Metabolic Engineering Of Escherichia Coli For Production Of 3’-sialyllactose And 6’-sialyllactose

Human milk oligosaccharides(HMOs)are mainly divided into neutral,fucosylated,and sialylated HMOs according to the difference of terminal modifications.6’-sialyllactose(6’-SL)and 3’-sialyllactose(3’-SL)were the most common sialylated oligosaccharides.They show a great potential in improving intestinal health,anti-adhesion,anti-viral and promoting brain development,and have been approved as Generally Recognized As Safe by the the American Food and Drug Administration and recognized as Novel Food by the European Food Safety Authority.Biosynthesis is the most common method for the industrial production of HMOs due to the advantages of high efficiency and low cost.In this study,we construct Escherichia coli cell factories for de novo synthesis of 3’-SL and 6’-SL,respectively.Several metabolic engineering strategies were used to make a gradual increase in 3’-SL and 6’-SL yields,including screening sialyltransferases,enhancing supply levels of key precursors,fine-tuning metabolic intensity,and optimizing of cofactor regeneration.The main conclusions of metabolic engineering of Escherichia coli for production of 3’-sialyllactose are as follows:(1)Construction of the de novo synthesis pathway for 3’-SL in engineered Escherichia coli.N-acetylneuraminic acid synthase(Neu B),UDP-N-acetylglucosamine 2-epimerase(Neu C),and N-acylneuraminate cytidylyltransferase(Neu A)derived from C.jejuni ATCC 43438,andα2,3-Sia T(NST)derived from Neisseria meningitidis MC58 were introduced into E.coli BL21(DE3)Δlac Z.Finally,the synthesis of 3’-SL was confirmed by UPLC-MS.To avoid the loss of carbon flow,the competing pathway genes were knocked out individually or in combination,including nan A(encoding N-acetylneuraminic acid lyase),nan T(encoding Nacetylneuraminic acid transporter)and nan K(encoding N-acetylmannosamine kinase).Ultimately,the yield of the recombinant strain EZAK1 in shake-flask cultivation reached 1.90g/L.(2)Screening α2,3-Sia Ts from different species and comparing the effects for 3’-SL synthesis in vivo.We screened α2,3-Sia Ts from Pasteurella multocida PM70,Vibrio sp.JTFAJ-16 and Photobacterium sp.JT-ISH-224,and modified them based on previous in vitro experiments,obtained p CD-Pm0、p CD-t Pm0、p CD-Pm0M、p CD-Vs16、p CD-t Vs16 and p CD-Ps2.Strain EZAK5 harboring plasmids p ET-1BCA and p CD-Vs16,produced 2.62 g/L 3’-SL.(3)Fine-tuning the expression intensity of key pathway genes to increase the supply levels of precursor.UDP-Glc NAc and CMP-Neu5 Ac are key precursors of the 3’-SL,phosphoglucosamine mutase gene(glm M),UDP-N-acetylglucosamine pyrophosphorylase gene(glm U),and glucosamine-6-phosphate synthase gene(glm S*)are responsible for the synthesis of UDP-Glc NAc,CMP-N-acetylneuraminic acid synthase gene(neu A)is responsible for the synthesis of CMP-Neu5 Ac.The expression intensity of the above genes was regulated in combination using plasmids to enhance the metabolic flow in the direction of the target product.Finally,the 3’-SL shake-flask yield of recombinant strain EZAK17 reached 3.19 g/L.(4)Strengthening the conversion of CMP-Neu5 Ac to 3’-SL by integrating of Vs16 into chromosome,and the large-scale production potential of 3’-SL was demonstrated through fedbatch cultivation: An additional individual copy of Vs16 gene was inserted into the rec A locus of the chromosome of strain EZAK,generating strain EZAKS.Through the combination of plasmid expression and genomic integration expression of Vs16 gene,the highest yield of recombinant strain ES2 reached 4.51g/L.The total production of 3’-SL intracellularly and extracellularly by the ES2 strain reached 31.4 g/L at 38.5 h in a 5-L fermentor.The main conclusions of metabolic engineering of Escherichia coli for production of 6’-sialyllactose are as follows:(1)The de novo synthesis pathway of 6’-SL was constructed in engineered Escherichia coli,and the effects of competitive pathways knock-out were explored.p ET-1BCA and p CDST6 were introduced into host EZ and the generation of 6’-SL was verified by UPLC-MS.To compare the effects of each competitive pathway inactivation on key precursor and product synthesis,N-acetylneuraminic acid(Neu5Ac)and 6’-SL synthesis pathway genes were introduced into seven knockout hosts,respectively.For Neu5 Ac synthesis,the product was barely detectable in the fermentation broth of hosts without nan K inactivation;for 6’-SL synthesis,the inactivation of nan K and nan A together strengthen metabolic flux toward 6’-SL synthesis through a synergistic effect.Finally,the shake flask yield of ZAK1 strain reached 1.21g/L.(2)The effective α2,6-Sia Ts were screened and integrated into genome of EZAK strain,and expression levels of α2,6-Sia T and genes of CMP-Neu5 Ac synthetic pathway were finetuned: To enhance the sialylation effect for 6’-SL in vivo synthesis,α2,6-Sia Ts derived from Photobacterium sp.JT-ISH-224 and Photobacterium leiognathi JT-SHIZ-119 were synthesized and modified based on the results of in vitro experiments to obtain ST6,t ST6,t ST6 M,PL119,and t PL119.Ultimately,the yield of PL119-expressing strain ZAK4 was enhanced by 60%compared to the control ZAK1,reaching 1.60 g/L.To explore the appropriate expression intensity of key pathway enzymes,PL119 was integrated into the rec A locus of the strain EZAK genome and the supply level of UDP-Glc NAc was enhanced.The 6’-SL titer of strain ZAKS2 harboring plasmids p ET-1BCA and p RSF-MUS* was further increased to 2.12 g/L.(3)Construction of a cofactor regeneration system to enhance the yield of 6’-SL: The conversion of Neu5 Ac to CMP-Neu5 Ac is catalyzed by Neu A requiring the participation of the cofactor CTP.We constructed a cofactor regeneration strategy by coupling the ATP and CTP cycling.The yield of strains overexpressing cmk,ndk,and cmk-ndk was enhanced compared to the control,and then,the expression of cmk,ndk and cmk-ndk was optimized by different plasmid copy number combinations.Finally,the shake-flask yield of strain ZAKS17 reached2.72 g/L.After 80 h of fed-batch fermentation in a 5-L fermenter,the extracellular titer of 6’-SL reached 22.45 g/L,and only 0.8 g/L residues were detected intracellularly,with the dry cell weight(DCW)of 40 g/L,and a yield of 0.64 mol/mol lactose.