Biosensor-based engineering of biosynthetic pathways.

Biosynthetic pathways provide an enzymatic route from inexpensive renewable resources to valuable metabolic products such as pharmaceuticals and plastics. Designing these pathways is challenging due to the complexities of biology. Advances in the design and construction of genetic variants has enabled billions of cells, each possessing a slightly different metabolic design, to be rapidly generated.

Engineering an allosteric transcription factor to respond to new ligands.

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery.

Synthetic biosensors for precise gene control and real-time monitoring of metabolites.

Characterization and standardization of inducible transcriptional regulators has transformed how scientists approach biology by allowing precise and tunable control of gene expression. Despite their utility, only a handful of well-characterized regulators exist, limiting the complexity of engineered biological systems. We apply a characterization pipeline to four genetically encoded sensors that respond to acrylate, glucarate, erythromycin and naringenin.

Evolution-guided optimization of biosynthetic pathways.

Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers.

Anti-glycophorin single-chain Fv fusion to low-affinity mutant erythropoietin improves red blood cell-lineage specificity.

The presence of erythropoietin (Epo) receptors on cells besides red blood cell precursors, such as cancer cells or megakaryocyte precursors, can lead to side effects during Epo therapy including enhanced tumor growth and platelet production. It would be ideal if the action of Epo could be limited to erythroid precursors. To address this issue, we constructed single-chain variable fragment (scFv)-Epo fusion proteins that used the anti-glycophorin 10F7 scFv amino-terminal to Epo analogues that would have minimal activity alone.