Enzyme engineering for functional lipids synthesis: recent advance and perspective

Abstract

Functional lipids, primarily derived through the modification of natural lipids by various processes, are widely acknowledged for their potential to impart health benefits. In contrast to chemical methods for lipid modification, enzymatic catalysis offers distinct advantages, including high selectivity, mild operating conditions, and reduced byproduct formation. Nevertheless, enzymes face challenges in industrial applications, such as low activity, stability, and undesired selectivity. To address these challenges, protein engineering techniques have been implemented to enhance enzyme performance in functional lipid synthesis. This article aims to review recent advances in protein engineering, encompassing approaches from directed evolution to rational design, with the goal of improving the properties of lipid-modifying enzymes. Furthermore, the article explores the future prospects and challenges associated with enzyme-catalyzed functional lipid synthesis.

Keywords

Functional lipids / Biocatalysis / Protein engineering / Activity / Selectivity / Stability

References

[1] Adi Goldenzweig SJF. Principles of protein stability and their application in computational design. Annu Rev Biochem, 2018, 87: 105-129.

[2] Ahrari F, Yousefi M, Habibi Z, Mohammadi M. Application of undecanedicarboxylic acid to prepare cross-linked enzymes (CLEs) of Rhizomucor miehei lipase (RML); Selective enrichment of polyunsaturated fatty acids. J Mol Catal, 2022, 520: 112172-112182.

[3] Akil E, da Adejanildo S, Pereira TE-B, Amaral PFF, Torres AG. Efficient production of bioactive structured lipids by fast acidolysis catalyzed by Yarrowia lipolytica lipase, free and immobilized in chitosan-alginate beads, in solvent-free medium. Int J Biol Macromol, 2020, 163: 910-918.

[4] Aldai N, de Renobales M, Barron LJR, Kramer JKG. What are the trans fatty acids issues in foods after discontinuation of industrially produced trans fats? Ruminant products, vegetable oils, and synthetic supplements. Eur J Lipid Sci Technol, 2013, 115: 1378-1401.

[5] Almeida FLC, Castro MPJ, Travália BM, Forte MBS. Trends in lipase immobilization: bibliometric review and patent analysis. Process Biochem, 2021, 110: 37-51.

[6] Arnold FH. Directed evolution: bringing new chemistry to life. Angew Chem Int Ed Engl, 2018, 57: 4143-4148.

[7] Baek M, DiMaio F, Anishchenko I, Dauparas J, Ovchinnikov S, Lee GR, Wang J, Cong Q, Kinch LN, Schaeffer RD, Millán C, Park H, Adams C, Glassman CR, DeGiovanni A, Pereira JH, Rodrigues AV, van Dijk AA, Ebrecht AC, Opperman DJ, Sagmeister T, Buhlheller C, Pavkov-Keller T, Rathinaswamy MK, Dalwadi U, Yip CK, Burke JE, Garcia KC, Grishin NV, Adams PD, Read RJ, Baker D. Accurate prediction of protein structures and interactions using a three-track neural network. Science, 2021, 373: 871-876.

[8] Bell EL, Smithson R, Kilbride S, Foster J, Hardy FJ, Ramachandran S, Tedstone AA, Haigh SJ, Garforth AA, Day PJR, Levy C, Shaver MP, Green AP. Directed evolution of an efficient and thermostable PET depolymerase. Nat Catal, 2022, 5: 673-681.

[9] Bergamo P, Luongo D, Miyamoto J, Cocca E, Kishino S, Ogawa J, Tanabe S, Rossi M. Immunomodulatory activity of a gut microbial metabolite of dietary linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, associated with improved antioxidant/detoxifying defences. J Funct Foods, 2014, 11: 192-202.

[10] Bernal C, Rodríguez K, Martínez R. Integrating enzyme immobilization and protein engineering: an alternative path for the development of novel and improved industrial biocatalysts. Biotechnol Adv, 2018, 36: 1470-1480.

[11] Biermann U, Bornscheuer UT, Feussner I, Meier MAR, Metzger JO. Fatty acids and their derivatives as renewable platform molecules for the chemical industry. Angew Chem Int Ed Engl, 2021, 60: 20144-20165.

