Reza Khankal
1. Understanding xylose uptake in Escherichia coli under conditions of high xylose concentrations
2. Characterizing the influence of a cAMP-independent CRP mutant on metabolism and xylitol production in E. coli
Efficient microbial conversion of biomass into renewable fuels and value-added chemicals remains an important goal in biotechnology. Xylose, which is the second most abundant sugar in nature and a major constituent of hemicellulose in lignocellulosic biomass and wastes, is a good target for these microbial processes. Escherichia coli is capable of utilizing a wide range of sugars as carbon and energy sources and producing native metabolites and non-native compounds. In E. coli, xylose uptake occurs primarily through a high-affinity, ATP-binding cassette transporter (xylFGH), although a second, low-affinity proton symporter (xylE) is also present. The efficiency of xylose utilization in this organism is therefore suboptimal due to energetic requirements for xylose uptake. E. coli has been engineered to uptake xylose and reduce it to xylitol in the presence of glucose by expressing xylose reductase (XR) and a cAMP-independent CRP mutant (CRP*). These studies showed that while xylose is negligibly metabolized by wild-type E. coli in the presence of glucose (classic diauxic growth), low levels of xylose transport and xylitol production are possible in wild-type E. coli expressing XR. Xylose transport and xylitol production are greatly improved in crp* mutant strains. These results indicate that either the native xylose transporters are not tightly controlled by CRP or additional transport mechanisms exist.
Known xylose transporters in E.coli
In order to understand and ultimately engineer the energetics of xylose uptake and metabolism in E. coli, we would like to identify and characterize other promiscuous (or perhaps specific) transporters involved. Conversion of xylose to xylitol (which is secreted) provides a measure of xylose transport without requiring further xylose metabolism. With this system we are able to study the influence of different native and heterologous transporters (deleted or overexpressed) on xylose uptake under various conditions (e.g. in the presence of glucose) uncoupled from constraints associated with xylose metabolism or growth on other substrates.
Studies using strains deficient in one or both xyl transport systems demonstrate the existence of at least one other, low-affinity xylose uptake mechanism. This secondary uptake is not apparently affected by individual deletions in other known, promiscuous transporters. Our results indicate that xylose transport in the presence of glucose is not mediated by XylE or XylFGH (CRP control over these genes is tight), and secondary xylose transport is upregulated in the context of a crp* genotype, in addition to XylE and XylFGH. Changes in specific growth rate and yield in our transporter-modified strains are helping to understand and improve the energetics of xylose uptake. Transcription analyses will help us to identify genes which may be up-regulated or involved in xylose transport under different conditions (e.g. in the presence of xylose or a glucose-xylose mixture, xyl deletions, or CRP*). To further understand metabolism in the context of the crp* mutation, we are also analyzing the transcription profiles of crp* strains grown under various conditions and comparing these profiles to those of wild-type and crp mutant strains.
Overview of xylitol production in E.coli
Poster Presented at the Society for
Industrial Microbiology 2006 Annual Meeting
"Understanding and Improving Xylose Utilization in Escherichia coli".pdf