PMID- 38289384 OWN - NLM STAT- MEDLINE DCOM- 20240131 LR - 20240206 IS - 1432-0614 (Electronic) IS - 0175-7598 (Print) IS - 0175-7598 (Linking) VI - 108 IP - 1 DP - 2024 Jan 30 TI - Identification of a novel thermostable transaminase and its application in L-phosphinothricin biosynthesis. PG - 184 LID - 10.1007/s00253-024-13023-7 [doi] LID - 184 AB - Transaminase (TA) is a crucial biocatalyst for enantioselective production of the herbicide L-phosphinothricin (L-PPT). The use of enzymatic cascades has been shown to effectively overcome the unfavorable thermodynamic equilibrium of TA-catalyzed transamination reaction, also increasing demand for TA stability. In this work, a novel thermostable transaminase (PtTA) from Pseudomonas thermotolerans was mined and characterized. The PtTA showed a high specific activity (28.63 U/mg) towards 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO), with excellent thermostability and substrate tolerance. Two cascade systems driven by PtTA were developed for L-PPT biosynthesis, including asymmetric synthesis of L-PPT from PPO and deracemization of D, L-PPT. For the asymmetric synthesis of L-PPT from PPO, a three-enzyme cascade was constructed as a recombinant Escherichia coli (E. coli G), by co-expressing PtTA, glutamate dehydrogenase (GluDH) and D-glucose dehydrogenase (GDH). Complete conversion of 400 mM PPO was achieved using only 40 mM amino donor L-glutamate. Furthermore, by coupling D-amino acid aminotransferase (Ym DAAT) from Bacillus sp. YM-1 and PtTA, a two-transaminase cascade was developed for the one-pot deracemization of D, L-PPT. Under the highest reported substrate concentration (800 mM D, L-PPT), a 90.43% L-PPT yield was realized. The superior catalytic performance of the PtTA-driven cascade demonstrated that the thermodynamic limitation was overcome, highlighting its application prospect for L-PPT biosynthesis. KEY POINTS: * A novel thermostable transaminase was mined for L-phosphinothricin biosynthesis. * The asymmetric synthesis of L-phosphinothricin was achieved via a three-enzyme cascade. * Development of a two-transaminase cascade for D, L-phosphinothricin deracemization. CI - (c) 2024. The Author(s). FAU - Liu, Han-Lin AU - Liu HL AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. FAU - Yi, Pu-Hong AU - Yi PH AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. FAU - Wu, Jia-Min AU - Wu JM AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. FAU - Cheng, Feng AU - Cheng F AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. FAU - Liu, Zhi-Qiang AU - Liu ZQ AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. FAU - Jin, Li-Qun AU - Jin LQ AUID- ORCID: 0000-0002-6936-609X AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. jlq@zjut.edu.cn. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. jlq@zjut.edu.cn. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. jlq@zjut.edu.cn. FAU - Xue, Ya-Ping AU - Xue YP AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. FAU - Zheng, Yu-Guo AU - Zheng YG AD - Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, The National and Local, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. AD - Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China. LA - eng GR - 2020YFA0908400/National Key R&D Program of China/ GR - 2022R52041/Zhejiang Provincial Science and Technology Innovation Leading Talent program/ PT - Journal Article DEP - 20240130 PL - Germany TA - Appl Microbiol Biotechnol JT - Applied microbiology and biotechnology JID - 8406612 RN - EC 2.6.1.- (Transaminases) RN - 51276-47-2 (phosphinothricin) RN - 107-92-6 (Butyric Acid) RN - EC 1.1.1.47 (Glucose 1-Dehydrogenase) RN - 3KX376GY7L (Glutamic Acid) RN - 0 (Aminobutyrates) SB - IM MH - *Transaminases/genetics MH - *Escherichia coli/genetics MH - Butyric Acid MH - Glucose 1-Dehydrogenase MH - Glutamic Acid MH - *Aminobutyrates PMC - PMC10827958 OTO - NOTNLM OT - Asymmetric synthesis OT - Deracemization OT - Enzyme cascade OT - L-phosphinothricin OT - Thermostable transaminase COIS- The authors declare no competing interests. EDAT- 2024/01/30 12:42 MHDA- 2024/01/31 06:42 PMCR- 2024/01/30 CRDT- 2024/01/30 11:05 PHST- 2023/07/27 00:00 [received] PHST- 2024/01/18 00:00 [accepted] PHST- 2023/11/14 00:00 [revised] PHST- 2024/01/31 06:42 [medline] PHST- 2024/01/30 12:42 [pubmed] PHST- 2024/01/30 11:05 [entrez] PHST- 2024/01/30 00:00 [pmc-release] AID - 10.1007/s00253-024-13023-7 [pii] AID - 13023 [pii] AID - 10.1007/s00253-024-13023-7 [doi] PST - epublish SO - Appl Microbiol Biotechnol. 2024 Jan 30;108(1):184. doi: 10.1007/s00253-024-13023-7.