Controlling selectivity of modular microbial biosynthesis of butyryl-CoA-derived designer esters
Introduction
Esters are industrial platform chemicals with versatile applications as flavors, fragrances, solvents, and biofuels (Lee and Trinh, 2020). Microbial biosynthesis of esters from lignocellulosic biomass can potentially offer an alternative promising solution to the current petroleum-based process that is neither renewable nor sustainable (Chubukov et al., 2016; Seo et al., 2019). For bioenergy applications, short-chain (C6–C10) esters have recently been attracting attention as drop-in fuels due to their favorable properties such as high energy density (Layton and Trinh, 2016b), high hydrophobicity (Tai et al., 2015), and good compatibility with current infrastructures including engines, transport, and storage density (Contino et al., 2013a; Jenkins et al., 2013). For instance, ethyl valerate (C7) (Contino et al., 2013b), butyl butyrate (C8) (Chuck and Donnelly, 2014; Jenkins et al., 2013), butyl valerate (C9) (Contino et al., 2013a), and pentyl valerate (C10) (Contino et al., 2013a) are good fuel additives for gasolines while butyl butyrate (C8) (Chuck and Donnelly, 2014; Jenkins et al., 2013) and ethyl octanoate (C10) (Chuck and Donnelly, 2014) are considered as an alternative jet fuel.
In nature, eukaryotic cells utilize alcohol acetyltransferases (AATs) to condense an alcohol and acetyl-CoA to make acetate esters in a thermodynamically favorable reaction, as often found in plants and fruits for generating scents (D'Auria, 2006) or in fermenting yeasts for making flavors (Wyk et al., 2018). Inspired by nature, microbial biomanufacturing platforms (e.g., Escherichia coli and Clostridium acetobutylicum) have been engineered to make these acetate esters directly from fermentable sugars (Chacon et al., 2019; Feng et al., 2021; Horton and Bennett, 2006; Horton et al., 2003; Layton and Trinh, 2014, 2016a; Lee and Trinh, 2019; Rodriguez et al., 2014; Vadali et al., 2004). Remarkably, the substrate promiscuity of AATs also enables microbial biosynthesis of acylate esters beyond acetate esters including propionate esters (Layton and Trinh, 2016a), lactate esters (Lee and Trinh, 2019; Seo et al., 2021), butyrate esters (Feng et al., 2021; Layton and Trinh, 2014; Noh et al., 2018), pentanoate esters (Layton and Trinh, 2016a), and hexanoate esters (Layton and Trinh, 2016a). Therefore, harnessing diversity of AATs, acyl-CoAs, and alcohols can result in the de novo microbial biosynthesis of a vast library of esters from renewable feedstocks for useful applications.
To enable a systematic and rapid generation of microbial biocatalysts to produce various esters, a modular cell engineering framework has recently been developed (Garcia and Trinh, 2019a, 2019b, 2020; Trinh et al., 2015; Wilbanks et al., 2018). Each ester production strain can be assembled from an engineered modular chassis cell and exchangeable ester producing pathways known as production modules. Nevertheless, experimental implementation has been challenging due to the intrinsic complexity of these ester production modules requiring expression of multiple heterologous enzymes derived from bacteria, yeasts, and plants (Layton and Trinh, 2014, 2016a, 2016b; Lee and Trinh, 2019).
Critical to the effective microbial biosynthesis of a target designer ester is the availability of efficient and robust AATs and precursor metabolite pathways that are compatible with a microbial host (Seo et al., 2021). Selective microbial biosynthesis of acylate esters other than acetate esters is very challenging due to low availability of target acyl-CoAs and alcohols, a high intracellular pool of competing substrates (i.e., non-target acetyl-CoA and alcohols), and inefficient AATs. For instance, the microbial ester production is much less efficient for a butyryl-CoA-derived acylate ester (e.g., butyl butyrate (Feng et al., 2021; Layton and Trinh, 2014)) than for an acetate ester (e.g., isobutyl acetate (Tai et al., 2015; Tashiro et al., 2015), isoamyl acetate (Tai et al., 2015)), due to low product selectivity. In particular, equipped with a butyrate ester pathway, an engineered E. coli can generate two acyl-CoAs (i.e., acetyl-CoA, butyryl-CoA) and two alcohols (i.e., ethanol, butanol) from fermentable sugars that can be condensed to form two possible acetate esters (i.e., ethyl acetate (EA), butyl acetate (BA)) and two possible butyrate esters (i.e., ethyl butyrate (EB), butyl butyrate (BB)). Furthermore, effective microbial production of acylate esters has been hampered by the required expression of multiple heterologous enzymes that are not compatible with the host. Specifically, low solubility of eukaryotic AATs in a microbial host is a commonly observed problem (Tai et al., 2015; Zhu et al., 2015). Currently, innovative strategies to produce designer esters with high selectivity and efficiency in a microbial host are very limited.
