Production of sialyllactose

Abstract

Provided is a recombinant Corynebacterium glutamicum, which is genetically engineered to introduce an N-acetyl-mannosamine (ManNAc) synthetic pathway, and is genetically engineered to express a heterologous N-acetylneuraminic acid synthase, a heterologous CMP-N-acetylneuraminic acid synthase, a heterologous lactose transporter, and a heterologous sialyltransferase. Provided is a method for using the recombinant Corynebacterium glutamicum to produce sialyllactose.

Description

Production of sialyllactose

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202411075756.2, filed on August 6, 2024, which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biotechnology, and in particular to the production of sialyllactose using microorganisms. BACKGROUND

[0004] Human milk oligosaccharide (HMO) is a special oligosaccharide that exists in breast milk and plays an important role in the health of infants. Human milk oligosaccharide is a derivative of lactose, but is more complex in chemical structure than lactose. Studies have found that human milk oligosaccharide helps the development of the digestive system of infants, and can also regulate the immune system and promote the development of infant immunity. Human milk oligosaccharide also has the effect of probiotics, and plays an important role in maintaining the balance of intestinal flora. It has been reported that among HMOs, sialylated human milk oligosaccharides belong to a class of oligosaccharides in HMOs, and their main components are 3'-sialyllactose and 6'-sialyllactose, which account for about 10% of the components in HMOs.

[0005] Currently, the production methods of 3'-sialyllactose and 6'-sialyllactose mainly include enzymatic synthesis and microbial synthesis. The mainstream method is microbial synthesis, and the use of microbial bioengineering production methods can produce sialyllactose in large quantities from inexpensive raw materials through a simple process, so it is attracting attention as a method for producing sialyllactose that has the potential to be developed as a raw material for health functional foods and pharmaceuticals. Internationally, a variety of 3'-sialyllactose and 6'-sialyllactose production strains and production technologies derived from Escherichia coli have been published, but E. coli as a production host is known to have endotoxins, and is not an ideal host for food raw material production.

[0006] Corynebacterium glutamicum is a non-spore-forming, non-motile, gram-positive bacterium isolated from soil, with a short rod to rod shape, belonging to the Corynebacteriaceae family of the genus Corynebacterium, and has been certified by the U.S. Food and Drug Administration (FDA) as a "Generally Recognized as Safe" (GRAS) strain (Woo & Park, 2014; Lee et al., 2016). Corynebacterium glutamicum was first reported in 1957 for the fermentation production of L-glutamic acid (Kinoshita et al., 1957), and has been used for the production of amino acids such as L-lysine, L-isoleucine, L-valine, and L-arginine (Wendisch et al., 2016). SUMMARY

[0007] In one aspect, the present disclosure provides a recombinant Corynebacterium glutamicum genetically engineered to introduce a sialyllactose synthesis pathway. In some embodiments, the sialyllactose is 3'-sialyllactose or 6'-sialyllactose.

[0008] In some embodiments, the recombinant Corynebacterium glutamicum is genetically engineered to introduce a CMP-N-acetylneuraminic acid (CMP-Neu5Ac) synthesis pathway and genetically engineered to express an exogenous lactose transporter and an exogenous sialyltransferase. In some embodiments, the recombinant Corynebacterium glutamicum is genetically engineered to introduce an N-acetylmannosamine (ManNAc) synthesis pathway and genetically engineered to express an exogenous N-acetylneuraminic acid synthase, an exogenous CMP-N-acetylneuraminic acid synthase, an exogenous lactose transporter, and an exogenous sialyltransferase.

[0009] In some embodiments, the sialyltransferase is an a-2,3-sialyltransferase or an a-2,6-sialyltransferase.

[0010] In some embodiments, the a-2,3-sialyltransferase is an a-2,3-sialyltransferase derived from Pasteurella dagmatis, Neisseria meningitidis serotype L3, Vibrio sp. JT-FAJ-16, or Photobacterium multocida, or a functional variant thereof, and the a-2,6-sialyltransferase is an a-2,6-sialyltransferase derived from Photobacterium sp. JT-ISH-224, Photobacterium damselae, or Photobacterium leiognathi, or a functional variant thereof.

[0011] In some embodiments, the lactose transporter protein is a lactose permease, preferably a lactose permease (LacY) derived from E. coli, or a functional variant thereof.

[0012] In some embodiments, the N-acetylneuraminate synthase is an N-acetylneuraminate synthase derived from Campylobacter jejuni or Neisseria meningitidis, or a functional variant thereof.

[0013] In some embodiments, the CMP-N-acetylneuraminate synthase is a CMP-N- acetylneuraminate synthase derived from Campylobacter jejuni or Neisseria meningitidis, or a functional variant thereof.

[0014] In some embodiments, the recombinant C. glutamicum is genetically modified to express an exogenous UDP-N-acetylglucosamine 2-epimerase, thereby introducing an N- acetylmannosamine (ManNAc) synthesis pathway in the C. glutamicum. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase is a UDP-N-acetylglucosamine 2- epimerase derived from Campylobacter jejuni or Neisseria meningitidis, or a functional variant thereof. In some embodiments, the recombinant C. glutamicum further comprises one, two, or three of the following genetic modifications: (1) at least one genetic modification that eliminates or attenuates the action of an endogenous glucosamine-6-phosphate deaminase; (2) at least one genetic modification that eliminates or attenuates the action of one, two, or three enzymes selected from the group consisting of an endogenous N-acetylglucosamine-6-phosphate deacetylase, an endogenous N-acetylmannosamine kinase, and an endogenous N-acetylmannosamine-6-phosphate 2-epimerase; and (3) at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylneuraminate lyase.

[0015] In some embodiments, the recombinant C. glutamicum is genetically modified to express an exogenous glucosamine-6-phosphate N-acetyltransferase 1 and an exogenous N- acetylglucosamine 2-epimerase, thereby introducing an N-acetylmannosamine (ManNAc) synthesis pathway in the C. glutamicum.

[0016] In some embodiments, the glucosamine-6-phosphate N-acetyltransferase 1 is a glucosamine-6-phosphate N-acetyltransferase 1 derived from Saccharomyces cerevisiae or Caenorhabditis elegans, or a functional variant thereof.

[0017] In some embodiments, the N-acetylglucosamine 2-epimerase is a N-acetylglucosamine 2-epimerase derived from Synechocystis sp. PCC6803, Bacteroides ovatus, or Anabaena sp., or a functional variant thereof.

[0018] In some embodiments, the recombinant C. glutamicum further comprises one, two, three, or four of the following genetic modifications: (1) at least one genetic modification that eliminates or attenuates the action of an endogenous glucosamine-6-phosphate deaminase; (2) at least one genetic modification that eliminates or attenuates the action of one or both of an endogenous N-acetylmannosamine kinase and an endogenous N-acetylmannosamine-6-phosphate 2-epimerase; (3) at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylglucosamine-6-phosphate deacetylase; and (4) at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylneuraminic acid lyase.

[0019] In some embodiments, the at least one genetic modification that eliminates or attenuates the action of an endogenous glucosamine-6-phosphate deaminase comprises: a knockout or knockdown of an endogenous glucosamine-6-phosphate deaminase gene, and / or a knockout or knockdown of a native promoter of an endogenous glucosamine-6-phosphate deaminase gene.

[0020] In some embodiments, the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylglucosamine-6-phosphate deacetylase comprises: a knockout or knockdown of an endogenous N-acetylglucosamine-6-phosphate deacetylase gene, and / or a knockout or knockdown of a native promoter of an endogenous N-acetylglucosamine-6-phosphate deacetylase gene.

[0021] In some embodiments, the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylmannosamine kinase comprises: a knockout or knockdown of an endogenous N-acetylmannosamine kinase (NanK) gene, and / or a knockout or knockdown of a native promoter of an endogenous N-acetylmannosamine kinase gene.

[0022] In some embodiments, the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylmannosamine-6-phosphate 2-epimerase comprises: a knockout or knockdown of an endogenous N-acetylmannosamine-6-phosphate 2-epimerase gene, and / or a knockout or knockdown of a native promoter of an endogenous N-acetylmannosamine-6-phosphate 2-epimerase gene.

[0023] In some embodiments, the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylneuraminic acid lyase comprises: a knockout or knockdown of an endogenous N-acetylneuraminic acid lyase gene, and / or a knockout or knockdown of a native promoter of an endogenous N-acetylneuraminic acid lyase gene.

[0024] In some embodiments, the recombinant C. glutamicum further comprises one, two, or three of the following genetic modifications: (1) at least one genetic modification that enhances the action of glutamine-fructose-6-phosphate amidotransferase; (2) at least one genetic modification that enhances the action of phosphoglucosamine mutase; and (3) at least one genetic modification that enhances the action of N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme.

[0025] In some embodiments, the at least one genetic modification that enhances the action of glutamine-fructose-6-phosphate amidotransferase comprises increasing the activity of glutamine-fructose-6-phosphate amidotransferase, or overexpressing glutamine-fructose-6-phosphate amidotransferase. In some embodiments, the at least one genetic modification that enhances the action of glutamine-fructose-6-phosphate amidotransferase comprises replacing the endogenous glutamine-fructose-6-phosphate amidotransferase gene with an exogenous glutamine-fructose-6-phosphate amidotransferase gene having higher enzymatic activity, increasing the gene copy number of the glutamine-fructose-6-phosphate amidotransferase gene, and / or replacing the natural promoter of the endogenous glutamine-fructose-6-phosphate amidotransferase with a promoter having higher expression level. In some embodiments, the at least one genetic modification that enhances the action of glutamine-fructose-6-phosphate amidotransferase comprises introducing at least one copy of an exogenous glutamine-fructose-6-phosphate amidotransferase gene into the C. glutamicum; preferably, the exogenous glutamine-fructose-6-phosphate amidotransferase is glutamine-fructose-6-phosphate amidotransferase mutant GlmS*54 derived from E. coli.

[0026] In some embodiments, the at least one genetic modification that enhances the action of phosphoglucosamine mutase comprises increasing the activity of phosphoglucosamine mutase, or overexpressing phosphoglucosamine mutase. In some embodiments, the at least one genetic modification that enhances the action of phosphoglucosamine mutase comprises replacing the endogenous phosphoglucosamine mutase gene with an exogenous phosphoglucosamine mutase gene having higher enzymatic activity, increasing the gene copy number of the phosphoglucosamine mutase gene, and / or replacing the natural promoter of the endogenous phosphoglucosamine mutase with a promoter having higher expression level. In some embodiments, the at least one genetic modification that enhances the action of phosphoglucosamine mutase comprises introducing at least one copy of a phosphoglucosamine mutase gene into the C. glutamicum; preferably, the phosphoglucosamine mutase is phosphoglucosamine mutase from C. glutamicum.

[0027] In some embodiments, the at least one genetic modification that enhances the action of N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme comprises: increasing the activity of N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme, or overexpressing N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme. In some embodiments, the at least one genetic modification that enhances the action of N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme comprises: replacing the endogenous N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene with an exogenous N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene having higher enzymatic activity, increasing the gene copy number of N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene, and / or replacing the natural promoter of endogenous N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme with a promoter having higher expression level. In some embodiments, the at least one genetic modification that enhances the action of N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme comprises: introducing at least one copy of N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene into the C. glutamicum; preferably, the N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme is N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme from C. glutamicum.

[0028] In some embodiments, the recombinant C. glutamicum further comprises the following genetic modification: (1) at least one genetic modification that eliminates or attenuates the action of an enzyme encoded by nanH; and / or (2) at least one genetic modification that eliminates or attenuates the action of an enzyme encoded by nanP.

[0029] In some embodiments, the at least one genetic modification that eliminates or attenuates the action of an enzyme encoded by nanH comprises: knocking out or knocking down nanH, and / or knocking out or knocking down the natural promoter of nanH.

[0030] In some embodiments, the at least one genetic modification that eliminates or attenuates the action of an enzyme encoded by nanP comprises: knocking out or knocking down nanP, and / or knocking out or knocking down the natural promoter of nanP.

[0031] Another aspect of the present application provides a method for producing sialyllactose, comprising culturing any of the aforementioned recombinant C. glutamicum under suitable conditions, and recovering the synthesized sialyllactose.

[0032] In some embodiments, at least one carbon source and lactose are added to the culture medium for culturing the recombinant C. glutamicum. In some embodiments, the at least one carbon source is selected from the group consisting of glucose, fructose, sucrose, acetic acid, lactic acid, and succinic acid.

[0033] In some embodiments, the sialyllactose is 3'-sialyllactose or 6'-sialyllactose.

[0034] In some embodiments, the culturing of the recombinant C. glutamicum is batch culture, fed-batch culture, or continuous culture.

[0035] In some embodiments, at least one carbon source and / or lactose is supplemented during the culturing.

[0036] Yet another aspect of the present application provides use of any of the aforementioned recombinant C. glutamicum for producing sialyllactose. In some embodiments, the sialyllactose is 3'-sialyllactose or 6'-sialyllactose. BRIEF DESCRIPTION OF DRAWINGS

[0037] FIG. 1 is a schematic diagram of a synthesis pathway 1 of sialyllactose in C. glutamicum. The synthesis pathway shown in solid line represents a naturally occurring synthesis pathway in C. glutamicum, the synthesis pathway shown in dashed line represents an exogenously introduced synthesis pathway, and the synthesis pathway with "X" represents a synthesis pathway that is knocked out.

[0038] FIG. 2 is a schematic diagram of a synthesis pathway 2 of sialyllactose in C. glutamicum. The synthesis pathway shown in solid line represents a naturally occurring synthesis pathway in C. glutamicum, the synthesis pathway shown in dashed line represents an exogenously introduced synthesis pathway, and the synthesis pathway with "X" represents a synthesis pathway that is knocked out.

[0039] FIG. 3 is a growth and production curve of strain Cg6SL-4 in a fermenter. DETAILED DESCRIPTION

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0041] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict between the disclosure of the present specification and the incorporated references, the present specification will control. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0042] The terms "about" and "approximately," when used with a numerical variable, generally mean that the value of the variable is within a margin of error or within a range of values that is within the experimental error of the measurement or within a wider range of values (e.g., ±5% or ±10%) of the specified value.

[0043] The terms "comprise" or "comprising", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element but not the exclusion of any other steps or elements. "Consisting of" means an exclusion of any element not listed. "Consisting essentially of" means an exclusion of any element not listed, except for those that do not materially affect the basic and novel characteristics of the invention. The term "comprising" also includes the variants "consisting of" and "consisting essentially of."

[0044] When referring to a numerical range, the specific values of the upper and lower limits of the range are to be construed as specifically disclosed, as well as all intermediate ranges to be included within the scope of the disclosure. For example, a range of 1 to 10 is to be construed as specifically disclosing the values 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, as well as all intermediate ranges such as 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, and 9-10. Furthermore, any intermediate ranges, sub-ranges, and all individual values described in the numerical range can be excluded from the numerical range.

[0045] The term "and / or" should be understood to mean either one or both of the items it connects.

[0046] Provided herein are methods of producing sialyllactose (SL), including 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), using Corynebacterium glutamicum. Using Corynebacterium glutamicum as a chassis to produce sialyllactose has a large safety advantage, and surprisingly, the methods of producing sialyllactose using Corynebacterium glutamicum provided herein have a high sialyllactose production efficiency and yield.

[0047] Provided herein are recombinant Corynebacterium glutamicum that are genetically modified to comprise sialyllactose (e.g., 3'-sialyllactose and 6'-sialyllactose) synthesis pathway genes, which can be used to produce sialyllactose (e.g., 3'-sialyllactose and 6'-sialyllactose).

[0048] Sialyllactose can be produced from a donor substrate CMP-N-acetylneuraminic acid (CMP-Neu5Ac) and an acceptor substrate lactose by a sialyltransferase. The sialyltransferase can be an a-2,3-sialyltransferase (2,3-ST) that catalyzes the production of 3'-sialyllactose (3'-SL), or an a-2,6-sialyltransferase (2,6-ST) that catalyzes the production of 6'-sialyllactose (6'-SL).

[0049] In some embodiments, the recombinant C. glutamicum is genetically modified to comprise a synthetic pathway for CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Since wild-type C. glutamicum is naturally unable to uptake lactose, it does not contain an enzyme to transport lactose into the cell, thus, the C. glutamicum is also genetically engineered to express an exogenous lactose transporter to enable the C. glutamicum to perform the above-mentioned reaction to generate sialyllactose intracellularly.

