Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters
Introduction
Short-chain esters formulate volatile compounds commonly found in flowers, ripe fruits, and fermenting yeasts (Sugimoto et al., 2021; Sumby et al., 2010). Some of these esters are suggested to have an important ecological role in pollination (Knudsen and Tollsten, 1993). Industrially, these esters have versatile utility as flavors, fragrances, solvents, and biofuels. For instance, isoamyl acetate (3-methyl-1-butyl acetate) is known as banana oil with a global market value of $5 billion in 2019 (IndustryARC, 2019; IndustryResearch, 2021). An isomer of isoamyl acetate, ethyl valerate, is fully compatible for blending with gasoline or diesel (Lange et al., 2010), suggesting potential application of isoamyl acetate as drop-in biofuel. Currently, knowledge about the microbial biosynthesis of these molecules from renewable and sustainable feedstocks is limited, making it difficult to optimize their production without further interrogation.
Biologically, cells can synthesize an ester by condensing an alcohol and an acyl-CoA with an alcohol acyltransferase (AAT) (Layton and Trinh, 2014; Mason and Dufour, 2000). Due to the abundance and essentiality of acetyl-CoA in living cells, acetate esters are the most common esters found in nature. By activating one-, two-, or three-carbon recursive elongation via the recursive fatty acid biosynthesis (Liu et al., 2016; Youngquist et al., 2013), reverse beta-oxidation (Dellomonaco et al., 2011), or Ehrlich pathways (Atsumi et al., 2008; Zhang et al., 2008), it is possible to synthesize a large library of acetate esters containing unique alcohol moieties with linear, branched, saturated, unsaturated, even, and/or odd carbon chains (Layton and Trinh, 2016a, 2016b; Lee and Trinh, 2020). However, selective microbial biosynthesis of designer acetate esters at high efficiency has been an outstanding metabolic engineering problem. For instance, branched-chain acetate esters (e.g., isoamyl acetate) represent an important class of molecules that can be synthesized via the one-carbon recursive ketoacid elongation pathway (Connor et al., 2010; Connor and Liao, 2008). Starting from the precursor pyruvate, this pathway generates ketoacids that can be decarboxylated to aldehydes, reduced to branched-chain alcohols, and condensed to acetate esters. Isobutyl acetate is generated in the first cycle, followed by isoamyl acetate in the second cycle, and so on. Although microbial production of isoamyl acetate has been reported since early 2000s by the condensation of isoamyl alcohol and acetyl-CoA, production titers (<1 g/L) and selectivities (<30%) were relatively low (Abe and Horikoshi, 2005; Horton et al., 2003; Tai et al., 2015; Vadali et al., 2004a, 2004b).
Many confounding factors might negatively affect selective microbial biosynthesis of branched-chain acetate esters. In addition to the well-known toxicity of higher alcohols and esters (Wilbanks and Trinh, 2017) and required expression of multiple pathway enzymes (Tai et al., 2015), the recursive one-carbon elongation pathway generates intermediate alcohol byproducts (e.g., isobutanol) that compete with the target biosynthesis of esters (e.g., isobutyl acetate instead of isoamyl acetate) (Marcheschi et al., 2012; Zhang et al., 2008). Currently, understanding and controlling this recursive elongation pathway for efficient biosynthesis of target branched-chain acetate esters remains elusive. A cellular proteome constitutes ∼50% of dry cell weight, requiring a significant resource investment (Neidhardt et al., 1990). As rewiring cellular metabolism can severely impact overall proteome allocation, especially when multiple enzyme pathways are introduced and/or overexpressed, proteome allocation or reallocation must be considered to achieve optimal product production. This reallocation, however, is complex and poorly understood because it requires a precise control of the expression, specificities, and activities of multiple pathway enzymes in order to achieve optimal metabolic fluxes for selective microbial production of the target molecule (Lechner et al., 2016) and avoid metabolic burden (Glick, 1995; Wu et al., 2016).
In this study, we presented a systematic modular design approach to control proteome reallocation for the selective microbial biosynthesis of branched-chain acetate esters via manipulation of substrate specificity and expression level of multiple pathway enzymes. For proof-of-concept, we demonstrated the approach to enable selective production of isoamyl acetate over isobutyl acetate by controlling the one-carbon recursive ketoacid elongation pathway. Using quantitative proteomics, we shed light on pathway-level proteome reallocation, metabolic burden, and bottlenecks, which guided the effective metabolic rewiring for the efficient target ester biosynthesis.
