A protein-based biorefinery for bulk chemicals production

A biorefinery should ideally integrate biomass conversion processes to produce a range of fuels, power, materials, and chemicals from biomass. The term biorefinery is derived both from the raw material feedstock which is renewable biomass and also from the bioconversion processes often applied in the treatment and processing of the raw materials. Renewable sources are the basis of the alternative energy, fuels and compound obtained in a biorefinery-based way to meet the energy demand in a world where petrol fuels are becoming scarce and more expensive. The current biorefinery schemes are mainly based on hexose sugars (first generation) or lignocellulosic-based material (second generation) as feedstocks and depend on microbial cell factories for obtaining the products of interest. In most cases, the substrate utilization is not optimized jet: at the end of the process in addition to the main product, by-products such as huge quantity of lignin and some C5 sugars (deriving from incomplete utilization of lignocellulose) but also proteins (mainly the exhausted biomass at the end of the production) are still available. Focusing the attention on proteins, they are nowadays mainly absorbed as animal feed, but the quantities produced exceed the demand. Considering the implication related to GMO utilization and the increasing number of biorefineries, it can be stated that proteins could represent an interesting Abstract - 2 - “no-cost” starting material to produce more biofuel, bioproducts or biopower in a biorefinery-based way. This study has the aim to present some examples of production by using the protein side-stream of a biorefinery. In particular, we report the possible destiny of two aminoacids released from said proteins: glycine, to obtain butanol and isobutanol, and glutamate, to produce succinic acid. Butanol represents the most feasible alternative to gasoline, but, differently from ethanol, microbial fermentations are not enough developed to sustain the market demand. The natural producers, different species of the Gram positive group of Clostridia, present many and different limitations which are challenging the scientific communities in finding alternative hosts. Among the different alternative hosts, yeasts can present attractive features, being the most relevant the existence of industrial plants based on Saccharomyces cerevisiae fermentation for the production of ethanol. Up to now, in S. cerevisiae the production of butanol has been obtained by expressing heterologous genes from Clostridia. The best reported butanol titer is only 2.5 mg/L, probably mainly depending on problems related to the overexpression of heterologous prokaryotic enzymes. On the contrary, the isobutanol production is obtained at high level (0.63 g/L) in S. cerevisiae by using and optimizing an endogenous valine degradation pathway. We present an alternative and novel way to produce butanol and isobutanol using glycine as substrate, through a new identified and Abstract - 3 - biochemically characterized pathway. Starting from 15 g/L of glycine we have obtained 92 mg/L of butanol and 58 mg/L of isobutanol. Considering that the pathway has been just discovered and no optimization has been designed or applied, the obtained results has to be considered extremely positive. Since one of the most common stresses that microorganisms have to face during productions is the toxicity of the final product, we investigated also the possibility to use the glycine as protective agent for the cells. This idea originated from different studies that explain how the use of aminoacids, among which glycine, can help the cells to better respond to different stresses, such as high ethanol concentration and hydrogen peroxide. As never reported in literature, we have notably found that butanol causes a sort of oxidative stress, peroxidising the membrane lipids. Moreover, we have demonstrated that glycine can help the cells to better tolerate the presence of hydrogen peroxide, acetic acid, ethanol and butanol. The succinic acid occurs naturally in humans, animals, plants, and microorganisms as TCA cycle intermediate. Its use as a precursor to produce many important commodity chemicals used to make a wide assortment of products such as solvents, fiber and polymer production make it one of the most important relevant bulk chemicals. The microbial production of succinic acid via reductive TCA cycle using engineered Saccharomyces cerevisiae is nowadays on the market commercialised by Reverdia (Cassano Spinola, Italy). Abstract - 4 - Here we investigated the possibility to exploiting the glutamate degradation pathway through the γ-aminobutyric acid (GABA) intermediate as an additional way of obtaining succinate when a source of glutamate is feeded to the cells. Since this pathway, also called GAD/GABA shunt, is transcriptionally controlled, we transcriptionally up-regulated it through the constitutive overexpression of the involved genes, GAD1, UGA1 and UGA2. Even if the pathway was clearly overexpressed, as revealed by transcriptional analysis, the obtained amount of succinic acid did not reflect this modification. Further investigating the possible reason of that, we have demonstrated that, despite the higher transcription levels, the corresponding enzymatic activities of the pathway did not increased. We can argue that posttranslational regulations occur in the pathway, what at the moment impaired the initial aim of our work. However, the data add new and relevant information for the comprehension of the role and the regulation of this pathway in S. cerevisiae. It cannot be excluded that the production of succinic acid in yeasts might be in a future optimized by exploiting the GAD/GABA pathway, once it will be fully characterized. We have finally investigated the possibility to produce succinic acid in a strain of S. cerevisiae deleted in the gene encoding the enzyme glutamate synthase, GLT1. During this study we have tested different growing conditions, since the target gene of deletion is located in a critical node of the nitrogen sensing and utilization, which is related to cellular plasticity and adaptability in perturbed environment. We could demonstrate that Abstract - 5 - when cells are growing in low salts concentration medium, the deleted strain compared to wild type strain is able to accumulate more reduced products, such as succinic acid, malate/fumarate, glycerol and ethanol, indicating a sort of necessity to reduce the excess of NAD(P)H generated by the GLT1 deletion. Regarding this preliminary study, deeper investigations have to be performed to find a fine tuned way to intentionally redirect the metabolic flux and obtain the product of interest, which in turn could be succinic acid, but also malic or fumaric acid, or again ethanol, being this last the product that has the responsibility to prove that bio-based second generation processes can effectively and positively have an impact on our future society.