Construction And Optimization Of N-acetylneuraminic Acid Biosynthesis Pathway In Recombinant Escherichia Coli

N-acetylneuraminic acid（NeuAc）is a vital functional sugar with a multitude of benefits,including promoting neural development in infants,enhancing memory and learning capabilities,and boosting immunity.NeuAc finds widespread use in the food and pharmaceutical industries.Microbial fermentation,utilizing cost-effective and renewable resources for sustainable production,represents a potent strategy for NeuAc production.It offers significant economic,efficient,and environmentally-friendly advantages.However,the titer of NeuAc production still has considerable scope for improvement.Firstly,the instability of the plasmid-free expression of key genes poses a significant challenge.Secondly,the feedback inhibition of glucosamine-6-phosphate（GlcN-6-P）on glucosamine-6-phosphate synthase（Glm S）imposes limitations on the synthesis of NeuAc.Additionally,the Neu C pathway lacks systematic research and optimization.Furthermore,the competition pathways for NeuAc can lead to an inadequate carbon flux in the target pathway.This study addressed the challenges in N-acetylneuraminic acid（NeuAc）production by utilizing metabolic engineering principles and the Escherichia coli BL21（DE3）host.The research focused on four aspects:developing and applying regulatory elements,constructing the precursor（N-acetylmannosamine）ManNAc synthesis pathway,optimizing the NeuAc metabolic network,and validating production in a bioreactor.The resulting high-performance strain,which synthesizes NeuAc de novo from glycerol as a carbon source,was developed without the use of plasmid vectors,inducers,or antibiotics,significantly improving NeuAc synthesis efficiency.The study’s main research objectives are as follows:（1）Construction and characterization of E.coli terminator library:To achieve precise and efficient expression of key genes in the NeuAc biosynthesis pathway,a detection system was constructed with red fluorescent protein（m Kate）and yellow fluorescent protein（YFP）.Using a combination of genomic databases and literature reports,200 terminators were characterized for their termination efficiency.Strong terminators were found to upregulate upstream m Kate expression by 1.8-fold while downregulating YFP expression by 132-fold,effectively promoting upstream gene expression and inhibiting transcription read-through of downstream genes.Further optimization strategies were proposed to improve terminator efficiency and achieve bidirectional termination.As a result,18 tandem terminators and 20 bidirectional terminators were obtained.（2）Construction of a synthetic NeuAc precursor N-acetylmannosamine（ManNAc）producing host strain:Using CRISPR-Cas9 gene editing technology,E.coli BL21（DE3）was used as the starting strain to integrate UDP-N-acetylglucosamine-2-epimerase（Neu C）from Neisseria meningitidis into the genome and further knockout of the NeuAc degradation and transport related genes nag AB,nan ATEK and man XYZ to construct a NeuAc producing host strain capable of accumulating NeuAc extracellularly.To relieve the feedback inhibition of GlcN-6-P,a Glm S_A mutant was introduced to obtain the recombinant strain NBC2.Using glycerol as the carbon source,0.4 g·L^-1 of ManNAc was obtained after 48 h of fermentation in a microplate.（3）Optimization,design,and construction of a NeuAc high-yield chassis strain:First,a promoter replacement strategy was employed,and a combination of artificial synthetic terminators was used to optimize the expression level of the key enzyme Neu C,resulting in an increase in precursor ManNAc titer to 1.25 g·L^-1.Second,by combining a modular engineering strategy,the biosynthesis of UDP-GlcNAc was systematically optimized,further increasing the ManNAc titer by 7.16-fold to 8.95 g·L^-1.By comparing the effects of NeuB from N.meningitidis,Moritella viscosa and E.coli on the biosynthesis ability of NeuAc at different expression levels,N.meningitidis was identified as the optimal source of NeuB and recombinant strain NBC32 was obtained,with the NeuAc titer of 6.27 g·L^-1.Based on this,by combining the knockout of genes wec B,man A,and pyk A involved in the competitive pathway,the carbon flux competition for the target pathway was blocked,significantly increasing NeuAc titer to 9.65 g·L^-1.（4）Production validation in a 3 L bioreactor:the best performing NeuAc recombinant engineering strain NBC45 was selected for fed-batch fermentation in a 3 L bioreactor to verify its NeuAc production capacity.Using glycerol as the sole carbon source,after 48 h of fermentation,the highest NeuAc titer reached 33.6 g·L^-1,with a productivity of 0.7 g·L^-1·h^-1and a yield of 0.35 g·g^-1.