Periodic Reporting for period 4 - CUSTOM-SENSE (Custom-made biosensors – Accelerating the transition to a bio-based economy) Reporting period: 2019-11-01 to 2021-04-30 Summary of the context and overall objectives of the project How will we meet the globally growing demand for pharmaceutically active compounds, nutrients and fine chemicals and how can we produce them more sustainable? For decades, biotechnologists have been engineering microorganisms to produce valuable chemical products from sugars. However, a lack of knowledge regarding the host cell metabolism as well as long and laborious development times render this approach challenging to this day. A couple of years ago, biosensors emerged as powerful molecular tools to screen large cell libraries for improved production performance. Such biosensors convert the intracellular product concentration into a fluorescence signal and allow for mutant isolation when combined with a fluorescence-activated cell sorting. However, application of such biosensors is often limited by its specificity profile - either no biosensor sensing the product of interest is available, or the biosensor is rather unspecific.In CUSTOM-SENSE, we established a technology platform to directly engineer the specificity spectrum of biosensors. This allowed us not only to develop several new biosensors for biotechnologically interesting chemicals, but enabled us also to sharpen the specificity profile of a biosensor. These new custom-made biosensors were combined with fluorescence-activated cell sorting and directly applied to accelerate the directed evolution of enzymes for natural product synthesis, and to engineer a bacterium for the production of the essential amino acid histidine. Beyond applications, the developed biosensors also proved to to be valuable tools in basic science as they helped us to discover novel and unexpected functional links in the prokaryotic metabolism.With this technology at hand, laborious developments times for high-performance production strains could be significantly shortened, which would support Europe´s transition to more sustainable economy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This project yielded novel custom-made biosensors, which are, due to the new universal design, easy to modify and adapt to optimally work in the respective desired screening campaign. The biosensors can be easily transferred to other organisms, broadening their range of possible applications. The biosensors, specifically constructed in this project, helped in the context of different applications (protein engineering, pathway engineering and genome engineering) and will be of great use for us and colleagues active in these fields. Furthermore, we developed a novel approach to evolve biosensors via a "transition ligand" approach. This new technique will allow researchers to develop custom-made biosensors for their own applications. Biosensors of CUSTOM-SENSE make bacteria glow when they produce chemicals useful to us humans.