[0050] Fructose-6-phosphate is an intermediate product of carbon source metabolism present in almost all organisms, including C. glutamicum. Common carbon sources (such as glucose, glycerol, etc.) can be metabolized to form fructose-6-phosphate in the cell. For example, glucose enters the cell and is phosphorylated to form glucose-6-phosphate, which is catalyzed by phosphoglucoisomerase (Pgi) to form fructose-6-phosphate.

[0051] Salicose can be synthesized from fructose-6-phosphate (Fru-6-P) through a series of enzyme-catalyzed reactions, and the synthesis pathway thereof can include the following enzyme-catalyzed reactions: (1) generation of glucosamine-6-phosphate (GlcN-6-P) from fructose-6-phosphate (Fru-6-P) catalyzed by glutamine-fructose-6-phosphate amidotransferase (GlmS); (2) generation of glucosamine-1-phosphate (GlcN-1-P) from glucosamine-6-phosphate (GlcN-6-P) catalyzed by phosphoglucomutase (GlmM); (3) generation of UDP-N-acetylglucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) catalyzed by N-acetylglucosamine-1-phosphate urtidyltransferase and acetylglucosamine-1-phosphate acetyltransferase bifunctional enzyme (GlmU); (4) generation of N-acetylmannosamine (ManNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) catalyzed by UDP-N-acetylglucosamine 2-epimerase (NeuC); (5) generation of N-acetylneuraminic acid (Neu5Ac, sialic acid) from N-acetylmannosamine (ManNAc) catalyzed by N-acetylneuraminate synthase (NeuB); (6) generation of CMP-N-acetylneuraminic acid (CMP-Neu5Ac) from N-acetylneuraminic acid (Neu5Ac, sialic acid) catalyzed by CMP-N-acetylneuraminate synthase (NeuA); (7) generation of sialyllactose from CMP-N-acetylneuraminic acid (CMP-Neu5Ac) as a donor substrate and lactose as an acceptor substrate catalyzed by sialyltransferase. The sialyltransferase can be an α-2,3-sialyltransferase (2,3-ST) that catalyzes the generation of 3'-sialyllactose (3'-SL), or can be an α-2,6-sialyltransferase (2,6-ST) that catalyzes the generation of 6'-sialyllactose (6'-SL). This synthesis pathway is also referred to herein as synthesis pathway 1, as shown in FIG. 1.

[0052] The present inventors have found that wild-type C. glutamicum naturally contains enzymes capable of catalyzing the first three reactions (1), (2), and (3), and thus the first three reactions (1), (2), and (3) can be naturally performed in the cells of wild-type C. glutamicum, but wild-type C. glutamicum lacks enzymes required for the other reactions. Therefore, in order to establish synthesis pathway 1 described above in C. glutamicum, the enzymes lacking in C. glutamicum can be introduced into C. glutamicum in the form of exogenous enzyme genes to express the enzymes in C. glutamicum.

[0053] In some embodiments, to establish the above-mentioned synthetic pathway 1 in C. glutamicum, the C. glutamicum is genetically engineered to express an exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC), an exogenous N-acetylneuraminate synthase (NeuB), an exogenous CMP-N-acetylneuraminate synthase (NeuA), and an exogenous sialyltransferase, and the thus genetically engineered C. glutamicum can synthesize sialyllactose in the cell.

[0054] The C. glutamicum is also genetically engineered to express an exogenous lactose transporter protein to transport lactose into the cell, thereby enabling the above-mentioned step (7) reaction.

[0055] The above-mentioned steps (2) to (4) reactions in the synthetic pathway 1 can also be achieved by another synthetic pathway: (2-a) acetylglucosamine-6-phosphate (GlcNAc-6-P) is generated from glucosamine-6-phosphate (GlcN-6-P) by glucosamine-6-phosphate N-acetyltransferase 1 (Gnal); (3-a) acetylglucosamine (GlcNAc) is generated from acetylglucosamine-6-phosphate (GlcNAc-6-P); and (4-a) N-acetylmannosamine (ManNAc) is generated from acetylglucosamine (GlcNAc) by N-acetylglucosamine 2-epimerase (Age). Thus, from the above-mentioned step (1) reaction, steps (2-a) to (4-a) reactions, and the above-mentioned steps (5) to (7) reactions, a second synthetic pathway for sialyllactose, which is also referred to herein as synthetic pathway 2, can be constructed, as shown in FIG. 2.

[0056] For the synthetic pathway 2, the present inventors found that C. glutamicum naturally contains enzymes capable of catalyzing the steps (1) and (3-a) reactions, and thus the cell naturally performs the above-mentioned steps (1) and (3-a) reactions, but lacks enzymes required for other reactions. Therefore, to establish the above-mentioned synthetic pathway 2 in C. glutamicum, the missing enzymes can be introduced into C. glutamicum in the form of exogenous enzyme genes to express the enzymes in C. glutamicum.

[0057] In some embodiments, to establish the above-mentioned synthetic pathway 2 in C. glutamicum, the C. glutamicum is genetically engineered to express an exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal), an exogenous N-acetylglucosamine 2-epimerase (Age), an exogenous N-acetylneuraminate synthase (NeuB), an exogenous CMP-N-acetylneuraminate synthase (NeuA), and an exogenous sialyltransferase, and the thus genetically engineered C. glutamicum can synthesize sialyllactose in the cell.

[0058] The above-mentioned synthetic pathway 1 and synthetic pathway 2 have a common synthetic pathway from N-acetylmannosamine (ManNAc) to CMP-N-acetylneuraminic acid (CMP-Neu5Ac) or sialyllactose, while the synthetic pathway of N-acetylmannosamine (ManNAc) is different.

[0059] In some embodiments, the CMP-N-acetylneuraminic acid (CMP-Neu5Ac) synthetic pathway comprised in the genetically engineered C. glutamicum of the present application is achieved by introducing a N-acetylmannosamine (ManNAc) synthetic pathway, and introducing an exogenous N-acetylneuraminic acid synthase (NeuB) and an exogenous CMP-N-acetylneuraminic acid synthase (NeuA).

[0060] The present application provides a genetically engineered C. glutamicum, wherein a N-acetylmannosamine (ManNAc) synthetic pathway is introduced, and a synthetic pathway from N-acetylmannosamine (ManNAc) to sialyllactose is introduced, thereby synthesizing sialyllactose.

[0061] The starting strain of the genetically engineered C. glutamicum of the present application can be any subspecies or strain of C. glutamicum, including but not limited to C. glutamicum ATCC 13032, C. glutamicum ATCC 13869, C. glutamicum ATCC 14020, and C. glutamicum ATCC 14067. These strains can be purchased from the American ATCC or the German DSMZ preservation center.

[0062] Introducing a N-acetylmannosamine (ManNAc) to sialyllactose synthetic pathway into the C. glutamicum includes introducing an exogenous N-acetylneuraminic acid synthase (NeuB), an exogenous CMP-N-acetylneuraminic acid synthase (NeuA), an exogenous lactose transporter protein, and an exogenous sialyltransferase into the C. glutamicum, so that these enzymes are expressed in the C. glutamicum.

[0063] Introducing a N-acetylmannosamine (ManNAc) synthetic pathway into the C. glutamicum includes introducing an exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) into the C. glutamicum so that it is expressed in the C. glutamicum, or introducing an exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) and an exogenous N-acetylglucosamine 2-epimerase (Age) into the C. glutamicum so that these enzymes are expressed in the C. glutamicum.

[0064] One or more or all of the above-mentioned exogenous enzymes can be a homologous protein or a functional variant thereof derived from a microorganism or an animal, for example, a homologous protein or a functional variant thereof derived from a bacterium, a fungus, an alga, a protozoan, and / or a nematode, wherein the bacterium can be a gram-negative bacterium or a gram-positive bacterium. In some embodiments, one or more or all of the above-mentioned exogenous enzymes can be a homologous protein or a functional variant thereof derived from Escherichia sp., such as E. coli; Campylobacter sp., such as Campylobacter jejuni, such as Campylobacter jejuni ATCC 43438; Pasteurella sp., such as Pasteurella dagmatis; Neisseria sp., such as Neisseria meningitidis; vibrio sp., such as vibrio sp. JT-FAJ-16; Photobacterium sp., such as Photobacterium sp. JT-ISH-224, Photobacterium multocida, Photobacterium damselae, Photobacterium leiognathi; Bacteroides sp., such as Bacteroides ovatus; Saccharomyces cerevisiae, such as Saccharomyces cerevisiae S288C; Synechocystis sp., such as Synechocystis sp. PCC6803; Anabaena sp.; or Caenorhabditis sp., such as Caenorhabditis elegans.

[0065] In some embodiments, the N-acetylneuraminate synthase is N-acetylneuraminate synthase (NeuB) derived from Campylobacter jejuni or a functional variant thereof. In some embodiments, the N-acetylneuraminate synthase is N-acetylneuraminate synthase (NeuB) derived from Campylobacter jejuni ATCC 43438 or a functional variant thereof. In some embodiments, the N-acetylneuraminate synthase (NeuB) can also be N-acetylneuraminate synthase (NmNeuB) derived from Neisseria meningitidis or a functional variant thereof. In some embodiments, the N-acetylneuraminate synthase comprises an amino acid sequence as set forth in SEQ ID NO: 3 or a homologous protein thereof, or a functional variant thereof. In some embodiments, a gene sequence encoding the N-acetylneuraminate synthase comprises a nucleic acid sequence as set forth in SEQ ID NO: 4.

[0066] In some embodiments, the CMP-N-acetylneuraminate synthase is CMP-N- acetylneuraminate synthase (NeuA) derived from Campylobacter jejuni or a functional variant thereof. In some embodiments, the CMP-N-acetylneuraminate synthase is CMP-N-acetylneuraminate synthase (NeuA) derived from Campylobacter jejuni ATCC 43438 or a functional variant thereof. In some embodiments, the CMP-N-acetylneuraminate synthase can also be CMP-N-acetylneuraminate synthase (NmNeuA) derived from Neisseria meningitidis or a functional variant thereof. In some embodiments, the CMP-N-acetylneuraminate synthase comprises an amino acid sequence as set forth in SEQ ID NO: 7 or a homologous protein thereof, or a functional variant thereof. In some embodiments, a gene sequence encoding the CMP-N-acetylneuraminate synthase comprises a nucleic acid sequence as set forth in SEQ ID NO: 8.

[0067] In some embodiments, the a-2,3-sialyltransferase is an a-2,3-sialyltransferase derived from Pasteurella dagmatis or Neisseria meningitidis L3 serotype or a functional variant thereof. In some embodiments, the a-2,3-sialyltransferase is an a-2,3-sialyltransferase derived from Neisseria meningitidis L3 MC58 or a functional variant thereof. In some embodiments, the a-2,3-sialyltransferase can also be an a-2,3-sialyltransferase derived from vibrio sp. or Photobacterium multocida or a functional variant thereof. In some embodiments, the a-2,3-sialyltransferase can be an a-2,3-sialyltransferase derived from vibrio sp. JT-FAJ-16 or a functional variant thereof. In some embodiments, the a-2,3-sialyltransferase comprises an amino acid sequence as set forth in SEQ ID NO: 9 or a homologous protein thereof, or a functional variant thereof. In some embodiments, a gene sequence encoding the a-2,3-sialyltransferase comprises a nucleic acid sequence as set forth in SEQ ID NO: 10.

[0068] In some embodiments, the a-2,6-sialyltransferase is an a-2,6-sialyltransferase derived from Photobacterium sp. or a functional variant thereof. In some embodiments, the a-2,6-sialyltransferase is an a-2,6-sialyltransferase derived from Photobacterium sp. JT-ISH-224 or a functional variant thereof.

[0069] In some embodiments, the a-2,6-sialyltransferase can also be an a-2,6-sialyltransferase derived from Photobacterium damselae or from Photobacterium leiognathi or a functional variant thereof. In some embodiments, the a-2,6-sialyltransferase comprises an amino acid sequence as set forth in SEQ ID NO: 11 or a homologous protein thereof, or a functional variant thereof. In some embodiments, a gene sequence encoding the a-2,6-sialyltransferase comprises a nucleic acid sequence as set forth in SEQ ID NO: 12.

[0070] In some embodiments, the lactose transporter protein is a lactose permease, preferably a lactose permease (LacY) derived from Escherichia coli or a functional variant thereof. In some embodiments, the lactose transporter protein comprises an amino acid sequence as set forth in SEQ ID NO: 5 or a homolog thereof, or a functional variant thereof. In some embodiments, the gene sequence encoding the lactose transporter protein comprises a nucleic acid sequence as set forth in SEQ ID NO: 6.

[0071] In some embodiments, the UDP-N-acetylglucosamine 2-epimerase is a UDP-N-acetylglucosamine 2-epimerase (NeuC) derived from Campylobacter jejuni or a functional variant thereof. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase is a UDP-N-acetylglucosamine 2-epimerase (NeuC) derived from Campylobacter jejuni ATCC 43438 or a functional variant thereof. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase can also be a UDP-N-acetylglucosamine 2-epimerase (NmNeuC) derived from Neisseria meningitidis or a functional variant thereof. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase comprises an amino acid sequence as set forth in SEQ ID NO: 1 or a homolog thereof, or a functional variant thereof. In some embodiments, the gene sequence encoding the UDP-N-acetylglucosamine 2-epimerase comprises a nucleic acid sequence as set forth in SEQ ID NO: 2.

[0072] In some embodiments, the glucosamine-6-phosphate N-acetyltransferase 1 is a glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) derived from Saccharomyces cerevisiae or a functional variant thereof. In some embodiments, the glucosamine-6-phosphate N-acetyltransferase 1 is a glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) derived from Saccharomyces cerevisiae S288C or a functional variant thereof. In some embodiments, the glucosamine-6-phosphate N-acetyltransferase 1 can also be a glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) derived from Caenorhabditis elegans or a functional variant thereof. In some embodiments, the glucosamine-6-phosphate N-acetyltransferase 1 comprises an amino acid sequence as set forth in SEQ ID NO: 13 or a homologous protein thereof, or a functional variant thereof. In some embodiments, a gene sequence encoding the glucosamine-6-phosphate N-acetyltransferase 1 comprises a nucleic acid sequence as set forth in SEQ ID NO: 14.

[0073] In some embodiments, the N-acetylglucosamine 2-epimerase is an N-acetylglucosamine 2-epimerase (Age) derived from Synechocystis sp. or a functional variant thereof. In some embodiments, the N-acetylglucosamine 2-epimerase is an N-acetylglucosamine 2-epimerase (Age) derived from Synechocystis sp. PCC6803 or a functional variant thereof. In some embodiments, the N-acetylglucosamine 2-epimerase can also be an N-acetylglucosamine 2-epimerase (Age) derived from Bacteroides ovatus or from Anabaena sp. or a functional variant thereof. In some embodiments, the N-acetylglucosamine 2-epimerase comprises an amino acid sequence as set forth in SEQ ID NO: 15 or a homologous protein thereof, or a functional variant thereof. In some embodiments, a gene sequence encoding the N-acetylglucosamine 2-epimerase comprises a nucleic acid sequence as set forth in SEQ ID NO: 16.

[0074] Methods for genetically modifying host cells are well known to those skilled in the art. Genetic modification techniques can be used to introduce an exogenous nucleic acid sequence into a host cell (e.g. in the form of a plasmid), or to insert an exogenous nucleic acid sequence into the host cell genome or to delete an endogenous nucleic acid sequence, or to replace an endogenous nucleic acid sequence in the host cell genome with an exogenous nucleic acid sequence, in order to alter the genotype of the host cell and thereby alter its phenotype.

[0075] Expressing an exogenous protein (such as one or more enzymes as mentioned above) in a host cell can comprise introducing an exogenous gene into the host cell, in order to express a protein encoded by the exogenous gene in the host cell, which can be achieved for example by introducing a nucleic acid sequence comprising the exogenous gene (e.g. a vector) into the host cell. The nucleic acid sequence comprising the exogenous gene can be in linear form or in circular form, and can be single-stranded or double-stranded. The vector can be a self-replicating vector. The vector can be an episomal vector or an integrating vector. The vector can be for example a plasmid vector, a phage vector, a bacterial artificial chromosome, a transposition-based vector, or a CRISPR / Cas system-based vector, etc. The vector can also be a suicidal vector, such as a suicidal plasmid vector.