[12] Bornscheuer UT. Enzymes in lipid modification: past achievements and current trends. Eur J Lipid Sci Technol, 2014, 116: 1322-1331.

[13] Bornscheuer UT. Chapter 1—enzymes in lipid modification: an overview. Lipid modification by enzymes and engineered microbes, 2018, Champaign: AOCS Press, 1-9.

[14] Bornscheuer UT, Hauer B, Jaeger KE, Schwaneberg U. Directed evolution empowered redesign of natural proteins for the sustainable production of chemicals and pharmaceuticals. Angew Chem Int Ed Engl, 2019, 58: 36-40.

[15] Cao J, Wu R, Zhu F, Dong QH, Su E. How to improve the efficiency of biocatalysis in non-aqueous pure deep eutectic solvents: a case study on the lipase-catalyzed transesterification reaction. Biochem Eng J, 2022, 179: 108336-108349.

[16] Chen G, Khan IM, He WS, Li YX, Jin P, Campanella OH, Zhang HH, Huo YR, Chen Y, Yang HQ, Miao M. Rebuilding the lid region from conformational and dynamic features to engineering applications of lipase in foods: Current status and future prospects. Compr Rev Food Sci Food Saf, 2022, 21: 2688-2714.

[17] Chow JY, Nguyen GKT. Rational design of lipase ROL to increase its thermostability for production of structured TAGs. Int J Mol Sci, 2022, 23: 9515-9527.

[18] Damnjanović J, Matsunaga N, Adachi M, Nakano H, Iwasaki Y. Facile enzymatic synthesis of phosphatidylthreonine using an engineered Phospholipase D. Eur J Lipid Sci Technol, 2018, 120: 1800089-1800096.

[19] de Rodrigues CA, Barbosa MS, dos Santos JCB, Lisboa MC, Souza RL, Pereira MM, Lima ÁS, Soares CMF. Computational and experimental analysis on the preferential selectivity of lipases for triglycerides in Licuri oil. Bioprocess Biosyst Eng, 2021, 44: 2141-2151.

[20] Enespa CP, Singh DP. Sources, purification, immobilization and industrial applications of microbial lipases: an overview. Crit Rev Food Sci Nutr, 2022, 63: 6653-6686.

[21] Eser BE, Poborsky M, Dai R, Kishino S, Ljubic A, Takeuchi M, Jacobsen C, Ogawa J, Kristensen P, Guo Z. Rational engineering of hydratase from lactobacillus acidophilus reveals critical residues directing substrate specificity and regioselectivity. ChemBioChem, 2020, 21: 550-563.

[22] Ge FY, Chen G, Qian MJ, Xu C, Liu J, Cao JQ, Li XC, Hu D, Xu YS, Xin Y, Wang DL, Zhou J, Shi H, Tan ZB. Artificial intelligence aided lipase production and engineering for enzymatic performance improvement. J Agric Food Chem, 2023, 71: 14911-14930.

[23] Hayashi D, Mouchlis VD, Dennis EA. Omega-3 versus Omega-6 fatty acid availability is controlled by hydrophobic site geometries of phospholipase A2s. J Lipid Res, 2021, 62: 100113-100125.

[24] Hu R, Cui R, Tang Q, Lan D, Wang F, Wang Y. Enhancement of phospholipid binding and catalytic efficiency of Streptomyces klenkii Phospholipase D by increasing hydrophobicity of the active site loop. J Agric Food Chem, 2021, 69: 11110-11120.

[25] Huang J, Dai S, Chen X, Xu L, Yan J, Yang M, Yan Y. Alteration of chain-length selectivity and thermostability of Rhizopus oryzae lipase via virtual saturation mutagenesis coupled with disulfide bond design. Appl Environ Microbiol, 2023, 89: e0187822.

[26] Inoue A, Adachi M, Damnjanović J, Nakano H, Iwasaki Y. Direct enzymatic synthesis of 1-phosphatidyl-β-D-glucose by engineered phospholipase D. ChemistrySelect, 2016, 1: 4121-4125.

