Design And Construction Of Bacillus Subtilis Chassis Cell For Efficient Synthesis Of N-acetylneuraminic Acid

N-acetylneuraminic acid（Neu Ac）,or sialic acid,is the most abundant sialic acid in mammals.It plays an important role in improving brain development and cognition,enhancing immunity,which has been widely used as nutraceutical and pharmaceutical intermediates.At the same time,various derivatives of Neu Ac,especially for sialylated human milk oligosaccharides,also have important applications in the fields of food additives and medicine.At present,the industrial production methods of Neu Ac are mainly based on chemical extraction from Neu Ac-rich feedstocks and whole-cell biocatalysis using N-acetylglucosamine（Glc NAc）as substrate,which have inherent defects such as high cost,environmental pollution and allergen contamination.Therefore,the development of microbial chassis cells for the efficient synthesis of the platform compound Neu Ac is of great significance for efficient biosynthesis of various Neu Ac derivatives.Bacillus subtilis,generally regarded as food safe strain,is suitable for developing a chassis cell for efficient synthesis of Neu Ac.In this study,based on a Neu Ac-producing B.subtilis B6CG strain constructed in our laboratory,a B.subtilis chassis cell capable of efficiently synthesizing Neu Ac was constructed through four aspects,including developing gene expression element toolbox,analyzing and eliminating rate-limiting steps,balancing cell growth and bioproduction,and refactoring synthetic pathways.The main results of this study are as follows:（1）Through the analysis of B.subtilis transcriptome and proteome data,594 N-terminal coding sequences（NCS）with potential different strength were obtained.In above abtained 594NCS,96 were selected and tested by regulating the expression level of green fluorescent protein（GFP）,and an NCS library with expression strength accross 4 orders of magnitude were firstly obtained,which increased the expression level of GFP to 6.95-fold.According to the characteristics of dynamic expression pattern,NCS was divided into four different patterns,including growth-coupled,growth-delayed,consistent expression,and inhibitory NCS.Through statistical analysis,five existing mechanisms for NCS regulation cannot be directly used for NCS design,including N-terminal rule,m RNA secondary structure,thermodynamics and kinetics,AT content,and charged amino acid content.Based on analysis B.subtilis NCS sequence and corresbunding expression level,the abundance of certain specific amino acids was significantly correlated to the protein expression level.Based on this,the rational design of NCS were carried out,which increased the GFP expression level to 8.47-fold,and realized the up-or down-regulation of the target gene expression level through rational design.To simplify the rational design of NCS,a website was developed for automatic design of NCS（http://www.wjiangnan.com/dna/）.（2）The expression level of the key gene glucosamine-6-phosphate acetyltransferase（GNA1）was strengthened using natural and synthetic NCS respectively,and Neu Ac titer increased from 0.8 g/L to 2.0 g/L under the control of N abr B.Then,a kinetic model describing the 13-step enzymatic reaction of the Neu Ac synthesis pathway was constructed based on the Michaelis-Menten equation,and two potential key rate-limiting targets in the Neu Ac synthesis pathway were analyzed and obtained,which were 6-phosphofructokinase（Pfk A）and pyruvate kinase（Pyk）,respectively.For rapid validation of the rate-limiting step,additional intermediate metabolites for Neu Ac synthesis were added to the cell-free reaction system.The Neu Ac titer in the cell-free system increased significantly from 0.3 g/L to 0.6 g/L when phosphoenolpyruvate（PEP）was added,which verified the results of the kinetic model analysis.Therefore,the key genes pfk A and pyk were down-regulated and dynamically down-regulated by NCS,and the Neu Ac titer was increased 80%,to 3.6 g/L.（3）For balancing cell growth and product synthesis,a genetic code expansion technology was constructed in B.subtilis.By expressing the unnatural amino acid aminoacyl t RNA synthetase and t RNA mutant（Mut RNA）,incorporation of unnatural amino acid（O-methyltyrosine,OMe Y）for the TAG stop codon during gene translation is achieved.Through high-throughput sorting of Mut RNA promoter mutation library,copy number optimization,and synthetic promoter testing,a precise regulation tool of gene expression based on titrating unnatural amino acids was constructed,which can achieve 80-fold activation of gene expression.Using unnatural amino acids to control the expression levels of essential genes of B.subtilis respectively,including UDP-N-acetylpyruvylglucosamine reductase（Mur B）,two-component system regulatory protein（Wal R）and phosphatidic acid cytidine transferase（Cds A）,the precise control of the specific growth rate of the engineered strains was achieved,and the Neu Ac titer increased 33.3%to 4.8 g/L under the optimal balance of cell growth and Neu Ac synthesis.At the same time,the chassis cells obtained by genetic code expansion have an escape rate lower than 3.67?×?10^-10?escapees/c.f.u.,which avoids the propagation of engineered strains in nature and realizes efficient biocontainment of engineered strains.（4）To eliminate the severely accumulated by-product N-acetylglucosamine（Glc NAc）,the UDP-Glc NAc epimerase（Neu C）pathway without Glc NAc production was reconstructed and optimized.The expression levels of the key enzymes in the synthesis pathway of UDP-Glc NAc,the direct precursor of Neu Ac synthesis,were optimized using promoters of different strengths,including glutamine fructose 6-phosphate aminotransferase（Glm S）,phosphoglucosamine mutase（Glm M）,and UDP-N-acetylglucosamine pyrophosphorylase（Glm U）.The Neu Ac titer was increased to 5.9 g/L and the by-product Glc NAc was eliminated.In order to reduce the accumulation of the by-product N-acetylmannose（Man NAc）,the directed evolution of N-acetylneuraminic acid synthase（Neu B）,the co-expression of Neu B from different species and the enhancement of PEP supply were implemented.Man NAc decreased by 46.3%and Neu Ac titer increased 54.9%to 7.9 g/L.Using unnatural amino acids to control the specific growth rate of the strain,the maximum titer of Neu Ac in fed-batch fermentation in a 3 L fermenter was 21.8g/L,the yield was 0.04 g/g glucose and the productivity was 0.34 g/L/h.