Two‐stage carbon distribution and cofactor generation for improving l‐threonine production of Escherichia coli

Abstract

L-Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative L-threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of L-threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH).Here, a metabolic modification strategy of two-stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an L-threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into L-threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for L-threonine production, we developed an L-glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02-and 1.21-fold increase in L-threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions. K E Y W O R D Scarbon distribution, cofactor generation, Escherichia coli, L-threonine, metabolic engineering

Expression regulation of multiple key genes to improve l-threonine in Escherichia coli

“…Deletion of aspC led to generation of small cells with slow growth, while overexpression of aspC exerted the opposite effect [55]. Using a switch to make the glycolytic fluxes towards TCA cycle in the early stage, and to make the carbon flux be redirected into l-threonine synthetic pathway by inducing the expression of aspC, fdh, gdhA and pntAB in the production stage could increase l-threonine production [62]. The gene aspC following different threonine-enhancing promoters were inserted into the chromosome of TWF063, resulting in the strains TWF066, TWF067 and TWF068.…”

“…TRFC derived by repeated compound mutagenesis (DES plus UV) from E. coli K12 could use sucrose as the carbon source, it could produce 124.57 g/L l-threonine after 40 h cultivation, using the combined feeding strategy of pseudo-exponential feeding and glucose-stat feeding resulted in high cell density [68]. THPE5 uses a switch to make the glycolytic fluxes towards TCA cycle in the early stage, leading to the improved glucose utilization and growth performance; in the production stage the carbon flux is redirected into l-threonine synthetic pathway via a synthetic genetic circuit by inducing the expression of aspC, fdh, gdhA and pntAB to increase l-threonine production, resulting in 70.5 g/L l-threonine with a yield of 0.404 g/g glucose after 40 h cultivation [62]. JLTHR could produce 127.3 g/L l-threonine with a glucose conversion rate of 58.12% after adding betaine hydrochloride in the medium [4].…”

Deletion of arcA, iclR, and tdcC in Escherichia coli to improve l‐threonine production

“…Furthermore, disruption of arcA could activate TCA cycle, leading to improved NADH/NAD + ratio [40]. Improvement of NADH/NAD + ratio facilitated l-threonine accumulation at exponential phases [35] and the upregulation of zwf in TWF015 ( arcA cra) was also good for NADPH production. Reducing power availability is significant for the production of l-threonine and the resulting strain TWF015 produced more l-threonine as expected (Fig.…”

“…Genes gltA, icd, sdhA, and mdh relevant to TCA cycle were significantly upregulated in TWF010. Enhancement of TCA cycle could overproduce NADH [34], and the improvement of NADH/NAD + ratio might facilitate l-threonine accumulation at the exponential phase [35] (Fig. 3C).…”

Highly Efficient Synthesis of Glutathione via a Genetic Engineering Enzymatic Method Coupled with Yeast ATP Generation

“…The GSH produced from glucose, lactose, maltose, sucrose, and FDP was 19.67 mmol/L, 13.59 mmol/L, 16.69 mmol/L, 17.26 mmol/L, and 17.60 mmol/L, respectively. The other carbon sources need to be converted into ATP through the yeast cell glycolytic pathway [35], so the efficiency of synthesizing GSH from these carbon sources is not as high as the direct synthesis of GSH from ATP. However, ATP is so expensive that it is economically unfeasible to add ATP directly at an industrial scale.…”