Enhancement of α-ketoisovalerate production by relieving the product inhibition of L-amino acid deaminase from Proteus mirabilis

doi:10.1016/j.cjche.2020.04.009

Abstract

Abstract: L-Amino acid deaminase (LAAD) is a key enzyme in the deamination of L-valine (L-val) to produce α- ketoisovalerate (KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production. In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition. The four mutant PM-LAAD^M4 (PM-LAAD^{S98A/T105A/S106A/L341A}) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the “lid” structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^M4 has been applied to KIV production, the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.

Key words: Product inhibition, L-Amino acid deaminases, α-Ketoisovalerate, Bio-catalysis, Protein engineering

摘要： L-Amino acid deaminase (LAAD) is a key enzyme in the deamination of L-valine (L-val) to produce α- ketoisovalerate (KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production. In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition. The four mutant PM-LAAD^M4 (PM-LAAD^{S98A/T105A/S106A/L341A}) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the “lid” structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^M4 has been applied to KIV production, the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.

关键词: Product inhibition, L-Amino acid deaminases, α-Ketoisovalerate, Bio-catalysis, Protein engineering

Shanshan Pei, Xiaobo Ruan, Jia Liu, Wei Song, Xiulai Chen, Qiuling Luo, Liming Liu, Jing Wu. Enhancement of α-ketoisovalerate production by relieving the product inhibition of L-amino acid deaminase from Proteus mirabilis[J]. Chinese Journal of Chemical Engineering, 2020, 28(8): 2190-2199.

