D-阿洛酮糖是一种低热量的甜味剂,具有多种生理功能,如维持血糖平衡和调节脂质代谢等。目前,D-阿洛酮糖工业生产主要通过D-阿洛酮糖3-差向异构酶(D-allulose-3-epimerase,DAEase)催化D-果糖的差向异构反应实现,但DAEase的应用性能仍需进一步提升以满足日益增长的市场需求。定向进化作为一种有效的分子改造策略,其筛选方法是限制效率的关键因素之一。通过构建并优化D-阿洛酮糖特异型生物传感器,结合微孔板筛选,对来自Clostridium cellulolyticum H10的DAEase(CcDAEase)突变体文库进行超高通量筛选,最终获得了比活提高14.6%的优势突变体H209Q,其最适温度为60 ℃,最适pH值为7.5,并进一步解析了该突变体的构效关系。该研究证明了转录因子型生物传感器可以作为一种有效的定向进化筛选工具,为进一步优化DAEase的性能提供了可能性,亦为D-阿洛酮糖的工业制备提供了理论依据和技术支撑。
D-allulose is a low-calorie sweetener with a variety of physiological functions, such as maintaining blood glucose balance and regulating lipid metabolism. Currently, the industrial production of D-allulose is mainly achieved by catalyzing the epimerization of D-fructose by D-allulose 3-epimerase (DAEase), but the application performance of DAEase still needs to be further improved to meet the growing market demand.Directed evolution is an effective molecular modification strategy, but the screening method is one of the key factors limiting its efficiency.By constructing and optimizing the D-allulose-responsive biosensor, combing microtiter plate screening, the DAEase from Clostridium cellulolyticum H10 (CcDAEase) library was ultrahigh-throughput screened, and a dominant mutant H209Q with a 14.6% increase in specific activity was finally obtained, its optimum temperature and optimum pH were 60 ℃ and 7.5, respectively, and the structure-activity relationship of this mutant was further analyzed.This study proves that the transcription factor-based biosensor can be employed as an effective directed evolution screening tool, which provides the possibility to further optimize the performance of DAEase, and also provides a theoretical basis and technical support for the industrial preparation of D-allulose.
[1] ROGERS P J, HOGENKAMP P S, DE GRAAF C, et al.Does low-energy sweetener consumption affect energy intake and body weight? A systematic review, including meta-analyses, of the evidence from human and animal studies[J].International Journal of Obesity (2005), 2016, 40(3):381-394.
[2] KIMURA T, KANASAKI A, HAYASHI N, et al.D-Allulose enhances postprandial fat oxidation in healthy humans[J].Nutrition, 2017, 43:16-20.
[3] CHEN D, CHEN J J, LIU X Y, et al.Biochemical identification of a hyperthermostable L-ribulose 3-epimerase from Labedella endophytica and its application for D-allulose bioconversion[J].International Journal of Biological Macromolecules, 2021, 189:214-222.
[4] HOSSAIN A, YAMAGUCHI F, HIROSE K, et al.Rare sugar D-psicose prevents progression and development of diabetes in T2DM model Otsuka Long-Evans Tokushima Fatty rats[J].Drug Design, Development and Therapy, 2015, 9:525-535.
[5] NATSUME Y, YAMADA T, IIDA T, et al.Investigation of D-allulose effects on high-sucrose diet-induced insulin resistance via hyperinsulinemic-euglycemic clamps in rats[J].Heliyon, 2021, 7(9):e08013.
[6] BEERENS K, DESMET T, SOETAERT W.Enzymes for the biocatalytic production of rare sugars[J].Journal of Industrial Microbiology & Biotechnology, 2012, 39(6):823-834.
[7] ISHIDA Y, KAMIYA T, ITOH H, et al.Cloning and characterization of the D-tagatose 3-epimerase gene from Pseudomonas cichorii ST-24[J].Journal of Fermentation and Bioengineering, 1997, 83(6):529-534.
[8] HU M Y, LI M L, JIANG B, et al.Bioproduction of D-allulose:Properties, applications, purification, and future perspectives[J].Comprehensive Reviews in Food Science and Food Safety, 2021, 20(6):6012-6026.
[9] NIRANTAR S R.Directed evolution methods for enzyme engineering[J].Molecules.2021, 26(18):5599.
[10] XIAO D, ZHANG W, GUO X T, et al.A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR[J].Nature Communications, 2021, 12(1):7108.
[11] LIU Z Z, CHEN S, WU J.Advances in ultrahigh-throughput screening technologies for protein evolution[J].Trends in Biotechnology, 2023, 41(9):1168-1181.
[12] TELLECHEA-LUZARDO J, STIEBRITZ M T, CARBONELL P.Transcription factor-based biosensors for screening and dynamic regulation[J].Frontiers in Bioengineering and Biotechnology, 2023, 11:1118702.
[13] MACHADO L F M, DIXON N.Directed evolution of transcription factor-based biosensors for altered effector specificity[J].Methods in Molecular Biology, 2022, 2461:175-193.
[14] PANDI A, GRIGORAS I, BORKOWSKI O, et al.Optimizing cell-free biosensors to monitor enzymatic production[J].ACS Synthetic Biology, 2019, 8(8):1952-1957.
[15] ARMETTA J, BERTHOME R, CROS A, et al.Biosensor-based enzyme engineering approach applied to psicose biosynthesis[J].Synthetic Biology, 2019, 4(1):ysz028.
[16] MIYAZAKI K.MEGAWHOP cloning:A method of creating random mutagenesis libraries via megaprimer PCR of whole plasmids[J].Methods in Enzymology, 2011, 498:399-406.
[17] BOSSHART A, HEE C S, BECHTOLD M, et al.Directed divergent evolution of a thermostable d-tagatose epimerase towards improved activity for two hexose substrates[J].ChemBioChem, 2015, 16(4):592-601.
[18] LIU Z Z, LIU S H, JIA J Y, et al.Optimization of ultrahigh-throughput screening assay for protein engineering of D-allulose 3-epimerase[J].Biomolecules, 2022, 12(11):1547.
[19] ZHU J X, LI Y, WANG J Z, et al.Adaptive steered molecular dynamics combined with protein structure networks revealing the mechanism of Y68I/G109P mutations that enhance the catalytic activity of D-psicose 3-epimerase from Clostridium bolteae[J].Frontiers in Chemistry, 2018, 6:437.
[20] LIU Z Z, WANG Y F, LIU S H, et al.Boosting the heterologous expression of D-allulose 3-epimerase in Bacillus subtilis through protein engineering and catabolite-responsive element box engineering[J].Journal of Agricultural and Food Chemistry, 2022, 70(38):12128-12134.