[27] Kuchner OAF. Directed evolutionof enzyme catalysts. Trends Biotechnol, 1997, 15: 523-530.

[28] Lan D, Zhao G, Holzmann N, Yuan S, Wang J, Wang Y. Structure-guided rational design of a mono- and diacylglycerol lipase from Aspergillus oryzae: a single residue mutant increases the hydrolysis ability. J Agric Food Chem, 2021, 69: 5344-5352.

[29] Lee WJ, Zhang Z, Lai OM, Tan CP, Wang Y. Diacylglycerol in food industry: synthesis methods, functionalities, health benefits, potential risks and drawbacks. Trends Food Sci Technol, 2020, 97: 114-125.

[30] Li D, Han T, Xue J, Xu W, Xu J, Wu Q. Engineering fatty acid photodecarboxylase to enable highly selective decarboxylation of trans fatty acids. Angew Chem Int Ed Engl, 2021, 60: 20695-20699.

[31] Li L, Mao X, Deng F, Wang Y, Wang F. Improving both the thermostability and catalytic efficiency of phospholipase d from moritella sp. JT01 through disulfide bond engineering strategy. Int J Mol Sci, 2022, 23: 19-31.

[32] Li L, Wu W, Deng Z, Zhang S, Guan W. Improved thermostability of lipase Lip2 from Yarrowia lipolytica through disulfide bond design for preparation of medium-long-medium structured lipids. LWT, 2022, 166: 113786-113794.

[33] Li Z, Meng S, Nie K, Schwaneberg U, Davari MD, Xu H, Ji Y, Liu L. Flexibility regulation of loops surrounding the tunnel entrance in cytochrome P450 enhanced substrate access substantially. ACS Catal, 2022, 12: 12800-12808.

[34] Liu Y, Liu Q, Krivoruchko A, Khoomrung S, Nielsen J. Engineering yeast phospholipid metabolism for de novo oleoylethanolamide production. Nat Chem Biol, 2020, 16: 197-205.

[35] Lovelock SL, Crawshaw R, Basler S, Levy C, Baker D, Hilvert D, Green AP. The road to fully programmable protein catalysis. Nature, 2022, 606: 49-58.

[36] Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, Acosta DJ, Alexander BR, Cole HO, Zhang Y, Lynd NA, Ellington AD, Alper HS. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature, 2022, 604: 662-667.

[37] Madhavan A, Arun KB, Binod P, Sirohi R, Tarafdar A, Reshmy R, Kumar Awasthi M, Sindhu R. Design of novel enzyme biocatalysts for industrial bioprocess: Harnessing the power of protein engineering, high throughput screening and synthetic biology. Bioresour Technol, 2021, 325: 124617-124628.

[38] Maldonado MR, Alnoch RC, Marques J, de Almeida L, dos Santos A, Andretta AT, del Pilar R, Ropaín C, Maltempi E, de Souza D, Mitchell A, Krieger N. Key mutation sites for improvement of the enantioselectivity of lipases through protein engineering. Biochem Eng J, 2021, 172: 108047-108059.

[39] Markel U, Lanvers P, Sauer DF, Wittwer M, Dhoke GV, Davari MD, Schiffels J, Schwaneberg U. A Photoclick-based high-throughput screening for the directed evolution of decarboxylase OleT. Chem Eur J, 2021, 27: 954-958.

[40] Martins PA, Trobo-Maseda L, Lima FA, de Morais JWG, De Marco JL, Salum TFC, Guisán JM. Omega-3 production by fish oil hydrolysis using a lipase from Burkholderia gladioli BRM58833 immobilized and stabilized by post-immobilization techniques. Biochem Biophys Rep, 2022, 29: 101193-101202.

[41] Mazurenko S, Prokop Z, Damborsky J. Machine learning in enzyme engineering. ACS Catal, 2019, 10: 1210-1223.

[42] McDaniel MA, Maier SF, Einstein GO. "Brain-specific" nutrients: a memory cure?. Nutrition, 2003, 19: 957-975.

[43] Micha R, Mozaffarian D. Trans fatty acids: effects on metabolic syndrome, heart disease and diabetes. Nat Rev Endocrinol, 2009, 5: 335-344.

