title: An experimental modeling of trinomial bioengineering- crp, rDNA, and transporter engineering within single cell factory for maximizing two-phase bioreduction.
abstract: A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363mM with highest turnover number (112.77±3.95s-1). Solitary recombineering of such catalytic cell reproduced CHBE 161.04g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp (crp*, F136I) into this heterologous E. coli host yielded CHBE 477.54g/L/gDCW. Furthermore, using nerolidol as exogenous cell transporter, the CHBE productivity has been towered to 710.88g/L/gDCW. The CHBE production has thus been upscaled to 8-12 times than those reported so far. qRT-PCR studies revealed that both membrane efflux channels such as acrAB as well as ROS scavenger genes such as ahpCF have been activated by engineering crp. Moreover, membrane protecting genes such as manXYZ together with solvent extrusion associated genes such as glpC have been upregulated inside mutant host. Although numerous proteins have been investigated to convert COBE to CHBE; this is the first approach to use engineering triad involving crp engineering, recombinant DNA engineering and transporter engineering together for improving cell performance during two-phase biocatalysis.
authors.affiliation: Dr. B.C. Roy College of Pharmacy & Allied Health Sciences, Durgapur, WB, India. Electronic address: souvik_basak1@yahoo.com.
authors.forename: Souvik
authors.initials: S
authors.lastname: Basak
authors.affiliation: Dr. B.C. Roy College of Pharmacy & Allied Health Sciences, Durgapur, WB, India.
authors.forename: Sumanta Kumar
authors.initials: SK
authors.lastname: Ghosh
authors.affiliation: Nanoscience and Nanotechnology Centre, Department of Chemistry, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India.
authors.forename: Vinay Deep
authors.initials: VD
authors.lastname: Punetha
authors.affiliation: Department of Biomedical Engineering, School of Engineering, University of Bridgeport, CT, USA.
authors.forename: Ashish N
authors.initials: AN
authors.lastname: Aphale
authors.affiliation: Department of Biomedical Engineering, School of Engineering, University of Bridgeport, CT, USA; Department of Mechanical Engineering, School of Engineering, University of Bridgeport, CT, USA.
authors.forename: Prabir K
authors.initials: PK
authors.lastname: Patra
authors.affiliation: Nanoscience and Nanotechnology Centre, Department of Chemistry, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India. Electronic address: ngsahoo@yahoo.co.in.
authors.forename: Nanda Gopal
authors.initials: NG
authors.lastname: Sahoo
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000090:Acetoacetates / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D001711:Biotransformation / Q000187:drug effects; D002087:Butyrates / Q000378:metabolism; D041221:Candida glabrata / Q000201:enzymology / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D012717:Sesquiterpenes / Q000494:pharmacology
chemical_list: D000085:Acetates; D000090:Acetoacetates; D002087:Butyrates; D004275:DNA, Ribosomal; D012717:Sesquiterpenes; C121244:ethyl 4-chloro-3-hydroxybutanoate; C006848:butyl acetate; C111982:ethyl 4-chloro-3-oxobutanoate; D000429:Alcohol Oxidoreductases; C037055:nerolidol
keywords: Biotransformation; Cell permeation enhancer; Cyclic AMP receptor protein; Global transcription machinery engineering; Organic solvent stress