Metabolic engineering of Escherichia coli to enhance acetol production from glycerol

Abstract

Acetol, a C3 keto alcohol, is an important intermediate used to produce polyols and acrolein. To enhance acetol production from glycerol by Escherichia coli, a mutant (HJ02) was constructed by replacing the native glpK gene with the allele from E. coli Lin 43 and overexpression of yqhD, which encodes aldehyde oxidoreductase YqhD that converts methylglyoxal to acetol. Compared to the control strain without the glpK replacement, HJ02 had 5.5 times greater acetol production and a 53.4 % higher glycerol consumption rate. Then, glucose was added as a co-substrate to enhance NADPH availability and the ptsG gene was deleted in HJ02 (HJ04) to alleviate carbon catabolite repression, which led to a 30 % increase in the NADPH level and NADPH/NADP(+). Consequently, HJ04 accumulated up to 1.20 g/L of acetol, which is 69.0 % higher than that of HJ02. Furthermore, the gapA gene in HJ04 was silenced by antisense RNA (HJ05) to further enhance acetol production. The acetol concentration produced by HJ05 reached 1.82 g/L, which was 2.1 and 1.5 times higher than that of HJ02 and HJ04.Real-time PCR analysis indicates that glucose catabolism was rerouted from glycolysis to the oxidative pentose phosphate pathway in HJ05.

Elucidation of the co-metabolism of glycerol and glucose in Escherichia coli by genetic engineering, transcription profiling, and 13C metabolic flux analysis

“…13 C-MFA revealed that glucose shifted flux from the glycolytic pathway to the oxidative PP pathway to increase NADPH availability in the Δ ptsGglpK* mutant, demonstrating the benefit of co-utilization of glycerol and glucose for NADPH supply. In our previous work, we engineered an acetol overproducing strain (HJ05) through overexpressing of yqhD and silencing of gapA from the Δ ptsGglpK* mutant [12]. Because the aldehyde oxidoreductase encoded by yqhD utilizes NADPH as a cofactor [57], NADPH availability is one important factor for the acetol production.…”

“…Another obstacle to glycerol utilization is the shortage of NADPH formation because minimal glycolytic flux is reverted from GAP upwards to the oxidative pentose phosphate (PP) pathway. Because NADPH is an important cofactor needed for the production of useful metabolites, co-fermentation of glycerol with glucose has been proposed as an efficient process [4, 12, 13], especially for promoting 1,3-propanediol fermentation for the industrial scale production by DuPont [14]. However, glucose utilization prevents the metabolism of glycerol because of carbon catabolite repression (CCR) [15].…”

“…The part PEP not consumed in glucose transport was canalized to shikimate pathway [17], which is a very important route for the synthesis of aromatic amino acids and natural products [19]. Therefore, we constructed a CCR-negative Δ ptsGglpK* mutant by both blocking a key glucose transporter gene ptsG and replacing the native glpK with glpK22 from E. coli Lin 43 to enhance the glycerol conversion [12]. This mutant can co-consume glycerol and glucose with a faster glycerol assimilation rate than glucose assimilation rate [12].…”

“…Therefore, we constructed a CCR-negative Δ ptsGglpK* mutant by both blocking a key glucose transporter gene ptsG and replacing the native glpK with glpK22 from E. coli Lin 43 to enhance the glycerol conversion [12]. This mutant can co-consume glycerol and glucose with a faster glycerol assimilation rate than glucose assimilation rate [12]. To reveal the metabolic potential of the Δ ptsGglpK* mutant, it is necessary to understand its flux distributions and regulation of the co-metabolism of glycerol and glucose in the mutant.…”

Enhanced trans‐2,3‐dihydro‐3‐hydroxyanthranilic acid production by pH control and glycerol feeding strategies in engineered Pseudomonas chlororaphis GP72

“…The conversion of glycerol to high‐value products through microbial fermentation has appeared as a promising approach. Over the past several years, glycerol has been successfully utilized as a carbon source to produce acetol, bioethanol, D‐lactic acid, 1, 2‐propanediol, poly (3‐hydroxybutyrate), shikimate, succinate, and 3‐hydroxy propionic acid etc …”