Microbial Characterization Facility

Lead Project PI: Adam P. Arkin
Lead Campus: UC Berkeley

Bacteria are an attractive alternative to yeast for industrial level fuel ethanol production and perhaps the production of other types of fuels such as lipids and diesel. While, currently, few bacteria are close to the efficiency of fungi in the production of ethanol, recent findings show that rational engineering can greatly increase output. Bacteria like Zymomonas mobilis, a successful model microbe for ethanol production, have been moderately engineered and selected for industrial output of ethanol. However, there have been no systematic genomic or high-throughput genetic studies to elucidate how other metabolic and stress pathways affect the process in bacteria. Rational engineering of bacteria to optimize ethanol production or any fuel molecule requires a thorough, systems-level understanding of the how bacterial metabolism, gene regulation, and stress response influence this process. Our project will develop a facility for these studies and apply them to key biofuel relevant organisms starting with Zymomonas mobilis.

Building on our long expertise in the systems biology of microbes, we are building an experimental and computational comparative microbial systems biology infrastructure that we can apply widely to identify, understand and engineer the central pathways in bacteria and fungi that are important to biomass production and fermentation into biofuels. The approach combines functional genomics in the form of gene expression and metabolic analysis, high throughput genetics, and quantitative dynamic analysis of pathway function using highly controlled and reproducibly perturbed microbial cultures for elucidating the interactions between individual genes, pathways, and the environment. The core experimental technologies involve extensive use of custom high-density tiling microarrays for genome annotation and inference of regulatory elements, custom oligonucleotide microarrays for gene expression, metabolite analysis, and the parallel, quantitative analysis of thousands of sequence defined mutants using a molecular barcoding strategy. In addition to providing genetic resources for any microorganism, our proposed facility will enable automated sample preparation as well as automated, high-throughput microbial experiments (using liquid handling robotics and microplate readers). These experimental efforts will be supported by the development of a sophisticated computational infrastructure whose beginnings can be found in the MicrobesOnline (http://microbesonline.org) and RegTransBase (http//regtransbase.lbl.gov). For this project, these tools will be deepened with more genomes, better metabolic and stress gene annotation, regulatory network curation and inference, and functional genomic analysis tools, and tools for metabolic inference and engineering. Together, the facility will generate a quantitative, systems-level view of microbial physiology that will serve as the blueprint for the rational engineering of these organisms to meet bioenergy challenges.

Our first target is to use this framework to improve ethanol production in Zymomonas mobilis. Until very recently, the only commercial fermentations have relied on yeast and other super-fungi that have been identified as excellent producers of ethanol from C5 sugars and other carbon sources. However, for some time it has been recognized that the facultative anaerobe Zymomonas mobilis can have a higher yield and faster specific rate of ethanol production when compared to yeast. In addition, yeast has a higher aeration cost, a high biomass production, and low temperature and ethanol tolerance compared to Zymomonas. Its yield of ethanol from sugar, derived from the Entner-Doudoroff pathway, is around 96%, the same as yeast. Because of its industrial potential, DuPont and others have dedicated themselves to scaling up Zymomonas for commercial fermentation of lignocellulosic residues such as corn stover. Zymomonas has already, for over a decade, been a target of metabolic engineering for utilization of diverse C5 sugars such as xylose and arabinose that are present in lignocellulosic hydrolysates.

A number of components in hydrolysates can inhibit the growth and ethanol production of bacteria and yeast. While in Z. mobilis the major inhibitor is usually acetate, other products such as vanillin, syringaldehyde, hydroxymethyl-furfural and furfural all show significant inhibitory effects. These inhibitors, and ethanol itself, have differential effects on growth and on ethanol production and have different behaviors depending on the sugars being utilized. In fact, the osmotic stress of sugars themselves can have a strong affect on the productivity of fermentation and different sugars have different uptake rates and saturation points. Further, the growth mode of Z. mobilis can have a strong effect on its productivity. Immobilized and suspended cultures have very different productivities and the use of flocculent cultures can increase volumetric productivity by as much as ten-fold. Efforts to optimize Z. mobilis for production are limited because a detailed understanding of all the physiological factors affecting metabolism including stress responses and inessential secondary metabolic pathways are not currently available. Identifying the genetic factors that contribute to these effects is a main aim of this proposal.