[0076] The vector can comprise an exogenous gene expression cassette, which can include an exogenous gene and regulatory sequences operably linked thereto that can direct expression of the exogenous gene in a suitable host cell. The regulatory sequences can include, but are not limited to, promoters, enhancers, terminators, and other expression control elements. The promoter can be a constitutive promoter to allow continuous expression of the exogenous gene, or an inducible promoter to allow induced expression of the exogenous gene upon addition of an inducer. Regulatory sequences suitable for use in C. glutamicum are well known to those skilled in the art, for example, promoters suitable for use in C. glutamicum include, but are not limited to, inducible promoters such as Ptac, Plac, Ptrc, and the like, or constitutive promoters such as Psod, PcspB, Ptuf, and PgapA, and the like; other promoters derived from C. glutamicum and variants thereof, such as Pcg2195 and the like (Wei L, et al., Appl Microbiol Biotechnol. 2018 May; 102(9): 4117-4130, hereby incorporated by reference in its entirety) ; PdapA and variants thereof such as Pdap-A16 (Vasicova P, et al., J Bacteriol. 1999 Oct; 181(19): 6188-91, hereby incorporated by reference in its entirety); Pdap-A16-1 and variants thereof Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, and the like (Duan, Yanting et al., ACS Synthetic Biology. 2021, 10, 38-48, hereby incorporated by reference in its entirety); and promoters engineered based on these promoters, such as promoters obtained by combining different promoters with different translation initiation elements, such as BCD (bicistronic design) (Mutalik, V., et al., Nat Methods 10, 354-360 (2013)). In some embodiments, the expression of the exogenous gene uses the Psod promoter, which comprises a nucleic acid sequence as set forth in SEQ ID NO: 80. In some embodiments, the expression of the exogenous gene uses the Pdape11-BCD5 promoter, which comprises a nucleic acid sequence as set forth in SEQ ID NO: 81. In some embodiments, the expression of the exogenous gene uses the Pcg2195 promoter, which comprises a nucleic acid sequence as set forth in SEQ ID NO: 82.

[0077] The vector can also comprise one or more selectable marker genes that allow for easy selection of transformed, transfected or transduced cells, such as genes that provide resistance to antibiotics, heavy metals and / or are negative selection marker genes (e.g. the sacB gene) and the like.

[0078] Vectors that can be used to introduce an exogenous nucleic acid sequence in C. glutamicum are well known to the person skilled in the art and include, but are not limited to, the pBL1, pEKEx1, pEKEx2, pXMJ19, pJC1, pHM1519, pVWEx1, pZ8-1, pECTAC-K99, pECTAC-XK99E, pECTAC-XC99E, pECTAC-XT99A, pNG2, pAPE12 plasmid vectors or plasmid vectors based thereon, pK18mobsacB or suicide plasmid vectors based thereon.

[0079] The exogenous gene can be present in and expressed from an episomal vector (e.g. an episomal plasmid) after being introduced into the host cell, for example by introducing an expression vector (e.g. a plasmid vector) comprising the expression cassette of the exogenous gene into the host cell. The exogenous gene can also be integrated into the genome of the host cell for expression, for example by introducing an integrative plasmid vector (which can comprise homology arms for integration into the host cell genome by homologous recombination, for example) comprising the exogenous gene into the host cell, a phage vector, a CRISPR / Cas system or a transposon system (e.g. Piggybac or Sleeping Beauty system) into the host cell. The exogenous gene can be randomly integrated into the host cell or can be targeted to integrate into a suitable site in the host cell (e.g. by homologous recombination or a CRISPR / Cas system). The expression cassette comprising the exogenous gene can be randomly or targeted integrated into the host cell genome or the exogenous gene can be targeted to integrate into a suitable site in the host cell to make use of the host cell’s endogenous regulatory sequences for expression of the exogenous gene.

[0080] Methods for expressing multiple exogenous genes in a host cell are well known to those skilled in the art. For example, a separate transcriptional unit can be constructed for each exogenous gene, each with a separate promoter and transcribed into a separate mRNA, and appropriate promoters can be selected for different genes as desired. For another example, two or more (e.g., two, three, or four) exogenous genes can be included in the same transcriptional unit, sharing the same promoter. Each of the multiple exogenous genes in the same transcriptional unit can be preceded by a ribosome binding site (RBS) to facilitate translation of each gene individually, with the multiple genes being transcribed and translated in a polycistronic fashion. The multiple transcriptional units can be included in different vectors separately or in the same vector. The transcriptional units can be introduced into the host cell by the methods described above.

[0081] Suitable methods for introducing an exogenous nucleic acid sequence (e.g., a vector) into a host cell are known to those skilled in the art, including but not limited to calcium phosphate transfection, protoplast fusion, electroporation, liposomes, lipid nanoparticles, microinjection, naked DNA or RNA (e.g., mRNA) transfection, plasmid vector transformation, phage vector transduction, etc.

[0082] The genetic engineering techniques described above can also be used to knock out or knock down an endogenous gene, so that the host cell does not have a functional protein encoded by the endogenous gene or so that the protein encoded by the endogenous gene has reduced activity. This can be achieved, for example, by deleting all or part of the sequence of the endogenous gene, mutating the endogenous gene, or inserting an exogenous sequence into the endogenous gene. These genetic engineering techniques can also be used to alter the regulatory sequences (e.g., promoters) of an endogenous gene to increase or decrease expression of the endogenous gene. Those skilled in the art are able to select appropriate methods for genetically engineering a host cell based on the host cell and the exogenous or endogenous nucleic acid sequences used (A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989) and Ausubel et al, eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1997)).

[0083] In some embodiments, the gene encoding any one or more of the above-described exogenous enzymes is integrated into the chromosome of the C. glutamicum or is contained in a free expression vector (e.g., a plasmid vector) that is introduced into the C. glutamicum. In some embodiments, the expression of the gene encoding any one or more of the above-described exogenous enzymes is initiated using a promoter derived from C. glutamicum. In some embodiments, the expression of the gene encoding any one or more of the above-described exogenous enzymes is initiated using the Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter.

[0084] In some embodiments, the gene of any one or more of the above-described exogenous enzymes is codon-optimized for C. glutamicum.

[0085] In some embodiments, any one or more of the above-described exogenous enzymes is introduced into the C. glutamicum by transforming the C. glutamicum with a plasmid vector comprising a gene expression cassette of any one or more of the above-described exogenous enzymes. In some embodiments, the gene expression cassette of any one or more of the above-described exogenous enzymes comprises a promoter operably linked to the gene of any one or more of the above-described exogenous enzymes, which is a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB.

[0086] In some embodiments, the gene encoding any one or more of the above-mentioned exogenous enzymes can be integrated into the ISCgl (e.g., ISCgl a, ISCgl b, ISCgl c, and / or ISCgl e) and / or ISCg2 locus (e.g., ISCg2b, ISCg2c, ISCg2d, ISCg2e, and / or ISCg2f) locus, poxB locus, and / or tnp21a (ISCg21a) locus in the C. glutamicum chromosome. In some embodiments, the ISCgl (e.g., ISCgl a, ISCgl b, ISCgl c, and / or ISCgl e) and / or ISCg2 locus (e.g., ISCg2b, ISCg2c, ISCg2d, ISCg2e, and / or ISCg2f) locus, poxB locus, and / or tnp21a (ISCg21a) locus in the C. glutamicum chromosome is knocked out, and the gene encoding any one or more of the above-mentioned exogenous enzymes is introduced at the position of the knocked-out locus.

[0087] In some embodiments, when part of the sequence or the entire sequence of the enzyme gene involved in the deletion of the competing metabolic pathway of the intermediate metabolite and / or the sialoside enzyme gene as described below is deleted, the gene of any one, two, or three of the above-mentioned exogenous enzymes can also be integrated into the position of the sequence of the deleted enzyme gene (i.e., glucosamine-6-phosphate deaminase (NagB) gene, N-acetylglucosamine-6-phosphate deacetylase (NagA) gene, N-acetylmannosamine kinase (NanK) gene, N-acetylmannosamine-6-phosphate 2-epimerase (NanE) gene, and / or N-acetylneuraminidase (NanA) gene, and / or nanH and / or nanP).

[0088] In some embodiments, a gene encoding an exogenous N-acetylneuraminate synthase (NeuB) is integrated into the chromosome of C. glutamicum or contained in a free expression vector (e.g., a plasmid vector) that is introduced into C. glutamicum. In some embodiments, expression of the exogenous N-acetylneuraminate synthase (NeuB) gene is initiated using a promoter derived from C. glutamicum. In some embodiments, expression of the exogenous N-acetylneuraminate synthase (NeuB) gene is initiated using a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the gene for the exogenous N-acetylneuraminate synthase (NeuB) is codon optimized for C. glutamicum. In some embodiments, the exogenous N-acetylneuraminate synthase (NeuB) is introduced into C. glutamicum by transforming C. glutamicum with a plasmid vector comprising an exogenous N-acetylneuraminate synthase (NeuB) gene expression cassette. In some embodiments, the exogenous N-acetylneuraminate synthase (NeuB) gene expression cassette comprises a promoter operably linked to an exogenous N-acetylneuraminate synthase (NeuB) gene, the promoter being a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, the gene encoding an exogenous N-acetylneuraminate synthase (NeuB) is integrated into the ISCg2f locus in the chromosome of C. glutamicum. In some embodiments, the ISCg2f locus in the chromosome of C. glutamicum is knocked out and a gene for an exogenous N-acetylneuraminate synthase (NeuB) is introduced at the location of the knocked out locus.

[0089] In some embodiments, a gene encoding an exogenous CMP-N-acetylneuraminate synthase (NeuA) is integrated into the chromosome of C. glutamicum or contained in a free expression vector (e.g., a plasmid vector) that is introduced into C. glutamicum. In some embodiments, expression of the exogenous CMP-N-acetylneuraminate synthase (NeuA) gene is initiated using a promoter derived from C. glutamicum. In some embodiments, expression of the exogenous CMP-N-acetylneuraminate synthase (NeuA) gene is initiated using a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the gene for the exogenous CMP-N-acetylneuraminate synthase (NeuA) is codon optimized for C. glutamicum. In some embodiments, the exogenous CMP-N-acetylneuraminate synthase (NeuA) is introduced into C. glutamicum by transforming C. glutamicum with a plasmid vector comprising an exogenous CMP-N-acetylneuraminate synthase (NeuA) gene expression cassette. In some embodiments, the exogenous CMP-N-acetylneuraminate synthase (NeuA) gene expression cassette comprises a promoter operably linked to an exogenous CMP-N-acetylneuraminate synthase (NeuA) gene, the promoter being a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, a gene encoding an exogenous CMP-N-acetylneuraminate synthase (NeuA) is integrated into the ISCg2d locus in the chromosome of C. glutamicum. In some embodiments, the ISCg2d locus in the chromosome of C. glutamicum is knocked out and a gene for an exogenous CMP-N-acetylneuraminate synthase (NeuA) is introduced at the location of the knocked out locus.

[0090] In some embodiments, the gene encoding the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) is integrated into the chromosome of the C. glutamicum or contained in a free expression vector (such as a plasmid vector) that is introduced into the C. glutamicum. In some embodiments, the expression of the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) gene is initiated using a promoter derived from C. glutamicum. In some embodiments, the expression of the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) gene is initiated using Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the gene for the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) is codon optimized for C. glutamicum. In some embodiments, the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) gene is introduced into the C. glutamicum by transforming the C. glutamicum with a plasmid vector comprising an exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) gene expression cassette. In some embodiments, the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) gene expression cassette comprises a promoter operably linked to the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) gene, which is Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, the gene encoding the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) is integrated into the ISCg2d locus in the C. glutamicum chromosome. In some embodiments, the ISCg2d locus in the C. glutamicum chromosome is knocked out and the gene for the exogenous sialyltransferase (such as an a-2,3-sialyltransferase or an a-2,6-sialyltransferase) is introduced at the location of the knocked out locus.

[0091] In some embodiments, the gene encoding the exogenous lactose transporter is integrated into the chromosome of the C. glutamicum or is contained in a free expression vector (e.g., a plasmid vector) that is introduced into the C. glutamicum. In some embodiments, the expression of the exogenous lactose transporter gene is initiated using a promoter derived from C. glutamicum. In some embodiments, the expression of the exogenous lactose transporter gene is initiated using the Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdapel l-BCD5 promoter. In some embodiments, the gene for the exogenous lactose transporter is codon-optimized for C. glutamicum. In some embodiments, the exogenous lactose transporter is introduced into the C. glutamicum by transforming the C. glutamicum with a plasmid vector comprising an exogenous lactose transporter gene expression cassette. In some embodiments, the exogenous lactose transporter gene expression cassette comprises a promoter operably linked to the exogenous lactose transporter gene, and the promoter is a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdapel l-BCD5 promoter. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, the gene encoding the exogenous lactose transporter is integrated into the poxB locus in the chromosome of the C. glutamicum. In some embodiments, the poxB locus in the chromosome of the C. glutamicum is knocked out, and the gene for the exogenous lactose transporter is introduced at the location of the knocked-out locus.

[0092] In some embodiments, a gene encoding an exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) is integrated into the chromosome of C. glutamicum or contained in a free expression vector (e.g., a plasmid vector) that is introduced into C. glutamicum. In some embodiments, expression of the exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) gene is initiated using a promoter derived from C. glutamicum. In some embodiments, expression of the exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) gene is initiated using a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the gene for the exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) is codon optimized for C. glutamicum. In some embodiments, the exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) gene is introduced into C. glutamicum by transforming C. glutamicum with a plasmid vector comprising an exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) gene expression cassette. In some embodiments, the exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) gene expression cassette comprises a promoter operably linked to an exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) gene, wherein the promoter is a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, the gene encoding an exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) is integrated into the ISCg2f locus in the chromosome of C. glutamicum. In some embodiments, the ISCg2f locus in the chromosome of C. glutamicum is knocked out and a gene for an exogenous UDP-N-acetylglucosamine 2-epimerase (NeuC) is introduced at the location of the knocked out gene.

[0093] In some embodiments, a gene encoding an exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) is integrated into the chromosome of C. glutamicum or contained in a free expression vector (e.g., a plasmid vector) that is introduced into C. glutamicum. In some embodiments, expression of the exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) gene is initiated using the Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the gene for the exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) is codon optimized for C. glutamicum. In some embodiments, the exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) gene is introduced into C. glutamicum by transforming C. glutamicum with a plasmid vector comprising an exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) gene expression cassette. In some embodiments, the exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) gene expression cassette comprises a promoter operably linked to the exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) gene, and the promoter is a Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, the gene encoding an exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) is integrated into the tnp21a locus in the chromosome of C. glutamicum. In some embodiments, the tnp21a locus in the chromosome of C. glutamicum is knocked out, and the gene for the exogenous glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) is introduced at the location of the knocked out gene.

[0094] In some embodiments, a gene encoding an exogenous N-acetylglucosamine 2-epimerase (Age) is integrated into the chromosome of C. glutamicum or contained in a free expression vector (e.g., a plasmid vector) that is introduced into C. glutamicum. In some embodiments, expression of the exogenous N-acetylglucosamine 2-epimerase (Age) gene is initiated using the Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdap-e11-BCD5 promoter. In some embodiments, the gene for the exogenous N-acetylglucosamine 2-epimerase (Age) is codon optimized for C. glutamicum. In some embodiments, the exogenous N-acetylglucosamine 2-epimerase (Age) gene is introduced into C. glutamicum by transforming C. glutamicum with a plasmid vector comprising an exogenous N-acetylglucosamine 2-epimerase (Age) gene expression cassette. In some embodiments, the exogenous N-acetylglucosamine 2-epimerase (Age) gene expression cassette comprises a promoter operably linked to the exogenous N-acetylglucosamine 2-epimerase (Age) gene, the promoter being Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdap-e11-BCD5. In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, the gene encoding an exogenous N-acetylglucosamine 2-epimerase (Age) is integrated into the tnp21a locus in the C. glutamicum chromosome. In some embodiments, the tnp21a locus in the C. glutamicum chromosome is knocked out and a gene for an exogenous N-acetylglucosamine 2-epimerase (Age) is introduced at the location of the knocked out gene.

[0095] In some embodiments, 2 or more (e.g., 2, 3, or 4) of the above-mentioned exogenous genes can be introduced into the C. glutamicum in the same transcriptional unit. In some embodiments, the UDP-N-acetylglucosamine 2-epimerase (NeuC) gene and the N- acetylneuraminate synthase (NeuB) gene are introduced into the C. glutamicum in the same transcriptional unit. In some embodiments, the CMP-N-acetylneuraminate synthase (NeuA) gene and the sialyltransferase gene are introduced into the C. glutamicum in the same transcriptional unit. In some embodiments, the glucosamine-6-phosphate N-acetyltransferase 1 (Gnal) gene and the N- acetylglucosamine 2-epimerase (Age) gene are introduced into the C. glutamicum in the same transcriptional unit.

