CUSTOM-SENSE has resulted in multiple tangible results. The universal biosensor design has been applied to several regulators, and we could show that constitutive promoters for controlling transcriptional regulator expression can be used for sensor fine-tuning. This is an important first step before functional biosensors can be transferred to new host system. In addition, we could develop a CRISPR-Cas System for our host system, which also aided during our biosensor-development.
Regarding pSenHcaR for quantification of cinnamic acid, a high-throughput (HT) screening method has been developed and implemented that was used successfully for discovering variants with altered ligand specificities. In addition, a HT-screening method was designed and developed to discover improved ammonia lyase variants.
Furthermore, the pSenHcaR-regulator turned out to be very evolvable. We successfully designed and used a novel strategy to obtain various biosensor variants with different specificities by using a completely new "transition ligand" approach. By following this strategy, four biosensor with a very different specificity profile could be obtained.
Furthermore, a malonyl-CoA biosensor was developed, which enabled us to generated bacterial strains with an enhanced intracellular malonyl-CoA availability. Patenting these findings delayed the publication of the results, but gave us the chance to isolate more productive strain variants starting from the already improved variant of the first round. We are currently doing the reconstruction of the identified mutagenic hot spots to also finalize a publication. The future will show, whether these findings can be exploited. Malonyl-CoA is key precursor of many plant natural products with pharmacological activities. Hence, we hope that the beneficial genomic mutations identified and filed for a patent application by us, have a positive impact on the formation of these valuable compounds. If so, they will be hopefully introduced into large-scale production strains constructed for commercialization.
A highly promising LysG-variant, now allowing for the detection of L-histidine was discovered and the regulator variant was characterized in detail. Subsequently, this biosensor was used in a screening for L-histidine accumulating C. glutamicum variants. Such production strains are of great interest, as microbial production strains have not been developed for this valuable amino acid yet. The manuscript describing the development of the engineered pSenLys-biosensor with a focused ligand spectrum (pSenHis) was published in Nature Communications and well-received by scientists of the field.
In addition to the publications and the patent, the results of CUSTOM-SENSE were communicated to a broader scientific audience. Since 2017, the two-week lab course taught by the PI at the RWTH Aachen University contains a larger biosensor-focused experiment to directly teach the biosensor concepts developed in CUSTOM-SENSE to the new generation of scientists. Furthermore, since 2016, the PI teaches at the advanced course “Advanced Course Microbial Physiology and Fermentation Technology” at the University of Delft, The Netherlands, once a year. The audience is very international (USA, EU, UK) and typically takes part at this course at the scientist-level. In this course, the PI introduces biosensors as novel tools for microbial strain development and always relates to the scientific work performed within CUSTOM-SENSE. In addition to the special courses and workshops, all CUSTOM-SENSE coworkers presented their work in the form of oral talks or posters at more than 20 conferences. Furthermore, CUSTOM-SENSE has been presented to a broader audience at the “Open Day” of the Forschungszentrum Jülich and in the news magazine of the Helmholtz-society.
