Efficient and Single Cell-Based Bioethanol Production - Medical Science Assignment Help

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Better processes for biofuel generation are required for effective mitigation of climate change. Lignocellulosic biomass provides an abundant potential source for producing bioethanol but is limited by difficulties in providing all enzymatic activities required for the conversion of lignocellulose to bioethanol in a single setting. Here, we describe further characterisation of a novel yeast, Vegemita bioethanolium, that is highly efficient in the production of bioethanol from simple sugars. The Pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH) and Laccase enzymes that enable this high-level bioethanol capacity were introduced into Saccharomyces cerevisiae, a yeast well known for ethanol production. We additionally engineered these cells to express genes for lignocellulose-digesting enzymes, which were isolated from the Yummyosus fairybreadus bacterium that normally feeds on lignocellulosic biomass, providing an endogenous source of simple sugars. Thus, we have generated an incredibly efficient bioethanol-producing organism that will put humankind back on a sustainable track. Introduction Mitigation of climate change requires rapid adaption to renewable and clean sources of energy including biofuels. A current method involves the production of bioethanol using anaerobic yeast fermentation of simple sugars such as xylose, sucrose, glucose, and maltose. The sources of these simple sugars include starch and molasses, which raises concerns about deforestation for new plantations and the use of land that would otherwise be used for food production. The generation of biofuels from other forms of biomass, such as wood and algae, holds great promise but is limited by toxic inhibitory compounds formed during anaerobic fermentation and the inability of yeast to completely digest cell-wall lignocellulose to simple sugars (1). While many bacterial species possess lignocellulose hydrolytic activity, no single organism possesses all metabolic pathways required for complete and efficient digestion of lignocellulose to bioethanol (2). We previously published the discovery of a novel soil bacterium in the Firmicutes phylum, Yummyosus fairybreadus, which completely hydrolyses lignocellulose from a range of sources into simple sugars (3). Mutagenesis screens identified two key types of enzyme activities that can together generate a complete lignocellulose hydrolytic pathway. The first enzyme activity is encoded by a set of hemicellulases that break down long chain polysaccharides. The second type of enzyme is glycosyl hydrolase activity, which is encoded by a set of enzymes that utilise different sugar backbones. These genes are expressed in a series of operons that we have named the 100s’n’1000s operons, with each operon encoding a hemicellulase and glycosyl hydrolase specific for a particular sugar backbone. For example, the structural genes of the maltose 100s’n’1000s operon encode enzymes that can digest maltose-based lignocellulose into monomeric maltose molecules. Each 100s’n’1000s operon is under negative-repressible control where the repressor is active only in the presence of the sugar that the operon is specific for (e.g maltose). In another previous study (4), we outlined the discovery of a novel yeast, Vegemita bioethanolium that possesses the ability to produce ethanol at a greater rate and at higher levels per cell than the A. Mateur et al., 2021: Submitted to Journal of Molecular Biology, Griffith University for peer review. yeast that is traditionally used for bioethanol production, Saccharomyces cerevisiae. While these activities were observed using media containing a range of simple sugars, V. bioethanolium grows very slowly compared to S. cerevisiae and is thus not a practical candidate for industrial production of bioethanol. Sequencing of the V. bioethanolium genome allowed the identification of genes encoding Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), which are key sequential enzymes for ethanol production during fermentation under anaerobic conditions. We also identified a gene encoding Laccase, a key enzyme for the detoxification of inhibitory compounds produced during anaerobic fermentation. We hypothesised that expressing the V. bioethanolium ADH, PDC and Laccase enzymes in S. cerevisiae using recombinant expression techniques will allow very efficient bioethanol production. Moreover, introduction of an appropriate set of bacterial lignocellulose digestive enzymes into the same yeast will allow complete and efficient catabolism of lignocellulose to bioethanol by a single organism. Here we describe expression analyses and cloning experiments that have allowed us to assemble and express both Y. fairybreadus lignocellulose hydrolytic enzymes and highly efficient ethanol-producing V. bioethanolium enzymes within the same S. cerevisiae cells. Thus, we have engineered the most efficient bioethanol-producing organism ever known to man. Methods V. bioethanolium Growth Assays: A single colony of V. bioethanolium was inoculated into yeast extract peptone dextrose (YPD) broth and grown for 48 hours at 25°C and 200 RPM on a rotary shaker. 100 µl of the liquid yeast culture was spread on agar plates containing Yeast Nitrogen Base (YNB) containing 20g/L xylose, sucrose, glucose, and maltose or no sugar and incubated for 24 hours at 25°C. Protein expression in E. coli: Single colonies from the agar plates were grown in 2YT broth containing Kanamycin (50 µg/ml) at 37°C with shaking. The culture was induced using 1 mM isopropyl-1-thio-βD-galactopyranoside (IPTG) at an optical density (OD) of 600nm. Expression continued for 4 h before cells were harvested and inclusion bodies were dissolved in 6 M guanidine hydrochloride for 2 h at room temperature. The proteins were purified via Glutathione-sepharose agarose column using the N- terminal GST tag. The protein was eluted with reduced glutathione under optimized conditions. The eluted protein was renatured (folded properly so that it was active) in refolding buffer (100 mM Tris, pH 8.0, 0.5 M L-arginine-HCl, 2 mM EDTA and 0.9 mM oxidized glutathione) for antibody production.

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