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

   https://doi.org/10.1038/s41467-021-27852-x  

   Zhang, Yanfei ; Cortez, Jeremy D. ; Hammer, Sarah K. ; Carrasco-López, César ; García Echauri, Sergio Á. ; Wiggins, Jessica B. ; Wang, Wei ; Avalos, José L. ( January 2022 , Nature Communications)

   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.
2. Dynamic and tunable metabolite control for robust minimal-equipment assessment of serum zinc

   https://doi.org/10.1038/s41467-019-13454-1  

   McNerney, Monica P. ; Michel, Cirstyn L. ; Kishore, Krishi ; Standeven, Janet ; Styczynski, Mark P. ( December 2019 , Nature Communications)

   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.
3. Elucidation of Sequence–Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli

   Kim, Nancy M. ; Sinnott, Riley W. ; Rothschild, Lily N. ; Sandoval Nicholas R. ( October 2022 , 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-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 σ⁵⁴-dependent promoter biosensor in the context of themore »host. This work can enable high-throughput screening methods for strain development.« less
4. Elucidation of Sequence–Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli

   https://doi.org/10.3389/fbioe.2022.821152  

   Kim, Nancy M. ; Sinnott, Riley W. ; Rothschild, Lily N. ; Sandoval, Nicholas R. ( February 2022 , 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 54more »-dependent promoter biosensor in the context of the host. This work can enable high-throughput screening methods for strain development.« less
5. A high-throughput skim-sequencing approach for genotyping, dosage estimation and identifying translocations

   https://doi.org/10.1038/s41598-022-19858-2  

   Adhikari, Laxman ; Shrestha, Sandesh ; Wu, Shuangye ; Crain, Jared ; Gao, Liangliang ; Evers, Byron ; Wilson, Duane ; Ju, Yoonha ; Koo, Dal-Hoe ; Hucl, Pierre ; et al ( October 2022 , Scientific Reports)

   The development of next-generation sequencing (NGS) enabled a shift from array-based genotyping to directly sequencing genomic libraries for high-throughput genotyping. Even though whole-genome sequencing was initially too costly for routine analysis in large populations such as breeding or genetic studies, continued advancements in genome sequencing and bioinformatics have provided the opportunity to capitalize on whole-genome information. As new sequencing platforms can routinely provide high-quality sequencing data for sufficient genome coverage to genotype various breeding populations, a limitation comes in the time and cost of library construction when multiplexing a large number of samples. Here we describe a high-throughput whole-genome skim-sequencing (skim-seq) approach that can be utilized for a broad range of genotyping and genomic characterization. Using optimized low-volume Illumina Nextera chemistry, we developed a skim-seq method and combined up to 960 samples in one multiplex library using dual index barcoding. With the dual-index barcoding, the number of samples for multiplexing can be adjusted depending on the amount of data required, and could be extended to 3,072 samples or more. Panels of doubled haploid wheat lines (Triticum aestivum, CDC Stanley x CDC Landmark), wheat-barley (T.aestivumxHordeum vulgare) and wheat-wheatgrass (Triticum durum x Thinopyrum intermedium) introgression lines as well as known monosomicmore »wheat stocks were genotyped using the skim-seq approach. Bioinformatics pipelines were developed for various applications where sequencing coverage ranged from 1 × down to 0.01 × per sample. Using reference genomes, we detected chromosome dosage, identified aneuploidy, and karyotyped introgression lines from the skim-seq data. Leveraging the recent advancements in genome sequencing, skim-seq provides an effective and low-cost tool for routine genotyping and genetic analysis, which can track and identify introgressions and genomic regions of interest in genetics research and applied breeding programs.

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