Genome-scale metabolic modelling enables deciphering ethanol metabolism via the acrylate pathway in the propionate-producer Anaerotignum neopropionicum.

BACKGROUND: Microbial production of propionate from diluted streams of ethanol (e.g., deriving from syngas fermentation) is a sustainable alternative to the petrochemical production route. Yet, few ethanol-fermenting propionigenic bacteria are known, and understanding of their metabolism is limited. Anaerotignum neopropionicum is a propionate-producing bacterium that uses the acrylate pathway to ferment ethanol and CO2 to propionate and acetate. In this work, we used computational and experimental methods to study the metabolism of A. neopropionicum and, in particular, the pathway for conversion of ethanol into propionate. RESULTS: Our work describes iANEO_SB607, the first genome-scale metabolic model (GEM) of A. neopropionicum. The model was built combining the use of automatic tools with an extensive manual curation process, and it was validated with experimental data from this and published studies. The model predicted growth of A. neopropionicum on ethanol, lactate, sugars and amino acids, matching observed phenotypes. In addition, the model was used to implement a dynamic flux balance analysis (dFBA) approach that accurately predicted the fermentation profile of A. neopropionicum during batch growth on ethanol. A systematic analysis of the metabolism of A. neopropionicum combined with model simulations shed light into the mechanism of ethanol fermentation via the acrylate pathway, and revealed the presence of the electron-transferring complexes NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn) and acryloyl-CoA reductase-EtfAB, identified for the first time in this bacterium. CONCLUSIONS: The realisation of the GEM iANEO_SB607 is a stepping stone towards the understanding of the metabolism of the propionate-producer A. neopropionicum. With it, we have gained insight into the functioning of the acrylate pathway and energetic aspects of the cell, with focus on the fermentation of ethanol. Overall, this study provides a basis to further exploit the potential of propionigenic bacteria as microbial cell factories.

Upgrading dilute ethanol to odd-chain carboxylic acids by a synthetic co-culture of Anaerotignum neopropionicum and Clostridium kluyveri.

BACKGROUND: Dilute ethanol streams generated during fermentation of biomass or syngas can be used as feedstocks for the production of higher value products. In this study, we describe a novel synthetic microbial co-culture that can effectively upgrade dilute ethanol streams to odd-chain carboxylic acids (OCCAs), specifically valerate and heptanoate. The co-culture consists of two strict anaerobic microorganisms: Anaerotignum neopropionicum, a propionigenic bacterium that ferments ethanol, and Clostridium kluyveri, well-known for its chain-elongating metabolism. In this co-culture, A. neopropionicum grows on ethanol and CO2 producing propionate and acetate, which are then utilised by C. kluyveri for chain elongation with ethanol as the electron donor. RESULTS: A co-culture of A. neopropionicum and C. kluyveri was established in serum bottles with 50 mM ethanol, leading to the production of valerate (5.4 ± 0.1 mM) as main product of ethanol-driven chain elongation. In a continuous bioreactor supplied with 3.1 g ethanol L-1 d-1, the co-culture exhibited high ethanol conversion (96.6%) and produced 25% (mol/mol) valerate, with a steady-state concentration of 8.5 mM and a rate of 5.7 mmol L-1 d-1. In addition, up to 6.5 mM heptanoate was produced at a rate of 2.9 mmol L-1 d-1. Batch experiments were also conducted to study the individual growth of the two strains on ethanol. A. neopropionicum showed the highest growth rate when cultured with 50 mM ethanol (µmax = 0.103 ± 0.003 h-1) and tolerated ethanol concentrations of up to 300 mM. Cultivation experiments with C. kluyveri showed that propionate and acetate were used simultaneously for chain elongation. However, growth on propionate alone (50 mM and 100 mM) led to a 1.8-fold reduction in growth rate compared to growth on acetate. Our results also revealed sub-optimal substrate use by C. kluyveri during odd-chain elongation, where excessive ethanol was oxidised to acetate. CONCLUSIONS: This study highlights the potential of synthetic co-cultivation in chain elongation processes to target the production of OCCAs. Furthermore, our findings shed light on to the metabolism of odd-chain elongation by C. kluyveri.

The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum.

