Researchers in Professor José Avalos’s lab at Princeton University have developed a new technique to isolate high-producing strains of yeast, identify mutant enzymes with enhanced activity, and construct biosynthetic pathways for production of isobutanol and isopentanol. The tool makes the genetic selection process thousands of times faster and can supercharge the development of advanced biofuels.

An open-access paper on their work is published in the journal Nature Communications.

The metabolism of branched chain amino acids (BCAAs), including valine, leucine, and isoleucine is central to microbial production of many valuable products. Among microbial hosts that produce BCAAs and BCAA-derived compounds, the yeast Saccharomyces cerevisiae is a favored industrial organism due to its genetic tractability, resistance to phage contamination, and ability to grow at low pH and high alcohol concentrations. However, the rate at which genetic diversity can be introduced in strains greatly outpaces the rate at which they can be screened for production; and, unfortunately, there are currently no eukaryotic biosensors specific for individual BCAAs and their derived products, which could be used to develop high-throughput screens and accelerate strain development.

… There is great interest in producing BCHAs [branched-chain higher alcohols], as they are among the top ten advanced biofuels identified by the U.S. Department of Energy for their potential to boost gasoline engine efficiency, and can be upgraded to renewable jet fuel. BCHA production can be enhanced by overexpressing enzymes from the Ehrlich degradation pathway. In this pathway, α-ketoacid decarboxylases (α-KDCs) and alcohol dehydrogenases (ADHs) convert the α-ketoacid precursors α-KIV, α-KIC, and α-K3MV to isobutanol, isopentanol, and 2-methyl-1-butanol, respectively.

Here, we report the development and application of a genetically encoded biosensor for eukaryotic BCAA metabolism and BCHA biosynthesis. The biosensor is based on the α-IPM-dependent function of Leu3p to control expression of GFP. Two biosensor configurations enable high-throughput screening for improved isobutanol or isopentanol production. These two configurations differ mechanistically, by monitoring α-IPM as either a by-product of isobutanol or a precursor of isopentanol. We apply the biosensor to isolate high-producing strains, identify mutant enzymes with enhanced activity, and construct biosynthetic pathways for production of isobutanol and isopentanol in both mitochondria and cytosol. This biosensor has the potential to accelerate the development of yeast strains to produce BCHAs as well as other products derived from BCAA metabolism.

—Zhang et al.

The research began with the challenge of speeding up the development of yeast strains for isobutanol and isopentanol production. The rate at which scientists can introduce genetic diversity in yeast greatly outpaces the rate at which they can screen each strain to find those with increased biofuel production. Researchers thus had to figure out which genes to turn on or off and what enzymes or proteins were beneficial to the process using very slow, laborious and expensive methods.

The researchers genetically engineering the cells to produce a fluorescent protein when they were making chemicals for biofuels. Scientists could then use the fluorescence as a sensor to look for production.

We essentially harnessed this transcription factor so that when the cell is producing more biofuel, it also turns on production of a fluorescent protein. It’s doing what it normally does, but now we can see the cell respond to enhanced metabolic activity. We are no longer blind. Now, we’re measuring hundreds of thousands of strains per minute. This is a several-orders-of-magnitude faster way to identify better strains.

—José Avalos, corresponding author

Resources

• Zhang, Y., Cortez, J.D., Hammer, S.K. et al. (2022) “Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production.” Nat Commun 13, 270 doi: 10.1038/s41467-021-27852-x