{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:59:05Z","timestamp":1787029145590,"version":"build-2736575974"},"reference-count":34,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2024,9,16]],"date-time":"2024-09-16T00:00:00Z","timestamp":1726444800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Postdoctoral Innovation Project of Shandong Province","award":["DCX-ZG-202203010"],"award-info":[{"award-number":["DCX-ZG-202203010"]}]},{"name":"Postdoctoral Innovation Project of Shandong Province","award":["Q20221002"],"award-info":[{"award-number":["Q20221002"]}]},{"name":"Science and Technology Research Project of the Education Department of Hubei Province","award":["DCX-ZG-202203010"],"award-info":[{"award-number":["DCX-ZG-202203010"]}]},{"name":"Science and Technology Research Project of the Education Department of Hubei Province","award":["Q20221002"],"award-info":[{"award-number":["Q20221002"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>(R)-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The ChKRED20 is a robust NADH-dependent ketoreductase identified from Chryseobacterium sp. CA49. We obtained a ChKRED20 mutant (M12) through directed evolutionary screening of ChKRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to (R)-1, 3-BDO. So far, both ChKRED20 and its mutants have been expressed in intracellular in E. coli, the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in P. pastoris, and the target protein yield was 302 mg\/L in shake-flask fermentation and approximately 3.5 g\/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 \u00b0C and had high activity within a broad pH range of 6.0\u20138.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to (R)-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of (R)-1, 3-BDO with excellent stereoselectivity (ee &gt; 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of (R)-1, 3-BDO, and the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of ChKRED20 or its mutants, which may have applications in industrial settings.<\/jats:p>","DOI":"10.3390\/molecules29184393","type":"journal-article","created":{"date-parts":[[2024,9,16]],"date-time":"2024-09-16T10:56:57Z","timestamp":1726484217000},"page":"4393","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Heterologous Expression of Ketoreductase ChKRED20 Mutant in Pichia pastoris and Bioreductive Production of (R)-1, 3-Butanediol"],"prefix":"10.3390","volume":"29","author":[{"given":"Wanping","family":"Chen","sequence":"first","affiliation":[{"name":"School of Pharmacy, Qingdao University, Qingdao 266021, China"},{"name":"Enzymaster (Ningbo) Bio-Engineering Co., Ltd., Ningbo 315100, China"},{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Sun","sequence":"additional","affiliation":[{"name":"Enzymaster (Ningbo) Bio-Engineering Co., Ltd., Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinwei","family":"Wu","sequence":"additional","affiliation":[{"name":"Enzymaster (Ningbo) Bio-Engineering Co., Ltd., Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenni","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chin-Yu","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sitong","family":"Liu","sequence":"additional","affiliation":[{"name":"Enzymaster (Ningbo) Bio-Engineering Co., Ltd., Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2589-7347","authenticated-orcid":false,"given":"Haibin","family":"Chen","sequence":"additional","affiliation":[{"name":"Enzymaster (Ningbo) Bio-Engineering Co., Ltd., Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baoguo","family":"Sun","sequence":"additional","affiliation":[{"name":"Enzymaster (Ningbo) Bio-Engineering Co., Ltd., Ningbo 315100, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingxin","family":"Dong","sequence":"additional","affiliation":[{"name":"School of Pharmacy, Qingdao University, Qingdao 266021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,9,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1016\/S1381-1177(00)00032-1","article-title":"Industrial production of (R)-1,3-butanediol by new biocatalysts","volume":"11","author":"Matsuyama","year":"2001","journal-title":"J. Mol. Catal. B Enzym."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"H466","DOI":"10.1152\/ajpheart.00486.2021","article-title":"Low-dose 1,3-butanediol reverses age-associated vascular dysfunction independent of ketone body \u03b2-hydroxybutyrate","volume":"322","author":"McCarthy","year":"2022","journal-title":"Am. J. Physiology. Heart Circ. Physiol."