In this study, we presented systematic design and engineering approaches to tackle the current challenges of microbial biosynthesis of designer esters. As a proof-of-study, we demonstrated the microbial biosynthesis of designer butyryl-CoA-derived esters (i.e., BA, EB, BB) with high selectivity in an engineered modular E. coli cell. Specifically, we first developed a combinatorial modular design of the butyryl-CoA-derived ester biosynthesis pathways for rapid construction and optimization. Next, we optimized the culture conditions for expression of multiple pathway enzymes including culture temperatures and inducer concentrations to enhance and balance metabolic fluxes toward the synthesis of the target esters. To further improve the compatibility of the engineered pathways with the modular cell, we screened combinatorial strategies of protein solubilization including codon optimization, use of fusion tags, and/or co-expression of chaperones to improve the soluble expression of multiple pathway enzymes (e.g., AATs). Finally, we characterized the engineered ester-producing strains under anaerobic conditions with pH-adjustment to achieve the enhanced production of designer esters with high selectivity. Overall, this study presents a generalizable framework for engineering modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable biomass feedstocks.
Section snippets
Results
Designing a general framework to build exchangeable ester production modules for the de novo microbial biosynthesis of designer butyryl-CoA-derived esters. To generate the de novo microbial biosynthesis of butyryl-CoA-derived esters from fermentable sugars in E. coli (Fig. 1a), three major pathways are required including i) acyl-CoA synthesis pathway (butyryl-CoA, acetyl-CoA), ii) alcohol synthesis pathway (ethanol, butanol), and iii) ester synthesis pathway (AAT). Biosynthesis of acetyl-CoA is
Discussion
In this study, we reported the development of a generalizable framework to engineer a modular microbial platform for anaerobic production of butyryl-CoA-derived esters from fermentable sugars. Using the modular design approach, each ester production strain can be generated from an engineered modular (chassis) cell and an exchangeable ester production module in a plug-and-play fashion. This study focused on engineering exchangeable ester production modules to be compatible with the chassis cell
Methods
Strains and plasmids. The list of strains and plasmids used in this study are presented in Table 1. Briefly, E. coli TOP10 strain was used for molecular cloning. Except for EcJWBA15, TCS083 △fadE (DE3) (Layton and Trinh, 2014) was used as a host strain. For EcJWBA15, TCS095 (DE3) (Wilbanks et al., 2018) was used as a host strain. A set of duet vectors including pACYCDuet-1, pETDuet-1, and pRSFDuet-1 were used as plasmid backbones for constructing a library of BA, EB, and BB production modules.
Credit author statement
Jong-Won Lee: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, review & editing. Cong T. Trinh: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Formal analysis, Investigation, Visualization, Writing - original draft, review & editing.
Author contributions
CTT conceived and supervised this study. JWL and CTT designed the experiments, analyzed the data, and drafted the manuscript. JWL performed the experiments. Both authors read and approved the final manuscript.
Acknowledgments
This research was financially supported in part by the NSF CAREER award (NSF#1553250), the DOE BER award (DE-SC0022226), and the DOE subcontract grant (DE-AC05-000R22725) by the Center of Bioenergy Innovation, the U.S. Department of Energy Bioenergy Research Center funded by the Office of Biological and Environmental Research in the DOE Office of Science, and the U.S. Department of Energy Joint Genome Institute. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE
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