i need to find 8 molecular biological errors in the journal article critique attached. Any findings will be helpful. Exclude the introductory paragraph
Microsoft Word - Journal Critique Article 2021.docx A. Mateur et al., 2021: Submitted to Journal of Molecular Biology, Griffith University for peer review. Efficient and Single Cell-Based Bioethanol Production A. Matuer, O. Dear, B. Lyndlie, C. Airless and N. Competent Department of Science, Cornflakes University, Bris Vegas, Australia Corresponding author: W. Schroder. School of Environment and Science, Griffith University, Australia:
[email protected] Abstract 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-β- D-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. Generation of anti-Laccase antibodies: Antibodies against the V. bioethanolium Laccase protein were raised in mice by immunisation with recombinant protein in Fruend’s adjuvant. Female 4 week-old CD1 mice were used in accordance with the guidelines set forth in the National Institutes of Health manual Guide for the Care and Use of laboratory Animals. 5 µg of recombinant protein in 100 µl of phosphate-buffered saline (PBS) plus adjuvant was injected subcutaneously at three-week intervals. Non-heparinised whole blood was collected from the lateral tail vein of the mice prior to immunisation (negative control) and 14 days after the last injection of protein. The blood was allowed to coagulate and was then spun. Sera containing the antibodies was prepared, aliquoted and frozen at -20°C. Western Blot: Recombinant proteins were resolved by 12% SDS-PAGE and transferred onto Immobilon P membrane (Millipore Corp, Bedford, MA) and immunoblotted by using enhanced chemiluminescence (ECL) Western blotting protocol (Amersham International, Buckinghamshire, England). Membranes were incubated overnight at 5°C in 50 mM Tris, 150 mM NaCl, 0.1% Tween 20 A. Mateur et al., 2021: Submitted to Journal of Molecular Biology, Griffith University for peer review. (pH 8.0) (TBS-T) containing 10% (w/v) nonfat dry milk, followed by a 1 hr incubation with the primary antibody diluted in TBS-T, 5% (w/v) dry milk (TTM), one wash with TBS-T, two with high-salt TTM (0.5 M NaCl), and two with TTM. Horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunoresearch Laboratories, Incorporated) was diluted 1:50,000 in TTM and added to the membranes for 45 min. The membranes were washed once with TBS-T, twice with high-salt TTM, three times with TTM containing 0.5% Triton X-100, and three times with TBS-T. Reverse Transcription: RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. First strand total cDNA synthesis was performed in a 20-μl reaction containing 1 μg total RNA, 500 μM 2′-deoxynucleoside 5′-triphosphates, 200 ng random hexamer oligonucleotides, 1× Superscript First Strand buffer, 10 mM DTT, and 200 U Superscript III (Invitrogen). The cDNA was stored at -80 °C until use. Quantitative RT-PCR: Primers used for qRT-PCR are shown in Appendix Table 1. The amplification reaction mixture of 20 μl contained 0.1 μg randomly primed cDNA, 0.5 μM each primer pair, and 10 μl 2× Platinum SYBR Green Quantitative PCR Supermix-UDG (Invitrogen). Cycling conditions were as follows: one cycle of 50°C for 2 min and one cycle of 95°C for 2 min, followed by 45 cycles of 94°C for 5 s, 60°C for 10 s, and 72°C for 40 s. The real-time PCR was performed using a Rotor-Gene 3000 PCR machine (Corbett Research, Mortlake, New South Wales, Australia). The data were analyzed with Rotor-Gene Real Time Analysis software (Corbett Research). Each sample was analyzed in duplicate and normalised to actin mRNA PCR amplification: The specific primers used to amplify the open reading frame (ORF) from each gene of interest are indicated below (Table 1). To facilitate cloning, restriction enzyme sites were introduced in the forward and reverse primers. Platinum SuperFi II DNA Polymerase–High-Fidelity PCR Enzyme was used to amplify each gene as per the manufacturer’s protocol with 50 ng of V. bioethanolium cDNA or genomic Y. fairybreadus DNA used as the template. Amplicons were electrophoresed on a 1.5% agarose gel with a 100bp Ladder Molecular Weight Marker (Fisher scientific). The PCR-amplified fragment was purified using the QIAquick PCR Purification Kit (Qiagen) and cloned in-frame with both the N-terminal green fluorescent protein (GFP) and the C-terminal His tag. E. coli DH5α cells were transformed with the recombinant plasmid and plated onto Luria Bertani (LB) agar plates containing Kanamycin (50 µg/ml) or Ampicillin (100 µg/ml) as appropriate for the plasmid. Plates were incubated at 37°C overnight and then stored at 4°C. Table 1. PCR Primers used for cloning target (rest. enzyme tag) Forward primer: 5’ to 3’ (rest. enzyme tag) Reverse primer: 5’ to 3’ Product size V. bioethanolium PDC (EcoRI) nngaattcATGGGGGC CCCCCGGGG (BamHI) nnggatccTTATTTT TATTTTTATTTTTATTTT 1048 V. bioethanolium ADH (EcoRI) nngaattc ATGGACAC GGTCTAGCAGATCG (BamHI) nnggatccCTAGTCG CGTGATATGATTTA 1084 V. bioethanolium Laccase (BamHI) nnggatccATGGTATC CTGTATCGGAACAT (XhoI) nnctcgagTTAAATTTA ATAATGAGAATGTC 835 Y. fairybreadus xylose- specific 100s’n’1000s operon (HindIII) nnAAGCTTnnATGGT