Engineering new-to-nature biochemical conversions by combining fermentative metabolism with respiratory modules

The study addresses the limitation that classical fermentations must internally balance redox equivalents, restricting feasible substrate–product pairs. The authors hypothesize that an obligate aerobic fermentative metabolism can be engineered that preserves an intact electron transport chain (ETC) yet blocks all endogenous electron transfer into quinones, enabling the modular reintroduction of specific quinone-reducing steps to selectively dispose of excess electrons with oxygen. The purpose is to create a controllable respiro‑fermentative growth mode that rebalances otherwise unbalanced fermentations (e.g., converting more reduced substrates like glycerol to products such as lactate or isobutanol) while retaining the high-yield advantages of fermentation. This has significance for expanding industrial bio-production beyond traditional redox-balanced constraints.

Prior work enforced aerobic fermentation by disrupting the ETC (e.g., deleting terminal oxidases or NADH dehydrogenases and ubiquinone biosynthesis), yielding obligate fermenters useful for production and for evolving oxygenases, but still confined to redox-balanced substrate–product combinations. Attempts to broaden anaerobic fermentations of reduced substrates like glycerol used external electron acceptors, complex co-substrates (e.g., tryptone/yeast extract), or electro-fermentation via electrodes, which pose scalability or process control issues. Microaerobic strategies use limiting oxygen to permit redox balancing without full respiration, but industrial O2 gradients hamper tight control. Therefore, a genetic design allowing precise, module-based respiratory electron disposal while maintaining fermentative flux is needed to unlock unbalanced fermentations under robust aerobic conditions.

- Design of an obligately fermentative strain with intact ETC: Using the E. coli iML1515 model and EcoCyc curation, the authors identified quinone-reducing reactions. They deleted the two main NADH dehydrogenases (Δndh ΔnuoEFG) and an additional set of quinone-dependent dehydrogenases and putative NAD(P)H:quinone oxidoreductases forming potential NAD(P)H-driven “mini-cycles” to quinones (targets included ldhA/dld, mqo, putA, glpD, glpABC, glcDEF, dadA, ihgO, sdhABCD, lldD, poxB, fadE, kefF, wrbA, mdaB/ygiN, yieF; 14 relevant reactions). The resulting strain was termed NNmini. - Phenotyping NNmini: Aerobic and anaerobic growth on glucose gradients was measured (OD600, growth rates, biomass yields). Extracellular metabolites were quantified (ion chromatography). Intracellular NAD(H) and NADP(H) were measured by LC-MS/MS; ATP/adenylate levels quantified. NADH/NAD ratios compared to wild type. Specific glucose uptake rates were determined using YSI analyzer measurements and growth data. - Alternative minimal-deletion strategy: ΔubiCA in a Δndh ΔnuoEFG background was tested; reversion to respiratory growth occurred via mutations (e.g., in ubiE and dld promoter), so this route was abandoned for modularity and robustness. - Growth improvement via supplementation: Hypothesizing acetyl-CoA limitation under aerobic fermentation due to Pdh inhibition at high NADH and inactivity of Pfl, the authors supplemented NNmini cultures with acetate, pyruvate, or casamino acids (CAAs) and quantified effects on growth rate/biomass. - 13C tracing: NNmini grown on glucose with 13C2-acetate; proteinogenic amino acids analyzed to determine incorporation (LC-MS), testing whether acetate supplies acetyl-CoA and lower TCA intermediates only. - Controlled respiro-fermentation module: Genomic reintegration of glpD (glycerol-3-phosphate:ubiquinone oxidoreductase) into NNmini to enable glycerol utilization with electron transfer to ubiquinone/oxygen, while main metabolism remains fermentative to lactate. Growth on glycerol was characterized; lactate and minor byproducts quantified; intracellular ATP measured. - Non-native product module: For isobutanol production from glycerol, NNmini ΔldhA + glpD was transformed with the pIBA4 plasmid (iBUOH pathway; IPTG-inducible). Growth and product formation (isobutanol, ethanol) were quantified (GC-MS/MS for isobutanol; enzymatic kits for ethanol/acetate; residual glycerol by HPAEC). Cultivations were run at 30 °C in sealed headspace vials to manage volatility. Standard media, growth conditions, sampling, and analytical methods (LC-MS/MS, IC, HPAEC) are detailed, including calibration and instrument parameters.

