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.

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Human milk oligosaccharides (HMOs) attract considerable interest in recent years because of their particular role in infant health. Lacto-N-tetraose (LNT), one of the most abundant HMOs, has been commercially added in the infant formula as a functional fortifier. In this study, a novel LNT-producing β-1,3-galactosyltransferase (β-1,3-GalT) from Pseudogulbenkiania ferrooxidans was screened from 14 putative candidates, and a highly LNT-producing metabolically engineered Escherichia coli strain was constructed based on a previously constructed lacto-N-triose II (LNT II)-producing strain, by strengthening UDP-galactose synthesis and introduction of P. ferrooxidans β-1,3-GalT. The engineered strain produced 3.11 and 25.49 g/L LNT in shake-flask and fed-batch cultivation, with the molar conversion ratio of LNT II to LNT of 88.15 and 85.09%, respectively. The productivity and specific yield of LNT in fed-batch cultivation were measured to be 0.61 g/L·h and 0.76 g/g dry cell weight, respectively. To the best of our knowledge, it is the highest LNT yield ever reported.

The occurrence of mycotoxin zearalenone (ZEN) and its derivatives has been a severe global threat to food and animals. In addition to the chemical and physical degradation methods, a powerful biocatalyst is urgently required for the detoxification of ZEN. Here, an efficient ZEN-degrading lactonase from Gliocladium roseum, named ZENG, was identified for the first time. The recombinant ZENG exhibited the highest activity at pH 7.0 and 38 °C. In addition, the recombinant enzyme showed a high degrading performance toward ZEN and its toxic derivatives α-zearalenol (α-ZOL) and α-zearalanol (α-ZAL), with the specific activities as 315, 187, and 117 units/mg, respectively. To meet the industrial demands, attempts were also made to enhance the thermostability of ZENG using a structure-based modification. Three double-site mutants, including H134L/S136L, H134F/S136F, and H134I/S134I, in the position between the cap and core catalytic domain of ZENG were designed. Finally, the thermostability of both H134L/S136L and H134F/S136F displayed a significant improvement compared to the wild-type enzyme.