Efficient Production Of N-acetylneuraminic Acid By Recombinant Bacillus Subtilis Via Whole Cell Catalysis

N-acetylneuraminic acid?NeuAc?is the main form of sialic acid in humans,which mainly occupies the terminal positions of glycoprotein,glycolipids and oligosaccharides and plays important roles in regulating biological recognition,cellular immunity,and diseases.NeuAc is also one of the most important monomers of sialylated human milk oligosaccharides,which is used as a nutraceutical to enhance immunity and improve brain development in infants.In addition,derivatives of NeuAc can be used as anti-cancer,anti-adhesion and anti-viral drug,and as a nanocarrier-specific treatment for diseases in medical treatment.With the significant increasing demand for food and medicinal application of NeuAc,higher requirements for the production of NeuAc are required.However,the lower production efficiency and high environmental pollution of traditional extraction and chemical synthesis methods limit the supply of NeuAc,while the fermentation process produces NeuAc with a low yield,and the host strain Escherichia coli for whole-cell biocatalysis can produce endotoxin that may contaminate NeuAc during production.All above questions limit the green production of NeuAc and its applications in food and medicine.In this study,we have developed a method for the production of NeuAc by food-grade strain of Bacillus subtilis via whole-cell biocatalysis.The main results of this work are summarized as follows:?1?A heterologous pathway for the synthesis of NeuAc was constructed in B.subtilis with N-acetylglucosamine?GlcNAc?and pyruvate as substrate.Firstly,we cloned age?encoding the N-acetylglucosamine 2-epimerase?from Anabaena sp.CH1 and nanA?encoding the NeuAc aldolase?from E.coli K12 to the pP43NMK plasmid.And seven different strong constitutive promoters were selected to control the expression of AGE and NanA.Finally,we constructed a series of recombinant strains with different promoters to to control AGE and NanA expression.The recombinant strain B6CG/p43AN which expressed AGE and NanA under the control of P₄₃ promoter produced the highest titer of NeuAc(32.84 g?L^-1)via whole cell catalysis with 1.2mol·L^-1 pyruvate and 0.4 mol·L^-1 GlcNAc for 48 h.And the molar conversion of GlcNAc was26.55%.?2?To search for a better source of the nanA gene that coding high catalytic efficiency NanA,the nanA originating from E.coli K12,Corynebacterium.glutamicum ATCC 13869 and Staphylococcus.hominis EFS20452.1 was selected based on literature mining.When the shnanA?NeuAc aldolase?originated from S.hominis was used for constructing NeuAc synthetic pathway,the recombinant strain B6CG/p43ANsh produced 46.04 g?L^-1 NeuAc,which increased by 40.2%compared with the recombinant strain expressing NanA originating from E.coli K12.?3?In order to increase the expression level of NeuAc aldolase?ShNanA?,ten N-terminal coding sequences of highly expressed protein in B.subtilis was selected.And the N-terminal coding sequences was fused to the 5'-end of shnanA to screen for up-regulating shnanA expression.Finally,N1-tufA was the most effective N-terminal coding sequences for up-regulating shnanA expression,which led to improved NeuAc production reaching 56.82 g?L^-1by recombinant strain B6CG/p43ANshN1-tufA.?4?The problem of the consumption of pyruvate of B.subtilis itself was solved by blocking the formation of the by-product acetoin by knocking out the alsS and alsD genes.The obtained recombinant B6CGSD/p43ANshN1-tufA produced 68.75 g?L^-1 NeuAc,and the molar conversion rate of GlcNAc is 55.57%.Finally,we optimized the cultivation time of the recombinant strain B6CGSD/p43ANshN1-tufA and the conditions of the whole-cell biocatalysis including the pH,the temperature,the category and concentration of surfactant,the OD₆₀₀ of strain,and substrate concentration.Finally,the titer of NeuAc was increased to 93.78g?L^-1.The conversion rate of GlcNAc was 50.54%.