[0096] In some embodiments, the recombinant C. glutamicum can be further engineered to eliminate or attenuate the competing metabolic pathways of the above-mentioned intermediate metabolites in the synthesis pathway 1 or the synthesis pathway 2, to enhance the flux of these intermediate metabolites into the above-mentioned synthesis pathway 1 or the synthesis pathway 2, thereby enhancing the use of these intermediate metabolites for the synthesis of the target product, sialyllactose. The competing metabolic pathways refer to the metabolic pathways that are not included in the above-mentioned synthesis pathway 1 or the synthesis pathway 2, in which the intermediate metabolites are involved, and which produce other substances from the intermediate metabolites, which are not any intermediate metabolites or products involved in the synthesis pathway 1 or the synthesis pathway 2.

[0097] In some embodiments, the intermediate metabolites of the synthesis pathway 1 include fructose-6-phosphate (Fru-6-P), glucosamine-6-phosphate (GlcN-6-P), glucosamine-1-phosphate (GlcN-1-P), UDP-acetylglucosamine (UDP-GlcNAc), N-acetylmannosamine (ManNAc), N-acetylneuraminic acid (Neu5Ac, sialic acid), CMP-N-acetylneuraminic acid (CMP-Neu5Ac), lactose.

[0098] In some embodiments, the intermediate metabolites of the synthesis pathway 2 include fructose-6-phosphate (Fru-6-P), glucosamine-6-phosphate (GlcN-6-P), acetylglucosamine-6-phosphate (GlcNAc-6-P), acetylglucosamine (GlcNAc), N-acetylmannosamine (ManNAc), N-acetylneuraminic acid (Neu5Ac, sialic acid), CMP-N-acetylneuraminic acid (CMP-Neu5Ac), lactose.

[0099] As shown in FIG. 1 and FIG. 2, through analysis of the Corynebacterium glutamicum genome, it was found that for the intermediate metabolites in the synthesis pathway 1 or the synthesis pathway 2, the following competitive metabolic pathways catalyzed by the naturally existing enzymes in the Corynebacterium glutamicum exist: (1) endogenous glucosamine-6-phosphate deaminase (NagB) which can catalyze glucosamine-6-phosphate (GlcN-6-P) to generate fructose-6-phosphate (Fru-6-P); (2) endogenous N-acetylmannosamine kinase (NanK) which can catalyze N-acetylmannosamine (ManNAc) to generate acetylmannosamine-6-phosphate (ManNAc-6-P); (3) endogenous N-acetylmannosamine-6-phosphate 2-epimerase (NanE) which can catalyze the reversible reaction between acetylmannosamine-6-phosphate (ManNAc-6-P) and acetylglucosamine-6-phosphate (GlcNAc-6-P); (4) endogenous N-acetylglucosamine-6-phosphate deacetylase (NagA) which can catalyze acetylglucosamine-6-phosphate (GlcNAc-6-P) to generate glucosamine-6-phosphate (GlcN-6-P); and (5) endogenous N-acetylneuraminic acid lyase (NanA) which can catalyze the reversible reaction between N-acetylmannosamine (ManNAc) and N-acetylneuraminic acid (Neu5Ac, sialic acid).

[0100] Therefore, in some embodiments, on the basis of the aforementioned genetic modification, the Corynebacterium glutamicum can be further genetically modified to eliminate or weaken the action of one or more endogenous enzymes involved in the aforementioned competitive metabolic pathways. In some embodiments, the Corynebacterium glutamicum is genetically modified to eliminate or weaken the action of all five endogenous enzymes involved in the aforementioned competitive metabolic pathways.

[0101] Therefore, in some embodiments, the recombinant Corynebacterium glutamicum can comprise the following genetic modifications:

[0102] (1) at least one genetic modification capable of eliminating or weakening the action of glucosamine-6-phosphate deaminase (NagB), thereby preventing or reducing the reaction from proceeding in the direction from glucosamine-6-phosphate (GlcN-6-P) to generate fructose-6-phosphate (Fru-6-P);

[0103] (2) at least one genetic modification capable of eliminating or weakening the action of one, two or three enzymes selected from the group consisting of N-acetylglucosamine-6-phosphate deacetylase (NagA), N-acetylmannosamine kinase (NanK) and N-acetylmannosamine-6-phosphate 2-epimerase (NanE), thereby preventing or reducing the reaction from proceeding in the direction from N-acetylmannosamine (ManNAc) to generate glucosamine-6-phosphate (GlcN-6-P);

[0104] (3) at least one genetic modification that eliminates or attenuates the function of one or both of N-acetylmannosamine kinase (NanK) and N-acetylmannosamine-6-phosphate 2-epimerase (NanE), thereby preventing or reducing the reaction proceeding in the direction from N-acetylmannosamine (ManNAc) toward the production of acetylglucosamine-6-phosphate (GlcNAc-6-P);

[0105] (4) at least one genetic modification that eliminates or attenuates the function of N-acetylglucosamine-6-phosphate deacetylase (NagA), thereby preventing or reducing the reaction proceeding in the direction from acetylglucosamine-6-phosphate (GlcNAc-6-P) toward the production of glucosamine-6-phosphate (GlcN-6-P); and / or

[0106] (5) at least one genetic modification that eliminates or attenuates the function of N-acetylneuraminic acid lyase (NanA), thereby preventing or reducing the reaction proceeding in the direction from N-acetylneuraminic acid (Neu5Ac, sialic acid) toward the production of N-acetylmannosamine (ManNAc).

[0107] In some embodiments, the recombinant C. glutamicum comprises one or more or all of the above-described (1), (2), and (5) genetic modifications to eliminate or attenuate the competing metabolic pathways of the above-described intermediate metabolites of synthesis pathway 1.

[0108] In some embodiments, the recombinant C. glutamicum comprises one or more or all of the above-described (1), (3), (4), and (5) genetic modifications to eliminate or attenuate the competing metabolic pathways of the above-described intermediate metabolites of synthesis pathway 1.

[0109] For any one or more endogenous enzymes involved in the above-described competing metabolic pathways, the genetic modification that eliminates or attenuates the enzyme includes a genetic modification that partially or completely inactivates the endogenous gene encoding the enzyme, including but not limited to: knocking out or knocking down the endogenous gene, e.g., deleting all or part of the sequence of the endogenous gene, mutating the endogenous gene, inserting an exogenous sequence into the endogenous gene, to inactivate or attenuate the enzyme activity encoded by the endogenous gene. The genetic modification that partially or completely inactivates the endogenous gene encoding the enzyme can also include knocking out or knocking down the natural regulatory sequence (e.g., natural promoter) of the endogenous gene, e.g., deleting all or part of the sequence of the regulatory sequence, mutating the regulatory sequence, inserting an exogenous sequence into the regulatory sequence, to partially or completely inactivate the regulatory sequence, to eliminate or reduce the expression of the endogenous gene.

[0110] The above gene modification techniques are well known to those skilled in the art, for example, can be achieved by introducing an integrative plasmid vector (the plasmid can comprise homology arms to integrate into a specified site in the host cell genome by homologous recombination, for example) or a targeted genome editing technology (such as CRISPR / Cas system) into the host cell. In some embodiments, the above gene modification can be achieved by transforming C. glutamicum with a recombination nucleic acid molecule (such as a plasmid vector) with homology arms to replace part or all of the endogenous gene by homologous recombination to achieve partial or complete deletion of the endogenous gene, or to replace part or all of the natural promoter to achieve partial or complete deletion of the natural promoter. In some embodiments, the recombination nucleic acid molecule with homology arms used comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB.

[0111] In some embodiments, the glucosamine-6-phosphate deaminase (NagB) comprises an amino acid sequence as set forth in SEQ ID NO: 85. In some embodiments, the gene encoding the glucosamine-6-phosphate deaminase (NagB) comprises a nucleic acid sequence as set forth in SEQ ID NO: 86.

[0112] In some embodiments, the N-acetylglucosamine-6-phosphate deacetylase (NagA) comprises an amino acid sequence as set forth in SEQ ID NO: 83. In some embodiments, the gene encoding the N-acetylglucosamine-6-phosphate deacetylase (NagA) comprises a nucleic acid sequence as set forth in SEQ ID NO: 84.

[0113] In some embodiments, the N-acetylglucosamine-6-phosphate deacetylase (NagA) comprises an amino acid sequence as set forth in SEQ ID NO: 83. In some embodiments, the gene encoding the N-acetylglucosamine-6-phosphate deacetylase (NagA) comprises a nucleic acid sequence as set forth in SEQ ID NO: 84.

[0114] In some embodiments, the N-acetylglucosamine-6-phosphate deacetylase (NagA) comprises an amino acid sequence as set forth in SEQ ID NO: 83. In some embodiments, the gene encoding the N-acetylglucosamine-6-phosphate deacetylase (NagA) comprises a nucleic acid sequence as set forth in SEQ ID NO: 84.

[0115] In some embodiments, the N-acetylneuraminidase (NanA) comprises an amino acid sequence as set forth in SEQ ID NO: 87. In some embodiments, the gene encoding the N- acetylmannosamine kinase (NanK) comprises a nucleic acid sequence as set forth in SEQ ID NO: 88.

[0116] When different strains of C. glutamicum are used as host cells, the amino acid sequences or the gene sequences of the glucosamine-6-phosphate deaminase (NagB), the N- acetylglucosamine-6-phosphate deacetylase (NagA), the N-acetylmannosamine kinase (NanK), the N-acetylmannosamine-6-phosphate 2-epimerase (NanE), and / or the N- acetylneuraminidase (NanA) can be different from the above-mentioned sequences, but it is understood that these enzymes also encompass their homologous proteins in different strains of C. glutamicum, which have the same activity as the above-mentioned enzymes and have a similar structure, for example, have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the recombinant C. glutamicum can be further engineered to enhance the reactions naturally present in C. glutamicum in the sialyllactose synthesis pathway 1 or synthesis pathway 2, for example, the (1), (2), and / or (3) step reactions in synthesis pathway 1, or the (1) and / or (3-a) step reactions in synthesis pathway 2, which can be achieved by enhancing the action of the enzymes involved in these reactions.

[0117] In some embodiments, the recombinant C. glutamicum can further comprise one, two, or three of the following genetic modifications:

[0118] (1) at least one genetic modification to enhance the action of glutamine-fructose-6-phosphate aminotransferase (GlmS) to strengthen the reaction of generating glucosamine-6-phosphate (GlcN-6-P) from fructose-6-phosphate (Fru-6-P);

[0119] (2) at least one genetic modification to enhance the action of phosphoglucosamine mutase (GlmM) to strengthen the reaction of generating glucosamine-1-phosphate (GlcN-1-P) from glucosamine-6-phosphate (GlcN-6-P); and

[0120] (3) at least one genetic modification to enhance the action of N-acetylglucosamine-1-phosphate urtidyltransferase and glucosamine-1-phosphate acetyltransferase bifunctional enzyme (GlmU) to strengthen the reaction of generating UDP-N-acetylglucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P).

[0121] For any one or more of the enzymes described above, genetic modifications that enhance the action of the enzyme can include increasing the activity of the enzyme and / or overexpressing the enzyme. Increasing the activity of the enzyme can include, for example, mutating the endogenous enzyme gene to increase its enzymatic activity, or replacing the endogenous enzyme gene with an exogenous enzyme gene that has higher enzymatic activity, which can be an enzyme derived from a different species than the host cell, or a mutant of a wild-type enzyme. Overexpressing the enzyme can include, for example, increasing the gene copy number of the enzyme gene, and / or replacing the natural promoter of the enzyme with a promoter that has higher expression levels. In some embodiments, increasing the gene copy number of the enzyme includes introducing an exogenous enzyme gene into the host cell without changing the expression of the endogenous enzyme gene, which can be 1, 2, 3, or more copies, which can have the same or different sequence as the endogenous enzyme, for example, the exogenous enzyme can be derived from the same species as the host cell, or the exogenous enzyme can have higher enzymatic activity than the endogenous enzyme, for example, can be an enzyme derived from a different species than the host cell, or a mutant of a wild-type enzyme.

[0122] In some embodiments, the recombinant C. glutamicum comprises a genetic modification that increases the copy number of one, two, or three of the glutamine- fructose-6-phosphate amidotransferase (GlmS) gene, the phosphoglucosamine mutase (GlmM) gene, the N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) gene.

[0123] In some embodiments, the genetic modification that increases the copy number of the glutamine-fructose-6-phosphate amidotransferase (GlmS) gene comprises introducing a glutamine-fructose-6-phosphate amidotransferase (GlmS) into the C. glutamicum. In some embodiments, the introduced glutamine-fructose-6-phosphate amidotransferase (GlmS) gene is a gene of a glutamine-fructose-6-phosphate amidotransferase (GlmS) derived from C. glutamicum or a functional variant thereof. In some embodiments, the introduced glutamine-fructose-6-phosphate amidotransferase (GlmS) has higher enzymatic activity than the endogenous glutamine-fructose-6-phosphate amidotransferase (GlmS) of C. glutamicum. In some embodiments, the introduced exogenous glutamine-fructose-6-phosphate amidotransferase (GlmS) is a glutamine-fructose-6-phosphate amidotransferase mutant GlmS*54 derived from E. coli MG1655. In some embodiments, the GlmS*54 comprises an amino acid sequence as set forth in SEQ ID NO: 17. In some embodiments, the gene of the GlmS*54 comprises a nucleic acid sequence as set forth in SEQ ID NO: 18.

[0124] In some embodiments, the genetic modification that increases the copy number of the phosphoglucosamine mutase (GlmM) gene comprises introducing a phosphoglucosamine mutase (GlmM) gene into the C. glutamicum. In some embodiments, the phosphoglucosamine mutase (GlmM) gene is a gene for a phosphoglucosamine mutase (GlmM) or a functional variant thereof derived from C. glutamicum. In some embodiments, the introduced phosphoglucosamine mutase (GlmM) gene has the same sequence as the enzyme encoded by the endogenous phosphoglucosamine mutase (GlmM) gene in the C. glutamicum. In some embodiments, the phosphoglucosamine mutase (GlmM) comprises an amino acid sequence as set forth in SEQ ID NO: 95 or a functional variant thereof. In some embodiments, the gene for the phosphoglucosamine mutase (GlmM) comprises a nucleic acid sequence as set forth in SEQ ID NO: 96.

[0125] In some embodiments, the genetic modification that increases the copy number of the N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) gene comprises introducing a N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) gene into the C. glutamicum. In some embodiments, the N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) gene is a gene for a N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) or a functional variant thereof derived from C. glutamicum. In some embodiments, the introduced N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) gene has the same sequence as the enzyme encoded by the endogenous N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) gene in the C. glutamicum. In some embodiments, the N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) comprises an amino acid sequence as set forth in SEQ ID NO: 93 or a functional variant thereof. In some embodiments, the gene for the N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) comprises a nucleic acid sequence as set forth in SEQ ID NO: 94.

[0126] In some embodiments, any one, two, or three of the above-described glutamine- fructose-6-phosphate aminotransferase (GlmS) gene, phosphoglucosamine mutase (GlmM) gene, and N-acetylglucosamine-1 -phosphate urtidylyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme (GlmU) gene introduced into C. glutamicum is integrated into the chromosome of C. glutamicum or is contained in a free expression vector (e.g., a plasmid vector) introduced into C. glutamicum.

[0127] In some embodiments, expression of any one, two, or three of the above-described enzyme genes is initiated using a promoter derived from C. glutamicum. In some embodiments, expression of any one, two, or three of the above-described enzyme genes is initiated using the Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter.

[0128] In some embodiments, any one, two, or three of the above-described enzyme genes is codon-optimized for C. glutamicum.

[0129] In some embodiments, any one or more of the above-described enzymes is introduced into C. glutamicum by transforming C. glutamicum with a plasmid vector comprising an expression cassette for any one, two, or three of the above-described enzyme genes. In some embodiments, the expression cassette for any one, two, or three of the above-described enzyme genes comprises a promoter operably linked to any one, two, or three of the above-described enzyme genes, which is the Psod, PcspB, Ptuf, PgapA, Pcg2195, PdapA, Pdap-A16, Pdap-A16-1, Pdap-e10, Pdap-e11, Pdap-e12, Pdap-e10-35, or Pdape11-BCD5 promoter. In some embodiments, any two or three of the above-described enzyme genes are introduced into C. glutamicum contained in the same transcriptional unit.

