Man-made genes help yeast turn agricultural waste biomass into methyl halides
US researchers have developed a new way to engineer microorganisms to use biomass to produce methyl halides, simple chemicals used as agricultural fumigants and precursor molecules for complex chemicals and fuels.
Processing non-food biomass into fuels and high-value chemicals could provide a sustainable alternative to petrochemicals. With this in mind, a team of researchers used a new approach to engineer yeasts and bacteria to produce methyl halides from sources such as corn stover, bagasse and switchgrass.
Chris Voigt and colleagues at the University of California, San Francisco, used a technique called synthetic metagenomics to discover new enzymes that could produce methyl halides.
Synthetic metagenomics has been made possible by the gene sequencing revolution and recent advances in DNA synthesis technology. ’Databases of sequence information are growing very fast,’ says Voigt, ’but it’s not always clear what the functions of these sequences are, so it’s now a question of how to harness that information, and that’s where DNA synthesis really comes in.’ Synthesis is now cheap enough that researchers can trawl the database for anything that looks like it might code for a desired enzyme, synthesise all the the resulting sequences and screen them for function.
This allows researchers to exploit enzymes from organisms which would be hard to culture in the lab, or which aren’t even expressed naturally. ’We can retrieve the DNA without ever touching the organism,’ explains Voigt, ’so it’s becoming much closer to an information-based science.’
Certain algae, fungi and seaweeds naturally produce methyl halides using enzymes called methyl halide transferases (MHTs). Unfortunately, these are unsuitable for industrial application owing to difficulties in culturing and poor yields. By searching sequence databases, however, the team found 89 different gene sequences that were similar to known MHTs, coming from a variety of bacteria, plants and fungi.
Wolfgang Streit, from the University of Hamburg, is impressed by the results: ’The fascinating thing is that, in the database, only one of these genes was annotated as a methyl halide transferase, and they have pulled out all these other pieces of DNA and shown that they are also transferases.’ He adds that it would be even more useful if the team could persuade the microbes to selectively halogenate or methylate more complex, valuable molecules.
When the team synthesised all 89 genes and expressed them in E. coli bacteria to test their ability to produce methyl halides from sugars, the performance was very varied, with most enzymes favouring production of either methyl bromide or iodide. The enzyme from the shore-living beachwort plant, Batis maritima, gave the highest yields of methyl chloride, bromide and iodide, making it the most versatile of the bunch.
To make a more robust process, the team inserted the gene into brewer’s yeast, which is more tolerant of the methyl halide products and well-suited to scale-up of the process. ’We coupled the yeast with a bacterium from a French landfill, which is very good at degrading different forms of cellulosic biomass,’ explains Voigt. ’It can eat waste materials like sugar cane bagasse and corn stover, and it has the unique ability to excrete a large portion of what it eats in the form of acetate. The yeast can take that acetate and turn it into methyl halide, so there’s a symbiotic relationship because the bacterium is inhibited by the acetate it produces. The yeast removes that toxic acetate, so they grow to a very stable concentration in the bioreactor.’
Because the methyl halides produced are volatile, they can simply be condensed out of the gas stream coming out of the top of the reactor. This makes the process very streamlined, and it is very easy to harvest and purify the products without having to disturb the reactors.
Now that they have proven the principle of their synthetic metagenomics approach, Voigt plans to apply it to a host of other problems. ’We eventually want to start combining enzymes that are not present together [in nature], to put together whole pathways that are capable of all sorts of transformations.’
et alJ. Am. Chem. Soc. 2009 DOI:10.1021/ja809461u