(188g) Enhancing Butanol Tolerance of Escherichia coli Via Solo Gene Reveals Hydrophobic Interaction of Multi-Tasking Chaperone Secb | AIChE

(188g) Enhancing Butanol Tolerance of Escherichia coli Via Solo Gene Reveals Hydrophobic Interaction of Multi-Tasking Chaperone Secb

Authors 

Xu, G. - Presenter, Jiangnan University

Enhancing butanol tolerance of Escherichia coli via solo gene reveals
hydrophobic interaction of multi-tasking chaperone SecB

Guochao Xu, Anning Wu, Lin Xiao, Ye Ni



1
The Key Laboratory of Industrial Biotechnology,
Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi
214122, Jiangsu, People¡¯s Republic of China

* Corresponding author: guochaoxu@jiangnan.edu.cn; yni@jiangna.edu.cn.

Synthetic
biology is becoming a promising alternative to synthetic chemistry and has
enormous implications for the production of natural products, fine and bulk
chemicals, drugs and biofuels etc (1). Escherichia
coli
has proved to be one of the most important platform microorganism for
synthetic biology, considering its diverse genetic engineering tools for
pathway and module reconstruction, genomic sequence information for metabolic
engineering and rational design, and recently developed computational tools for
process optimization (2). E. coli has
been employed in the synthesis of organic solvent-like chemicals including
gasoline, diesel, and aviation fuel etc. It is widely known that toxicity of
metabolites (e.g., alcohols, aldehydes, ketones and carboxylic acids) to E. coli is one of the main bottlenecks
for achieving maximum production of biofuels. Under merely 1.0% butanol (v/v),
the growth of E. coli could be
severely inhibited (3). Engineering host strain for enhanced product tolerance
is often envisaged as one of the effective approaches to improve biofuel
production.

Molecular chaperones of E. coli
were mined for enhancing butanol tolerance of host cells. Thirty chaperones
were overexpressed in E. coli JM109,
and SecB recombinant strain was identified with the highest butanol tolerance.
The tolerance rate (T) of E. coli overexpressing SecB was
determined to be 9.14% at 1.2% butanol, which was 3.35-fold of the control
strain. Random mutagenesis of SecB was implemented, and mutant SecBT10A was
identified from 2800 variants rendering host the highest butanol tolerance.
Saturation mutagenesis on T10 site revealed that hydrophobic residues were
required for high butanol tolerance of E.
coli
. Compared with wild-type (WT) SecB, the T of SecBT10A strain was further increased from 9.14% to
14.4% at 1.2% butanol, which was 5.37-fold of control strain. Remarkably, E. coli engineered with SecBT10A
could tolerate as high as 1.8% butanol. The binding affinity of SecBT10A
toward model substrate preMBP was 12-fold of that of WT SecB as determined by
isothermal titration calorimetry. Residue T10 locates at the entrance of
hydrophobic substrate binding groove of SecB, and might play important role in
recognition and binding of cargo proteins. In summary, this study provides
evidence for the application of SecB chaperone in engineering butanol tolerance
of host strains.


Figure 1. Effect of SecB and SecBT10A in
enhancing the organic solvent tolerance of E.
coli
.

[1] Keasling, J. D. Metab. Eng., 2012, 3, 189¨C195.

[2] Fischer, C. R.; Klein-Marcuschamer, D.;
Stephanopoulos, G. Metab. Eng., 2008, 10, 295¨C304.

[3] Segura, A.; Molina, L.; Fillet, F. et
al., Curr. Opin. Biotechnol., 2012, 23, 415¨C421.