[0130] In some embodiments, the plasmid vector comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB. In some embodiments, any one, two or three of the aforementioned enzyme genes can be integrated into the ISCgl (e.g., ISCgl a, ISCgl b, ISCgl c and / or ISCgl e) and / or ISCg2 locus (e.g., ISCg2b, ISCg2c, ISCg2d, ISCg2e and / or ISCg2f) loci, poxB locus and / or tnp21a (ISCg21a) locus in the C. glutamicum chromosome. In some embodiments, the ISCgl (e.g., ISCgl a, ISCgl b, ISCgl c and / or ISCgl e) and / or ISCg2 locus (e.g., ISCg2b, ISCg2c, ISCg2d, ISCg2e and / or ISCg2f) loci, poxB locus and / or tnp21a (ISCg21a) locus in the C. glutamicum chromosome are knocked out, and any one, two or three of the aforementioned enzyme genes are introduced at the position of the knocked-out loci. In some embodiments, any one, two or three of the aforementioned enzyme genes can also be integrated into the position of the sequence of the aforementioned deleted competing metabolic pathway enzymes involved in the intermediate metabolite (i.e., glucosamine-6-phosphate deaminase (NagB) gene, N-acetylglucosamine-6-phosphate deacetylase (NagA) gene, N-acetylmannosamine kinase (NanK) gene, N-acetylmannosamine-6-phosphate 2-epimerase (NanE) gene and / or N-acetylneuraminate lyase (NanA) gene).

[0131] In addition, the present inventors have also found that there are two naturally occurring sialoside hydrolase (also known as sialidase) genes, nanH (also known as Cgl1556 or cg1756, GenBank Accession No. BAB98949.1) and nanP (also known as Cgl2650 or cg2935, GenBank Accession No. BAC00044.1) in the C. glutamicum genome, which encode proteins that can be classified as glycoside hydrolases and can degrade the product sialyllactose. Elimination or attenuation of the action of these enzymes can further improve the yield of sialyllactose.

[0132] In some embodiments, the recombinant C. glutamicum can further comprise a genetic modification that eliminates or attenuates the action of an endogenous sialoside hydrolase, which can include the following genetic modifications:

[0133] (1) at least one genetic modification that eliminates or attenuates the action of the enzyme encoded by nanH; and / or

[0134] (2) at least one genetic modification that eliminates or attenuates the action of a nanP-encoded enzyme.

[0135] Genetic modifications that eliminate or attenuate the action of nanH- and / or nanP-encoded enzymes include partial or complete inactivation of nanH and / or nanP, including but not limited to: knocking out or knocking down nanH and / or nanP, e.g., deleting all or part of the sequence of nanH and / or nanP, mutating nanH and / or nanP, and / or inserting an exogenous sequence into nanH and / or nanP. Genetic modifications that partially or completely inactivate nanH and / or nanP can also include knocking out or knocking down the natural regulatory sequence (e.g., the natural promoter) of nanH and / or nanP, e.g., deleting all or part of the sequence of the regulatory sequence, mutating the regulatory sequence, and / or inserting an exogenous sequence into the regulatory sequence to partially or completely inactivate the regulatory sequence to eliminate or reduce the expression of nanH and / or nanP. The above genetic modification techniques are well known to those skilled in the art, e.g., can be achieved by introducing an integrating plasmid vector (which can comprise homology arms to integrate into the host cell genome at a specified site by homologous recombination, for example) or a targeted genome editing technology (such as the CRISPR / Cas system) into the host cell. In some embodiments, the above genetic modification can be achieved by transforming C. glutamicum with a recombination nucleic acid molecule (e.g., a plasmid vector) with homology arms to replace part or all of nanH and / or nanP by homologous recombination to achieve partial or complete deletion of nanH and / or nanP, or to replace part or all of the natural promoter to achieve partial or complete deletion of the natural promoter. In some embodiments, the recombination nucleic acid molecule with homology arms used comprises a positive selection marker and a negative selection marker. In some embodiments, the positive selection marker is a kanamycin resistance gene. In some embodiments, the negative selection marker is sacB.

[0136] In some embodiments, the nanH comprises a nucleic acid sequence as set forth in SEQ ID NO: 98. In some embodiments, the nanH-encoded enzyme comprises an amino acid sequence as set forth in SEQ ID NO: 97.

[0137] In some embodiments, the nanP comprises a nucleic acid sequence as set forth in SEQ ID NO: 100. In some embodiments, the nanP-encoded enzyme comprises an amino acid sequence as set forth in SEQ ID NO: 99

[0138] When different strains of C. glutamicum are used as host cells, the sequences of the nanH and / or nanP or the enzymes encoded thereby can differ from the sequences described above, but it is understood that these enzymes also encompass their homologous proteins in different strains of C. glutamicum which have the same activity as the enzymes described above and have a similar structure, e.g. at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity over the amino acid sequence of the enzyme.

[0139] The present application also provides a method for producing sialyllactose, the method comprising culturing the recombinant C. glutamicum under suitable conditions to synthesize sialyllactose. In some embodiments, the method further comprises recovering the sialyllactose.

[0140] The recombinant C. glutamicum can be cultured using any suitable medium, such as those well known to those skilled in the art. In some embodiments, at least one carbon source is included in the medium, which can be selected from, but not limited to, glucose, fructose, sucrose, acetic acid, lactic acid, and / or succinic acid, etc. The carbon source can be supplemented at appropriate times during the culturing of the recombinant C. glutamicum. In some embodiments, the carbon source is present in the medium at a concentration of 10 g / L to 50 g / L, such as 20 g / L to 50 g / L, 30 g / L to 50 g / L, 10 g / L to 40 g / L, 20 g / L to 40 g / L, 30 g / L to 40 g / L, 10 g / L to 30 g / L, or 20 g / L to 30 g / L.

[0141] In some embodiments, lactose can be included in the medium. Lactose can be supplemented at appropriate times during the culturing of the host cells. In some embodiments, the initial lactose concentration in the medium is 10 g / L to 50 g / L, such as 20 g / L to 50 g / L, 10 g / L to 40 g / L, 20 g / L to 40 g / L, 10 g / L to 30 g / L, or 20 g / L to 30 g / L. Lactose can be added to the medium at the beginning of culturing the cells, or it can be added to the medium after the cells have been cultured for a period of time, such as 5 to 30 hours, such as 5 to 25 hours, 5 to 20 hours, 10 to 30 hours, 10 to 25 hours, 10 to 20 hours, 15 to 25 hours, 15 to 20 hours, or 20 to 25 hours.

[0142] In some embodiments, the carbon source and / or lactose can be supplemented to the medium during the culturing (fermentation), such as starting 5-20 hours, such as 10-20 hours or 10-15 hours after the start of the culturing (fermentation).

[0143] When the gene or genes encoding an enzyme or enzymes comprised in the host cell are expressed in an inducible manner, an inducer is added during the cell cultivation process to induce expression of the enzyme(s).

[0144] The cultivation of the host cell can be a batch fermentation, i.e. using a closed culture system with a specific medium at the beginning of the fermentation and using specific temperature, pressure, aeration and other environmental conditions to optimize growth, without addition of nutrients and without discharge of the fermentation broth during cultivation of the cells. The cultivation of the host cell can also be a fed-batch fermentation, i.e. nutrients are added intermittently or continuously during the fermentation, but without discharge of the fermentation broth. The cultivation of the host cell can also be a continuous fermentation, i.e. nutrients are added continuously and the fermentation broth is discharged continuously, so that the volume of the broth in the fermentation system is kept constant. The fermentation of the host cell can also be a combination of two or three of the above-mentioned fermentation modes.

[0145] Suitable conditions for the cultivation of the recombinant C. glutamicum include suitable temperature, pH, dissolved oxygen, osmotic pressure, and other conditions. Suitable conditions can differ depending on the different host cell, which can be readily determined by one skilled in the art. In some embodiments, the cultivation step is performed at about 25°C to about 35°C, preferably at about 28°C to about 32°C, more preferably at about 30°C. In some embodiments, the cultivation step is performed at about pH 6.0 to about pH 8.0, preferably at about pH 6.5 to about pH 7.5, more preferably at about pH 7.0. In some embodiments, the cultivation step is performed at about 10% to about 50% dissolved oxygen, more preferably at about 20% to about 40% dissolved oxygen.

[0146] In some embodiments, the cultivation step is performed for about 12 hours or more, for example about 36 hours or more, about 48 hours or more, about 53 hours or more, about 60 hours or more, or about 72 hours or more; about 12 hours or less, for example about 36 hours or less, about 48 hours or less, about 53 hours or less, about 60 hours or less, or about 72 hours or less.

[0147] The production process of the present application can produce about 150 mg / L or more of 3'-sialyllactose after batch fermentation for 48 hours, for example about 250 mg / L or more, about 580 mg / L or more, about 620 mg / L or more, about 660 mg / L or more, or about 680 mg / L or more.

[0148] The production method of the present application can produce about 190 mg / L or more of 6'-sialyllactose after 48 hours of batch fermentation, for example, about 300 mg / L or more, about 640 mg / L or more, about 680 mg / L or more, about 720 mg / L or more, or about 730 mg / L or more.

[0149] The production method of the present application produces up to about 17.3 g / L of 6'-sialyllactose in a fed-batch fermentation.

[0150] The sialyllactose synthesized by the genetically engineered C. glutamicum of the present application can be transported naturally by the cell to the extracellular space. In some embodiments, the production method of the present application includes recovering the synthesized sialyllactose from the culture medium and / or from the host cells, for example, the product can be recovered from the supernatant of the culture medium and / or cell lysate. Cell lysis can be performed by chemical or physical methods known in the art. The term "recovery" refers to the isolation or further purification of the sialyllactose produced by the recombinant C. glutamicum of the present application from other components in the host cell culture. The term "purification" refers to the removal of impurities and unwanted byproducts, such as cells, ions, salts, and / or other saccharides other than the desired sialyllactose.

[0151] Purification can be performed by techniques known to those skilled in the art. For example, the product can be purified from the culture medium by methods known to those skilled in the art, for example, by column chromatography using activated charcoal, elution with a concentration gradient of ethanol, or by size exclusion chromatography or ion exchange chromatography. Purity can be assessed by any known method, such as thin layer chromatography or other electrophoretic or chromatographic techniques generally known in the art.

[0152] Definitions of terms

[0153] The term "gene" refers to a nucleotide sequence that encodes a gene product. The gene product can be a protein or a ribonucleic acid.

[0154] The term "nucleic acid," "nucleic acid sequence," or "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, including DNA or RNA, including linear or circular DNA or RNA.

[0155] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. The enzymes in the present application are proteins that are capable of catalyzing a chemical reaction of a substrate.

[0156] The term "recombinant" when used with reference to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, a recombinant cell expresses genes that it does not normally express, or expresses native genes at abnormal times or in abnormal amounts; or expresses a native gene but in a different form.

[0157] The terms "genetically engineered," "genetically modified," "engineered," "engineered" mean the sequence of a polynucleotide or polypeptide or the sequence of a gene contained by a host cell is altered by human manipulation to contain a sequence that does not naturally occur in the polynucleotide, polypeptide, or host cell. When a host cell is "engineered" to display a certain characteristic (e.g., to contain a gene encoding a certain protein, to express a certain protein, or to overexpress a certain gene), it is meant that the host cell did not have that characteristic prior to the engineering, but does have that characteristic after the engineering.

[0158] The term "synthetic pathway" refers to a series of reactions controlled and catalyzed by enzymes that result in the synthesis of a chemical. The term "synthetic pathway gene" refers to a gene that encodes an enzyme that catalyzes a reaction in a series of reactions comprising a synthetic pathway, which gene, when expressed in a host cell, results in the enzyme catalyzing the synthesis of a particular chemical.

[0159] The term "exogenous" with reference to a host cell means a substance or molecule that originates or is produced by a source other than the host cell. An "exogenous gene" or "exogenous enzyme" means a nucleic acid that is not a gene or enzyme that naturally occurs in the cell, but rather is introduced into the cell by artificial means. The sequence of an exogenous gene or exogenous enzyme can be the same as or different from an endogenous sequence that naturally occurs in the cell. A gene or enzyme that is different from an endogenous sequence that naturally occurs in the cell can be referred to as a heterologous gene or heterologous enzyme, which can be derived from the same or a different strain or species as the host cell. Herein, unless otherwise indicated, reference to an "exogenous gene" or "exogenous enzyme" includes the case of a "heterologous gene" or "heterologous enzyme."

[0160] The term "endogenous" refers to a gene or protein (e.g., a wild-type gene or wild-type enzyme) or a synthetic pathway that naturally occurs in a host cell.

[0161] The term "wild-type" refers to a naturally occurring organism or cell, or a nucleic acid sequence that occurs in a naturally occurring organism or cell, or a protein expressed by a naturally occurring organism or cell.

[0162] The term "naturally occurring" refers to a nucleic acid sequence, amino acid sequence, complex, pathway, or cell that occurs in nature without human intervention.

[0163] The term "derived from", when applied to a protein or gene sequence, refers to a protein or gene sequence that has the same structure or sequence as a protein or gene sequence naturally occurring in a particular organism, and is not limited to being isolated directly from the organism.

[0164] The term "mutant" refers to a polypeptide having one or more insertions, deletions, and / or substitutions of amino acids relative to a parent polypeptide. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of one or several (e.g., 1-5) amino acids adjacent to an amino acid occupying a position. The mutant retains at least one activity of the parent polypeptide, but can vary in the level of activity, e.g., the mutant can be unchanged or improved in at least one activity or property relative to the parent polypeptide.

[0165] The term "parent" refers to a polypeptide that is changed to produce a mutant, and the parent can be a naturally occurring (wild-type) polypeptide or a mutant thereof.

[0166] The term "functional variant", when applied to a polypeptide or protein, refers to a polypeptide having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a parent polypeptide, and having the same or substantially the same function as the parent polypeptide. A functional variant can also refer to a polypeptide having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) insertions, deletions, and / or substitutions of amino acids compared to a parent polypeptide, and having the same or substantially the same function as the parent polypeptide.

[0167] The term "sequence identity" refers to the percentage of nucleotide or amino acid residues of two or more sequences that are the same when the sequences are aligned to maximize sequence match, i.e., taking into account gaps and insertions. Alignment and calculation of percent sequence identity of two sequences can be performed by any suitable computer program known in the art. Such programs include, but are not limited to, BLAST, ALIGN, ClustalW, EMBOSS Needle, etc. For example, a global alignment of sequences can be performed based on the Needleman-Wunsch algorithm (Needleman, Saul B.; and Wunsch, Christian D. (1970), "A general method applicable to the search for similarities in the amino acid sequence of two proteins", Journal of Molecular Biology 48(3): 443-53), which is available from http: / / www.ebi.ac.uk / Tools / psa / and can be used to align sequences using default parameters. Local alignments can be performed with BLAST (Basic Local Alignment Search Tool), which was first described by Altschul et al. (1990) J. Mol. Biol. 215; 403. Biol. 215; 403-410, the BLAST alignment tool is available from the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / / ) and can be used to align sequences using default parameters, for example.

[0168] The term "vector" refers to a means for facilitating or enabling the transfer of a nucleic acid fragment from one environment to another, such as a host cell. A vector allows for the insertion of another nucleic acid fragment therein to enable replication of the inserted fragment.

[0169] The term "expression vector" refers to a vector used to express a gene product, which typically includes one or more expression control sequences for controlling and regulating transcription and / or translation of a gene sequence encoding a product to be expressed.

[0170] The term "expression cassette" refers to a nucleotide sequence comprising a relevant nucleic acid under the control of and operably linked to an appropriate promoter or other regulatory element, so as to be transcribed in a host cell.

[0171] The term "transcriptional unit" refers to a nucleic acid sequence containing one or more genes to be transcribed. The genes within a transcriptional unit are operably linked to each other in such a way that all genes within the unit are under the transcriptional control of the same promoter and / or enhancer and can thereby be transcribed to produce more than one protein or product.

[0172] The term "operably linked" means that the regulatory sequences required for expression of a coding sequence are positioned in the DNA molecule in proper positions relative to the coding sequence, so as to affect expression of the coding sequence.

[0173] The term "host cell" refers to any cell containing an exogenous nucleic acid sequence. In the present application, the host cell is a C. glutamicum containing an exogenous nucleic acid sequence.

[0174] The term "knockout" refers to the genetic manipulation of a cell or organism such that it does not produce a functional product encoded by the gene.

[0175] The term "knockdown" refers to the genetic manipulation of a cell or organism such that the functional activity of a functional product encoded by the gene produced by the cell or organism is reduced.

[0176] The term "elimination or reduction of the action of an enzyme" refers to the elimination or reduction of a reaction catalyzed by the enzyme, which can be embodied in the absence of production of a product of a reaction catalyzed by the enzyme or a reduction in the amount of a product produced by a reaction catalyzed by the enzyme.

