1,3-N-acetylglucosaminyltransferase (LgtA) was introduced to construct an LNT II-producing base strain. To increase the titer and yield of LNT II, combinatorial optimization of the copy number and the ribosomal binding site sequence was performed to tune the gene expression strength and translation rates of the pathway enzymes. Next, multipoint mutations were introduced to glucosamine-6-phosphatesynthase (GlmS) to relieve the feedback inhibition. Then, a series of engineered strains were constructed by blocking the futile pathways by the deletion of the relevant genes. Finally, the culture conditions were optimized. LNT II titer was improved step-by-step from 0.53 to 5.52 g/L in shake-flask cultivations. Fed-batch culture of the final engineered strain produced 46.2 g/L of LNT II, with an LNT II productivity and content of 0.77 g/(L•h) and 0.95 g/g dry cell weight, respectively.

Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin

“…The overexpression of heterologous LgtA from N. meningitides (NmlgtA) in the E. coli strain achieves the generation of LNT II, as this enzyme catalyzes the transfer of the GlcNAc residue of UDP-GlcNAc to lactose, which has been reported by numerous studies [ 9 , 29 , 30 ]. Using this strategy, Zhu et al successfully constructed the biosynthesis pathway for LNT II production by introducing heterologous NmLgtA in the E. coli strain when using glycerol as the carbon source [ 16 ]. In their study, glycerol must be converted to Fru-6-P through a series of chemical reactions, after which the generated Fru-6-P is converted to GlcN-6-P by GlmS, an initiating enzyme responsible for catalyzing the key step for LNT II production from Fru-6-P.…”

“…All primers used in this study are listed in Additional file 1 : Table S2 and synthesized by GENEWIZ (Suzhou, China). The lgtA from Neisseria meningitidis was codon-optimized by Sangon Biotech (the sequence is shown in Additional file 1 : Table S3) and constructed in three plasmid vectors: pRSFDuet-1, pETDuet-1, and pCDFDuet-1 to obtain plasmids: pRSF- lgtA , pET- lgtA, and pCDF- lgtA , respectively [ 16 ]. Due to the incompatibility of the same plasmids, antibiotic resistance of corresponding vectors was replaced by chloramphenicol (Cm), generating pCDFDuet-1(Cm R ), pETDuet-1(Cm R ), and pRSFDuet-1(Cm R ).…”

“…On the other hand, using glycerol as carbon source and energy, Baumgartner et al constructed an LNT II-producing strain by metabolically engineering a β-galactosidase-negative E. coli (Δ lacZ ) via chromosomal integration of LgtA, resulting in 2.465 g L −1 of LNT II [ 15 ]. Similarly, Zhu et al overexpressed GlmS (glucosamine-6-phosphate synthase), GlmM (glucosamine synthase), GlmU (UDP- N -acetylglucosamine pyrophosphorylase), and LgtA in E. coli (Δ nagB Δ lacZ Δ wecB ), and optimized the gene expression strength and translation rates, forming 46.2 g L −1 of LNT II using glycerol and β-lactose as a carbon source and substrate in a 5-L fed-batch fermentation [ 16 ]. In contrast to the above, LNT II production using metabolically engineered cells, McArthur et al prepared LNT II by employing Nahk, GlmU, and LgtA enzymes using GlcNAc substrate through the one-pot multienzyme (OPME) strategy in vitro [ 9 ].…”

“…The E. coli strain cannot synthesize LNT II due to the lack of a naturally endogenous synthesis pathway of LNT II in the cells. However, the genome-scale metabolic network analysis indicates that the E. coli strain can naturally synthesize the precursor of LNT II, UDP-GlcNAc, under three consecutive biocatalysis processes via GlmS, GlmM, and GlmU from Fru-6-P, which is an important intermediate of the glycolytic pathway that provides the possibility for constructing the biosynthesis pathway for LNT II production in E. coli cells [ 16 ]. In this study, the biosynthesis pathway for LNT II production was first redesigned by introducing heterologous LgtA from Neisseria meningitides (NmlgtA) in E. coli , based on the GlcNAc as a sole carbon source and β - lactose as the substrate.…”

Cell-Free Biosynthesis System: Methodology and Perspective of in Vitro Efficient Platform for Pyruvate Biosynthesis and Transformation

“…In recent decades, the modification of intracellular metabolic pathways based on metabolic engineering has generated a large number of engineered strains producing high yields of various target products. − These engineered strains contain enhanced target-product synthetic pathways and weakened branch pathways to maximize carbon flux to the target product. Although a large number of successfully engineered strains have been created based on metabolic engineering, − the workload of strain modification is rather difficult. Some compounds, such as 1,3-propanediol, account for more than 100 person-years from strain modification to economical production and industrial application .…”