Researchers at the US Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) have identified microbial genes that can improve both the tolerance and the production of isopentanol in engineered strains of Escherichia coli. Isopentenol is a five-carbon (C5) alcohol that is a highly promising candidate for biogasoline, but, like other short-chained alcohols, is toxic to E.coli at commercial levels of fuel production.
Aindrila Mukhopadhyay, a chemist who directs the host engineering program for JBEI’s Fuels Synthesis Division, led a study in which transcriptomic data and a synthetic metabolic pathway were used to identify several genes that not only improve tolerance but also production of isopentenol in E.coli. MetR, the methionine biosynthesis regulator, improved the titer for isopentenol production by 55%, while MdlB, the ABC transporter, facilitated a 12% improvement in isopentenol production.
The toxicity to microbes of many of the best candidate compounds for advanced biofuels presents a “production versus survival” conundrum. (Accordingly, the Mukhopadhyay Group has also separately been investigating the engineering of microbial transport systems, such as efflux pumps, to export a broad range of substrates including solvents and to provide a direct engineering route to relieve fuel accumulation-related stress to and improve production titer. The group has used a high-throughput approach to create a library of efflux pumps in E. coli that can confer tolerance to many candidate fuels.)
In order for microbial biofuel production to be cost effective, yields must exceed native microbial tolerance levels, necessitating the development of solvent-tolerant microbial strains. In parallel with improved tolerance it is also crucial that we improve production.
Our study demonstrates that microbial tolerance engineering using transcriptomics data can be used to identify target genes that improve fuel production. Our targets include a regulator for amino acid biosynthesis, and an ABC transporter protein, the first native transporter that improves tolerance to a short-chain alcohol.—Aindrila Mukhopadhyay
Mukhopadhyay, who also holds an appointment with the Lawrence Berkeley National Laboratory Physical Biosciences Division, is the corresponding author of a paper describing this study in press in the open access journal mBio. Co-authors are Heather Jensen, Jee Loon Foo, Robert Dahl, Kevin George, Jay Keasling, Taek Soon Lee and Susanna Leong.
|The overexpression of E. coli genes displaying tolerance to isopentenol increased production of this leading biogasoline candidate in a production strain of the microbes. Click to enlarge.|
To this end, Mukhopadhyay and her group in this new study used transcriptomic data—a measurement of differential expression of gene transcripts in a given genome—to identify 40 E. coli genes that showed increase when exposed to externally added isopentenol. These genes were then overexpressed in E. coli to evaluate their potential for improving isopentenol tolerance. Genes conferring isopentenol tolerance were then co-expressed individually with an isopentenol production metabolic pathway in E. coli to determine which would increase productivity as well.
Mukhopadhyay and her group are especially eager to further investigate the MdlB transporter, which they believe, as the first native transporter gene shown to improve production of a short-chain alcohol, will provide a valuable new avenue for host engineering in biogasoline production.
The critical point is that you must first identify the genes that can serve as engineering targets, and then test them to find which ones work best. Now that we have identified MdlB as a target, we are going to examine it in great depth to see how can we improve its function and optimize its use in a production microbe.—Aindrila Mukhopadhyay
This research was supported by the DOE Office of Science. JBEI is a DOE Office of Science Bioenergy Research Center led by Berkeley Lab.
Foo JL, Jensen HM, Dahl RH, George K, Keasling JD, Lee TS, Leong SSJ, Mukhopadhyay A (2014) “Improving microbial bio-gasoline production in Escherichia coli using tolerance engineering,” mBio, in press