skip to main content


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

Branched-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 fromSaccharomyces cerevisiaeto 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.

 
more » « less
Award ID(s):
1751840
NSF-PAR ID:
10361601
Author(s) / Creator(s):
; ; ; ; ; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Nature Communications
Volume:
13
Issue:
1
ISSN:
2041-1723
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    The development of fast and affordable microbial production from recombinant pathways is a challenging endeavor, with targeted improvements difficult to predict due to the complex nature of living systems. To address the limitations in biosynthetic pathways, much work has been done to generate large libraries of various genetic parts (promoters, RBSs, enzymes, etc.) to discover library members that bring about significantly improved levels of metabolite production. To evaluate these large libraries, high throughput approaches are necessary, such as those that rely on biosensors. There are various modes of operation to apply biosensors to library screens that are available at different scales of throughput. The effectiveness of each biosensor-based method is dependent on the pathway or strain to which it is applied, and all approaches have strengths and weaknesses to be carefully considered for any high throughput library screen. In this review, we discuss the various approaches used in biosensor screening for improved metabolite production, focusing on transcription factor-based biosensors.

     
    more » « less
  2. ABSTRACT  
    more » « less
  3. Abstract

    The lipid-derived hormones jasmonates (JAs) play key functions in a wide range of physiological and developmental processes that regulate growth, secondary metabolism and defense against biotic and abiotic stresses. In this connection, biosynthesis, tissue-specific distribution, metabolism, perception, signaling of JAs have been the target of extensive studies. In recent years, the involvement of JAs signaling pathway in the regulation of growth and adaptive responses to environmental challenges has been further examined. However, JAs-mediated mechanisms underlying the transition from ‘growth mode’ to ‘adaptive mode’ remain ambiguous. Combined analysis of transgenic lines deficient in JAs signaling in conjunction with the data from JAs-treated plants revealed the function of these hormones in rewiring of central metabolism. The collective data illustrate JAs-mediated decrease in the levels of metabolites associated with active growth such as sucrose, raffinose, orotate, citrate, malate, and an increase in phosphorylated hexoses, responsible for the suppression of growth and photosynthesis, concurrent with the induction of protective metabolites, such as aromatic and branched-chain amino acids, and aspartate family of metabolites. This finding provides an insight into the function of JAs in shifting the central metabolism from the production of growth-promoting metabolites to protective compounds and expands our understanding of the role of JAs in resource allocation in response to environmental challenges.

     
    more » « less
  4. Abstract

    Gas fermentation has emerged as a technologically and economically attractive option for producing renewable fuels and chemicals from carbon monoxide (CO) rich waste streams. LanzaTech has developed a proprietary strain of the gas fermentating acetogenClostridium autoethanogenumas a microbial platform for synthesizing ethanol, 2,3‐butanediol, and other chemicals. Bubble column reactor technology is being developed for the large‐scale production, motivating the investigation of multiphase reactor hydrodynamics. In this study, we combined hydrodynamics with a genome‐scale reconstruction ofC. autoethanogenummetabolism and multiphase convection–dispersion equations to compare the performance of bubble column reactors with and without liquid recycle. For both reactor configurations, hydrodynamics was predicted to diminish bubble column performance with respect to CO conversion, biomass production, and ethanol production when compared with bubble column models in which the gas phase was modeled as ideal plug flow plus axial dispersion. Liquid recycle was predicted to be advantageous by increasing CO conversion, biomass production, and ethanol and 2,3‐butanediol production compared with the non‐recycle reactor configuration. Parametric studies performed for the liquid recycle configuration with two‐phase hydrodynamics showed that increased CO feed flow rates (more gas supply), smaller CO gas bubbles (more gas–liquid mass transfer), and shorter column heights (more gas per volume of liquid per time) favored ethanol production over acetate production. Our computational results demonstrate the power of combining cellular metabolic models and two‐phase hydrodynamics for simulating and optimizing gas fermentation reactors.

     
    more » « less
  5. Abstract Background

    The increasing prevalence of plastic waste combined with the inefficiencies of mechanical recycling has inspired interest in processes that can convert these waste streams into value-added biomaterials. To date, the microbial conversion of plastic substrates into biomaterials has been predominantly limited to polyhydroxyalkanoates production. Expanding the capabilities of these microbial conversion platforms to include a greater diversity of products generated from plastic waste streams can serve to promote the adoption of these technologies at a larger scale and encourage a more sustainable materials economy.

    Results

    Herein, we report the development of a new strain ofPseudomonasbacteria capable of converting depolymerized polyethylene into high value bespoke recombinant protein products. Using hexadecane, a proxy for depolymerized polyethylene, as a sole carbon nutrient source, we optimized media compositions that facilitate robust biomass growth above 1 × 109 cfu/ml, with results suggesting the benefits of lower hydrocarbon concentrations and the use of NH4Cl as a nitrogen source. We genomically integrated recombinant genes for green fluorescent protein and spider dragline-inspired silk protein, and we showed their expression inPseudomonas aeruginosa, reaching titers of approximately 10 mg/L when hexadecane was used as the sole carbon source. Lastly, we demonstrated that chemically depolymerized polyethylene, comprised of a mixture of branched and unbranched alkanes, could be converted into silk protein byPseudomonas aeruginosaat titers of 11.3 ± 1.1 mg/L.

    Conclusion

    This work demonstrates a microbial platform for the conversion of a both alkanes and plastic-derived substrates to recombinant, protein-based materials. The findings in this work can serve as a basis for future endeavors seeking to upcycle recalcitrant plastic wastes into value-added recombinant proteins.

     
    more » « less