Details

Autor(en) / Beteiligte

• Basak, Souvik
• Ghosh, Sumanta Kumar
• Punetha, Vinay Deep
• Aphale, Ashish N.
• Patra, Prabir K.
• Sahoo, Nanda Gopal

Titel

An experimental modeling of trinomial bioengineering- crp, rDNA, and transporter engineering within single cell factory for maximizing two-phase bioreduction

Ist Teil von

• International journal of biological macromolecules, 2017-02, Vol.95, p.818-825

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2017

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• •First approach to combine crp, recombinant DNA and transporter engineering for improved bioproduction in two-phase system.•Genomic crp remained integrated to foster basal protein expression.•Nerolidol is used to improve substrate (COBE) uptake inside biocatalyst.•Bioproductivity climbed to 710.88g/L per g of dry cell weight, 8∼12 times than those reported so far.•qRT-PCR analyses revealed activation of both solvent tolerant and antioxidant machineries inside cell. 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.