Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an l-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate l-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

Protein–carbohydrate interactions play crucial roles in biology. Understanding and modifying these interactions is of major interest for fighting many diseases. We took a synthetic biology approach and incorporated noncanonical amino acids into a bacterial lectin to modulate its interactions with carbohydrates. We focused on tryptophan, which is prevalent in carbohydrate binding sites. The exchange of the tryptophan residues with analogs fluorinated at different positions resulted in three distinctly fluorinated variants of the lectin from Ralstonia solanacearum. We observed differences in stability and affinity toward fucosylated glycans and rationalized them by X-ray and modeling studies. While fluorination decreased the aromaticity of the indole ring and, therefore, the strength of carbohydrate–aromatic interactions, additional weak hydrogen bonds were formed between fluorine and the ligand hydroxyl groups. Our approach opens new possibilities to engineer carbohydrate receptors.

To efficiently use lignocellulosic biomass hydrolysates as fermentation media for bioethanol production, besides being capable of producing significant amount of ethanol, the fermenting host should also meet the following two requirements: (1) resistant to the inhibitory compounds formed during biomass pretreatment process, (2) capable of utilizing C5 sugars, such as xylose, as carbon source. In our laboratory, a screening was conducted on microorganisms collected from environmental sources for their tolerance to hydrolysate inhibitors. A unique resistant strain was selected and identified as Pichia anomala (Wickerhamomyces anomalus), deposited as CBS 132101. The strain is able to produce ethanol in various biomass hydrolysates, both with and without oxygen. Besides, the strain could assimilate xylose and use nitrate as N source. These physiological characteristics make P. anomala an interesting strain for bioethanol production from lignocellulosic biomass hydrolysates.