[0177] The term "enhancement of the action of an enzyme" refers to the enhancement of a reaction catalyzed by the enzyme, which can be embodied in an increase in the amount of a product produced by a reaction catalyzed by the enzyme.

[0178] The term "enzyme activity" refers to the ability of an enzyme to catalyze the conversion of a substrate to a product, which can be evaluated by the amount of product produced.

[0179] The term "overexpression" refers to the expression of a gene product or polypeptide in a host cell in an amount greater than that prior to genetic manipulation. If the host cell did not contain a particular gene product prior to genetic manipulation, "overexpression" can also refer to any detectable expression of the particular gene product resulting from its introduction into the host cell.

[0180] The term "native promoter" refers to the promoter of a gene that naturally exists in a host cell.

[0181] The term "homologous protein" refers to a protein having similar activity and / or similar structure to a protein of interest (e.g., a reference protein). In some embodiments, a homologous protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference protein.

[0182] The term "glutamine-fructose-6-phosphate amidotransferase" refers to an enzyme that catalyzes the formation of glucosamine-6-phosphate (GlcN-6-P) from fructose-6-phosphate (Fru-6-P). The glutamine-fructose-6-phosphate amidotransferase in C. glutamicum can be referred to as GlmS.

[0183] The term "phosphoglucosamine mutase" refers to an enzyme that catalyzes the formation of glucosamine-1 -phosphate (GlcN-1 -P) from glucosamine-6-phosphate (GlcN-6-P). The phosphoglucosamine mutase in C. glutamicum can be referred to as GlmM.

[0184] The term "N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 - phosphate acetyltransferase bifunctional enzyme" refers to an enzyme that catalyzes the formation of N-acetylglucosamine-1 -phosphate (GlcNAc-1 -P) from glucosamine-1 -phosphate (GlcN-1 -P), followed by further formation of UDP-N-acetylglucosamine (UDP-GlcNAc). The N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme in C. glutamicum can be referred to as GlmU.

[0185] The term "UDP-N-acetylglucosamine 2-epimerase" refers to an enzyme that catalyzes the formation of N-acetylmannosamine (ManNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc). The UDP-N-acetylglucosamine 2-epimerase in C. jejuni can also be referred to as NeuC.

[0186] The term "N-acetylneuraminate synthase" refers to an enzyme that catalyzes the formation of N-acetylneuraminic acid (Neu5Ac, sialic acid) from N-acetylmannosamine (ManNAc). The N-acetylneuraminate synthase in C. jejuni can also be referred to as NeuB.

[0187] The term "CMP-N-acetylneuraminate synthase" refers to an enzyme that catalyzes the formation of CMP-N-acetylneuraminic acid (CMP-Neu5Ac) from N-acetylneuraminic acid (Neu5Ac, sialic acid). The CMP-N-acetylneuraminate synthase in C. jejuni can also be referred to as NeuA.

[0188] The term "sialyltransferase" refers to a polypeptide that is capable of catalyzing the transfer of a sialic acid residue from a donor substrate to an acceptor substrate. The donor substrate of a sialyltransferase is typically CMP-N-acetylneuraminic acid (CMP-Neu5Ac), and the acceptor substrate is typically lactose. The term "sialyltransferase" is to be understood herein to include wild-type sialyltransferases and functional variants thereof. A sialyltransferase can also be referred to as ST herein.

[0189] The term "a-2,3-sialyltransferase" refers to an enzyme that catalyzes the linkage of a sialic acid residue to the end of a sugar chain (e.g., the end of a lactose) in an a-2,3 glycosidic linkage. An a-2,3-sialyltransferase can also be referred to herein as a 2,3-ST.

[0190] The term "a-2,6-sialyltransferase" refers to an enzyme that catalyzes the linkage of a sialic acid residue to the end of a sugar chain (e.g., the end of a lactose) in an a-2,6 glycosidic linkage. An a-2,6-sialyltransferase can also be referred to herein as a 2,6-ST.

[0191] The term "lactose transporter" refers to any protein expressed in a microorganism that is capable of transporting (shuttling) lactose across the cytoplasmic membrane. The lactose permease of E. coli can also be referred to as LacY.

[0192] The term "glucosamine-6-phosphate N-acetyltransferase 1" refers to an enzyme that catalyzes the formation of acetylglucosamine-6-phosphate (GlcNAc-6-P) from glucosamine-6-phosphate (GlcN-6-P). The glucosamine-6-phosphate N-acetyltransferase 1 of S. cerevisiae can also be referred to as Gnal.

[0193] The term "N-acetylglucosamine 2-epimerase" refers to an enzyme that catalyzes the formation of N-acetylmannosamine (ManNAc) from N-acetylglucosamine (GlcNAc). The N-acetylglucosamine 2-epimerase of C. reinhardtii can also be referred to as Age.

[0194] The term "glucosamine-6-phosphate deaminase" refers to an enzyme that catalyzes the formation of fructose-6-phosphate (Fru-6-P) from glucosamine-6-phosphate (GlcN-6-P). The glucosamine-6-phosphate deaminase of C. glutamicum can also be referred to as NagB.

[0195] The term "N-acetylmannosamine kinase" refers to an enzyme that catalyzes the formation of acetylmannosamine-6-phosphate (ManNAc-6-P) from N-acetylmannosamine (ManNAc). The N-acetylmannosamine kinase of C. glutamicum can also be referred to as NanK.

[0196] The term "N-acetylmannosamine-6-phosphate 2-epimerase" refers to an enzyme that catalyzes the reversible reaction between acetylmannosamine-6-phosphate (ManNAc-6-P) and acetylglucosamine-6-phosphate (GlcNAc-6-P). The N-acetylmannosamine-6-phosphate 2-epimerase of C. glutamicum can also be referred to as NanE.

[0197] The term "N-acetylglucosamine-6-phosphate deacetylase" refers to an enzyme that catalyzes the formation of glucosamine-6-phosphate (GlcN-6-P) from acetylglucosamine-6-phosphate (GlcNAc-6-P). The N-acetylglucosamine-6-phosphate deacetylase of C. glutamicum can also be referred to as NagA.

[0198] The term "N-acetylneuraminate lyase" refers to an enzyme that catalyzes the reversible reaction between N-acetylmannosamine (ManNAc) and N-acetylneuraminic acid (Neu5Ac, sialic acid). The endogenous N-acetylneuraminate lyase of C. glutamicum can also be referred to as NanA.

[0199] It will be appreciated that the sequence of homologous proteins in different organisms, including different species or different strains of the same species, for enzymes having a particular activity, is readily available to one skilled in the art, for example by searching in public databases such as GenBank.

[0200] It is known in the art that enzymes and their encoding genes having the same biological activity can have different names, which are sometimes related to the microorganism from which the enzyme is derived. The enzymes described herein are intended to encompass enzymes having the defined function, and should encompass enzymes having the defined function from any organism, including microorganisms, animals, etc., unless otherwise specified.

[0201] In the present application, it will be appreciated that when an enzyme is referred to by its abbreviation in parentheses after its name, the abbreviation should not be considered as limiting to a particular sequence or a particular source of the enzyme, but is merely illustrative, for ease of reference and understanding.

[0202] Unless otherwise indicated, herein nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0203] The present application is further described by the following examples which should not be construed as limiting the present application.

[0204] The reagents used in the following examples are commercially available unless otherwise specified. The molecular biology experimental methods not specifically mentioned in the examples were performed according to the specific methods listed in J. Sambrook, Molecular Cloning: A Laboratory Manual, Third Edition, or according to the instructions of the kits and products.

[0205] Example 1 Materials and Methods

[0206] Two different media were used for cultivation of the strains, i.e. seed medium LBHI and fermentation medium FM20.

[0207] The seed culture medium LBHI consisted of the following components: yeast extract 5 g / L, peptone 2.5 g / L, NaCl 5 g / L, and brain heart infusion (BHI) 18.5 g / L.

[0208] The seed culture medium LBHI+K15 (also known as LBHI+kan) consists of the following components: LBHI medium and kanamycin 15 mg / L.

[0209] The components of the LBHI+sucrose medium are as follows: LBHI medium, sucrose 10g / L.

[0210] The fermentation medium FM20 consists of the following components: glucose 30 g / L, lactose 20 g / L, peptone 4 g / L, yeast extract 2 g / L, (NH4)2SO4 10 g / L, urea 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4 0.25 g / L, MOPS 42 g / L, CaCl2 10 mg / L, biotin 0.2 mg / L, protocatechuic acid 0.03 mg / L, FeSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, ZnSO4·7H2O 1 mg / L, CuSO4 0.2 mg / L, NiCl2·6H2O 0.02 mg / L, pH 7.0.

[0211] The seed culture medium was sterilized by autoclaving (121°C, 20 min), and the fermentation medium was sterilized by filtration (0.22 μm filter membrane). If necessary, antibiotics (e.g., kanamycin) were added to make the medium selective. An additional 2% agar was added to the medium when preparing solid plates.

[0212] During fermentation, colonies from the agar plates were picked and cultured overnight on LBHI seed medium. After overnight culture, the strain was inoculated at a 5% inoculum into 96-well plates containing fermentation medium. The plates were incubated at 30°C, 900 rpm, and 80% humidity for 48 hours. The entire culture was then incubated at 80°C for 15 minutes, centrifuged, and the supernatant was collected to determine the product titer.

[0213] Detection of product titer: 3’-SL and 6’-SL were analyzed by Agilent HPLC-RID, which determines the concentration of the substance by analyzing the change in the refractive index of the mobile phase as the sample passes through. All sugars were separated using an Agilent Acquity UPLC BEH Amide column (1.7 μm 2.1*150 mm Waters, USA) and a mobile phase containing 670 mL of acetonitrile, 330 mL of ultrapure water, 0.254 g of ammonium acetate, 1 ml of acetic acid at an isocratic flow rate of 0.3 ml / min, an injection volume of 1 μl, a column temperature of 35°C, and a differential detector temperature of 35°C.

[0214] Genetic modification of C. glutamicum: sacB modification method was used, specifically, a plasmid based on pK18mobsacB was used, and by homologous recombination, the sucrose enzyme encoded by the sacB gene of B. subtilis was used as a counter-selection marker. This enzyme catalyzes the hydrolysis of sucrose and the synthesis of high molecular weight fructose polymers, called levans, and when the sacB gene is expressed in C. glutamicum, the strain cannot grow in sucrose-containing medium. The genetic modification procedure was performed according to the reference (Lothar Eggeling and Michael Bott, Handbook of Corynebacterium glutamicum, 1st Edition, 2005).

[0215] Kanamycin resistance of recombinant bacteria and sacB gene sucrose plate counter- screening method: foreign plasmids were electroporated into strains and plated on LBHI+kan plates. Clones were selected from LBHI+kan plates and cultured overnight in LBHI-kan medium, and the bacterial solution after overnight culture was plated on LBHI+sucrose plates and cultured until colony formation. The colonies grown on LBHI+sucrose plates were spotted on LBHI-kan plates and LBHI+kan plates, and the colonies that did not grow on LBHI+kan plates but grew on LBHI-kan plates were confirmed by PCR using primers.

[0216] Plasmid construction was performed according to the recombinant cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, Catalog No: C113-02) for primer design and seamless assembly, and the plasmid was verified by PCR and Sanger sequencing to confirm the correct sequence. The correct plasmid was used for strain genetic modification or gene expression.

[0217] The promoters used for the introduced exogenous genes or the promoters used for the overexpression of endogenous genes were derived from the endogenous promoters of the C. glutamicum strain itself or from non-patent literature, such as P., et al. Journal of Bacteriology, 1999, 181(19): 6188-6191; Duan Y, et al. ACS Synthetic Biology, 2021, 10(1): 38-48; Mutalik V K, et al. Nature Methods, 2013, 10(4): 354-360, and the like.

[0218] Construction of synthetic pathway 1 of 3'-sialyllactose and 6'-sialyllactose producing strain

[0219] Construction of C. glutamicum producing 3'-sialyllactose and 6'-sialyllactose by sacB engineering: According to the product synthesis pathway of Fig. 1, exogenous genes were introduced.

[0220] Firstly, the gene of UDP-N-acetylglucosamine 2-epimerase NeuC (SEQ ID NO: 1) from Campylobacter jejuni ATCC43438 and the gene of N-acetylneuraminate synthase NeuB (SEQ ID NO: 3) from Campylobacter jejuni ATCC43438 were integrated: using primer pair 24945 / 24946 and 24952 / 24953, respectively, PCR amplification was performed to obtain homologous arms Iscg2f-Up and Iscg2f-Down (PCR system used 2xPhanta Max Master Mix (Dye Plus), Vazyme) with Corynebacterium glutamicum ATCC 13032 genome as template; using primer pair 24947 / 22076, Psod promoter (SEQ ID NO: 80) fragment was amplified from Corynebacterium glutamicum ATCC 13032 genome; using primer pair 24948 / 24949, neuB gene fragment was amplified from the plasmid containing codon-optimized neuB gene; using primer pair 24950 / 24951, neuC gene fragment was amplified from the plasmid containing codon-optimized neuC gene; plasmid pK18mobsacB was digested with restriction enzymes EcoRI and Hind III to obtain linearized fragment line-pK18mobsacB; the above six fragments: Iscg2f-Up, Iscg2f-Down, Psod, neuB, neuC, line-pK18mobsacB were assembled by using recombination cloning kit (ClonExpress Multis One Step Cloning Kit, Vazyme, item number: C113-02) for seamless assembly, the reaction system and reaction conditions were in accordance with the instructions of the kit, and after the seamless assembly was completed, T1 competent cells were transformed to obtain recombinant plasmid pK18mobsacB-neuB-neuC. The correct plasmid was verified by electroporation into Corynebacterium glutamicum ATCC 13032, and plated on LBHI+kan plate. Three clones were selected from the LBHI+kan plate and cultured overnight in LBHI medium without antibiotics, and the bacterial liquid after overnight culture was plated on LBHI+sucrose plate and cultured at 30°C until colony formation. The colonies grown on the LBHI+sucrose plate were spotted on LBHI plate without antibiotics and LBHI+kan plate. The colonies that did not grow on the LBHI+kan plate but grew on the LBHI plate without antibiotics were confirmed by PCR using primers. The correct strain was sent for sequencing for further confirmation of the sequence. The correct strain was named 13032-neuBC.

[0221] The gene of lactose permease LacY (SEQ ID NO: 5) derived from E. coli MG1655 was integrated in 13032-neuBC. The homologous arms poxB-Up and poxB-Down were amplified from Corynebacterium glutamicum ATCC 13032 genome using primer pairs 7342 / 24967 and 24970 / 22030, respectively; the promoter fragment Psod (SEQ ID NO: 80) was amplified from Corynebacterium glutamicum ATCC 13032 genome using primer pair 24968 / 22076; the lacY gene fragment was amplified from a plasmid containing the codon-optimized lacY gene using primer pair 24969 / 701; the plasmid pK18mobsacB was digested with restriction enzymes EcoRI and Hind III to obtain the linearized fragment line-pK18mobsacB; the five fragments above were assembled by seamless cloning kit, the reaction system and reaction conditions were according to the kit instruction, after the completion of the seamless assembly, the T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-lacY. The verified plasmid was electroporated into the previously obtained recombinant Corynebacterium glutamicum strain 13032-neuBC, and the kanamycin resistance and sacB gene sucrose plate counter-screening method used in the previously described gene integration method of NeuC and NeuB were used to obtain the strain 13032-neuBC-lacY with lacY inserted.

[0222] A 3'-sialyllactose (3'-SL) synthesis strain Cg3SL was constructed by integrating the gene of CMP-N-acetylneuraminic acid synthase NeuA (SEQ ID NO: 7) from Campylobacter jejuni ATCC 43438 and the gene of a-2,3-sialyltransferase (2,3-ST) (SEQ ID NO: 9) from Pasteurella dagmatis into 13032-neuBC-lacY. The homologous arms Iscg2d-Up and Iscg2d-Down were amplified from the genome of Corynebacterium glutamicum ATCC 13032 using primer pairs 24756 / 24957 and 24961 / 8928, respectively; the Psod promoter (SEQ ID NO: 80) fragment was amplified from the genome of Corynebacterium glutamicum ATCC 13032 using primer pair 25002 / 22076; the 2,3-ST gene fragment was amplified from a plasmid containing the codon-optimized 2,3-ST gene using primer pair 25006 / 24988; the neuA gene fragment was amplified from a plasmid containing the codon-optimized neuA gene using primer pair 24765 / 24960; the plasmid pK18mobsacB was digested with restriction enzymes EcoRI and Hind III to obtain a linearized fragment line-pK18mobsacB; the six fragments were assembled using a recombination cloning kit according to the instructions of the kit, and the T1 competent cells were transformed after the assembly was completed to obtain the recombinant plasmid pK18mobsacB-2,3-ST-neuA. The verified plasmid was electroporated into the previously obtained recombinant strain of Corynebacterium glutamicum 13032-neuBC-lacY, and the 3'-sialyllactose synthesis strain Cg3SL with the inserted 2,3-ST and neuA genes was obtained by using the kanamycin resistance and sacB gene sucrose plate counter-screening method in the previously described gene integration method of NeuC and NeuB.

