Insight into metabolic pathways of the potential biofuel producer, Paenibacillus polymyxa ICGEB2008

Abstract

Background: Paenibacillus polymyxa is a facultative anaerobe known for production of hydrolytic enzymes and various important biofuel molecules. Despite its wide industrial use and the availability of its genome sequence, very little is known about metabolic pathways operative in the Paenibacillus system. Here, we report metabolic insights of an insect gut symbiont, Paenibacillus polymyxa ICGEB2008, and reveal pathways playing an important role in the production of 2,3-butanediol and ethanol.

Result: We developed a metabolic network model of P. polymyxa ICGEB2008 with 133 metabolites and 158 reactions. Flux balance analysis was employed to investigate the importance of redox balance in ICGEB2008. This led to the detection of the Bifid shunt, a pathway previously not described in Paenibacillus, which can uncouple the production of ATP from the generation of reducing equivalents. Using a combined experimental and modeling approach, we further studied pathways involved in 2,3-butanediol and ethanol production and also demonstrated the production of hydrogen by the organism. We could further show that the nitrogen source is critical for metabolite production by Paenibacillus, and correctly quantify the influence on the by-product metabolite profile of ICGEB2008. Both simulations and experiments showed that metabolic flux is diverted from ethanol to acetate production when an oxidized nitrogen source is utilized.

Conclusion: We have created a predictive model of the central carbon metabolism of P. polymyxa ICGEB2008 and could show the presence of the Bifid shunt and explain its role in ICGEB2008. An in-depth study has been performed to understand the metabolic pathways involved in ethanol, 2,3-butanediol and hydrogen production, which can be utilized as a basis for further metabolic engineering efforts to improve the efficiency of biofuel production by this P. polymyxa strain.

Keywords: 2,3-Butanediol; Ethanol; Metabolic engineering; Metabolic modeling; Paenibacillus polymyxa; Redox metabolism.

Fig. 1

Product profile of P. polymyxa ICGEB2008 under anaerobic condition of growth with two different nitrogen sources. The experiments were performed in triplicate by growing the culture in 50 ml medium for 24 h and analyzing the extracellular metabolites via HPLC. The results represent average and standard deviation of data from three biological replicates

Fig. 2

Representation of the predicted flux distributions with nitrate (red arrows) and ammonia (blue arrows) nutrition. Thickness of arrows is proportional to flux values. The two main differences are the use of reactions producing NADH under nitrate nutrition and the employment of FHL as reductant valve during ammonia nutrition. Nitrate reduction is represented by two different processes in the model. Either nitrate is used as final acceptor in the electron transfer chain yielding nitrite, or as source for the NIR + NAR reduction to ammonia

Fig. 3

Scan over a range of maintenance ATP required per biomass carbon for the model without formate hydrogen lyase (FHL) activity. One unit of biomass refers to one carbon of newly produced P. polymyxa. Markers show the experimental values for biomass, ethanol and 2,3-butanediol production

Fig. 4

a Scan over the strength of an additional electron sink to identify the amount of additional reduction required to predict the experimentally observed by-product formation. Predicted ratio of ethanol/butanediol shifts towards 2,3-butanediol in response to oxidized medium. Positive values indicate additional flux through the DEHOG reaction and thus either an increased demand of reductant or a more reduced biomass. Markers show the experimental values for biomass, ethanol and 2,3-butanediol production. The simulated outputs fit well to these when using FHL and an adjusted redox demand of +0.7/biomass carbon (see text). b Scan over a range of maintenance ATP required per biomass carbon as in Fig. 3, but with FHL and an additional reductant requirement of +0.7/biomass carbon, as determined by Fig. 4a)

Fig. 5

ATP requirement scan with nitrate as nitrogen source. The model predicted ammonium production, which was experimentally confirmed for growth of P. polymyxa ICGEB2008 on nitrate. Constraining the total ammonium production in the model to experimentally observed values, the model predicts a decrease in ethanol production to zero and an increase in acetate formation, which is in good qualitative agreement with experimental data. The simulations suggest that the maintenance ATP requirement per biomass almost doubles on nitrate when compared to ammonium nutrition