Glucaric acid is an organic dibasic acid that plays an important role in medicine, food, clothing, textile and chemical industry. It is known as one of the top value-added chemicals from biomass. The synthesis of glucaric acid mainly relies on chemical oxidation and biological methods, but the production of glucaric acid by chemical oxidation has disadvantages such as low yield, many by-products, and unfriendly environment problems. The biosynthesis of glucaric acid is more environmentally friendly, and can realize low-cost production of glucaric acid. The biosynthetic pathway of glucaric acid had been successfully constructed in Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae. At present, the production of glucaric acid has been greatly improved by metabolic engineering and synthetic biology methods, but the activity and stability of the key enzyme in the synthesis pathway of glucaric acid, inositol oxygenase MIOX4, has not been improved efficiently. In order to improve the activity of MIOX4, S. cerevisiae was used as a chassis microorganism in this study. The S. cerevisiae BY4741opi1Δ (BY4741 strain with knockout of OPI1 gene) was the starting strain in this experiment. Taking advantage of the fact that S. cerevisiae could use its own inositol-1-phosphate synthase (Ino1) to convert glucose-6-phosphate into inositol-1-phos- phate, which is then converted to inositol by its own inositol monophosphatase (Inm1/2), the inositol oxygenase miox4 gene from Arabidopsis thaliana and the uronate dehydrogenase udh gene from Pseudomonas syringae were exogenously expressed on the genome of BY4741opi1Δ strain to construct the glucaric acid metabolic pathway. Firstly, the udh gene was integrated into the terminator region of OPI1 of BY4741opi1Δ strain. Then the structure simulation of the inositol oxygenase MIOX4 from the Arabidopsis thaliana genome was performed to predict its catalytic active sites and ligand binding sites to identify the region of the key amino acids. The sequences of the key region were randomly mutated by two rounds of error-prone PCR and the mutated miox4* was integrated into the promoter region of OPI1 of BY4741opi1Δ strain to construct its mutant library. Taking advantage of the fact that the E. coli glucaric acid biosensor plasmid of R7M10, expressing both transcription activator CdaR and reporter gene green fluorescent protein GFP, can display the concentration of glucaric acid as fluorescence intensity, it was used as a high-throughput screening tool to screen the MIOX4 mutant library. As a result, 60 strains with higher fluorescence were obtained from the preliminary screen. Subsequently, they were further verified by shake flask fermentation and 24 strains were obtained whose glucaric acid production were higher than the control. Sequencing analysis showed that the four potential MIOX4 mutants (S133R, K88R, E72V, T40P) were obtained from the mutant library which increased the production of glucaric acid by 53%, 30%, 21%, and 17% compared with the control strains, respectively. Finally, by analyzing the protein structures of the mutant MIOX4 (S133R) with the highest increase in glucaric acid production and wild type MIOX4, it was found that the mutation site R133 increased the steric distance between the site and the substrate inositol, which reduced the steric hindrance and thus increased its catalytic ability of inositol. This study further proves that MIOX4 plays an important role in the biosynthesis of glucaric acid and will provide foundation for the subsequent research on the structure and function of MIOX and the further improvement of glucaric acid production.
[1] SINGH J, GUPTA K P.Induction of apoptosis by calcium D-glucar- ate in 7, 12-dimethyl benz[a]anthracene-exposed mouse skin[J].Journal of Environmental Pathology, Toxicology and Oncology, 2007, 26(1):63-73.
[2] KIELY D E, CHEN L, LIN T H.Hydroxylated nylons based on unprotected esterified D-glucaric acid by simple condensation reactions[J].Journal of the American Chemical Society, 1994, 116(2):571-578.
[3] BOZELL J J, PETERSEN G R.Technology development for the pro- duction of biobased products from biorefinery carbohydrates-the US Department of Energy’s “ Top10 ” revisited[J].Green Chemistry, 2010, 12(4):539.
[4] SMITH T N, HASH K, DAVEY C L, et al.Modifications in the nitric acid oxidation of D-glucose[J].Carbohydrate Research, 2012, 350:6-13.
[5] MOON T S, YOON S H, LANZA A M, et al.Production of glucaric acid from a synthetic pathway in recombinant Escherichia coli[J].Applied and Environmental Microbiology, 2009, 75(3):589-595.
[6] GUPTA A, HICKS M A, MANCHESTER S P, et al.Porting the synthetic D-glucaric acid pathway from Escherichia coli to Saccharo- myces cerevisiae[J].Biotechnology Journal,2016, 11(9):1 201-1 208.
