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Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G.

ABSTRACT: Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate-dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host β-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).

SUBMITTER: Lin B

PROVIDER: S-EPMC4538657 | biostudies-other | 2015 Aug

REPOSITORIES: biostudies-other

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Project description:The bacterial lifestyle is plastic, requiring transcriptional, translational, and metabolic tailoring for survival. These dynamic cellular processes are energy intensive; therefore, flexible energetics is requisite for adaptive plasticity. An intricate network of complementary and supplementary pathways exists in bacterial energy metabolism. There are two main entry points for electrons in the aerobic electron transport system, NADH dehydrogenase (NDH) and succinate dehydrogenase (SDH), receiving electrons from NADH and succinate, respectively. Aerobic bacterial phyla have a non-proton-pumping NADH dehydrogenase, which is often the primary dehydrogenase under aerobiosis. Here, we report adaptive changes supporting growth restoration in an Escherichia coli strain lacking the primary dehydrogenase. Growth optimization is achieved by reducing the activity of succinate dehydrogenase, and thus we demonstrate a physiological discord between proton-pumping NADH dehydrogenase and succinate dehydrogenase in supporting growth. Beyond the fundamental understanding of the bioenergetic network, identifying this compensatory feature provides impetus to rational antimicrobial combinations for targeting the non-proton-pumping dehydrogenase. IMPORTANCE Energy generation pathways are a potential avenue for the development of novel antibiotics. However, bacteria possess remarkable resilience due to the compensatory pathways, which presents a challenge in this direction. NADH, the primary reducing equivalent, can transfer electrons to two distinct types of NADH dehydrogenases. Type I NADH dehydrogenase is an enzyme complex comprising multiple subunits and can generate proton motive force (PMF). Type II NADH dehydrogenase does not pump protons but plays a crucial role in maintaining the turnover of NAD+. To study the adaptive rewiring of energy metabolism, we evolved an Escherichia coli mutant lacking type II NADH dehydrogenase. We discovered that by modifying the flux through the tricarboxylic acid (TCA) cycle, E. coli could mitigate the growth impairment observed in the absence of type II NADH dehydrogenase. This research provides valuable insights into the intricate mechanisms employed by bacteria to compensate for disruptions in energy metabolism.

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Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G.

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