- Obligate aerobic fermentative phenotype: NNmini grew slowly and with low biomass in aerobic glucose cultures, secreted lactate exclusively at 1.78 ± 0.2 mol lactate/mol glucose, and showed an ~16-fold increase in NADH/NAD versus wild type. Aerobic growth rate on 20 mM glucose: 0.0329 ± 0.0011 h^-1 vs wild type 0.6502 ± 0.0052 h^-1. Specific glucose uptake (aerobic): NNmini 14.19 ± 1.54 mmol gCDW^-1 h^-1 (comparable to fermentative wild type), indicating fermentative metabolism. - Anaerobic phenotype: On 20 mM glucose, wild type 0.5095 ± 0.0041 h^-1 vs NNmini 0.4185 ± 0.0033 h^-1; both secreted acetate, formate, ethanol (hetero-fermentative), showing NNmini’s aerobic homolactic mode is less growth-efficient than native anaerobic fermentation. - ΔubiCA alternative: Despite initial fermentative behavior, mutations restored respiration, and acetate byproduct formed; approach unsuitable for modular respiratory reintegration. - Growth limitations and supplementation: Evidence for acetyl-CoA limitation under aerobic fermentation: supplementation improved NNmini growth on 10 mM glucose—acetate (max OD600 ~0.8 at 4 mM; max µ = 0.277 h^-1), CAAs (max OD600 1.825; µ = 0.315 ± 0.014 h^-1), pyruvate (max OD600 0.47; µ = 0.108 ± 0.003 h^-1). 13C2-acetate incorporation was detected only in leucine, proline, and arginine, confirming acetate use to acetyl-CoA and lower TCA up to α-ketoglutarate (blocked further by ΔsdhCDAB and Δmqo). - Controlled respiro-fermentation module (glpD): NNmini + glpD grew aerobically on glycerol (40 mM glycerol µ = 0.126 ± 0.014 h^-1) and converted glycerol to lactate with yield 0.85 ± 0.14 mol/mol. Minor acetate byproduct at higher glycerol (e.g., 1.25 ± 0.05 mM at 20 mM glycerol). Intracellular ATP was ~2.6-fold higher in NNmini + glpD on glycerol than NNmini on glucose, indicating some respiratory ATP generation from QH2 reoxidation. - Isobutanol production from glycerol: In NNmini ΔldhA + glpD + pIBA, growth on glycerol was achieved with IPTG induction; isobutanol reached 2.012 ± 0.059 mM from 5 mM glycerol (~80% of theoretical, noting evaporation). Ethanol byproduct was detected, likely from AdhA side activity. Growth was slower than lactate-producing ancestor, consistent with heterologous pathway burden and multi-enzyme redox recycling requirements.

Eliminating all endogenous routes transferring electrons to quinones created an obligately fermentative E. coli that, even under aerobic conditions, must excrete reduced products to recycle NADH. Retaining an intact ETC enabled reintroduction of selected quinone-dependent enzymes as respiratory modules to offload specific electron pairs to oxygen in a controlled manner. Reintegration of GlpD provided a single respiratory step that balanced the excess reducing equivalents from glycerol catabolism, enabling robust aerobic conversion of glycerol to lactate with near-stoichiometric yields and modest respiratory ATP gain without restoring full respiratory growth. Redirecting the fermentative sink from lactate to isobutanol validated that non-native product pathways can replace native fermentation routes while remaining growth-coupled under controlled respiro‑fermentative conditions. The data indicate that aerobic fermentative growth in NNmini is limited by acetyl-CoA supply due to Pdh inhibition at high NADH and the inactivity of Pfl in oxygen, as supplementation with acetate or CAAs increased growth substantially and 13C2‑acetate labeling localized to acetyl-CoA–derived biomass precursors. These results support the hypothesis that modular respiratory reinsertion can selectively rebalance fermentations and broaden feasible substrate–product combinations beyond redox-balanced pairs, offering new routes for valorizing reduced substrates like glycerol. Future optimization may include relieving acetyl-CoA bottlenecks (e.g., engineering Pdh to reduce NADH inhibition, reintegrating pyruvate oxidase, or introducing phosphoketolase), and tuning global regulation or applying adaptive laboratory evolution to improve resource allocation under aerobic fermentation.

The authors introduce a modular metabolic design that combines obligate fermentative metabolism with selective respiratory modules to rebalance electron equivalents using oxygen. They engineered NNmini, an E. coli strain that homolactically ferments glucose aerobically, and demonstrated controlled respiro‑fermentation by reintegrating GlpD to enable aerobic glycerol-to-lactate fermentation near stoichiometrically, with some respiratory ATP gain. Replacing lactate formation with a heterologous isobutanol pathway established aerobic, growth-coupled isobutanol production from glycerol. This platform lifts the constraint of redox-balanced substrate–product pairs in fermentations and expands the accessible bioconversion space for industrial processes, including glycerol valorization. Future work should focus on alleviating acetyl-CoA limitations, optimizing heterologous pathway expression and burden, and employing regulatory rewiring or adaptive evolution to enhance growth and productivities.

- Aerobic fermentative growth of NNmini shows low growth rates and biomass yields compared to both wild type respiration and anaerobic fermentation, likely due to regulatory mismatches and acetyl-CoA limitations (Pdh inhibition at high NADH, Pfl inactivity in O2). - An alternative minimal-deletion strategy (ΔubiCA with Δndh ΔnuoEFG) was unstable, with mutations restoring respiration and acetate byproduct formation, and is incompatible with modular respiratory reintegration. - The isobutanol production strain exhibited slower growth and submaximal yields, potentially limited by pathway burden, enzyme expression balance, and product volatility (evaporation). - The current design does not address global regulatory reprogramming (e.g., ArcAB, FNR), which may cause unnecessary respiratory gene expression under aerobic fermentation, increasing metabolic burden. - Process-scale considerations (e.g., oxygen gradients) are acknowledged, and while the genetic design seeks to decouple from tight microaerobic control, further validation in bioreactors is needed.