Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

Hyeongmin Seo; Jong-Won Lee; Sergio Garcia; Cong T Trinh

doi:10.1186/s13068-019-1583-8

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

Biotechnol Biofuels. 2019 Oct 15:12:245. doi: 10.1186/s13068-019-1583-8. eCollection 2019.

Authors

Hyeongmin Seo^#^{1

2}, Jong-Won Lee^#^{3

2}, Sergio Garcia^{1

2}, Cong T Trinh^{1

3

2}

Affiliations

¹ 1Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, TN USA.
² 3Center for Bioenergy Innovation (CBI), Oak Ridge National Laboratory, Oak Ridge, TN USA.
³ 2Bredesen Center for Interdisciplinary Research and Graduate Education, The University of Tennessee, Knoxville, TN USA.

^# Contributed equally.

Abstract

Background: Esters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows high-temperature fermentation with advantageous downstream product separation. However, due to the limited thermostability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum.

Results: In this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CAT_Sa) for enhanced isobutyl acetate production at elevated temperatures. We first analyzed the broad alcohol substrate range of CAT_Sa. Then, we targeted a highly conserved region in the binding pocket of CAT_Sa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CAT_Sa F97W, we demonstrated direct conversion of cellulose into isobutyl acetate by an engineered C. thermocellum at elevated temperatures.

Conclusions: This study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.

Keywords: Alcohol acetyltransferase; Chloramphenicol acetyltransferase; Clostridium thermocellum; Consolidated bioprocessing; Esters; Isobutyl acetate; Thermostability.