Adaptive laboratory evolution for improved tolerance of isobutyl acetate in Escherichia coli

Highlights

• E. coli was evolved for improved IBA tolerance, but also demonstrated higher titers.

• Three mutations (metH, rho, arcA) were responsible for improved IBA tolerance.

• Tolerance towards other acetate esters and acids was also demonstrated.

• The metH, rho mutant had 3.2-fold greater production than the parent strain.

Abstract

Previously, Escherichia coli was engineered to produce isobutyl acetate (IBA). Titers greater than the toxicity threshold (3 g/L) were achieved by using layer-assisted production. To avoid this costly and complex method, adaptive laboratory evolution (ALE) was applied to E. coli for improved IBA tolerance. Over 37 rounds of selective pressure, 22 IBA-tolerant mutants were isolated. Remarkably, these mutants not only tolerate high IBA concentrations, they also produce higher IBA titers. Using whole-genome sequencing followed by CRISPR/Cas9 mediated genome editing, the mutations (SNPs in metH, rho and deletion of arcA) that confer improved tolerance and higher titers were elucidated. The improved IBA titers in the evolved mutants were a result of an increased supply of acetyl-CoA and altered transcriptional machinery. Without the use of phase separation, a strain capable of 3.2-fold greater IBA production than the parent strain was constructed by combing select beneficial mutations. These results highlight the impact improved tolerance has on the production capability of a biosynthetic system.

Introduction

Microbial production has emerged as a promising alternative to traditional petroleum-dependent industries and natural extraction processes. Microbes can be engineered to produce a diverse variety of bioactive and industrially relevant chemicals from renewable feedstocks, such as sugars and CO₂ (Case and Atsumi, 2016; Keasling et al., 2021; Pontrelli et al., 2018; Sheng et al., 2020; Sun and Alper, 2020; Yang et al., 2021). The bioproduction of esters has been of particular interest due to their versatile commercial applications as flavorants, fragrances, solvents, coatings, and paints (Carroll et al., 2016). A promising target ester is isobutyl acetate (IBA), a volatile, fruity smelling compound with great potential as a drop-in biofuel. Compared to similar alcohols fuels, such as isobutanol (ISO), IBA's higher volatility and lower polarity and hygroscopicity allow it to be more easily separated from aqueous cell cultures (Rodriguez et al., 2014). These properties, along with its ubiquitous use in the fragrances and food industries, make IBA a valuable commodity chemical and have driven efforts to produce the compound biologically.

Previously, an efficient IBA production pathway was constructed in E. coli (Fig. 1) (Rodriguez et al., 2014; Tashiro et al., 2015). This pathway utilizes an alcohol-O-acyltransferase (ATF) (Verstrepen et al., 2003) enzyme that condenses acetyl-CoA and ISO, derived from the 2-keto acid-based pathway (Atsumi et al., 2008b), to form IBA (Rodriguez et al., 2014; Tai et al., 2015). A major challenge of biological IBA production is its inherent toxicity. The production strain JCL260 (Atsumi et al., 2008a), previously modified for ISO production, experiences severe growth-inhibition at 3 g/L IBA. This drastically limits titers unless IBA is separated from the culture, such as by removal from the headspace via gas-stripping or from the media via extraction using an organic bilayer. Using hexadecane as a bilayer and an orthogonal acetate assimilating pathway for increased acetyl-CoA generation, titers (20 g/L) greater than the toxicity threshold (3 g/L) were achieved (Tashiro et al., 2015). While these systems greatly increase IBA titers, they also introduce greater production costs and complexities to large scale production. One potential solution to IBA toxicity lies in its low water solubility. At ∼8 g/L, IBA innately forms a bilayer in aqueous solutions (Riemenschneider, 2000). Therefore, production utilizing natural bilayer formation is theoretically possible if E. coli can be adapted to grow under saturating IBA conditions.

ALE is a powerful tool for optimizing strain tolerance against environmental stressors (Dragosits and Mattanovich, 2013; Lee and Kim, 2020; Portnoy et al., 2011). Through adaptation, desired phenotypes are achieved through the accumulation of mutations overtime to cope with a selective pressure, such as toxicity. These mutations result in improved cellular fitness under stress typically by changing gene expression, altering enzyme function, or re-wiring regulation (Conrad et al., 2011). Often, these changes are synergistic combinations that would otherwise be inaccessible or overlooked by rational design. Previously, ALE has been used to alleviate toxicity for several compounds in various organisms, including ethanol (Yomano et al., 1998), 3-hydroxypropionic acid (Nguyen-Vo et al., 2019) and ISO (Atsumi et al., 2010) in E. coli, propionic acid (Xu et al., 2019) in Saccharomyces cerevisiae, and p-coumaric and ferulic acid (Mohamed et al., 2020) in Pseudomonas putida KT2440. These cases highlight the power and versatility of ALE for the overproduction of toxic commodities in microbial hosts.

In this study, an ALE serial transfer method was employed to evolve the ISO production host strain, JCL260, and subsequently 22 IBA-tolerant mutants, M01-23, were isolated. Excitingly, these evolved mutants also demonstrated increased IBA titers without the use of phase separation. To elucidate the mutations beneficial to tolerance and production, the entire genomes of JCL260 and M01-23 were sequenced. The identified mutations were reconstructed individually and in combination in JCL260 to test for enhanced tolerance and production. The metH, rho, and arcA mutation combination alleviated IBA toxicity while the metH, rho combination significantly boosted titers. These reconstructed strains were capable of better tolerance and higher production, respectively, than the evolved strains.