[44] Nielsen J, Keasling JD. Engineering cellular metabolism. Cell, 2016, 164: 1185-1197.

[45] Ogawa J. New lipid science in our inner ecosystem. Eur J Lipid Sci Technol, 2015, 117: 577-578.

[46] Pacheco BJS, Domingues O, Reina MP, de Baptista Álvaro, Neto GS, Andrade S, Veloso A, de Paula, . Improved synthesis of dietary triglycerides by using lipase supported on clay carriers. Biotechnol J, 2022, 17: e2100491-2100498.

[47] Prabhavathi Devi BLA, Gangadhar KN, Prasad RBN, Sugasini D, Rao YPC, Lokesh BR. Nutritionally enriched 1,3-diacylglycerol-rich oil: low calorie fat with hypolipidemic effects in rats. Food Chem, 2018, 248: 210-216.

[48] Qi N, Liu J, Song W, Liu J, Gao C, Chen X, Guo L, Liu L, Wu J. Rational design of phospholipase d to improve the transphosphatidylation activity for phosphatidylserine synthesis. J Agric Food Chem, 2022, 70: 6709-6718.

[49] Qu G, Li A, Acevedo-Rocha CG, Sun Z, Reetz MT. The crucial role of methodology development in directed evolution of selective enzymes. Angew Chem Int Ed Engl, 2020, 59: 13204-13231.

[50] Rathi PC, Fulton A, Jaeger K-E, Gohlke H. Application of rigidity theory to the thermostabilization of lipase a from bacillus subtilis. Plos Comput Biol, 2016, 12: e1004754.

[51] Reetz MT, Carballeira JD. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc, 2007, 2: 891-903.

[52] Reetz MT, Bocola M, Carballeira JD, Zha D, Vogel A. Expanding the range of substrate acceptance of enzymes: combinatorial active-site saturation test. Angew Chem Int Ed Engl, 2005, 44: 4192-4196.

[53] Samantha A, Damnjanović J, Iwasaki Y, Nakano H, Vrielink A. Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: insights into plasticity of substrate binding and activation. Biochem J, 2021, 478: 1749-1767.

[54] Soni S. Trends in lipase engineering for enhanced biocatalysis. Biotechnol Appl Biochem, 2022, 69: 265-272.

[55] Soni S, Sathe SS, Sheth RR, Tiwari P, Vadgama R-KN, Odaneth AA, Lali AM, Chandrayan SK. N-terminal domain replacement changes an archaeal monoacylglycerol lipase into a triacylglycerol lipase. Biotechnol Biofuels, 2019, 12: 110-120.

[56] Sulciner ML, Serhan CN, Gilligan MM, Mudge DK, Chang J, Gartung A, Lehner KA, Bielenberg DR, Schmidt B, Dalli J, Greene ER, Gus-Brautbar Y, Piwowarski J, Mammoto T, Zurakowski D, Perretti M, Sukhatme VP, Kaipainen A, Kieran MW, Huang S, Panigrahy D. Resolvins suppress tumor growth and enhance cancer therapy. J Exp Med, 2018, 215: 115-140.

[57] Sun Z, Liu Q, Qu G, Feng Y, Reetz MT. Utility of B-factors in protein science: interpreting rigidity, flexibility, and internal motion and engineering thermostability. Chem Rev, 2019, 119: 1626-1665.

[58] Tang Q, Lan D, Yang B, Khan FI, Wang Y. Site-directed mutagenesis studies of hydrophobic residues in the lid region of T1 lipase. Eur J Lipid Sci Technol, 2017, 119: 1600107-1600114.

[59] Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, Yuan D, Stroe O, Wood G, Laydon A, Zidek A, Green T, Tunyasuvunakool K, Petersen S, Jumper J, Clancy E, Green R, Vora A, Lutfi M, Figurnov M, Cowie A, Hobbs N, Kohli P, Kleywegt G, Birney E, Hassabis D, Velankar S. AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res, 2022, 50: D439-D444.

