skip to main content

Explore Research Products in the NSF-PAR

Title: Effective use of biosensors for high-throughput library screening for metabolite production
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
NSF-PAR ID:
10360972
Author(s) / Creator(s):
;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Journal of Industrial Microbiology and Biotechnology
Volume:
48
Issue:
9-10
ISSN:
1367-5435
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ( , Journal of Industrial Microbiology and Biotechnology)
    Abstract  

    Recent work in biosensors has shown promise to enable high throughput searches through large genetic libraries. However, just as physiological limitations and lack of in-depth mechanistic knowledge can prevent us from achieving high titers in microbial systems; similar roadblocks can appear in the application of biosensors. Here, we characterized a previously developed transcription-factor (ExuR) based galacturonate biosensor for its other cognate ligand, glucuronate. Though we saw an ideal response to glucuronate from the biosensor in controlled and ideal experimental circumstances, these results began to deviate from a well-behaved system when we explored the application of the sensor to different MIOX homologs. Through modifications to circuit architecture and culture conditions, we were able to decrease this variation and use these more optimal conditions to apply the biosensor for the separation of two closely related MIOX homologs.

     One-Sentence Summary

    In this work, a transcription-factor biosensor was investigated for its potential to screen a library of myo -inositol oxygenase variants while seeking to mitigate the impact the production pathway appeared to have on the biosensor.

     
    more » « less
  2. ; ; ; ; ; ; ; ( , Nature Communications)
    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
  3. ; ; ; ; ( , Nature Communications)
    Abstract

    Bacterial biosensors can enable programmable, selective chemical production, but difficulties incorporating metabolic pathways into complex sensor circuits have limited their development and applications. Here we overcome these challenges and present the development of fast-responding, tunable sensor cells that produce different pigmented metabolites based on extracellular concentrations of zinc (a critical micronutrient). We create a library of dual-input synthetic promoters that decouple cell growth from zinc-specific metabolite production, enabling visible cell coloration within 4 h. Using additional transcriptional and metabolic control methods, we shift the response thresholds by an order of magnitude to measure clinically relevant zinc concentrations. The resulting sensor cells report zinc concentrations in individual donor serum samples; we demonstrate that they can provide results in a minimal-equipment fashion, serving as the basis for a field-deployable assay for zinc deficiency. The presented advances are likely generalizable to the creation of other types of sensors and diagnostics.

     
    more » « less
  4. ; ; ; ( , Frontiers in Bioengineering and Biotechnology)
    Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ 54 -dependent, butanol-responsive TF–promoter pair, BmoR-P BMO derived from Thauera butanivorans , at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ 54 -dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development. 
    more » « less
  5. ; ; ; ( , Frontiers in bioengineering and biotechnology)
    Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ54-dependent, butanol-responsive TF–promoter pair, BmoR-PBMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized PBMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in PBMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ54-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email