Cloning, expression and molecular evolution of Komagataella phaffii ketoreductase with enhanced catalytic activity Mohd Imran Shah, Kavitha Ramaswamy, Sangita Venkataraman, Sankaranarayanan Meenakshisundaram Preparative Biochemistry and Biotechnology, 2026 The synthesis of optically active alcohols is vital for the pharmaceutical and fine chemical industries, yet traditional chemical methods generally lack efficiency and stereoselectivity. Although ketoreductases are a more environmentally friendly option, many of them need to have their catalytic performance improved to satisfy industrial demands. This study focused on engineering Komagataella phaffii ketoreductase (KpKR), the first characterized ketoreductase from this yeast, by applying an innovative combination of error-prone PCR and ribosome display for rapid directed evolution. Through the use of kinetic analysis, molecular docking, homology modeling, and substrate specificity assays, five evolved variants (M1-M5) were identified and characterized. Compared to a range of microbial ketoreductases, such as Bacillus sp. ECU0013 and yeast-derived enzymes, the KpKR variants demonstrated efficient catalytic performance; notably, M5 showed enhanced catalytic efficiency (kcat/km = 199.58 s-1mM-1) toward ketoesters, while M1 demonstrated remarkable activity for halogenated substrates, outperforming Bacillus enzymes by thousands-fold. Structural investigation identified key C-terminal changes that likely contributed to improved active site accessibility. Furthermore, sequence analysis confirmed KpKR as a member of the short-chain dehydrogenase/reductase (SDR) family. These results underscore the innovative utility of ribosome display in enzyme engineering and establish the evolved KpKR variants as powerful, highly efficient biocatalysts suitable for challenging pharmaceutical synthesis.
Screening Method for high yielding S-Adenosyl-L-Methionine in recombinant pichia pastows strains, based on selection pressure and gene copy number Asian Journal of Microbiology Biotechnology and Environmental Sciences, 2015
Cloning, expression and molecular evolution of Komagataella phaffii ketoreductase with enhanced catalytic activity MI Shah, K Ramaswamy, S Venkataraman, S Meenakshisundaram Preparative Biochemistry & Biotechnology 56 (2), 247-260 , 2026 2026 Citations: 1
Cloning, Expression and Characterisation of Newly Mined Ketoreductase in Komagataella phaffii MI Shah, KSC Bose, S Meenakshisundaram Applied Biochemistry and Microbiology 61 (5), 844-855 , 2025 2025
ENHANCED PRODUCTION OF STEM BROMELAIN IN Pichia pastoris BY CO EXPRESSION OF UNFOLDED PROTEIN RESPONSE ACTIVATOR GENE HAC1P S Varadharaj, J Krishnan, MI Shah, M Sankaranarayanan Protein Expression and Purification, 106804 , 2025 2025
A metabolic-engineering framework approach via fed-batch fermentation for enhancing glucaric acid production in Komagataella phaffii J Krishna, KSC Bose, S Varadharaj, M Sankaranarayanan Enzyme and Microbial Technology 187, 110627 , 2025 2025 Citations: 6
Genome-scale metabolic model analysis of Pichia pastoris for enhancing the production of S -adenosyl- l -methionine K Subash Chandra Bose, MI Shah, J Krishna, M Sankaranarayanan Bioprocess and Biosystems Engineering 46 (10), 1471-1482 , 2023 2023 Citations: 14
Targeted gene manipulation of Leloir pathway genes for the constitutive expression of β-galactosidase and its transgalactosylation product galacto-oligosaccharides from … V Ponnusamy, M Sankaranarayanan Enzyme and Microbial Technology 169, 110263 , 2023 2023 Citations: 13
Enhancement of fructan extraction from garlic and fructooligosaccharide purification using an activated charcoal column R Shalini, J Krishna, M Sankaranarayanan, U Antony LWT 148, 111703 , 2021 2021 Citations: 25
