Engineering Escherichia coli for high-level production of propionate

Abstract

Mounting environmental concerns associated with the use of petroleum-based chemical manufacturing practices has generated significant interest in the development of biological alternatives for the production of propionate. However, biological platforms for propionate production have been limited to strict anaerobes, such as Propionibacteria and select Clostridia. In this work, we demonstrated high-level heterologous production of propionate under microaerobic conditions in engineered Escherichia coli. Activation of the native Sleeping beauty mutase (Sbm) operon not only transformed E. coli to be propionogenic (i.e., propionate-producing) but also introduced an intracellular "flux competition" between the traditional C2-fermentative pathway and the novel C3-fermentative pathway. Dissimilation of the major carbon source of glycerol was identified to critically affect such "flux competition" and, therefore, propionate synthesis. As a result, the propionogenic E. coli was further engineered by inactivation or overexpression of various genes involved in the glycerol dissimilation pathways and their individual genetic effects on propionate production were investigated. Generally, knocking out genes involved in glycerol dissimilation (except glpA) can minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway. For optimal propionate production with high C3:C2-fermentative product ratios, glycerol dissimilation should be channeled through the respiratory pathway and, upon suppressed solventogenesis with minimal production of highly reduced alcohols, the alternative NADH-consuming route associated with propionate synthesis can be critical for more flexible redox balancing. With the implementation of various biochemical and genetic strategies, high propionate titers of more than 11 g/L with high yields up to 0.4 g-propionate/g-glycerol (accounting for ~50 % of dissimilated glycerol) were achieved, demonstrating the potential for industrial application. To our knowledge, this represents the most effective engineered microbial system for propionate production with titers and yields comparable to those achieved by anaerobic batch cultivation of various native propionate-producing strains of Propionibacteria.

Microbial Propionic Acid Production

“…However, this operon lacks a methylmalonyl-CoA epimerase [73] and should not be functional. Nevertheless, several groups have reported propionate production from overexpression of the operon in complex media [82][83][84]. We suspect that the reported propionate production is a result a ghost-peak interfering with propionate quantification [45].…”

Construction of a novel anaerobic pathway in Escherichia coli for propionate production

“…Anaerobic fermentation condition was realized by creation of vacuum followed by flushing with pure nitrogen for 3 times, and then the reactor was sealed with a butyl rubber cap in anaerobic chamber. Thus, fermentation by propionibacteria has various limitations, such as nitrogen flux for maintaining anaerobic condition [15], slow growth,costly complex culture media and lack of metabolic engineering tools for strain improvement [17], which make this technology not economically applicable.…”

“…There was only one report we found concerning production of propionate by engineered cell factories [17]. Akawi Lamees et al knocked out genes involved in glycerol dissimilation (except glpA) to minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway for more flexible redox balancing, and finally achieved propionate titers of more than 11 g/L with yields up to 0.4 g-propionate/g-glycerol in complex fermentation medium and well-controlled micro aerobic condition.…”

“…Akawi Lamees et al knocked out genes involved in glycerol dissimilation (except glpA) to minimize levels of solventogenesis and shift more dissimilated carbon flux toward the C3-fermentative pathway for more flexible redox balancing, and finally achieved propionate titers of more than 11 g/L with yields up to 0.4 g-propionate/g-glycerol in complex fermentation medium and well-controlled micro aerobic condition. And their work was mainly focused on engineering of glycerol utilization [17]. There were also researches concerning products involved Sbm cycle.…”

Engineering Escherichia coli for Microbial Production of Butanone

“…It should be noted that butanone (and to a lesser extent acetone) can also be used as an intermediate for the biological conversion of other value-added products such as ethyl esters (40) and secondary alcohols such as 2-butanol (5) and isopropanol (41). Lastly, in addition to medium-chain methyl ketones, CoA-dependent elongation pathways can be applied for the biosynthesis of several other long-chain oleochemicals, such as fatty acid ethyl esters, fatty alcohols and amines, paraffins, and olefins (42,43), thus expanding the scope of whole-cell biocatalytic platforms.…”