},{"key":"ref_3","first-page":"165","article-title":"Microbial Cell Factories for Diol Production","volume":"155","author":"Sabra","year":"2016","journal-title":"Adv. Biochem. Eng. Biotechnol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1946","DOI":"10.1021\/acssynbio.1c00144","article-title":"Metabolic Engineering of Escherichia coli for High-Yield Production of (R)-1,3-Butanediol","volume":"10","author":"Liu","year":"2021","journal-title":"ACS Synth. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1016\/j.jbiosc.2012.11.025","article-title":"Improvement of (R)-1,3-butanediol production by engineered Escherichia coli","volume":"115","author":"Kataoka","year":"2013","journal-title":"J. Biosci. Bioeng."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1080\/09168451.2014.891933","article-title":"Enhancement of (R)-1,3-butanediol production by engineered Escherichia coli using a bioreactor system with strict regulation of overall oxygen transfer coefficient and pH","volume":"78","author":"Kataoka","year":"2014","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1945","DOI":"10.1007\/s00253-016-7947-0","article-title":"Single mutations of ketoreductase ChKRED20 enhance the bioreductive production of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol","volume":"101","author":"Zhao","year":"2017","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1016\/j.cbi.2014.09.024","article-title":"The aldo-keto reductases (AKRs): Overview","volume":"234","author":"Penning","year":"2015","journal-title":"Chem. Biol. Interact."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1249","DOI":"10.1007\/s00253-007-0957-1","article-title":"Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: Promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction","volume":"75","author":"Itoh","year":"2007","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1333","DOI":"10.1111\/1751-7915.14249","article-title":"A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol","volume":"16","author":"Guo","year":"2023","journal-title":"Microb. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/j.enzmictec.2019.03.001","article-title":"Structure-guided engineering of ChKRED20 from Chryseobacterium sp. CA49 for asymmetric reduction of aryl ketoesters","volume":"125","author":"Li","year":"2019","journal-title":"Enzym. Microb. Technol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.molcatb.2014.01.009","article-title":"Identification of ketone reductase ChKRED20 from the genome of Chryseobacterium sp. CA49 for highly efficient anti-Prelog reduction of 3,5-bis(trifluoromethyl)acetophenone","volume":"102","author":"Liu","year":"2014","journal-title":"J. Mol. Catal. B Enzym."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3567","DOI":"10.1007\/s00253-015-7200-2","article-title":"Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20","volume":"100","author":"Zhao","year":"2016","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1113","DOI":"10.1007\/s00253-012-3986-3","article-title":"High-level expression, purification, and enzymatic characterization of truncated poly(vinyl alcohol) dehydrogenase in methylotrophic yeast Pichia pastoris","volume":"97","author":"Jia","year":"2013","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/bs.mie.2021.07.004","article-title":"Expression of proteins in Pichia pastoris","volume":"660","author":"Mastropietro","year":"2021","journal-title":"Methods Enzymol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"55","DOI":"10.2174\/0929866527666200625203240","article-title":"Expression, Purification and Characterization of Recombinant Human Coagulation Factor XIIIa in Pichia Pastoris","volume":"28","author":"Cheng","year":"2021","journal-title":"Protein Pept. Lett."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1007\/978-1-0716-2399-2_10","article-title":"Engineering of Promoters for Gene Expression in Pichia pastoris","volume":"2513","author":"Vogl","year":"2022","journal-title":"Methods Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Guangbo, Y., Min, S., Wei, S., Lixin, M., Chao, Z., Yaping, W., and Zunxi, H. (2021). Heterologous expression of nattokinase from B. subtilis natto using Pichia pastoris GS115 and assessment of its thrombolytic activity. BMC Biotechnol., 21.","DOI":"10.1186\/s12896-021-00708-4"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"W159","DOI":"10.1093\/nar\/gkac394","article-title":"CB-Dock2: Improved protein-ligand blind docking by integrating cavity detection, docking and homologous template fitting","volume":"50","author":"Liu","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1093\/protein\/8.2.127","article-title":"LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions","volume":"8","author":"Wallace","year":"1995","journal-title":"Protein Eng."