References

[1] U.T. Bornscheuer, B. Hauer, K.E. Jaeger, U. Schwaneberg, Directed evolution empowered redesign of natural proteins for the sustainable production of chemicals and pharmaceuticals, Angew. Chem. Int. Ed. 58(1) (2019) 36-40.
[2] H.X. Ying, J. Wang, T. Shi, Y.L. Zhao, P.K. Ouyang, K.Q. Chen, Engineering of lysine cyclodeaminase conformational dynamics for relieving substrate and product inhibitions in the biosynthesis of L-pipecolic acid, Catal. Sci. Technol. 9(2) (2019) 398-405.
[3] A. Currin, M.S. Dunstan, L.O. Johannissen, K.A. Hollywood, M. Vinaixa, A.J. Jervis, N. Swainston, N.J.W. Rattray, J.M. Gardiner, D.B. Kell, E. Takano, H.S. Toogood, N.S. Scrutton, Engineering the "missing link" in biosynthetic (-)-menthol production:bacterial isopulegone isomerase, ACS Catal. 8(3) (2018) 2012-2020.
[4] Y.H. Choi, J.H. Kim, B.S. Park, B.G. Kim, Solubilization and iterative saturation mutagenesis of α 1,3-fucosyltransferase from Helicobacter pylori to enhance its catalytic efficiency, Biotechnol. Bioeng. 113(8) (2016) 1666-1675.
[5] D. Shen, Y.J. Xu, S.L. Shi, A bulky chiral N-heterocyclic carbene palladium catalyst enables highly enantioselective suzuki-miyaura cross-coupling reactions for the synthesis of biaryl atropisomers, J. Am. Chem. Soc. 141(37) (2019) 14938-14945.
[6] Y. Zhou, G. Wang, P.C. Wang, Z.Y. Li, T.Q. Yue, J.B. Wang, P. Zou, Expanding APEX2 substrates for proximity-dependent labeling of nucleic acids and poteins in living cells, Angew. Chem. Int. Ed. 58(34) (2019) 11763-11767.
[7] J.Q. Duan, B.B. Li, Y.C. Qin, Y.J. Dong, J. Ren, G.Y. Li, Recent progress in directed evolution of stereoselective monoamine oxidases, Bioresour Bioprocess. 6(1) (2019) 37.
[8] A.X. Cheng, P. Zhang, B. Wang, D.D. Yang, X.T. Duan, Y.L. Jiang, T. Xu, Y. Jiang, J.H. Shi, C.T. Ding, G. Wu, Z.H. Sang, Q. Wu, H. Wang, M. Wu, Z.Y. Zhang, X. Pan, Y.Y. Pan, P. Gao, H.F. Zhang, C.Z. Zhou, J. Guo, Z.Y. Yang, Aurora-A mediated phosphorylation of LDHB promotes glycolysis and tumor progression by relieving the substrate-inhibition effect, Nat. Chem. 10(2019) 5566.
[9] Y.P. Shang, Q. Chen, A.T. Li, S. Quan, J.H. Xu, H.L. Yu, Attenuated substrate inhibition of a haloketone reductase via structure-guided loop engineering, J. Biotechnol. 308(2020) 141-147.
[10] K. Larue, M. Melgar, V.J.J. Martin, Directed evolution of a fungal β-glucosidase in Saccharomyces cerevisiae, Biotechnol. Biofuels. 9(2016) 52.
[11] K.M. Trollope, J.F. Gorgens, H. Volschenk, Semirational directed evolution of loop regions in Aspergillus japonicus β-fructofuranosidase for improved fructooligosaccharide production, Appl. Environ. Microbiol. 81(20) (2015) 7319-7329.
[12] X. Hu, S. Robin, S. O'Connell, G. Walsh, J.G. Wall, Engineering of a fungal β-galactosidase to remove product inhibition by galactose, Appl. Environ. Microbiol. 87(5) (2010) 1773-1782.
[13] X. Wang, Y.Q. Jiang, M.B. Wu, L. Zhu, L.R. Yang, J.B. Lin, Semi-rationally engineered variants of S-adenosylmethionine synthetase from Escherichia coli with reduced product inhibition and improved catalytic activity, Enzym. Microb. Technol. 129(2019) 109355.
[14] T. Borner, G. Rehn, C. Grey, P. Adlercreutz, A process concept for high-purity production of amines by transaminase-catalyzed asymmetric synthesis:Combining enzyme cascade and membrane-assisted ISPR, Org. Process. Res. Dev. 19(7) (2015) 793-799.
[15] P. Wang, Y.S. Wang, Z.G. Su, Microbial production of propionic acid with propionibacterium freudenreichii using an anion exchanger-based in situ product recovery (ISPR) process with direct and indirect contact of cells, Appl. Biochem. Biotechnol. 166(4) (2012) 974-986.
[16] R.K. Singh, M.K. Tiwari, R. Singh, J.K. Lee, From protein engineering to immobilization:Promising strategies for the upgrade of industrial enzymes, Int. J. Mol. Sci. 14(2013) 1232-1277.
[17] J.T. Dafoe, A.J. Daugulis, In situ product removal in fermentation systems:Improved process performance and rational extractant selection, Biotechnol. Lett. 36(1) (2014) 443-460.
[18] O. Fellechner, M. Blatkiewicz, I. Smirnova, Reactive separations for in situ product removal of enzymatic reactions:A review, Chemie. Ingenieur. Technik. 91(11) (2019) 1522-1543.
[19] Y.S. Song, X.D. Wang, J.G. Zhang, Enhancement of bioconversion gangliosides to monosialotetrahexosylganglioside by in situ sialic acid removal and recovery, Can. J. Chem. Eng. 97(10) (2019) 2617-2620.
[20] G. Molla, R. Melis, L. Pollegioni, Breaking the mirror:L-amino acid deaminase, a novel stereoselective biocatalyst, Biotechnol. Adv. 35(6) (2017) 657-668.
[21] M.F. Mccarty, Toward a wholly nutritional therapy for type 2 diabetes, Med. Hypotheses 54(3) (2000) 483-487.
[22] G. Kuhlman, J.A. Roth, P.J. Flakoll, M.J. Vandehaar, S. Nissen, Effects of dietary leucine, α-ketoisocaproate and isovalerate on antibody production and lymphocyte blastogenesis in growing lambs, J. Nutr. 118(12) (1988) 1564-1569.
