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

作者：Basak Souvik^*; Ghosh Sumanta Kumar; Punetha Vinay Deep; Aphale Ashish N; Patra Prabir K; Sahoo Nanda Gopal^*

来源：International Journal of Biological Macromolecules, 2017, 95: 818-825.

DOI：10.1016/j.ijbiomac.2016.12.001

• Biotransformation
• Global transcription machinery engineering
• Cyclic AMP receptor protein
• Organic solvent stress
• Cell permeation enhancer

A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regeneratingE. coli host to convert Ethyl-4-chloro-3-oxobutanoate (CUBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363 mM with highest turnover number (112.77 +/- 3.95 s(-1)). Solitary recombineering of such catalytic cell reproduced CHBE 161.04 g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp (crp*, F136I) into this heterologous E. coli host yielded CHBE 477.54 g/L/gDCW. Furthermore, using nerolidol as exogenous cell transporter, the CHBE productivity has been towered to 710.88 g/L/gDCW. The CHBE production has thus been upscaled to 8-12 times than those reported so far. gRT-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 CUBE 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.

• 出版日期2017-2