MICRO$EC: Cost Effective, Whole-Genome Sequencing

While the feasibility of whole human genome sequencing was proven by the success of the Human Genome Project several years ago, the prevalence of personal genome sequencing in the medical industry is still elusive due to its unrealistic cost and time requirements. Micro$eq is a startup company with the goal of overcoming these limitations by sequencing a minimum of 12 complete human genomes per day at an error rate less than ten parts in million at a profitable market price of less than US$1000 per genome. To overcome the technology bottlenecks hindering current biotech companies from achieving these target throughput, error rate, and market price goals, Micro$eq has developed an innovative sequencing technique that uses shortread fragments with high coverage on a microfluidics platform. Short, amplified DNA fragments are generated from an input of customer saliva. 6 base pair(bp) sequence hybridization is used for sequencing each of the DNA fragments individually. The results are these hydridization reads are then assembled via de Bruijn graph theory and the graphical reconstructions of each fragment’s sequence are then assembled to a complete genome via shotgun sequencing with an expected error rate less than 1 in 100,000bp. Upon the completion of financial analysis, both a small-scale business model producing 72 genomes per day at US$999 per genome, and a largescale business model producing 52.2 genomes per year at a market price of US$299 per genome were found to be profitable, yielding Micro$eq investors return margins of ~90% and 300% for the small and large scale models, respectively. With a market price Micro$eq offers personal genome sequencing at one-tenth of its nearest potential competitor’s cost. Additionally, its ability for bulk-sequencing allows it to profitably venture into the previously untapped Pharmaceutical Industry market sector, enabling the creation of large-scale genome databases which are the next step forward in the quest for truly personalized

Neither 1 G nor 2 G fuel ethanol: setting the ground for a sugarcane-based biorefinery using an iSUCCELL yeast platform

First-generation (1 G) fuel ethanol production in sugarcane-based biorefineries is an established economic enterprise in Brazil. Second-generation (2 G) fuel ethanol from lignocellulosic materials, though extensively investigated, is currently facing severe difficulties to become economically viable. Some of the challenges inherent to these processes could be resolved by efficiently separating, and partially hydrolysing the cellulosic fraction of the lignocellulosic materials into the disaccharide cellobiose. Here we propose an alternative biorefinery, where the sucrose-rich stream from the 1 G process is mixed with a cellobiose-rich stream in the fermentation step. The advantages of mixing are threefold: 1) decreased concentrations of metabolic inhibitors that are typically produced during pretreatment and hydrolysis of lignocellulosic materials; 2) decreased cooling times after enzymatic hydrolysis prior to fermentation; 3) decreased availability of free glucose for contaminating microorganisms and undesired glucose repression effects. The iSUCCELL platform will be built upon the robust Saccharomyces cerevisiae strains currently present in 1 G biorefineries, which offer competitive advantage in non-aseptic environments, and into which intracellular hydrolyses of sucrose and cellobiose will be engineered. It is expected that high yields of ethanol can be achieved in a process with cell recycling, lower contamination levels and decreased antibiotic use, when compared to current 2 G technologies

Energy Homeostasis in Cellobiose Utilizing Saccharomyces cerevisiae

Plant biomass is a promising renewable starting material for chemical and fuel derivations. Complete consumption of sugar components in the plant cell wall is essential for a cost effective process design. The yeast Saccharomyces cerevisiae can be engineered to co-consume cellobiose and xylose, a scheme that allows simultaneous consumption of sugar content from cellulose and hemicellulose polysaccharides, decreasing overall process time. As the reactions are expected to occur in anaerobic conditions given the large-scale nature of the process, cellobiose phosphorylase (CBP), an enzyme that catalyzes intracellular cellobiose breakdown in anaerobic bacteria was studied and showed energetic benefits in anaerobic conditions.However, xylose was identified as a mixed-inhibitor and a substrate forming a byproduct of the CBP reaction. Protein engineering of CBP partially relieved the negative impact of xylose. A single distal mutation on CBP altered its susceptibility to xylose, potentially by modifying spatial conformation of CBP. In parallel, a novel xylose synthetic pathway involving xylulose-1-phosphate was tested, in an attempt to increase the xylose consumption rate, decrease cellular xylose and its negative impact on CBP. However, the existing xylose utilization pathway outperformed the synthetic pathway, likely because exogenous gene integration interfered with the existing regulatory network. Finally, cellobiose consumption was used as a minimal 2-gene synthetic biology pathway to explore cell physiology. This pathway induced drastic changes in cellular metabolites, allowing for the identification of novel regulation controlling ATP levels in cells. Uncoupling extracellular glucose sensors Snf3/Rgt2 from carbon utilization revealed a novel role of Snf3/Rgt2 in ATP regulation at the transcriptional level, potentially via the master regulator Gcn4. This regulation acted independently of proton pumping by Pma1, which is a known to be a major source of ATP consumption in yeast cells, but whose regulation remains to be determined. Together, these two pathways set an upper bound on ATP concentrations needed for optimal fermentation

