| Glutaric acid is a C5 dicarboxylic acid mainly used in the production of polyesters and polyamides.At present,a variety of biosynthetic pathways have been developed.However,there are some problems such as complex steps,high consumption of cofactors and low synthesis efficiency.In this study,firstly,the biosynthesis of glutaric acid precursor lysine was increased using rational metabolic modification and i ML1515 model.Secondly,a new glutaric acid synthesis pathway,namely the AMA pathway,was successfully designed.Subsequently,the catalytic mechanism of the rate-limiting enzyme ALDH was resolved and modified by protein engineering.In addition,transcriptomic data analysis revealed that cbp A could improve glutaric acid tolerance.Finally,the efficient production of glutaric acid was achieved by optimization of plasmid and pathway enzyme expression.The main studies were as follows:（1）Increasing precursor lysine biosynthesis:First,strain E.coli Lys1 was constructed based on metabolic engineering strategies commonly used in the literature for enhancing the lysine pathway.second,the genomic metabolic model i ML1515 was used to predict key gene targets for lysine biosynthetic pathway reorganization and metabolic modification was performed,and the obtained resultant E.coli Lys5 titer,yield,and production were 195.9 g·L-1,0.7 g·g-1 glucose,and 5.4 g·L-1·h-1.（2）Design,construction and validation of glutaric acid biosynthesis pathway:The AMA pathway was successfully designed based on Meta Cyc and BRENDA database search,and the feasibility of the cascade pathway was demonstrated by HPLC and LC-MS analysis.Meanwhile,the AMA pathway was introduced into strain E.coli Lys5 to construct strain E.coli AMA01,and the titer,yield and production of glutaric acid reached 51.6 g·L-1,0.3 g·g-1glucose and 0.1 g·L-1·h-1,respectively,after fermentation;and 24.8 g·L-1 glutaraldehyde by-product accumulation was detected.（3）Determination of rate-limiting enzyme ALDH,crystal structure resolution and mechanism analysis:Firstly,ALDH was determined to be the rate-limiting enzyme based on the study of enzyme activity and initial reaction rate.Second,the protein crystals of ALDH were successfully obtained through a series of protein crystallization conditions screening and optimization.By molecular docking,the ternary conformation of the substrate glutaraldehyde and cofactor NAD+with ALDH was obtained.Finally,the catalytic mechanism of ALDH was determined by experiments and calculations.（4）Rational design to improve the catalytic activity of ALDH:Based on the analysis of the energy barriers in the catalytic process of ALDH,the key issues affecting the catalytic efficiency were identified as the initial rate and hydrogen negative transfer,respectively.The mutant Mu5(ALDHI90C/I212C/N94S/P95N/G210T)was obtained after protein engineering modification,and the fermentation test of E.coli AMA02 containing Mu5 mutant strain showed that the titer,yield and production intensity of glutaric acid were 72.5 g·L-1,0.4 g·g-1glucose and 1.5 g·L-1·h-1,respectively.（5）Identification of glutaric acid tolerance targets:Seven highest up-regulated genes were identified by transcriptome data analysis and overexpressed in E.coli AMA02,respectively.Among them,the cbp A overexpression strain（named E.coli AMA03）showed the best tolerance;the cbp A genome was integrated into the glutaric acid degradation gene csi D to construct strain E.coli AMA04,and the glutaric acid titer,yield and production intensity were 82.6 g·L-1,0.4 g·g-1 glucose and 1.7 g·L-1·h-1 after fermentation,respectively.（6）Efficient production of glutaric acid:Firstly,the metabolic burden of the dual plasmid system was reduced and the pathway enzyme was constructed as a single plasmid system（p ETM6R1-ALDH-AAS-MAO）to obtain E.coli AMA05;secondly,the expression level of the pathway enzyme was optimized,and the titer,yield and intensity of glutaric acid production by E.coli AMA06 were 88.4 g·L-1,0.4 g·g-1 and 1.8 g·L-1·h-1,respectively. |
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