The Wood-Ljungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (Ald-Adh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum ∆adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the Adh-Ald route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to ∆adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these 'stored assets' for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.

Conversion of Carbon Monoxide to Chemicals Using Microbial Consortia.

Syngas, a gaseous mixture of CO, H2 and CO2, can be produced by gasification of carbon-containing materials, including organic waste materials or lignocellulosic biomass. The conversion of bio-based syngas to chemicals is foreseen as an important process in circular bioeconomy. Carbon monoxide is also produced as a waste gas in many industrial sectors (e.g., chemical, energy, steel). Often, the purity level of bio-based syngas and waste gases is low and/or the ratios of syngas components are not adequate for chemical conversion (e.g., by Fischer-Tropsch). Microbes are robust catalysts to transform impure syngas into a broad spectrum of products. Fermentation of CO-rich waste gases to ethanol has reached commercial scale (by axenic cultures of Clostridium species), but production of other chemical building blocks is underexplored. Currently, genetic engineering of carboxydotrophic acetogens is applied to increase the portfolio of products from syngas/CO, but the limited energy metabolism of these microbes limits product yields and applications (for example, only products requiring low levels of ATP for synthesis can be produced). An alternative approach is to explore microbial consortia, including open mixed cultures and synthetic co-cultures, to create a metabolic network based on CO conversion that can yield products such as medium-chain carboxylic acids, higher alcohols and other added-value chemicals.

Synthetic co-cultures: novel avenues for bio-based processes.

In nature, microorganisms live in multi-species communities allowing microbial interactions. These interactions are lost upon establishing a pure culture, increasing the metabolic burden and limiting the metabolic potential of the isolated microbe. In the past years, synthetic microbial co-cultivation, using well-defined consortia of two or more microbes, was increasingly explored for innovative applications in biotechnology. As such, interspecies interactions take place without the complexity of an open mixed culture, minimizing undesired side reactions. Ultimately, synthetic co-cultivation allows to take well-characterized microbes 'off-the-shelf' to create ecosystems with improved process capabilities. This review highlights some of the recent developments on co-cultivation, focusing on waste-to-chemicals conversions. It also addresses fundamental knowledge on microbial interactions deriving from these studies, which is important to further develop our ability to engineer functional co-cultures for bioproduction.

Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids.

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate. The model suggests that one strategy to optimize the production of medium-chain fatty-acids from syngas would be the addition of succinate. According to the prediction, addition of succinate would increase the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in a flux of up to 60 % of electrons into hexanoate. Another potential way to further optimize butyrate and hexanoate production would be an increase of C. autoethanogenum ethanol production. Blocking either acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) activity or formate transport, in the C. autoethanogenum metabolic model could potentially lead to an up to 150 % increase in ethanol production.

Metabolic shift induced by synthetic co-cultivation promotes high yield of chain elongated acids from syngas.

Bio-catalytic processes for sustainable production of chemicals and fuels receive increased attention within the concept of circular economy. Strategies to improve these production processes include genetic engineering of bio-catalysts or process technological optimization. Alternatively, synthetic microbial co-cultures can be used to enhance production of chemicals of interest. It remains often unclear however how microbe to microbe interactions affect the overall production process and how this can be further exploited for application. In the present study we explored the microbial interaction in a synthetic co-culture of Clostridium autoethanogenum and Clostridium kluyveri, producing chain elongated products from carbon monoxide. Monocultures of C. autoethanogenum converted CO to acetate and traces of ethanol, while during co-cultivation with C. kluyveri, it shifted its metabolism significantly towards solventogenesis. In C. autoethanogenum, expression of the genes involved in the central carbon- and energy-metabolism remained unchanged during co-cultivation compared to monoculture condition. Therefore the shift in the metabolic flux of C. autoethanogenum appears to be regulated by thermodynamics, and results from the continuous removal of ethanol by C. kluyveri. This trait could be further exploited, driving the metabolism of C. autoethanogenum to solely ethanol formation during co-cultivation, resulting in a high yield of chain elongated products from CO-derived electrons. This research highlights the important role of thermodynamic interactions in (synthetic) mixed microbial communities and shows that this can be exploited to promote desired conversions.