Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid

Abstract

An in vivo method of producing isoamyl acetate and succinate simultaneously has been developed in Escherichia coli to maximize yields of both high value compounds as well as maintain the proper redox balance between NADH and NAD(+). Previous attempts at producing the ester isoamyl acetate anaerobically did not produce the compound in high concentrations because of competing pathways and the need for NAD(+) regeneration. The objective of this study is to produce succinate as an example of a reduced coproduct to balance the ratio of NADH/NAD(+) as a way of maximizing isoamyl acetate production. Because the volatility of the two compounds differs greatly, the two could be easily separated in an industrial setting. An ldhA, adhE double mutant strain (SBS110MG) served as the control strain to test the effect of an additional ackA-pta mutation as found in SBS990MG. Both strains overexpressed the two heterologous genes pyruvate carboxylase and alcohol acetyltransferase (for ester production). The triple mutant SBS990MG was found to produce higher levels of both isoamyl acetate and succinate. At the optimal condition of 25 degrees C, the culture produced 9.4 mM isoamyl acetate and 45.5 mM succinate. SBS990MG produced 36% more ester and over 700% more succinate than SBS110MG. In addition, this study demonstrated that a significantly higher isoamyl acetate concentration can be attained by simultaneously balancing the carbon and cofactor flow; the isoamyl acetate concentration of 9.4 mM is more than seven times higher than an earlier report of about 1.2 mM.

Single-Cell Biorefinery

“…However, under aerobic conditions, 3-hydroxyaldehydrate (3-HPA) accumulates as a toxic metabolite. 67 Because the volatility of isoamyl acetate and succinate differs greatly, they can be easily separated in an industrial setting. Therefore, a coproduction strategy was developed through which the 3-hydroxypropionic acid (3-HP) generated via the oxidative pathway was coproduced with 1,3-PDO generated via the reductive pathway.…”

Limiting and excreting metabolites of succinate production in S.cerevisiae using flux balance analysis

“…The two pathways are known as α-ketoglutarate oxidation and fumarate reduction. Both pathways are via TCA cycle inside the S.cerevisiae [6,7,8,9,10,11,12]. Inside the pathways, there are variety of metabolites and reactions as well, which actually affect the growth production of succinate in yeast.…”

“…There are two main pathways in S.cerevisiae that can lead to the formation of succinate, which are α-ketoglutarate oxidation and fumarate reduction under via tricarboxylic cycle acid (TCA cycle) [6,7,8,9,10,11,12]. Both pathways are inside the mitochondrion.…”

“…This pathway converts acetyl-CoA to citrate, isocitrate and finally to succinate. In reductive TCA cycle, succinate is produced under anaerobic conditions, where it derived from phosphoenolpyruvate, via some intermediate that include the oxaloacetate, malate and fumarate [6,7,8,9,10,11,12]. Fig.…”

Improved Succinate Production by Metabolic Engineering

“…The glyoxylate cycle is essentially active under aerobic conditions upon adaptation to growth on acetate (Figure 1(b)). The glyoxylate pathway operates as a cycle to convert 2 mol acetyl CoA to 1 mol succinate [9]. The equation of glyoxylate pathway is: $\begin{array}{c}\mathrm{2}\text{Acetyl}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{CoA}+\mathrm{2}{\text{H}}_{\mathrm{2}}\text{O}+{\text{NAD}}^{+}\\ \hspace{1em}\hspace{1em}\to \text{Succinate}+\mathrm{2}\text{CoASH}+\text{NADH}+\mathrm{2}{\text{H}}^{+}\end{array}$ Since the conversion of glucose to succinate via glyoxylate pathway generates NADH, this route alone is not sufficient to balance the electrons.…”