Development And Application Of Biosensors Highly Responsive To N-acetylneuraminic Acid In Bacillus Subtilis

N-acetylneuraminic acid（NeuAc）is a member of the most common sialic acids（SAs）family in nature.It has various physiological functions such as anticancer,antiviral,promoting nervous system and bone development,and is widely used as a pharmaceutical intermediate and nutritional chemical.Bacillus subtilis,as a food safety grade model strain,has a clear genetic background and mature genetic manipulation tools,making it an excellent host for producing NeuAc.Biosensors that respond to target products are increasingly used in metabolic engineering to improve the synthesis efficiency of target products by screening efficient pathway enzyme mutants,balancing the expression of pathway enzymes,and regulating synthesis heterogeneity.At present,the reported biosensors in B.subtilis that respond to NeuAc have limited dynamic range and low activation times,which limits the screening of enzyme mutants with high NeuAc production and recombinant strains of B.subtilis.In this study,we first constructed B.subtilis with different NeuAc transport abilities,and based on this,designed and constructed biosensors that can efficiently respond to NeuAc in B.subtilis.Next,the NeuAc synthesis pathway was introduced into B.subtilis,and a mutant library was constructed by randomizing the ribosome binding site（RBS）and N-terminal coding sequence（NCS）of the N-acetylneuraminic acid synthase Neu B.Then,combined with biosensors and flow cytometry,strains with increased NeuAc production were screened,which proved that the constructed biosensors responding to NeuAc could be used for ultrahigh throughput screening of high-yield NeuAc strains.The main results are as follows:（1）A series of B.subtilis with different NeuAc transport abilities were constructed.First,it was verified that B.subtilis did not have the ability to transport extracellular NeuAc into cells,so we selected NeuAc transport proteins Nan C and Nan T from Escherichia coli,tested their ability to transport extracellular NeuAc by adding NeuAc externally,further optimized the expression of Nan T,and obtained four strains BSP1,BSP2,BSP9 and BSP7 with different NeuAc transport capabilities,which were used to respond to the construction of NeuAc biosensors.（2）Biosensors capable of efficiently responding to N-acetylneuraminic acid in B.subtilis were constructed.Firstly,insert the binding sites of Nan R from Bifidobacterium breve into different positions of the constitutive promoter,and screen for active heterozygous promoters without the addition of NeuAc;Next,introduce Nan R and use B.subtilis with different NeuAc transport abilities to select heterozygous promoters that can respond to NeuAc;Finally,a series of biosensors with wide dynamic range and high activation times were obtained by optimizing the expression level of Nan R,among which P535-N2 had the widest dynamic range,ranging from 180 to 20245 a.u.;P566-N2 has the highest activation multiple,up to 122 times.（3）An N-acetylneuraminic acid synthesis pathway was constructed in B.subtilis and the biosensor was successfully applied to screen B.subtilis with increased NeuAc production.Firstly,the feedback inhibition of endogenous glm S in B.subtilis was relieved through promoter and 5’UTR substitution.Next,neu C derived from Neisseria meningitidis was introduced,and the genes nag BB and nag BA related to the glucosamine 6-phosphate（Glc N6P）catabolism pathway were knocked out.Then,the p P43NMK plasmid was used as the vector to express Econeu B from Escherichia coli,and a strain with a yield of 2.02 g·L^-1 of NeuAc was successfully obtained.Randomized RBS and NCS of Econeu B on p P43NMK plasmid using degenerate primers,and constructed a size of approximately 9×10⁵ RBS and NCS libraries;Then,combined with the biosensor P535-N2 and flow cytometry for initial screening,4beneficial mutants were finally obtained through well plate re screening.Among them,the NeuAc production of BSNB3 reached 3.07 g·L^-1,which was 52%higher than the starting strain,demonstrating the applicability of this biosensor in guiding the production of NeuAc by B.subtilis.