Developing Clostridia as Cell Factories for Short- and Medium-Chain Ester Production

Abstract

Short- and medium-chain volatile esters with flavors and fruity fragrances, such as ethyl acetate, butyl acetate, and butyl butyrate, are usually value-added in brewing, food, and pharmacy. The esters can be naturally produced by some microorganisms. As ester-forming reactions are increasingly deeply understood, it is possible to produce esters in non-natural but more potential hosts. Clostridia are a group of important industrial microorganisms since they can produce a variety of volatile organic acids and alcohols with high titers, especially butanol and butyric acid through the CoA-dependent carbon chain elongation pathway. This implies sufficient supplies of acyl-CoA, organic acids, and alcohols in cells, which are precursors for ester production. Besides, some Clostridia could utilize lignocellulosic biomass, industrial off-gas, or crude glycerol to produce other branched or straight-chain alcohols and acids. Therefore, Clostridia offer great potential to be engineered to produce short- and medium-chain volatile esters. In the review, the efforts to produce esters from Clostridia via in vitro lipase-mediated catalysis and in vivo alcohol acyltransferase (AAT)-mediated reaction are comprehensively revisited. Besides, the advantageous characteristics of several Clostridia and clostridial consortia for bio-ester production and the driving force of synthetic biology to clostridial chassis development are also discussed. It is believed that synthetic biotechnology should enable the future development of more effective Clostridia for ester production.

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO

“…Besides formation of ethyl acetate, Atf1 is known to have broad alcohol specificity and is able to catalyze formation of other acetate esters, including butyl acetate and hexyl acetate [ 13 , 14 , 32 , 33 ]. To investigate whether an acetogen expressing Atf1 also has potential for biosynthesis of other acetate esters from CO, C. autoethanogenum [P Thl -Atf1] was grown on CO supplemented with either butanol or hexanol and was compared to a reference condition with and without ethanol supplementation (Fig.…”

“…Furthermore, a yield of 5% was reported for ethyl acetate production from glucose by E. coli BW25113 Δ ackA Δ ldhA (DE3) expressing Sce Atf1 [ 11 ]. On the other hand, use of the Sce Atf1 is associated with high-level production of other short-chain esters such as butyl acetate [ 13 ]. For example, up to ca.…”

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO

“…Alcohol doping shows potential of Atf1 for ester production from CO Besides formation of ethyl acetate, Atf1 is known to have broad alcohol speci city and is able to catalyze formation of other acetate esters, including butyl acetate and hexyl acetate [14,15,30,31]. To investigate whether an acetogen expressing Atf1 also has potential for biosynthesis of other acetate esters from CO, C. autoethanogenum [P Thl -Atf1] was grown on CO supplemented with either butanol or hexanol and was compared to a reference condition with and without ethanol supplementation (Fig.…”

“…For example, 0.1 mM ethyl acetate was produced with supplementation of 100 mM ethanol in E. coli TOPO expressing Atf1 [13], and 5% of the maximum pathway yield for ethyl acetate was achieved in the E. coli BW25113 ΔackAΔldhA (DE3) expressing Atf1 [11]. On the other hand, Atf1 is associated with high-level production of other short-chain esters such as butyl acetate, with, for example, production up to 175 mM in engineered Clostridium saccharoperbutylacetonicum [14,15]. So far only sugar-based microbial production platforms have been explored for short-chain ester production.…”

Inorganic Carbon Assimilation and Electrosynthesis of Platform Chemicals in Bioelectrochemical Systems (BESs) Inoculated with Clostridium saccharoperbutylacetonicum N1-H4

“…It has been demonstrated that the assimilation of CO 2 can give bacteria some advantages, such as dissipating the excess of reducing equivalents produced by metabolic processes or accumulating organic molecules as energy and carbon storage [ 6 ]. Among the microorganisms able to use inorganic sources of carbon, clostridia have been extensively investigated over the years due to their metabolic potential for industrial applications, with significant results [ 10 , 11 ]. Different strategies for CO 2 capture, conversion, improvement, and control have been developed, involving genetic engineering, synthetic biology, metabolic engineering, and more recently, microbial electrosynthesis (MES) [ 11 , 12 , 13 ], which operates at the nexus of microbiology and electrochemistry to reduce CO 2 to value-added products [ 12 , 13 ].…”