Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C

Abstract

Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L(-1) h(-1)). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate.

Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

“…All chromosomal modifications were conducted using a λ-red recombinase-mediated two-step method as previously described (26). Additional details are included in SI Materials and Methods.…”

“…S4 C and D). We were not able to replace xylR with its mutant alleles in XW043 using a λ-red recombinase-mediated two-step method (26) presumably because the second copy of wild-type xylR tended to replace the selection marker due to a favored intrachromosomal recombination. Instead, using a λ-red recombinase-mediated intrachromosomal recombination approach (details in SI Materials and Methods), we successfully replaced the xylR mutations with a wild-type copy in both CM2 and TL1 strains.…”

Continuous succinic acid production from xylose by Actinobacillus succinogenes

“…Various microbial platforms have been explored for bio-production of SA with the best bench-scale performance achieved by Actinobacillus succinogenes [11][12][13][14], Anaerobiospirillum succiniciproducens [15,16], Mannheimia succiniciproducens [17][18][19], and engineered strains of Escherichia coli [20][21][22].…”

Succinic acid-producing biofilms of Actinobacillus succinogenes: reproducibility, stability and productivity

“…Due to the CO 2 fixation step in the production of SA it is theoretically possible to obtain 1.12 grams of SA per gram of glucose (van Heerden and Nicol 2013a). Modified E. coli batch fermentations have exceeded the 1 g g -1 threshold (Balzer et al 2013;Jantama et al 2008) although no report exists where this threshold has been exceeded with a continuous culture. A.…”