[0223] A 6'-sialyllactose (6'-SL) synthesis strain Cg6SL was constructed by integrating the gene of CMP-N-acetylneuraminic acid synthetase NeuA (SEQ ID NO: 7) from Campylobacter jejuni ATCC 43438 and the gene of a-2,6-sialyltransferase (2,6-ST) (SEQ ID NO: 11) from Photobacterium sp. JT-ISH-224 in 13032-neuBC-lacY. The homologous arms Iscg2d-Up and Iscg2d-Down were amplified from the genome of Corynebacterium glutamicum ATCC 13032 using primer pairs 24756 / 24957 and 24961 / 8928, respectively; the Psod promoter (SEQ ID NO: 80) fragment was amplified from the genome of Corynebacterium glutamicum ATCC 13032 using primer pair 25002 / 22076; the 2,6-ST gene fragment was amplified from the plasmid containing the codon-optimized 2,6-ST gene using primer pair 25003 / 24959; the neuA gene fragment was amplified from the plasmid containing the codon-optimized neuA gene using primer pair 24765 / 24960; the plasmid pK18mobsacB was digested with restriction enzymes EcoRI and Hind III to obtain a linearized fragment line-pK18mobsacB; the above six fragments were assembled using a recombination cloning kit according to the instructions of the kit, and after the completion of the seamless assembly, T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-2,6-ST-neuA. The verified plasmid was electroporated into the previously obtained Corynebacterium glutamicum recombinant strain 13032-neuBC-lacY, and the 6'-sialyllactose synthesis strain Cg6SL containing the inserted 2,6-ST and neuA genes was obtained by using the kanamycin resistance and sacB gene sucrose plate counter-selection method used in the aforementioned gene integration method of NeuC and NeuB.

[0224] The Cg3SL and Cg6SL strains were subjected to hole plate fermentation culture in the manner of Example 1, and the contents of the products 3'-SL and 6'-SL were measured to be 251 mg / L and 302 mg / L, respectively.

[0225] Example 33'-Sialyllactose and 6'-sialyllactose production strain construction of synthesis pathway 2

[0226] Construction of 3'-sialyllactose and 6'-sialyllactose production strain of Corynebacterium glutamicum by sacB modification method: According to the product synthesis pathway of FIG. 2, the exogenous genes were introduced.

[0227] Integrating the gene of glucosamine-6-phosphate N-acetyltransferase 1 Gnal (SEQ ID NO: 13) from Saccharomyces cerevisiae S288C and the gene slr1975 (SEQ ID NO: 15) of N-acetylglucosamine 2-epimerase Age from Synechocystis sp. PCC6803 into Corynebacterium glutamicum ATCC 13032: using primer pair 25599 / 25600 and 25606 / 25607, respectively, to amplify the homologous arms tnp21a-Up and tnp21a-Down from the genome of Corynebacterium glutamicum ATCC 13032 as a template; using primer pair 25601 / 24757 to amplify the Pdape11-BCD5 promoter fragment from a plasmid containing the Pdape11-BCD5 promoter (SEQ ID NO: 81); using primer pair 25602 / 25603 to amplify the gnal gene fragment from a plasmid containing the codon-optimized gnal gene; using primer pair 25604 / 25605 to amplify the slr1975 gene from a plasmid containing the codon-optimized slr1975 gene; using restriction enzymes EcoRI and Hind III to digest the plasmid pK18mobsacB to obtain the linearized fragment line-pK18mobsacB; using the recombination cloning kit to perform seamless assembly of the above six fragments, the reaction system and reaction conditions are according to the kit instructions, and after the seamless assembly is completed, the T1 competent cells are transformed to obtain the recombinant plasmid pK18mobsacB-GNA1-slr1975. The verified plasmid is electroporated into Corynebacterium glutamicum ATCC 13032, respectively, and the kanamycin resistance and sacB gene sucrose plate counter-screening method as described in Example 2 are used in succession to obtain the strain 13032-GNA1-slr1975 inserted with the gnal gene and the slr1975.

[0228] Integrating N-acetylneuraminate synthase NeuB (SEQ ID NO: 3) from Campylobacter jejuni ATCC 43438 into 13032-GNA1-slr1975: homologous arms Iscg2f-Up and Iscg2f-Down were amplified from the genome of C. glutamicum ATCC 13032 using primer pairs 24945 / 24946 and 24952 / 24953, respectively (PCR system used 2x Phanta Max Master Mix (Dye Plus), Vazyme); the Psod promoter fragment was amplified from the genome of C. glutamicum ATCC 13032 using primer pair 24947 / 22076; the neuB fragment was amplified from a plasmid containing the codon-optimized neuB gene using primer pair 24948 / 25611; the plasmid pK18mobsacB was digested with restriction enzymes EcoRI and Hind III to obtain the linearized fragment line-pK18mobsacB; the above five fragments were assembled using a recombination cloning kit, the reaction system and reaction conditions were performed according to the kit instructions, and after the seamless assembly was completed, T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-neuB. The verified plasmid was electroporated into the previously obtained C. glutamicum recombinant strain 13032-GNA1-slr1975, and the kanamycin resistance and sacB gene sucrose plate counter-screening method as described in Example 2 were used in succession to obtain the insertion strain 13032-GNA1-slr1975-neuB.

[0229] The gene of lactose permease LacY (SEQ ID NO: 5) of E. coli MG1655 was integrated into the genome of the 13032-GNA1-slr1975-neuB strain according to the method of integrating lacY described in Example 2 to obtain the strain 13032-GNA1-slr1975-neuB-lacY.

[0230] The gene for CMP-N-acetylneuraminate synthase NeuA (SEQ ID NO: 7) from Campylobacter jejuni ATCC 43438 and the gene for a-2,3-sialyltransferase (2,3-ST) (SEQ ID NO: 9) from Pasteurella dagmatis were integrated into the genome of the 13032-GNA1-slr1975-neuB-lacY strain according to the method described in Example 2 for integrating neuA and 2,3-ST to obtain 3'-sialyllactose-producing strain NCg3SL based on synthetic pathway 2. Similarly, the gene for CMP-N-acetylneuraminate synthase NeuA (SEQ ID NO: 7) from Campylobacter jejuni ATCC 43438 and the gene for a-2,6-sialyltransferase (2,6-ST) (SEQ ID NO: 11) from Photobacterium sp. JT-ISH-224 were integrated into the genome of the 13032-GNA1-slr1975-neuB-lacY strain according to the method described in Example 2 for integrating neuA and 2,6-ST to obtain 6'-sialyllactose-producing strain NCg6SL based on synthetic pathway 2.

[0231] The two strains were subjected to well-plate fermentation using the method in Example 1 and the product 3'-SL and 6'-SL were measured to be 151 mg / L and 192 mg / L, respectively.

[0232] Example 4 Knockout of intermediate metabolite-degrading genes (competing metabolic pathways) in Cg3SL and Cg6SL

[0233] To further improve the strain performance, the product synthesis pathway according to Fig. 1 was used. Using the sacB modification method, the genes of N-acetylglucosamine-6-phosphate deacetylase NagA (Cgl2645, GenBank Accession No. BAC00039.1), glucosamine-6-phosphate deaminase NagB (Cgl2644, GenBank Accession No. BAC00038.1), N-acetylneuraminate lyase NanA (Cgl2646, GenBank Accession No. BAC00040.1), N-acetylmannosamine kinase NanK (Cgl2647, GenBank Accession No. BAC00041.1), and N-acetylmannosamine-6-phosphate 2-epimerase NanE (Cgl2648, GenBank Accession No. BAC00042.1) present in C. glutamicum were knocked out. Since the five genes are adjacent to each other on the chromosome of C. glutamicum, the homologous arms nagAB-nanAKE-Up and nagAB-nanAKE-Down were obtained by PCR amplification using primer pairs 24972 / 24973 and 7153 / 7154, respectively, with the genome of C. glutamicum ATCC 13032 as the template; the plasmid pK18mobsacB was digested with restriction enzymes EcoRI and Hind III to obtain the linearized fragment line-pK18mobsacB; the three fragments were assembled seamlessly using a recombination cloning kit, and the reaction system and reaction conditions were performed according to the instructions of the kit. After the seamless assembly was completed, the T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-ΔnagAB-nanAKE. The verified plasmid was electroporated into the previously obtained recombinant C. glutamicum strains Cg3SL and Cg6SL, and the kanamycin resistance and sacB gene sucrose plate counter-screening method as described in Example 2 was used to obtain strains Cg3SL-1 and Cg6SL-1. The two strains were subjected to hole plate fermentation culture in the manner of Example 1, and the contents of products 3’-SL and 6’-SL were measured to be 588 mg / L and 644 mg / L, respectively, indicating that the knockout of the intermediate metabolite degradation gene can significantly improve the performance of the strain.

[0234] Example 5 Enhancement of glmSUM gene

[0235] In Cg3SL-1 and Cg6SL-1, glutamine-fructose-6-phosphate aminotransferase mutant GlmS*54 (SEQ ID NO: 17; as described in Deng et al. (Biochimie 88, 419-429 (2006)) derived from E. coli MG1655 and phosphoglucosamine mutase GlmM (Cgl0583, GenBank Accession No. BAB97976.1) and N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase bifunctional enzyme GlmU (Cgl0943, GenBank Accession No. BAB98336.1) overexpressed by Corynebacterium glutamicum itself: using primer pairs 24972 / 24973 and 25619 / 7154, respectively, PCR amplification was performed to obtain homologous arms nagAB-nanAKE-Up and nagAB-nanAKE-Down using Corynebacterium glutamicum ATCC 13032 genome as a template; using primer pair 24974 / 22076, a promoter fragment Psod (SEQ ID NO: 80) was amplified from Corynebacterium glutamicum ATCC 13032 genome; using primer pair 24975 / 24976, a glmS*54 gene fragment was amplified from a plasmid containing the codon-optimized glmS*54 gene; using primer pair 24977 / 2819, a promoter fragment Pcg2195 (SEQ ID NO: 82) was amplified from Corynebacterium glutamicum ATCC 13032 genome; using primer pair 23999 / 24394, a glmU gene fragment was amplified from Corynebacterium glutamicum ATCC 13032 genome; using primer pair 24395 / 22168, a glmM gene fragment was amplified from Corynebacterium glutamicum ATCC 13032 genome; plasmid pK18mobsacB was digested with restriction enzymes EcoRI and Hind III to obtain a linearized fragment line-pK18mobsacB; the above several fragments were assembled seamlessly using a recombination cloning kit, the reaction system and reaction conditions were performed according to the kit instructions, and after the seamless assembly was completed, T1 competent cells were transformed to obtain recombinant plasmid pK18mobsacB-glmS*54-glmU-glmM. The verified plasmid was electroporated into the previously obtained Corynebacterium glutamicum recombinant strains Cg3SL-1 and Cg6SL-1, respectively, and the kanamycin resistance and sacB gene sucrose plate counter-screening method as described in Example 2 were used to obtain strains Cg3SL-2 and Cg6SL-2. The two strains were subjected to hole plate fermentation culture in the manner of Example 1, and the product 3’-SL and 6’-SL contents were measured to be 621 mg / L and 689 mg / L, respectively. It was shown that the enhancement of glmSUM three genes could improve the production level of sialyllactose.

[0236] Knockout of possible degradation genes of the product of Example 6

[0237] By analyzing the whole genome of C. glutamicum, it was found that C. glutamicum contains sialate glycoside hydrolase genes nanH (Cgl1556, GenBank Accession No. BAB98949.1) and nanP (Cgl2650, GenBank Accession No. BAC00044.1). The proteins encoded by the two genes belong to glycoside hydrolase and can degrade sialyllactose product. Knockout plasmid was constructed: using C. glutamicum ATCC 13032 genome as template, homologous arms Cgl1556-Up and Cgl1556-Down were obtained by PCR amplification using primer pairs 9666 / 9667 and 9668 / 9669, respectively; plasmid pK18mobsacB was digested with restriction endonucleases EcoRI and Hind III to obtain linearized fragment line-pK18mobsacB; the above three fragments were assembled by recombination cloning kit, the reaction system and reaction conditions were carried out according to the kit instructions, and after the seamless assembly was completed, T1 competent cells were transformed to obtain recombinant plasmid pK18mobsacB-ΔCgl1556. Using C. glutamicum ATCC 13032 genome as template, homologous arms Cgl2650-Up and Cgl2650-Down were obtained by PCR amplification using primer pairs 9672 / 9673 and 9674 / 9675, respectively; plasmid pK18mobsacB was digested with restriction endonucleases EcoRI and Hind III to obtain linearized fragment line-pK18mobsacB; the above three fragments were assembled by recombination cloning kit, the reaction system and reaction conditions were carried out according to the kit instructions, and after the seamless assembly was completed, T1 competent cells were transformed to obtain recombinant plasmid pK18mobsacB-ΔCgl2650. Using the two plasmids, Cgl1556 and Cgl2650 in C. glutamicum strains Cg3SL-2 and Cg6SL-2 were single-knockout and double-knockout by SacB modification method to obtain strains Cg3SL-3 (Cg3SL-2ΔCgl1556), Cg3SL-4 (Cg3SL-2ΔCgl1556ΔCgl2650), Cg6SL-3 (Cg6SL-2ΔCgl1556), Cg6SL-4 (Cg6SL-2ΔCgl1556ΔCgl2650). The four strains were subjected to plate fermentation culture using the method in Example 1, and the product content was measured to be 665 mg / L, 686 mg / L, 725 mg / L, and 738 mg / L, respectively. It was proved that the knockout of the two genes could further improve the production level of sialyllactose.

[0238] Example 7 Fermenter production of Cg6SL-4

[0239] Cg6SL-4 was tested in a 2L fermentor with initial aeration rate of 1 VVM, ammonia was used to control pH = 7.0 ± 0.5, agitation and aeration were used to control dissolved oxygen at 30% ± 10, temperature was controlled at 30℃, fermentation medium FM20 was used as initial medium, after about 10h cultivation, a solution containing 550g / kg glucose and 40g / kg lactose was used for fed-batch, initial feeding rate was 1ml / L / h, then the feeding rate was gradually increased to 10ml / L / h. OD600 was tested by spectrophotometer. The product of fermentation process was tested by the method in Example 1. The fermentation results are shown in Figure 3. The results show that the OD reached about 260 around 53h, the product titer reached 15.6g / L, after that the OD slowly decreased, while the product titer continued to grow, reaching 17.3g / L at the end of 77h fermentation. The production of sialyllactose in C. glutamicum is a non-growth dependent phenotype, which is very advantageous for production, the product production stage does not require the simultaneous growth of the strain, has the advantages of saving carbon source, energy, improving productivity, etc., and has very good industrial application prospect. This is the highest production level of sialyllactose production strain using C. glutamicum reported so far.

