Recent advances and challenges in microbial production of human milk oligosaccharides

Human milk oligosaccharides(HMOs)are one of the major differences between livestock milk and human milk,and the prebiotic functions of HMOs have been verified through in vitro and clinical trials.The most abundant HMOs include 2′-fucysollactose(2′-FL),3-fucosyllactose(3-FL),lacto-N-neotetraose(LNnT)and lacto-N-tetraose(LNT);their application and synthesis have attracted wide attentions.In recent years,the biotechnological production of 2′-FL,3-FL,LNnT and LNT have emerged based on techniques such as whole-cell catalysis and fermentation.In particular,the development of metabolic engineering and synthetic biology methods and strategies have facilitated efficient biosynthesis of these HMOs.However,these advantages have not been systematically reviewed yet.In this review,we first discuss the structures and applications of HMOs;secondly,strategies of microbial synthesis of the most abundant 2′-FL,3-FL,LNnT and LNT are summarized and compared.Finally,challenges and perspectives of efficient microbial production of HMOs as well as strategies for overcoming the challenges are discussed.This review reveals the whole picture of recent development in HMOs microbial synthesis and can further facilitate the understanding of limiting factors,and further propose a few directions to promote the development of efficient production hosts.

Construction of an engineered Escherichia coli for effective synthesis of 2'-fucosyllactose via the salvage pathway

2'-Fucosyllactose(2'-FL)is one of the important functional oligosaccharides in breast milk.So far,few attempts on biosynthesis of 2'-FL by the salvage pathway have been reported.Herein,the salvage pathway enzyme genes were introduced into the E.coli BL21star(DE3)for synthesis of 2'-FL.The 2'-FL titer increased from 1.56 to 2.13 g/L by deleting several endogenous genes on competitive pathways.Theα-1,2-fucosyltransferase(WbgL)was selected,and improved the 2'-FL titer to 2.88 g/L.Additionally,the expression level of pathway enzyme genes was tuned through optimizing the plasmid copy number.Furthermore,the spatial distribution of WbgL was enhanced by fusing with the MinD C-tag.After optimizing the fermentation conditions,the 2'-FL titer reached to 7.13 g/L.The final strain produced 59.22 g/L of 2'-FL with 95%molar conversion rate of lactose and 92% molar conversion rate of fucose in a 5 L fermenter.These findings will contribute to construct a highly efficient microbial cell factory to produce 2'-FL or other HMOs.

Biosynthesis of Lacto-N-biose I from starch and N-acetylglucosamine via an in vitro synthetic enzymatic biosystem

Human milk oligosaccharides(HMOs)are very distinctive components in human milk and are beneficial for infant health.Lacto-N-biose I(LNB)is the core structural unit of HMOs,which could be used for the synthesis of other HMOs.In this study,an ATP-free in vitro synthetic enzymatic biosystem contained four thermostable enzymes(alpha-glucan phosphorylase from Thermococcus kodakarensis,UDP-glucose-hexose-1-phosphate uridylyltransferase from Thermotoga maritima,UDP-glucose 4-epimerase from T.maritima,lacto-N-biose phosphorylase from Clostridium thermobutyricum)were constructed for the biosynthesis of LNB from starch and N-acetylglucosamine(GlcNAc).Under the optimal conditions,0.75 g/L and 2.23 g/L LNB was produced from 1.1 g/L and 4.4 g/L GlcNAc and excess starch,with the molar yield of 39%and 29%based on the GlcNAc concentration,respectively,confirming the feasibility of this in vitro synthetic enzymatic biosystem for LNB synthesis and shedding light on the biosynthesis of other HMOs using LNB as the core structural unit from low cost polysaccharides.

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

Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.