Microbial biosynthesis of lactate esters

Abstract

Background: Green organic solvents such as lactate esters have broad industrial applications and favorable environmental profiles. Thus, manufacturing and use of these biodegradable solvents from renewable feedstocks help benefit the environment. However, to date, the direct microbial biosynthesis of lactate esters from fermentable sugars has not yet been demonstrated.

Results: In this study, we present a microbial conversion platform for direct biosynthesis of lactate esters from fermentable sugars. First, we designed a pyruvate-to-lactate ester module, consisting of a lactate dehydrogenase (ldhA) to convert pyruvate to lactate, a propionate CoA-transferase (pct) to convert lactate to lactyl-CoA, and an alcohol acyltransferase (AAT) to condense lactyl-CoA and alcohol(s) to make lactate ester(s). By generating a library of five pyruvate-to-lactate ester modules with divergent AATs, we screened for the best module(s) capable of producing a wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-introducing a pyruvate-to-lactate ester module and an alcohol (i.e., ethanol, isobutanol) module into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the microbial biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance ethyl lactate production as a proof-of-study, we re-modularized the pathway into (1) the upstream module to generate the ethanol and lactate precursors and (2) the downstream module to generate lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the metabolic fluxes of the upstream and downstream modules through plasmid copy numbers, promoters, ribosome binding sites, and environmental perturbation, we were able to probe and alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold. We found that AAT is the most rate-limiting step in biosynthesis of lactate esters likely due to its low activity and specificity toward the non-natural substrate lactyl-CoA and alcohols.

Conclusions: We have successfully established the biosynthesis pathway of lactate esters from fermentable sugars and demonstrated for the first time the direct fermentative production of lactate esters from glucose using an E. coli modular cell. This study defines a cornerstone for the microbial production of lactate esters as green solvents from renewable resources with novel industrial applications.

Keywords: Acetate ester; Alcohol acyltransferase; Escherichia coli; Ester; Ethyl lactate; Green solvent; Isobutyl lactate; Lactate ester; Modular cell.

Fig. 1

In vivo characterization of various alcohol acyltransferases for biosynthesis of lactate esters. a Biosynthesis pathways of lactate and acetate esters with external supply of alcohols. b Ester production of EcJW101, EcJW102, EcJW103, EcJW104, and EcJW105 harboring ATF1, ATF2, SAAT, VAAT, and atfA, respectively in high cell density cultures with various alcohol doping. Each error bar represents 1 standard deviation (s.d., n = 3). Symbols: n.d. not detected, n.s. not significant, *p < 0.073, and **p < 0.013 (Student’s t-test). c The library of esters produced. Green check marks indicate the esters produced in this study while red star marks indicate the esters produced for first time in engineered strains

Fig. 2

Design, construction, and validation of the lactate ester biosynthesis pathways in E. coli. a Engineered biosynthesis pathway of ethyl lactate from glucose and its production in high cell density culture of EcJW201. b Engineered biosynthesis pathway of isobutyl lactate from glucose and its production in high cell density culture of EcJW202. In a and b, all of the strains were induced at 0 h with 0.5 mM IPTG. Each error bar represents 1 s.d. (n = 3). c Production of ethyl lactate from glucose in pH-controlled batch fermentation of EcJW201. The strain was induced at 6 h with 0.5 mM IPTG. Each error bar represents 1 s.d. (n = 2)

Fig. 3

Combinatorial modular pathway optimization for enhanced ethyl lactate biosynthesis by varying plasmid copy number. a Re-modularization of the ethyl lactate biosynthesis pathway. Pyruvate-to-lactate ester and ethanol modules were re-modulated into upstream and downstream modules using plasmids with different copy numbers. b Ethyl lactate production, c OD₆₀₀, d Consumed glucose, e Acetate, f Lactate, g Ethanol, and h Ethyl acetate of EcJW106-108 and EcJW203-208 in high cell density cultures induced with various concentrations of IPTG. Green rectangle: low copy number plasmid (10); P15A: origin of pACYCDuet-1; blue rectangle: medium copy number plasmid (40); ColE1: origin of pETDuet-1; red rectangle: high copy number plasmid (100); RSF1030: origin of pRSFDuet-1; P_T7: T7 promoter; T_T7: T7 terminator. All of the strains were induced at 0 h with 0.01, 0.1, or 1.0 mM IPTG, respectively. Each error bar represents 1 s.d. (n = 3). Red arrows indicate the selected strain with an optimum concentration of IPTG for the further studies

Fig. 4

Probing and alleviating the potential metabolic bottlenecks of the upstream or downstream modules of EcJW204 by varying the strength of promoters and/or ribosome binding sites. a Design of synthetic operons for the upstream and downstream modules. For the upstream module, the T7 promoter in MCS2 and the RBS between T7 promoter in MCS2 and the start codon of pdc were replaced with the combination of P_AY1 or P_AY3 promoter and 0.3 or 0.03 a.u. RBS. For the downstream module, the RBS between T7 promoter in MCS1 and the start codon of pct gene and the RBS between T7 promoter in MCS2 and the start codon of VAAT gene were replaced with the combination of 90, 9000, or 90000 a.u. RBS and 90, 9000, or 90000au RBS, respectively. Production of ethyl lactate in high cell density cultures of b EcJW209-212 and c EcJW213-221. Green rectangle: low copy number plasmid (10); P15A: origin of pACYCDuet-1; red rectangle: high copy number plasmid (100); RSF1030: origin of pRSFDuet-1; P_T7: T7 promoter; T_T7: T7 terminator. All of the strains were induced at 0 h with 0.01 mM IPTG. Each error bar represents 1 s.d. (n = 3)

Fig. 5

a Total esters and b Composition of total esters produced in high cell density cultures of EcJW209-212 with or without addition of ethanol. c Ethyl lactate production of EcJW109-117 with addition of 2 g/L of lactate and ethanol. Red rectangle: high copy number plasmid (100); RSF1030: origin of pRSFDuet-1; P_T7: T7 promoter; T_T7: T7 terminator. All of the strains were induced at 0 h with 0.01 mM IPTG. Each error bar represents 1 s.d. (n = 3)