Energy Homeostasis in Cellobiose Utilizing Saccharomyces cerevisiae

Plant biomass is a promising renewable starting material for chemical and fuel derivations. Complete consumption of sugar components in the plant cell wall is essential for a cost effective process design. The yeast Saccharomyces cerevisiae can be engineered to co-consume cellobiose and xylose, a scheme that allows simultaneous consumption of sugar content from cellulose and hemicellulose polysaccharides, decreasing overall process time. As the reactions are expected to occur in anaerobic conditions given the large-scale nature of the process, cellobiose phosphorylase (CBP), an enzyme that catalyzes intracellular cellobiose breakdown in anaerobic bacteria was studied and showed energetic benefits in anaerobic conditions.However, xylose was identified as a mixed-inhibitor and a substrate forming a byproduct of the CBP reaction. Protein engineering of CBP partially relieved the negative impact of xylose. A single distal mutation on CBP altered its susceptibility to xylose, potentially by modifying spatial conformation of CBP. In parallel, a novel xylose synthetic pathway involving xylulose-1-phosphate was tested, in an attempt to increase the xylose consumption rate, decrease cellular xylose and its negative impact on CBP. However, the existing xylose utilization pathway outperformed the synthetic pathway, likely because exogenous gene integration interfered with the existing regulatory network. Finally, cellobiose consumption was used as a minimal 2-gene synthetic biology pathway to explore cell physiology. This pathway induced drastic changes in cellular metabolites, allowing for the identification of novel regulation controlling ATP levels in cells. Uncoupling extracellular glucose sensors Snf3/Rgt2 from carbon utilization revealed a novel role of Snf3/Rgt2 in ATP regulation at the transcriptional level, potentially via the master regulator Gcn4. This regulation acted independently of proton pumping by Pma1, which is a known to be a major source of ATP consumption in yeast cells, but whose regulation remains to be determined. Together, these two pathways set an upper bound on ATP concentrations needed for optimal fermentation

Cellobiose Consumption Uncouples Extracellular Glucose Sensing and Glucose Metabolism in Saccharomyces cerevisiae

Glycolysis is central to energy metabolism in most organisms, and is highly regulated to enable optimal growth. In the yeast Saccharomyces cerevisiae, feedback mechanisms that control flux through glycolysis span transcriptional control to metabolite levels in the cell. Using a cellobiose consumption pathway, we decoupled glucose sensing from carbon utilization, revealing new modular layers of control that induce ATP consumption to drive rapid carbon fermentation. Alterations of the beta subunit of phosphofructokinase (PFK2), H+-plasma membrane ATPase (PMA1), and glucose sensors (SNF3, RGT2) revealed the importance of coupling extracellular glucose sensing to manage ATP levels in the cell. Controlling the upper bound of cellular ATP levels may be a general mechanism used to regulate energy levels in cells, via a regulatory network that can be uncoupled from ATP concentrations under perceived starvation conditions

Cellobiose Consumption Uncouples Extracellular Glucose Sensing and Glucose Metabolism in Saccharomyces cerevisiae

Glycolysis is central to energy metabolism in most organisms, and is highly regulated to enable optimal growth. In the yeast Saccharomyces cerevisiae, feedback mechanisms that control flux through glycolysis span transcriptional control to metabolite levels in the cell. Using a cellobiose consumption pathway, we decoupled glucose sensing from carbon utilization, revealing new modular layers of control that induce ATP consumption to drive rapid carbon fermentation. Alterations of the beta subunit of phosphofructokinase (PFK2), H+-plasma membrane ATPase (PMA1), and glucose sensors (SNF3, RGT2) revealed the importance of coupling extracellular glucose sensing to manage ATP levels in the cell. Controlling the upper bound of cellular ATP levels may be a general mechanism used to regulate energy levels in cells, via a regulatory network that can be uncoupled from ATP concentrations under perceived starvation conditions

Bypassing the Pentose Phosphate Pathway: Towards Modular Utilization of Xylose.

The efficient use of hemicellulose in the plant cell wall is critical for the economic conversion of plant biomass to renewable fuels and chemicals. Previously, the yeast Saccharomyces cerevisiae has been engineered to convert the hemicellulose-derived pentose sugars xylose and arabinose to d-xylulose-5-phosphate for conversion via the pentose phosphate pathway (PPP). However, efficient pentose utilization requires PPP optimization and may interfere with its roles in NADPH and pentose production. Here, we developed an alternative xylose utilization pathway that largely bypasses the PPP. In the new pathway, d-xylulose is converted to d-xylulose-1-phosphate, a novel metabolite to S. cerevisiae, which is then cleaved to glycolaldehyde and dihydroxyacetone phosphate. This synthetic pathway served as a platform for the biosynthesis of ethanol and ethylene glycol. The use of d-xylulose-1-phosphate as an entry point for xylose metabolism opens the way for optimizing chemical conversion of pentose sugars in S. cerevisiae in a modular fashion

Additional file 1. of Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae

Supplementary figures

Relief of Xylose Binding to Cellobiose Phosphorylase by a Single Distal Mutation

Cellobiose phosphorylase (CBP) cleaves cellobioseabundant in plant biomassto glucose and glucose 1-phosphate. However, the pentose sugar xylose, also abundant in plant biomass, acts as a mixed-inhibitor and a substrate for the reverse reaction, limiting the industrial potential of CBP. Preventing xylose, which lacks only a single hydroxymethyl group relative to glucose, from binding to the CBP active site poses a spatial challenge for protein engineering, since simple steric occlusion cannot be used to block xylose binding without also preventing glucose binding. Using CRISPR-based chromosomal library selection, we identified a distal mutation in CBP, Y47H, responsible for improved cellobiose consumption in the presence of xylose. <i>In silico</i> analysis suggests this mutation may alter the conformation of the cellobiose phosphorylase dimer complex to reduce xylose binding to the active site. These results may aid in engineering carbohydrate phosphorylases for improved specificity in biofuel production, and also in the production of industrially important oligosaccharides