Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production

AbstractBranched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

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Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

10.1101/2020.03.08.982801 ◽

2020 ◽

Cited By ~ 1

Author(s):

Yanfei Zhang ◽

Sarah K. Hammer ◽

Cesar Carrasco-Lopez ◽

Sergio A. Garcia Echauri ◽

José L. Avalos

Keyword(s):

Amino Acid ◽

Amino Acid Metabolism ◽

Metabolic Flux ◽

Acid Metabolism ◽

Transcriptional Regulator ◽

Higher Alcohol ◽

Alcohol Production ◽

Branched Chain Amino Acid ◽

Branched Chain ◽

Genetically Encoded Biosensor

AbstractBranched-chain amino acid (BCAA) metabolism can be harnessed to produce many valuable chemicals. Among these, isobutanol, which is derived from valine degradation, has received substantial attention due to its promise as an advanced biofuel. While Saccharomyces cerevisiae is the preferred organism for isobutanol production, the lack of isobutanol biosensors in this organism has limited the ability to screen strains at high throughput. Here, we use a transcriptional regulator of BCAA biosynthesis, Leu3p, to develop the first genetically encoded biosensor for isobutanol production in yeast. Small modifications allowed us to redeploy Leu3p in a second biosensor for isopentanol, another BCAA-derived product of interest. Each biosensor is highly specific to isobutanol or isopentanol, respectively, and was used to engineer metabolic enzymes to increase titers. The isobutanol biosensor was additionally employed to isolate high-producing strains, and guide the construction and enhancement of mitochondrial and cytosolic isobutanol biosynthetic pathways, including in combination with optogenetic actuators to enhance metabolic flux. These biosensors promise to accelerate the development of enzymes and strains for branched-chain higher alcohol production, and offer a blueprint to develop biosensors for other products derived from BCAA metabolism.

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