Rational design of Shewanella sp. L-arabinose isomerase for D-galactose isomerase activity under mesophilic conditions AB Jayaraman, T Kandasamy, D Venkataraman Enzyme and Microbial Technology 147, 109796 , 2021 2021 Citations: 18
Pichia pastoris Protein Disulfide Isomerase (PDI1) promoter for heterologous protein production and its sequence characterization M Prattipati, K Ramakrishnan, M Sankaranarayanan Enzyme and Microbial Technology 140, 109633 , 2020 2020 Citations: 13
Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators K Ramakrishnan, M Prattipati, P Samuel, M Sankaranarayanan Applied Microbiology and Biotechnology 104 (18), 7841-7851 , 2020 2020 Citations: 36
Molecular evolution of single chain fragment variable (scFv) for diagnosis of lymphatic filariasis N Mahalakshmi, R Ravishankaran, R Kamatchi, N Sangith, P Kaliraj, ... Molecular Biology Reports 46 (5), 5409-5418 , 2019 2019 Citations: 8
High-cell density cultivation of recombinant Pichia pastoris harboring fungal endoinulinase and prebiotics applications of inulo-oligosaccharides M Surendarbabu, S Meenakshisundaram Journal of Environmental Biology 40 (1), 69-75 , 2019 2019 Citations: 1
Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris R Paul, S Karthik, P Vimalraj, S Meenakshisundaram, P Kaliraj Preparative Biochemistry and Biotechnology 48 (9), 823-833 , 2018 2018 Citations: 10
Formate co-feeding improved Candida antarctica Lipase B activity in Pichia pastoris C Jayachandran, BP Athiyaman, M Sankaranarayanan Res J Biotechnol 12, 29-36 , 2017 2017 Citations: 14
Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase C Jayachandran, B Palanisamy Athiyaman, M Sankaranarayanan Plos one 12 (7), e0181370 , 2017 2017 Citations: 29
Evaluation of immuno diagnostic assay for the exposure of stage specific filarial infection R Ravishankaran, RN Shridharan, LA Vishal, S Meenakshisundaram, ... Acta Parasitologica 61 (2), 232-240 , 2016 2016 Citations: 5
The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris PA Balakumaran, J Förster, M Zimmermann, J Charumathi, A Schmitz, ... BMC biotechnology 16 (1), 20 , 2016 2016 Citations: 7
Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis R Kamatchi, J Charumathi, R Ravishankaran, P Kaliraj, ... Journal of helminthology 90 (1), 74-80 , 2016 2016 Citations: 5
Modeling of Process Parameters for Enhanced Production of Coenzyme Q10 From Rhodotorula glutinis PA Balakumaran, S Meenakshisundaram Preparative Biochemistry and Biotechnology 45 (4), 398-410 , 2015 2015 Citations: 18
An evaluation of antigen capture assays for detecting active filarial antigens R Ravishankaran, NS Radhika, LA Vishal, S Meenakshisundaram, ... Journal of helminthology 89 (3), 352-358 , 2015 2015 Citations: 6
MOST CITED SCHOLAR PUBLICATIONS
Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris. AK Vadhana, P Samuel, RM Berin, J Krishna, K Kamatchi, ... Enzyme and microbial technology 52 (3), 177-183 , 2013 2013 Citations: 70
Tackling multiple antibiotic resistance in enteropathogenic Escherichia coli (EPEC) clinical isolates: a diarylheptanoid from Alpinia officinarum shows promising antibacterial … K Subramanian, C Selvakkumar, KS Vinaykumar, N Goswami, ... International journal of antimicrobial agents 33 (3), 244-250 , 2009 2009 Citations: 61
Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris P Samuel, AKP Vadhana, R Kamatchi, A Antony, S Meenakshisundaram Microbiological research 168 (10), 615-620 , 2013 2013 Citations: 55
Enhancing precursors availability in Pichia pastoris for the overproduction of S-adenosyl-l-methionine employing molecular strategies with process tuning HR Kant, M Balamurali, S Meenakshisundaram Journal of biotechnology 188, 112-121 , 2014 2014 Citations: 41