[60] Verdasco-Martín CM, Garcia-Verdugo E, Porcar R, Fernandez-Lafuente R, Otero C. Selective synthesis of partial glycerides of conjugated linoleic acids via modulation of the catalytic properties of lipases by immobilization on different supports. Food Chem, 2018, 245: 39-46.

[61] Verger R. ‘Interfacial activation’ of lipases: facts and artifacts. Trends Biotechnol, 1997, 15: 32-38.

[62] Wang S, Xu Y, Yu X-W. Propeptide in Rhizopus chinensis lipase: new insights into its mechanism of activity and substrate selectivity by computational design. J Agric Food Chem, 2021, 69: 4263-4275.

[63] Wang Z, Wen J, Zhang J, Deng J, Zhuang W, Liu J, Wang Z, Rao Y, Zhu Y, Ying H. Atomic insights into the mechanism of trace water influence on lipase catalysis in organic media. Chem Eng J, 2023, 464: 142610-142622.

[64] Watson JL, Juergens D, Bennett NR, Trippe BL, Yim J, Eisenach HE, Ahern W, Borst AJ, Ragotte RJ, Milles LF, Wicky BIM, Hanikel N, Pellock SJ, Courbet A, Sheffler W, Wang J, Venkatesh P, Sappington I, Torres SV, Lauko A, De Bortoli V, Mathieu E, Ovchinnikov S, Barzilay R, Jaakkola TS, DiMaio F, Baek M, Baker D. De novo design of protein structure and function with RFdiffusion. Nature, 2023, 620: 1089-1100.

[65] Woo J-M, Kang Y-S, Lee S-M, Park S, Park J-B. Substrate-binding site engineering of Candida antarctica Lipase B to improve selectivity for synthesis of 1-monoacyl-sn-glycerols. Biotechnol Bioprocess Eng, 2022, 27: 234-243.

[66] Wu C, Hong B, Jiang S, Luo X, Lin H, Zhou Y, Wu J, Yue X, Shi H, Wu R. Recent advances on essential fatty acid biosynthesis and production: clarifying the roles of Δ12/Δ15 fatty acid desaturase. Biochem Eng J, 2022, 178: 108306-108319.

[67] Wu L, Qin L, Nie Y, Xu Y, Zhao YL. Computer-aided understanding and engineering of enzymatic selectivity. Biotechnol Adv, 2022, 54: 107793-107812.

[68] Wu S, Xiang C, Zhou Y, Khan MSH, Liu W, Feiler CG, Wei R, Weber G, Hohne M, Bornscheuer UT. A growth selection system for the directed evolution of amine-forming or converting enzymes. Nat Commun, 2022, 13: 7458-7467.

[69] Xu Q, Tang Q, Xu Y, Wu J, Mao X, Li F, Wang S, Wang Y. Biotechnology in future food lipids: opportunities and challenges. Annu Rev Food Sci Technol, 2023, 14: 225-246.

[70] Yang Y, Arnold FH. Navigating the unnatural reaction space: directed evolution of heme proteins for selective Carbene and Nitrene transfer. Acc Chem Res, 2021, 54: 1209-1225.

[71] Yang Y, Wang J, Yang B, Lan D, Wang Y. Possible charged residue switch for acylglycerol selectivity of lipase MAS1. Appl Biochem Biotechnol, 2022, 194: 5119-5131.

[72] Yi J-J, Heo S-Y, Ju J-H, Oh B-R, Son WS, Seo J-W. Synthesis of 13R,20-dihydroxy-docosahexaenoic acid by site-directed mutagenesis of lipoxygenase derived from Oscillatoria nigro-viridis PCC 7112. Biochem Biophys Res Commun, 2020, 533: 893-898.

[73] Yore Mark M, Syed I, Moraes-Vieira Pedro M, Zhang T, Herman Mark A, Homan Edwin A, Patel Rajesh T, Lee J, Chen S, Peroni Odile D, Dhaneshwar Abha S, Hammarstedt A, Smith U, McGraw Timothy E, Saghatelian A, Kahn Barbara B. Discovery of a class of endogenous mammalian lipids with anti-diabetic and anti-inflammatory effects. Cell, 2014, 159: 318-332.