[23] T. Luetke-Eversloh, C.N.S. Santos, G. Stephanopoulos, Perspectives of biotechnological production of L-tyrosine and its applications, Appl. Microbiol. Biotechnol. 77(4) (2007) 751-762.
[24] U. Stottmeister, A. Aurich, H. Wilde, J. Andersch, S. Schmidt, D. Sicker, White biotechnology for green chemistry:Fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses, J. Ind. Microbiol. Biotechnol. 32(11-12) (2005) 651-664.
[25] Y.P. Wu, M.S. Zha, S. Yin, H.Q. Yang, J. Boutet, R. Huet, C.T. Wang, B.G. Sun, Novel method for L-methionine production catalyzed by the aminotransferase ARO8 from Saccharomyces cerevisiae, J. Agric. Food Chem. 66(24) (2018) 6116-6122.
[26] T. Wakamatsu, H. Sakuraba, M. Kitamura, Y. Hakumai, K. Fukui, K. Ohnishi, M. Ashiuchi, T. Ohshima, Structural insights into L-tryptophan dehydrogenase from a photoautotrophic cyanobacterium, Nostoc punctiforme, Appl. Environ. Microbiol. 83(2) (2017) e02710-e02716.
[27] S. Nakano, Y. Minamino, F. Hasebe, S. Ito, Deracemization and stereoinversion to aromatic D-amino acid derivatives with ancestral L-amino acid oxidase, ACS Catal. 9(11) (2019) 10152-10158.
[28] P. Nshimiyimana, L. Liu, G.C. Du, Engineering of L-amino acid deaminases for the production of α-keto acids from L-amino acids, Bioengineered. 10(1) (2019) 43-51.
[29] P. Motta, G. Molla, L. Pollegioni, M. Nardini, Structure-function relationships in Lamino acid deaminase, a flavoprotein belonging to a novel class of biotechnologically relevant enzymes, J. Biol. Chem. 291(20) (2016) 10457-10475.
[30] G.S. Hossain, J.H. Li, H.D. Shin, G.C. Du, M. Wang, L. Liu, J. Chen, One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli wholecell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris, PLoS One 9(12) (2014), e114291..
[31] A.S. Xiong, R.H. Peng, J. Zhuang, J.G. Liu, F. Gao, J.M. Chen, Z.M. Cheng, Q.H. Yao, Nonpolymerase-cycling-assembly-based chemical gene synthesis:Strategies, methods, and progress, Biotechnol. Adv. 26(2) (2008) 121-134.
[32] L.C. Wu, X.L. Guo, G.B. Wu, P.F. Liu, Z.D. Liu, Efficient enzymatic synthesis of α-keto acids by redesigned substrate-binding pocket of the L-amino acid deaminase (PmiLAAD), Enzym. Microb. Technol. 132(2020) 109393.
[33] D.Y. Travin, Z.L. Watson, M. Metelev, F.R. Ward, I.A. Osterman, I.M. Khven, N.F. Khabibullina, M. Serebryakova, P. Mergaert, Y.S. Polikanov, J.H.D. Cate, K. Severinov, Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition, Nat. Chem. 10(2019) 4563.
[34] S.S. Han, H.H. Kyeong, J.M. Choi, Y.K. Sohn, J.H. Lee, H.S. Kim, Engineering of the conformational dynamics of an enzyme for relieving the product inhibition, ACS Catal. 6(12) (2016) 8440-8445.
[35] R. Melis, E. Rosini, V. Pirillo, L. Pollegioni, G. Molla, In vitro evolution of an L-amino acid deaminase active on L-1-naphthylalanine, Catal. Sci. Technol. 8(20) (2018) 5359-5367.
[36] Y.X. Yuan, W. Song, J. Liu, X.L. Chen, Q.L. Luo, L.M. Liu, Production of α-ketoisocaproate and α-keto-β-methylvalerate by engineered L-amino acid deaminase, Chemcatchem. 11(10) (2019) 2464-2472.
[37] B.J. Wang, Z.X. Cao, C. Rovira, J.S. Song, S. Shaik, Fenton-derived OH radicals enable the MPnS enzyme to convert 2-hydroxyethylphosphonate to methylphosphonate:Insights from Ab initio QM/MM MD simulations, J. Am. Chem. Soc. 141(23) (2019) 9284-9291.
[38] D. Bedrov, J.P. Piquemal, O. Borodin, A.D. MacKerell, B. Roux, C. Schroder, Molecular dynamics simulations of ionic liquids and electrolytes using polarizable force fields, Chem. Rev. 119(13) (2019) 7940-7995.
[39] A.J. Cooper, J.Z. Ginos, A. Meister, Synthesis and properties of the α-keto acids, Chem. Rev. 83(3) (1983) 321-358.
[40] S. Pospisil, J. Kopecky, V. Prikrylova, Derepression and altered feedback regulation of valine biosynthetic pathway in analogue-resistant mutants of Streptomyces cinnamonensis resulting in 2-ketoisovalerate excretion, J. Appl. Microbiol. 85(1) (1998) 9-16.
[41] F.S. Krause, B. Blombach, B.J. Eikmanns, Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production, Appl. Environ. Microbiol. 76(24) (2010) 8053-8061.
[42] J.J. Gu, J.D. Zhou, Z.X. Zhang, C.H. Kim, B. Jiang, J.P. Shi, J. Hao, Isobutanol and 2-ketoisovalerate production by Klebsiella pneumoniae via a native pathway, Metab. Eng. 43(2017) 71-84.
[43] J. Buchholz, A. Schwentner, B. Brunnenkan, C. Gabris, S. Grimm, R. Gerstmeir, R. Takors, B.J. Eikmanns, B. Blombach, Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate, Appl. Environ. Microbiol. 79(18) (2013) 5566-5575.
[44] R.X. Li, H.G. Sakir, J.H. Li, H.D. Shin, G.C. Du, J. Chen, L. Liu, Rational molecular engineering of L-amino acid deaminase for production of α-ketoisovaleric acid from Lvaline by Escherichia coli, RSC Adv. 7(11) (2017) 6615-6621.