| 1 |
Welcome and Introductions | We will introduce ourselves, and then discuss the design, content, and objectives of the course. In addition, available resources to search the primary research literature will be discussed. |
| 2 |
Cellulose Breakdown Basics | Cellulose is an energy-rich polymer that is difficult to hydrolyze. In this session, we will discuss the enzymology and properties of various cellulases and hemicellulases that are required to break down plant cell walls. We will also address complications that arise from contaminants found within natural lignin-complexed biomass that limit enzyme activity. |
| 3 |
Cellulolytic Bacteria and Fungi | Nature is well-equipped to degrade cellulosic biomass. Some degredation normally occurs in microbiomes of high cellulose turnover (compost piles, pond sludge, herbivore digesta), where a consortium of microbes expressing different proteins work together to degrade the material. In this session, we will examine how anaerobic microbes hydrolyze cellulose by the expression and secretion of specialized enzymes. |
| 4 |
Transcriptional Regulation | Cellulolytic organisms generally do not expend the energy to make proteins that degrade cellulose if a simpler carbon source is present. Transcriptional regulation is at the heart of cellulolytic protein production in these organisms. We will discuss two fundamental studies that shed light on the mechanisms of this regulation in native cellulolytic microbes. |
| 5 |
Recombinant Cellulases | Organisms that naturally produce cellulose-degrading enzymes are notoriously difficult to culture and generally cannot be incorporated into a large-scale fermentation process. However, attractive genes can be introduced into model microbial systems to facilitate enzyme production and purification. We will discuss two relevant examples of heterologous cellulase expression from E. coli and S. cerevisiae, paying special attention to the titers and catalytic efficiency of enzymes produced in these different model systems. |
| 6 |
Synthetic Cellulosomes | Anaerobic rumen microbes are known to produce cellulosomes, which are large molecular weight complexes of many proteins that work together to break down plant cell wall material in a targeted manner. Recently, several research groups have attempted to emulate this architecture artificially in yeast. We will learn about the first successful study in which functional cellulosome chimeras were produced, as well as a strategy to construct and analyze free cellulosomes. |
| 7 |
Guest speaker: Industrial Biomass Processing and Hydrolysis | Prof. Chris Kaiser, who is a member of the Scientific Advisory Board at Mascoma® (a local biofuels company), will discuss issues related to industrial biomass processing and hydrolysis. |
| 8 |
Metabolic Engineering | Model microbes like yeast and E. coli have been well-adapted to industrial processing, but several difficulties exist that limit their ability to grow and proliferate while breaking down cellulosic biomass. We will focus on recent genetic approaches that have been implemented to decrease product inhibition in yeast. In addition, we will learn about methods to artificially construct entire metabolic pathways in E. coli to produce butanol. |
| 9 |
Engineering Cellulolytic Organisms | Considerable effort has been made to develop techniques that would adapt native cellulolytic organisms to industrial processing. We will discuss two examples from in which novel transformation procedures have been pioneered to achieve higher ethanol product titers. |
| 10 |
Directed Evolution and Protein Engineering | One method to increase the catalytic efficiency of cellulases is to alter the primary sequence of a known enzyme and screen for enhanced reactivity against cellulose. We will discuss computational and chemical mutagenesis approaches that have been used to increase the catalytic efficiency of known cellulases and will compare these approaches to each other. |
| 11 |
Identifying New Cellulases from Nature | To increase the economic feasibility of cellulosic biofuels, it is necessary to find better enzymes to hydrolyze cellulose that can be adapted for bioprocessing. However, conventional gene/protein discovery approaches are not well-suited for this purpose, as organisms that produce these enzymes are difficult to isolate. We will discuss two different strategies based on metagenomics, which have led to the discovery and evaluation of novel cellulases from soil and termite microbiomes. |
| 12 |
Oral Presentations | Students will present their research paper reviews, and we will wrap-up the course with a final discussion. |