Conservation of cellulase enzyme in biopolishing application of cotton fabrics VS Kumar, S Meenakshisundaram, N Selvakumar Journal of the Textile Institute 99 (4), 339-346 , 2008 2008 Citations: 40
Transcriptional control of gene expression in Pichia pastoris by manipulation of terminators K Ramakrishnan, M Prattipati, P Samuel, M Sankaranarayanan Applied Microbiology and Biotechnology 104 (18), 7841-7851 , 2020 2020 Citations: 36
Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris KS Babu, A Antony, T Muthukumaran, S Meenakshisundaram Protein expression and purification 57 (2), 201-205 , 2008 2008 Citations: 32
Cofactor engineering improved CALB production in Pichia pastoris through heterologous expression of NADH oxidase and adenylate kinase C Jayachandran, B Palanisamy Athiyaman, M Sankaranarayanan Plos one 12 (7), e0181370 , 2017 2017 Citations: 29
Single step intein-mediated purification of hGMCSF expressed in salt-inducible E. coli K Srinivasa Babu, T Muthukumaran, A Antony, SD Prem Singh Samuel, ... Biotechnology letters 31 (5), 659-664 , 2009 2009 Citations: 28
Enhancement of fructan extraction from garlic and fructooligosaccharide purification using an activated charcoal column R Shalini, J Krishna, M Sankaranarayanan, U Antony LWT 148, 111703 , 2021 2021 Citations: 25
Active site analysis of cis -epoxysuccinate hydrolase from Nocardia tartaricans using homology modeling and site-directed mutagenesis V Vasu, J Kumaresan, MG Babu, S Meenakshisundaram Applied microbiology and biotechnology 93 (6), 2377-2386 , 2012 2012 Citations: 21
Rational design of Shewanella sp. L-arabinose isomerase for D-galactose isomerase activity under mesophilic conditions AB Jayaraman, T Kandasamy, D Venkataraman Enzyme and Microbial Technology 147, 109796 , 2021 2021 Citations: 18
Modeling of Process Parameters for Enhanced Production of Coenzyme Q10 From Rhodotorula glutinis PA Balakumaran, S Meenakshisundaram Preparative Biochemistry and Biotechnology 45 (4), 398-410 , 2015 2015 Citations: 18
Genome-scale metabolic model analysis of Pichia pastoris for enhancing the production of S -adenosyl- l -methionine K Subash Chandra Bose, MI Shah, J Krishna, M Sankaranarayanan Bioprocess and Biosystems Engineering 46 (10), 1471-1482 , 2023 2023 Citations: 14
Formate co-feeding improved Candida antarctica Lipase B activity in Pichia pastoris C Jayachandran, BP Athiyaman, M Sankaranarayanan Res J Biotechnol 12, 29-36 , 2017 2017 Citations: 14
Glucose uptake kinetics of Saccharomyces cerevisiae monitored with a newly developed FIA SA Rothen, M Saner, S Meenakshisundaram, B Sonnleitner, A Fiechter Journal of biotechnology 50 (1), 1-12 , 1996 1996 Citations: 14
Targeted gene manipulation of Leloir pathway genes for the constitutive expression of β-galactosidase and its transgalactosylation product galacto-oligosaccharides from … V Ponnusamy, M Sankaranarayanan Enzyme and Microbial Technology 169, 110263 , 2023 2023 Citations: 13
Pichia pastoris Protein Disulfide Isomerase (PDI1) promoter for heterologous protein production and its sequence characterization M Prattipati, K Ramakrishnan, M Sankaranarayanan Enzyme and Microbial Technology 140, 109633 , 2020 2020 Citations: 13
Production, purification and diagnostic application of filarial recombinant protein WbSXP-1 expressed in salt inducible Escherichia coli S Janardhan, P Pandiaraja, S Thirugnanam, MN Balamurali, K Fernando, ... Journal of Industrial Microbiology and Biotechnology 34 (10), 675-683 , 2007 2007 Citations: 13
Cloning, large-scale production and characterization of fusion protein (P-TUFT-ALT-2) of Brugian abundant larval transcript-2 with tuftsin in Pichia pastoris R Paul, S Karthik, P Vimalraj, S Meenakshisundaram, P Kaliraj Preparative Biochemistry and Biotechnology 48 (9), 823-833 , 2018 2018 Citations: 10