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2778","DOI":"10.1021\/ci200227u","article-title":"LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery","volume":"51","author":"Laskowski","year":"2011","journal-title":"J. Chem. Inf. Model."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1007\/978-1-4939-7366-8_4","article-title":"YASARA: A Tool to Obtain Structural Guidance in Biocatalytic Investigations","volume":"1685","author":"Land","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/j.pep.2016.02.006","article-title":"High-level expression of Proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains","volume":"122","author":"Yang","year":"2016","journal-title":"Protein Expr. Purif."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1202","DOI":"10.4014\/jmb.1112.12026","article-title":"Synthesis and high expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115","volume":"22","author":"Kang","year":"2012","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1007\/s10529-011-0816-0","article-title":"High-level expression and characterization of a highly thermostable chitosanase from Aspergillus fumigatus in Pichia pastoris","volume":"34","author":"Chen","year":"2012","journal-title":"Biotechnol. Lett."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1271\/bbb.57.348","article-title":"Microbial Production of Optically Active 1,3-Butanediol from 4-Hydroxy-2-butanone","volume":"57","author":"Matsuyama","year":"1993","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1007\/s00253-012-3942-2","article-title":"Asymmetric synthesis of (R)-1,3-butanediol from 4-hydroxy-2-butanone by a newly isolated strain Candida krusei ZJB-09162","volume":"94","author":"Zheng","year":"2012","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2409","DOI":"10.1096\/fj.201800821RR","article-title":"Dietary R, S-1,3-butanediol diacetoacetate reduces body weight and adiposity in obese mice fed a high-fat diet","volume":"33","author":"Davis","year":"2019","journal-title":"FASEB J."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Shao, Y., Xue, C., Liu, W., Zuo, S., Wei, P., Huang, L., Lian, J., and Xu, Z. (2022). High-level secretory production of leghemoglobin in Pichia pastoris through enhanced globin expression and heme biosynthesis. Bioresour. Technol., 363.","DOI":"10.1016\/j.biortech.2022.127884"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Peng, Y., Wang, Y., Liu, X., Zhou, R., Liao, X., Min, Y., Ma, L., Wang, Y., and Rao, B. (2022). Expression and Surface Display of an Acidic Cold-Active Chitosanase in Pichia pastoris Using Multi-Copy Expression and High-Density Cultivation. Molecules, 27.","DOI":"10.3390\/molecules27030800"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Yu, F., Li, X., Wang, F., Liu, Y., Zhai, C., Li, W., Ma, L., and Chen, W. (2023). TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method. Front. Bioeng. Biotechnol., 11.","DOI":"10.3389\/fbioe.2023.1167534"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Deng, B., Yue, Y., Yang, J., Yang, M., Xing, Q., Peng, H., Wang, F., Li, M., Ma, L., and Zhai, C. (2023). Improving the activity and thermostability of PETase from Ideonella sakaiensis through modulating its post-translational glycan modification. Commun. Biol., 6.","DOI":"10.1038\/s42003-023-04413-0"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Wang, F., Wang, X., Yu, X., Fu, L., Liu, Y., Ma, L., and Zhai, C. (2015). High-level expression of endo-\u03b2-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0137929"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1016\/j.foodres.2005.08.006","article-title":"Buffer pH and pKa values as affected by added glycerol and sucrose","volume":"39","author":"Chuy","year":"2006","journal-title":"Food Res. Int."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/29\/18\/4393\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:57:17Z","timestamp":1760111837000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/29\/18\/4393"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,9,16]]},"references-count":34,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2024,9]]}},"alternative-id":["molecules29184393"],"URL":"https:\/\/doi.org\/10.3390\/molecules29184393","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,9,16]]}}}