[74] Yu D, Wang JB, Reetz MT. Exploiting designed oxidase-peroxygenase mutual benefit system for asymmetric cascade reactions. J Am Chem Soc, 2019, 141: 5655-5658.

[75] Zeng W, Guo L, Xu S, Chen J, Zhou J. High-throughput screening technology in industrial biotechnology. Trends Biotechnol, 2020, 38: 888-906.

[76] Zhang H, Chu W, Sun J, Liu Z, Huang WC, Xue C, Mao X. Combining cell surface display and DNA-shuffling technology for directed evolution of streptomyces phospholipase d and synthesis of phosphatidylserine. J Agric Food Chem, 2019, 67: 13119-13126.

[77] Zhang TT, Xu J, Wang YM, Xue CH. Health benefits of dietary marine DHA/EPA-enriched glycerophospholipids. Prog Lipid Res, 2019, 75: 100997-101020.

[78] Zhang M, Li Q, Lan X, Li X, Zhang Y, Wang Z, Zheng J. Directed evolution of Aspergillus oryzae lipase for the efficient resolution of (R, S)-ethyl-2-(4-hydroxyphenoxy) propanoate. Bioprocess Biosyst Eng, 2020, 43: 2131-2141.

[79] Zhang Z, Chen M, Xu W, Zhang W, Zhang T, Guang C, Mu W. Microbial phospholipase D: Identification, modification and application. Trends Food Sci Technol, 2020, 96: 145-156.

[80] Zhang H, Secundo F, Sun J, Mao X. Advances in enzyme biocatalysis for the preparation of functional lipids. Biotechnol Adv, 2022, 61: 108036-108050.

[81] Zhang Y, Dai P, Liu R, Liu W, Xiao A, Li J, Li G, Liu J. Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement. J Biotechnol, 2022, 355: 1-9.

[82] Zheng WL, Pu ZJ, Xiao LX, Xu G, Yang LR, Yu HR, Wu JP. Mutability-landscape-guided engineering of l-threonine aldolase revealing the prelog rule in mediating diastereoselectivity of C-C bond formation. Angew Chem Int Ed Engl, 2023, 62: e202213855.

[83] Zhu L, Fang SZ, Liu WW, Zhang H, Zhang YQ, Xie ZH, Yang PY, Wan JC, Gao BY, Yu L. The triacylglycerol structure and composition of a human milk fat substitute affect the absorption of fatty acids and calcium, lipid metabolism and bile acid metabolism in newly-weaned Sprague-Dawley rats. Food Funct, 2023, 14: 7574-7585.

[84] Zong L, Zhang Y, Shao Z, Ljubic A, Jacobsen C, Gao R, Eser BE, Wang Y, Guo Z. Selective and sustainable production of sub-terminal hydroxy fatty acids by a self-sufficient CYP102 enzyme from bacillus amyloliquefaciens. ChemBioChem, 2023, 24: e202300368.

[85] Zorn K, Oroz-Guinea I, Brundiek H, Bornscheuer UT. Engineering and application of enzymes for lipid modification, an update. Prog Lipid Res, 2016, 63: 153-164.

[86] Zorn K, Oroz-Guinea I, Brundiek H, Dörr M, Bornscheuer UT. Alteration of chain length selectivity of candida antarctica lipase A by semi-rational design for the enrichment of Erucic and Gondoic fatty acids. Adv Synth Catal, 2018, 360: 4115-4131.

[87] Zorn K, Oroz-Guinea I, Bornscheuer UT. Strategies for enriching erucic acid from Crambe abyssinica oil by improved Candida antarctica lipase A variants. Process Biochem, 2019, 79: 65-73.

[88] Zou XQ, Ye LF, He XC, Wu SB, Zhang H, Jin QZ. Preparation of DHA-rich medium- and long-chain triacylglycerols by lipase-catalyzed acidolysis of microbial oil from Schizochytrium sp with medium-chain fatty acids. Appl Biochem Biotechnol, 2020, 191: 1294-1314.

Funding

Natural Science Foundation of Sichuan Province(2023NSFSC0132)

Fundamental Research Funds for Central Universities of the Sichuan University(YJ202308)