[7] LIU Y, GONG X, WANG C, et al.Production of glucaric acid from myo-inositol in engineered Pichia pastoris[J].Enzyme and Microbial Technology, 2016, 91:8-16.
[8] LEE C C, KIBBLEWHITE R E, PAAVOLA C D, et al.Production of glucaric acid from hemicellulose substrate by rosettasome enzyme assemblies[J].Molecular Biotechnology, 2016, 58(7):489-496.
[9] SHIUE E, PRATHER K L J.Improving D-glucaric acid production from myo-inositol in E.coli by increasing MIOX stability and myo-inositol transport[J].Metabolic Engineering, 2014, 22:22-31.
[10] MOON T S, DUEBER J E, SHIUE E, et al.Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E.coli[J].Metabolic Engineering, 2010, 12(3):298-305.
[11] CHEN N, WANG J Y, ZHAO Y Y, et al.Metabolic engineering of Saccharomyces cerevisiae for efficient production of glucaric acid at high titer[J].Microbial Cell Factories, 2018, 17(1):67.
[12] 李杰, 赵运英, 邓禹.高效合成葡萄糖二酸酿酒酵母工程菌的构建[J].生物工程学报, 2022, 38(2):705-718.
LI J, ZHAO Y Y, DENG Y.Engineering Saccharomyces cerevisiae for efficient production of glucaric acid[J].Chinese Journal of Biotechnology, 2022, 38(2):705-718.
[13] PACKER M S, LIU D R.Methods for the directed evolution of proteins[J].Nature Reviews Genetics, 2015, 16(7):379-394.
[14] 李树, 张伟, 赵春梅.易错PCR技术定向进化褐藻胶裂解酶Alg-2的分析[J].食品科学, 2019, 40(4):146-151.
LI S, ZHANG W, ZHAO C M.Directed evolution of alginate lyase Alg-2 based on error prone PCR[J].Food Science, 2019, 40(4):146-151.
[15] LIU Y H, HU B, XU Y J, et al. Improvement of the acid stability of Bacillus licheniformis alpha amylase by error-prone PCR[J].Journal of Applied Microbiology, 2012, 113(3):541-549.
[16] BLATTNER F R, PLUNKETT III G, BLOCH C A, et al.The complete genome sequence of Escherichia coli K-12[J].Science, 1997, 277(5 331):1 453-1 462.
[17] 王毳, 刘叶, 巩旭, 等. 构建葡萄糖二酸指示系统筛选肌醇加氧酶突变株[J].生物工程学报, 2018, 34(11):1 772-1 783.
WANG C, LIU Y, GONG X, et al. Construction of a glucaric acid biosensor for screening myo-inositol oxygenase variants[J].Chinese Journal of Biotechnology, 2018, 34(11): 1 772-1 783.
[18] DING N N, YUAN Z Q, ZHANG X J, et al. Programmable crossribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor[J].Nucleic Acids Research, 2020, 48(18):10 602-10 613.
[19] YANG J Y, ROY A, ZHANG Y.BioLiP:A semi-manually curated database for biologically relevant ligand-protein interactions[J].Nucleic Acids Research, 2021, 41(D1):D1 096-D1 103.
[20] 王睿, 喻晓蔚, 沙冲, 等.定向进化-易错PCR方法提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力[J].生物工程学报, 2009, 25(12):1 892-1 899.
WANG R, YU X W, SHA C, et al.Increasing activity of Rhizopus chinensis CCTCC M201021 lipase by directed evolution-error prone PCR[J].Chinese Journal of Bioengineering, 2009, 25(12):1 892-1 899.
[21] MADAN B, MISHRA P.Directed evolution of Bacillus licheniformis lipase for improvement of thermostability[J].Biochemical Engineering Journal, 2014, 91:276-282.
[22] ROGERS J K, CHURCH G M.Genetically encoded sensors enable real-time observation of metabolite production[J].Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(9):2 388-2 393.
[23] KARKHANE A A, YAKHCHALI B, JAZII F R, et al. The effect of substitution of Phe181 and Phe182 with Ala on activity, substrate specificity and stabilization of substrate at the active site of Bacillus thermocatenulatus lipase[J].Journal of Molecular Catalysis B:Enzymatic, 2009, 61(3-4):162-167.
[24] 薄秀梅, 张荣丽, 徐洲.MEKK3蛋白K391位点突变对其影响的生物信息学分析及活性鉴定[J].科学技术与工程, 2020, 20(13):5 066-5 073.
BO X M, ZHANG R L, XU Z.Bioinformatics analysis and activity identification of MEKK3 protein K391 site mutation on its protein[J].Science Technology and Engineering, 2020, 20(13):5 066-5 073.
