Engineering the transmission efficiency of the noncyclic glyoxylate pathway for fumarate production in Escherichia coli

Abstract

Background Fumarate is a multifunctional dicarboxylic acid in the tricarboxylic acid cycle, but microbial engineering for fumarate production is limited by the transmission efficiency of its biosynthetic pathway. Results Here, pathway engineering was used to construct the noncyclic glyoxylate pathway for fumarate production. To improve the transmission efficiency of intermediate metabolites, pathway optimization was conducted by fluctuating gene expression levels to identify potential bottlenecks and then remove them, resulting in a large increase in fumarate production from 8.7 to 16.2 g/L. To further enhance its transmission efficiency of targeted metabolites, transporter engineering was used by screening the C 4 -dicarboxylate transporters and then strengthening the capacity of fumarate export, leading to fumarate production up to 18.9 g/L. Finally, the engineered strain E. coli W3110△4-P (H) CAI (H) SC produced 22.4 g/L fumarate in a 5-L fed-batch bioreactor. Conclusions In this study, we offered rational metabolic engineering and flux optimization strategies for efficient production of fumarate. These strategies have great potential in developing efficient microbial cell factories for production of high-value added chemicals.

Engineering the glyoxylate cycle for chemical bioproduction

“…However, aceBA overexpression dramatically slowed cell gowth, which demonstrated the importance of flux balance between the GS and oxiditive TCA cycle (Li et al, 2014). Chen et al engineered E. coli for fumarate production via the glyoxylate pathway (Chen et al, 2020). They explored the effect of pyruvate carboxylase (pyc), citrate synthase (gltA), aconitase (acnB), isocitrate lyase (aceA), and succinate dehydrogenase (sdh) overexpression on fumarate production, and found that pyruvate carboxylase and isocitrate lyase played crucial roles.…”

Engineering for an HPV 9-valent Vaccine using Genomic Constitutive Over-expression and Low Lipopolysaccharide Levels in Escherichia Coli Cells

“…Plasmids pCas (Addgene #62225) and pTargetF (Addgene #62226) were gifts from Dr. Jiang Yu (Shanghai Institutes for Biological Sciences). Plasmids for expressing the L1 proteins of various HPV types (6,11,16,18,31,33,45,52,58) were previously constructed and described elsewhere [31].…”

“…Indeed, this is a routine approach for the over-expression of recombinant proteins in eukaryotic cell systems, such as the CHO system for antibody production [16]. Many loci on the E. coli chromosome are appropriate insertion sites for exogenous genes, and several recombinant E. coli strains have been constructed for the production of valuable bio-products via the establishment of novel synthesis pathways [17][18][19]. Previous research suggests that there are three main factors to consider when constructing a chromosomally integrated expression strain.…”

“…Moreover, we investigated the effect of copy number on L1 production from the integrated expression cassettes. Finally, we constructed 9 recombinant E. coli strains for L1 proteins of HPV types 6,11,16,18,31,33,45,52,58, and demonstrated desirable purity, VLP morphology and antigenicity of the various L1 proteins.…”

Engineering Escherichia coli for efficient aerobic conversion of glucose to fumaric acid

“…The branching of the oxidative TCA cycle and GS occurs at the isocitrate level, and thus, the decreased activity of 2-ketoglutarate dehydrogenase does not affect the efficiency of succinic acid synthesis through GS reactions. The activation of the glyoxylate shunt in E. coli is generally achieved via inactivation of the transcriptional regulator IclR, which represses the expression of the aceBAK operon genes encoding GS enzymes, while the deletion of fumarase isozyme fumB and fumAC genes prevents fumaric to malic acid conversion [ [10] , [11] , [12] , 14 ]. The approaches aimed at further optimisation of fumaric acid production by the engineered strains include the following: 1) the inactivation of certain pathways of mixed-acid fermentation competitively consuming pyruvate and acetyl-CoA, the key precursor in target product biosynthesis, by the deletion of ldhA and ackA genes [ 10 , 11 , 14 ]; and 2) the increase in intracellular availability of phosphoenolpyruvate (PEP) for carboxylation through the modification of the glucose transport and phosphorylation system with the concomitant intensification of oxaloacetate (OAA) formation upon the overexpression of PEP-carboxylating phosphoenolpyruvate carboxylase, Ppc [ 10 –- 12 ].…”