Section snippets
Modular pathway design principles
The branched-chain acetate ester biosynthesis pathway is derived from pyruvate (Fig. 1a). Pyruvate is converted to 2-ketoisovalerate via the L-valine biosynthesis pathway (KIV submodule) then elongated to 2-ketoacids via the +1 recursive ketoacid elongation cycle mediated by the LeuABCD operon (ketoacid elongation submodule). The Ehrlich pathway (KDC submodule) converts 2-ketoacids to aldehydes and alcohols, then the alcohol acyltransferase pathway (AAT submodule) condenses alcohols and
Discussion
The one-carbon recursive elongation pathway is important for making branched-chain amino acids, aldehydes, alcohols, ketoacids, and esters. Due to this complex and highly branched pathway, controlling selective microbial biosynthesis of these target molecules has been an outstanding metabolic engineering problem. To address the problem, we developed a generalizable modular design framework to systematically tune selective microbial biosynthesis of branched-chain acetate esters that require the
Strains and plasmids
E. coli DH5α and BL21(DE3) were used for molecular cloning and ester production, respectively. The strains and plasmids used are listed in Table 1.
Media and cultivation
E. coli strains were grown in lysogeny broth (LB) medium or M9 hybrid medium containing glucose as a carbon source and 5 g/L yeast extract supplemented with 100 μg/mL ampicillin and/or 100 μg/mL spectinomycin and/or 50 μg/mL kanamycin when appropriate.
For the batch fermentation, cells were cultured microaerobically in a 125 mL screw-capped shake
CRediT authorship contribution statement
Hyeongmin Seo: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization. Richard J. Giannone: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – review & editing. Yung-Hun Yang: Resources, Writing – review & editing. Cong T. Trinh: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Supervision, Project administration, Funding acquisition, Writing – original draft,
Declaration of competing interest
The authors declare that they have no competing interests.
Acknowledgments
This research was financially supported in part by 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. We would also like to acknowledge the gene synthesis performed at the U.S. Department of Energy Joint Genome Institute. The work conducted by the U.S. Department of Energy Joint
References (57)
- et al.
Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant
Gene
(1995) - et al.
A set of recombineering plasmids for gram-negative bacteria
Gene
(2006) - et al.
Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis
FEMS Microbiol. Lett.
(2004) - et al.
Multiobjective strain design: a framework for modular cell engineering
Metab. Eng.
(2019) Metabolic load and heterologous gene expression
Biotechnol. Adv.
(1995)- et al.
Trends in floral scent chemistry in pollination syndromes: floral scent composition in moth-pollinated taxa
Bot. J. Linn. Soc.
(1993) - et al.
Engineering modular ester fermentative pathways in Escherichia coli
Metab. Eng.
(2014) - et al.
Microbial synthesis of a branched-chain ester platform from organic waste carboxylates
Metabol. Eng. Commun.
(2016) - et al.
Towards renewable flavors, fragrances, and beyond
Curr. Opin. Biotechnol.
(2020) - et al.
High production of fatty alcohols in Escherichia coli with fatty acid starvation
Microb. Cell Factories
(2016)
Differential mRNA stability controls relative gene expression within a polycistronic operon
Cell
Engineering promiscuity of chloramphenicol acetyltransferase for microbial designer ester biosynthesis
Metab. Eng.
Microbial modulation of aromatic esters in wine: current knowledge and future prospects
Food Chem.
Engineered biosynthesis of medium-chain esters in Escherichia coli
Metab. Eng.
Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli
Metab. Eng.
Metabolic burden: cornerstones in synthetic biology and metabolic engineering applications
Trends Biotechnol.
Production of medium chain length fatty alcohols from glucose in Escherichia coli
Metab. Eng.
Enhanced production of isoamyl alcohol and isoamyl acetate by ubiquitination-deficient Saccharomyces cerevisiae mutants
Cell. Mol. Biol. Lett.
Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels
Nature
The MetaCyc database of metabolic pathways and enzymes - a 2019 update
Nucleic Acids Res.
Construction and optimization of a heterologous pathway for protocatechuate catabolism in Escherichia coli enables bioconversion of model aromatic compounds
Appl. Environ. Microbiol.
3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation
Appl. Microbiol. Biotechnol.
Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol
Appl. Environ. Microbiol.
The fruit ripening-related gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis
J. Exp. Bot.
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
Proc. Natl. Acad. Sci. U. S. A.
Engineered reversal of the beta-oxidation cycle for the synthesis of fuels and chemicals
Nature
Enzymatic assembly of DNA molecules up to several hundred kilobases
Nat. Methods
Heterologous expression of the Saccharomyces cerevisiae alcohol acetyltransferase genes in Clostridium acetobutylicum and Escherichia coli for the production of isoamyl acetate
J. Ind. Microbiol. Biotechnol.
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