Indigo is an ancient dye uniquely capable of producing the signature tones in blue denim; however, the dyeing process requires chemical steps that are environmentally damaging. We describe a sustainable dyeing strategy that not only circumvents the use of toxic reagents for indigo chemical synthesis but also removes the need for a reducing agent for dye solubilization. This strategy utilizes a glucose moiety as a biochemical protecting group to stabilize the reactive indigo precursor indoxyl to form indican, preventing spontaneous oxidation to crystalline indigo during microbial fermentation. Application of a β-glucosidase removes the protecting group from indican, resulting in indigo crystal formation in the cotton fibers. We identified the gene coding for the glucosyltransferase PtUGT1 from the indigo plant Polygonum tinctorium and solved the structure of PtUGT1. Heterologous expression of PtUGT1 in Escherichia coli supported high indican conversion, and biosynthesized indican was used to dye cotton swatches and a garment.

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker's yeast Saccharomyces cerevisiae. From a single round of mutagenesis of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of the cis,cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion-of-function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a prokaryotic chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions.

The development of robust enzymes, in particular cellulases, is a key step in the success of biological routes to `second-generation' biofuels. The typical sources of the enzymes used to degrade biomass include mesophilic and thermophilic organisms. The endoglucanase J30 from glycoside hydrolase family 9 was originally identified through metagenomic analyses of compost-derived bacterial consortia. These studies, which were tailored to favor growth on targeted feedstocks, have already been shown to identify cellulases with considerable thermal tolerance. The amino-acid sequence of J30 shows comparably low identity to those of previously analyzed enzymes. As an enzyme that combines a well measurable activity with a relatively low optimal temperature (50°C) and a modest thermal tolerance, it offers the potential for structural optimization aimed at increased stability. Here, the crystal structure of wild-type J30 is presented along with that of a designed triple-mutant variant with improved characteristics for industrial applications. Through the introduction of a structural Zn²⁺ site, the thermal tolerance was increased by more than 10°C and was paralleled by an increase in the catalytic optimum temperature by more than 5°C.