[0240] The primer sequences used in the above examples are as follows:

[0241] The enzyme and promoter sequences used in the above examples are as follows:

[0242] SEQ ID NO: 1 (UDP-N-acetylglucosamine 2-epimerase NeuC of Campylobacter jejuni ATCC43438, amino acid sequence):

[0243] SEQ ID NO: 2 (codon-optimized UDP-N-acetylglucosamine 2-epimerase gene neuC of Campylobacter jejuni ATCC43438, nucleic acid sequence):

[0244] SEQ ID NO: 3 (N-acetylneuraminate synthase NeuB of Campylobacter jejuni ATCC43438, amino acid sequence):

[0245] SEQ ID NO: 4 (N-acetylneuraminate synthase gene neuB of Campylobacter jejuni ATCC 43438, nucleic acid sequence, codon optimized):

[0246] SEQ ID NO: 5 (lactose permease LacY of E. coli MG1655, amino acid sequence):

[0247] SEQ ID NO: 6 (lactose permease gene lacY of E. coli MG1655, nucleic acid sequence, codon optimized):

[0248] SEQ ID NO: 7 (CMP-N-acetylneuraminate synthase NeuA of Campylobacter jejuni ATCC 43438, amino acid sequence):

[0249] SEQ ID NO: 8 (CMP-N-acetylneuraminate synthase gene neuA of Campylobacter jejuni ATCC 43438, nucleic acid sequence, codon optimized):

[0250] SEQ ID NO: 9 (a-2,3-sialyltransferase of Pasteurella dagmatis, amino acid sequence):

[0251] SEQ ID NO: 10 (a-2,3-sialyltransferase gene of Pasteurella dagmatis, nucleic acid sequence, codon optimized):

[0252] SEQ ID NO: 11 (a-2,6-sialyltransferase of Photobacterium sp. JT-ISH-224, amino acid sequence):

[0253] SEQ ID NO: 12 (a-2,6-sialyltransferase gene of Photobacterium sp. JT-ISH-224, nucleic acid sequence, codon optimized):

[0254] SEQ ID NO: 13 (Glucosamine-6-phosphate N-acetyltransferase 1 Gnal of Saccharomyces cerevisiae S288C, amino acid sequence):

[0255] SEQ ID NO: 14 (Glucosamine-6-phosphate N-acetyltransferase 1 gene gnal of Saccharomyces cerevisiae S288C, codon-optimized, nucleic acid sequence):

[0256] SEQ ID NO: 15 (N-acetylglucosamine 2-epimerase Age of Synechocystis sp. PCC6803, amino acid sequence):

[0257] SEQ ID NO: 16 (N-acetylglucosamine 2-epimerase gene slr1975 of Synechocystis sp. PCC6803, codon-optimized, nucleic acid sequence):

[0258] SEQ ID NO: 17 (Glutamine-fructose-6-phosphate amidotransferase mutant GlmS*54 of E. coli MG1655, amino acid sequence):

[0259] SEQ ID NO: 18 (Glutamine-fructose-6-phosphate amidotransferase mutant GlmS*54 gene of E. coli MG1655, codon-optimized, nucleic acid sequence):

[0260] SEQ ID NO 80 (Psod promoter):

[0261] SEQ ID NO 81 (Pdape11-BCD5 promoter):

[0262] SEQ ID NO 82 (Pcg2195 promoter):

[0263] SEQ ID NO: 83 (N-acetylglucosamine-6-phosphate deacetylase NagA of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0264] SEQ ID NO: 84 (N-acetylglucosamine-6-phosphate deacetylase NagA of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0265] SEQ ID NO: 85 (glucosamine-6-phosphate deaminase NagB of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0266] SEQ ID NO: 86 (glucosamine-6-phosphate deaminase NagB of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0267] SEQ ID NO: 87 (N-acetylneuraminate lyase NanA of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0268] SEQ ID NO: 88 (N-acetylneuraminate lyase NanA of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0269] SEQ ID NO: 89 (N-acetylmannosamine kinase NanK of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0270] SEQ ID NO: 90 (N-acetylmannosamine kinase NanK of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0271] SEQ ID NO: 91 (N-acetylmannosamine-6-phosphate 2-epimerase NanE of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0272] SEQ ID NO: 92 (N-acetylmannosamine-6-phosphate 2-epimerase NanE of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0273] SEQ ID NO: 93 (N-acetylglucosamine-1 -phosphate urtidylyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme GlmU of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0274] SEQ ID NO: 94 (N-acetylglucosamine-1 -phosphate urtidylyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme GlmU of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0275] SEQ ID NO: 95 (phosphoglucomutase GlmM of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0276] SEQ ID NO: 96 (phosphoglucomutase GlmM of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0277] SEQ ID NO: 97 (sialate O-acetylesterase, nanH gene product, of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0278] SEQ IN NO: 98 (sialoside hydrolase gene nanH of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0279] SEQ IN NO: 99 (sialoside hydrolase, nanP gene product, of Corynebacterium glutamicum ATCC 13032, amino acid sequence)

[0280] SEQ IN NO: 100 (sialoside hydrolase gene nanP of Corynebacterium glutamicum ATCC 13032, nucleic acid sequence)

[0281] The embodiments of the present application are not limited to the above-described examples, and various changes and improvements in form and details can be made by those skilled in the art without departing from the spirit and scope of the present application, and these are considered to fall within the scope of the present application.

Claims

1. A recombinant Corynebacterium glutamicum genetically engineered to introduce a sialyllactose synthesis pathway.

2. The recombinant Corynebacterium glutamicum according to claim 1, genetically engineered to introduce an N-acetylmannosamine (ManNAc) synthesis pathway and genetically engineered to express an exogenous N-acetylneuraminic acid synthase, an exogenous CMP-N-acetylneuraminic acid synthase, an exogenous lactose transporter and an exogenous sialyltransferase.

3. The recombinant Corynebacterium glutamicum according to claim 2, wherein the sialyltransferase is an alpha-2,3-sialyltransferase or an alpha-2,6-sialyltransferase.

4. The recombinant Corynebacterium glutamicum according to claim 3, wherein the alpha-2,3-sialyltransferase is an alpha-2,3-sialyltransferase derived from Pasteurella dagmatis, Neisseria meningitidis serogroup L3, vibrio sp. JT-FAJ-16 or Photobacterium multocida or a functional variant thereof and the alpha-2,6-sialyltransferase is an alpha-2,6-sialyltransferase derived from Photobacterium sp. JT-ISH-224, Photobacterium damselae or Photobacterium leiognathi or a functional variant thereof.

5. The recombinant Corynebacterium glutamicum according to any one of claims 1 to 4, wherein the lactose transporter is a lactose permease, preferably a lactose permease (LacY) derived from Escherichia coli or a functional variant thereof.

6. The recombinant Corynebacterium glutamicum according to any one of claims 1 to 5, wherein the N-acetylneuraminic acid synthase is an N-acetylneuraminic acid synthase derived from Campylobacter jejuni or Neisseria meningitidis or a functional variant thereof.

7. The recombinant Corynebacterium glutamicum according to any one of claims 1 to 6, wherein the CMP-N-acetylneuraminic acid synthase is a CMP-N-acetylneuraminic acid synthase derived from Campylobacter jejuni or Neisseria meningitidis or a functional variant thereof.

8. The recombinant Corynebacterium glutamicum according to any one of claims 1 to 7, wherein the recombinant Corynebacterium glutamicum is genetically modified to express an exogenous UDP-N-acetylglucosamine 2-epimerase thereby introducing an N-acetylmannosamine (ManNAc) synthesis pathway in the Corynebacterium glutamicum.

9. The recombinant C. glutamicum of claim 8, wherein the UDP-N-acetylglucosamine 2-epimerase is a UDP-N-acetylglucosamine 2-epimerase derived from Campylobacter jejuni or Neisseria meningitidis or a functional variant thereof.

10. The recombinant C. glutamicum of claim 8 or 9, further comprising one, two or three of the following genetic modifications: (1) at least one genetic modification that eliminates or attenuates the action of an endogenous glucosamine-6-phosphate deaminase; (2) at least one genetic modification that eliminates or attenuates the action of one, two or three enzymes selected from the group consisting of an endogenous N-acetylglucosamine-6-phosphate deacetylase, an endogenous N-acetylmannosamine kinase and an endogenous N-acetylmannosamine-6-phosphate 2-epimerase; and (3) at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylneuraminate lyase.

11. The recombinant C. glutamicum of claim 10, wherein: the at least one genetic modification that eliminates or attenuates the action of an endogenous glucosamine-6-phosphate deaminase comprises a knockout or knockdown of an endogenous glucosamine-6-phosphate deaminase gene and / or a knockout or knockdown of a native promoter of an endogenous glucosamine-6-phosphate deaminase gene; the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylglucosamine-6-phosphate deacetylase comprises a knockout or knockdown of an endogenous N-acetylglucosamine-6-phosphate deacetylase gene and / or a knockout or knockdown of a native promoter of an endogenous N-acetylglucosamine-6-phosphate deacetylase gene; the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylmannosamine kinase comprises a knockout or knockdown of an endogenous N-acetylmannosamine kinase (NanK) gene and / or a knockout or knockdown of a native promoter of an endogenous N-acetylmannosamine kinase gene; the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylmannosamine-6-phosphate 2-epimerase comprises a knockout or knockdown of an endogenous N-acetylmannosamine-6-phosphate 2-epimerase gene and / or a knockout or knockdown of a native promoter of an endogenous N-acetylmannosamine-6-phosphate 2-epimerase gene; and / or the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylneuraminate lyase comprises a knockout or knockdown of an endogenous N-acetylneuraminate lyase gene and / or a knockout or knockdown of a native promoter of an endogenous N-acetylneuraminate lyase gene.

12. The recombinant C. glutamicum of any one of claims 1-7, wherein the recombinant C. glutamicum is genetically modified to express an exogenous glucosamine-6-phosphate N-acetyltransferase 1 and an exogenous N-acetylglucosamine 2-epimerase, thereby introducing an N-acetylmannosamine (ManNAc) synthesis pathway in the C. glutamicum.

13. The recombinant C. glutamicum of claim 12, wherein the glucosamine-6-phosphate N-acetyltransferase 1 is a glucosamine-6-phosphate N-acetyltransferase 1 derived from Saccharomyces cerevisiae or Caenorhabditis elegans or a functional variant thereof.

14. The recombinant C. glutamicum of claim 12 or 13, wherein the N-acetylglucosamine 2-epimerase is an N-acetylglucosamine 2-epimerase derived from Synechocystis sp. PCC6803, Bacteroides ovatus or Anabaena sp. or a functional variant thereof.

15. The recombinant C. glutamicum of any one of claims 12-14, further comprising one, two, three or four of the following genetic modifications: (1) at least one genetic modification that eliminates or attenuates the action of an endogenous glucosamine-6-phosphate deaminase; (2) at least one genetic modification that eliminates or attenuates the action of one or both of an endogenous N-acetylmannosamine kinase and an endogenous N-acetylmannosamine-6-phosphate 2-epimerase; (3) at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylglucosamine-6-phosphate deacetylase; and (4) at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylneuraminate lyase.

16. The recombinant C. glutamicum of claim 15, wherein At least one genetic modification that eliminates or attenuates the action of endogenous glucosamine-6-phosphate deaminase includes: the endogenous glucosamine-6-phosphate deaminase gene is knocked out or knocked down, and / or the native promoter of the endogenous glucosamine-6-phosphate deaminase gene is knocked out or knocked down; the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylglucosamine-6-phosphate deacetylase comprises knocking out or knocking down the endogenous N-acetylglucosamine-6-phosphate deacetylase gene, and / or knocking out or knocking down the native promoter of the endogenous N-acetylglucosamine-6-phosphate deacetylase gene; the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylmannosamine kinase comprises knocking out or knocking down the endogenous N-acetylmannosamine kinase gene, and / or knocking out or knocking down the native promoter of the endogenous N-acetylmannosamine kinase gene; the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylmannosamine-6-phosphate 2-epimerase comprises knocking out or knocking down the endogenous N-acetylmannosamine-6-phosphate 2-epimerase gene, and / or knocking out or knocking down the native promoter of the endogenous N-acetylmannosamine-6-phosphate 2-epimerase gene; the at least one genetic modification that eliminates or attenuates the action of an endogenous N-acetylneuraminate lyase comprises knocking out or knocking down the endogenous N-acetylneuraminate lyase gene, and / or knocking out or knocking down the native promoter of the endogenous N-acetylneuraminate lyase gene.

17. The recombinant C. glutamicum of any one of claims 1-16, further comprising one, two, or three of the following genetic modifications: (1) at least one genetic modification that enhances the action of glutamine- fructose-6-phosphate amidotransferase; (2) at least one genetic modification that enhances the action of phosphoglucosamine mutase; and (3) at least one genetic modification that enhances the action of N- acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme.

18. The recombinant C. glutamicum according to claim 17, wherein the at least one genetic modification that enhances the action of glutamine-fructose-6-phosphate amidotransferase comprises: increasing the activity of, or overexpressing, glutamine-fructose-6-phosphate amidotransferase; the at least one genetic modification that enhances the action of phosphoglucosamine mutase comprises: increasing the activity of, or overexpressing, phosphoglucosamine mutase; and / or the at least one genetic modification that enhances the action of N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme comprises: increasing the activity of, or overexpressing, N-acetylglucosamine-1 -phosphate urtidyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme.

19. The recombinant C. glutamicum according to claim 17 or 18, wherein the at least one genetic modification that enhances the action of glutamine-fructose-6-phosphate amidotransferase comprises: replacing the endogenous glutamine-fructose-6-phosphate amidotransferase gene with an exogenous glutamine-fructose-6-phosphate amidotransferase gene having higher enzymatic activity, increasing the gene copy number of the glutamine-fructose-6-phosphate amidotransferase gene, and / or replacing the natural promoter of the endogenous glutamine-fructose-6-phosphate amidotransferase with a promoter having a higher expression level.

20. The recombinant C. glutamicum according to any one of claims 17 to 19, wherein the at least one genetic modification that enhances the action of glutamine- fructose-6-phosphate amidotransferase comprises: introducing at least one copy of an exogenous glutamine-fructose-6-phosphate amidotransferase gene into the C. glutamicum; preferably, the exogenous glutamine-fructose-6-phosphate amidotransferase is a glutamine-fructose-6-phosphate amidotransferase mutant GlmS*54 derived from E. coli.

21. The recombinant C. glutamicum according to any one of claims 17 to 20, wherein the at least one genetic modification that enhances the action of phosphoglucomutase comprises: replacing the endogenous phosphoglucosamine mutase gene with an exogenous phosphoglucosamine mutase gene having higher enzymatic activity, increasing the gene copy number of the phosphoglucosamine mutase gene, and / or replacing the natural promoter of the endogenous phosphoglucosamine mutase with a promoter having a higher expression level.

22. The recombinant C. glutamicum according to any one of claims 17 to 21, wherein the at least one genetic modification that enhances the action of phosphoglucomutase comprises: introducing at least one copy of a phosphoglucosamine mutase gene into the C. glutamicum; preferably, the phosphoglucosamine mutase is a phosphoglucosamine mutase from C. glutamicum.

23. The recombinant C. glutamicum of any of claims 17-22, wherein the at least one genetic modification that enhances the action of N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme comprises: Replacing the endogenous N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene with an exogenous N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene having higher enzymatic activity, increasing the gene copy number of the N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene, and / or replacing the natural promoter of the endogenous N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme with a promoter having higher expression level.

24. The recombinant C. glutamicum according to any one of claims 17 to 23, wherein the at least one genetic modification that enhances the action of the N-acetylglucosamine- 1 -phosphate uridyltransferase and glucosamine- 1 -phosphate acetyltransferase bifunctional enzyme comprises: Introducing at least one copy of a N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme gene into the coryneform bacterium; preferably, the N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme is a N-acetylglucosamine-1 -phosphate uridyltransferase and glucosamine-1 -phosphate acetyltransferase bifunctional enzyme from a coryneform bacterium.

25. The recombinant coryneform bacterium of any one of claims 1 to 24, wherein the recombinant coryneform bacterium further comprises the following genetic modification: (1) at least one genetic modification that eliminates or reduces the effect of an enzyme encoded by nanH; and / or (2) at least one genetic modification that eliminates or reduces the effect of an enzyme encoded by nanP.

26. The recombinant coryneform bacterium of claim 25, wherein At least one genetic modification that eliminates or attenuates the effect of an enzyme encoded by nanH includes: nanH is knocked out or knocked down, and / or the natural promoter of nanH is knocked out or knocked down; and / or the at least one genetic modification that eliminates or reduces the effect of an enzyme encoded by nanP comprises: nanP is knocked out or knocked down, and / or the natural promoter of nanP is knocked out or knocked down.

27. A method for producing a sialyllactose, comprising culturing the recombinant coryneform bacterium of any one of claims 1 to 26 under suitable conditions, and recovering the synthesized sialyllactose.

28. The method of claim 27, wherein at least one carbon source and lactose are added to the culture medium used for culturing the recombinant coryneform bacterium.

29. The method of claim 28, wherein the at least one carbon source is selected from the group consisting of glucose, fructose, sucrose, acetic acid, lactic acid, and succinic acid.

30. The method of any one of claims 27 to 29, wherein the sialyllactose is 3’-sialyllactose or 6’-sialyllactose.

31. The method of any one of claims 27 to 30, wherein the culturing of the recombinant coryneform bacterium is batch culture, fed-batch culture, or continuous culture.

32. The method of any one of claims 27 to 31, wherein at least one carbon source and / or lactose is supplemented during the culturing.

33. Use of the recombinant coryneform bacterium of any one of claims 1 to 26 for producing a sialyllactose.

34. The use of claim 33, wherein the sialyllactose is 3’-sialyllactose or 6’-sialyllactose.

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