Report
Teaser, summary, work performed and final results
Periodic Reporting for period 3 - CUSTOM-SENSE (Custom-made biosensors – Accelerating the transition to a bio-based economy)
Teaser
How will we meet the globally growing demand for pharmaceutically active compounds, nutrients and fine chemicals when crude oil resources are dwindling? For decades, biotechnologists have beenengineering microorganisms to produce valuable compounds from sugar and biomass...
Summary
How will we meet the globally growing demand for pharmaceutically active compounds, nutrients and fine chemicals when crude oil resources are dwindling? For decades, biotechnologists have been
engineering microorganisms to produce valuable compounds from sugar and biomass. 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.
We want to establish a platform to engineer transcriptional biosensors for the intracellular detection of heterologous compounds in single cells. The application of these sensors in combination with flow
cytometry and next-generation sequencing will enable high-throughput engineering of microorganisms at the single-cell level with unprecedented speed and simplicity.
In the field of biotechnology, this new technology will be a powerful tool for the:
- accelerated directed evolution of genes and pathways in vivo
- functional integration of heterologous genes or whole synthetic pathways into the metabolism of microorganisms for the production of small valuable metabolites
- genome engineering of industrially relevant microorganisms
- adaptation of production strains to process conditions
Furthermore, during CUSTOM-SENSE, biosensors will also prove to be a valuable tool to answer questions in basic science because they will help to elucidate the function of unknown genes and aid the discovery of
novel and unexpected functional links in cellular metabolism.
Work performed
So far the project has resulted in multiple tangible results. The universal biosensor design has been applied to several regulators, and we could show that constitutive promotors for controlling transcriptional regulator expression can be used for sensor response fine-tuning. This is an important first step before functional biosensors can be transferred to new host system. The manuscript for the universal biosensor design is currently in preparation, but additional experiments to also show the positive effects of the modulated regulator gene expression are needed.
The FapR-based biosensor for malonyl-CoA-detection can be used to detect relative differences of intracellular malonyl-CoA concentrations. However, malonyl-CoA is difficult to quantify in cytoplasmatic extracts, but right as we write this report, we were successful to set up an LC-MS/MS method, which allows discrimination between different malonyl-CoA accumulating C. glutamicum strains. After validation of this new analytic protocol, we will plot obtained biosensor response signals to the intracellular malonyl-CoA concentration of various metabolically engineered C. glutamicum strains for further characterization of this promising biosensor. The manuscript focusing on the development of the malony-CoA biosensor and its application in the context of microbial plant natural product synthesis is planned for July, 2018
Regarding pSenHcaR for quantification of cinnamic acid, an extremely high-throughput screening method has been developed and implemented that was used successfully employed for discovering variants with an altered ligands specificity. In addition, an FACS-based HT-screening method was designed and developed to discover improved ammonia lyase variants. Several key properties of the FACS screening approach were optimized. Using inappropriate conditions might have prevented the discovery of improved enzyme variants in previous screening campaigns, and adaptation of the FACS screening conditions represents a promising starting point for biosensor screening campaigns in general. Currently, work is in progress for fine-tuning this biosensor-screening method to obtain significantly improved ammonia lyase variants and neglect false positive ammonia lyase variants. The enzyme variants already disovered variants are currently characterized in detail.
After finalizing these experiments the manuscript will be written (May, 2018)
A highly promising LysG-variant, now allowing for the detection of L-histidine was discovered and the regulator variant was characterized in great detail. Currently, this biosensor is used in a screening for L-histidine accumulating C. glutamicum variants. Such production strains would be 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) is almost finished (planned submission February 2018) and after finalization of the experiments regarding the pSenLys biosensor variants suitable for FACS-based screenings at elevated amino acid concentrations we are planning an additional manuscript.
Final results
This project will yield a novel family of 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, already aid in the proposed different applications (protein engineering, pathway engineering and genome engineering) and first results (improved ammonia lyases and improved histidine production strains) are available and first publications are currently prepared.
In the second half of the project we now continue to also improve our comparative genome-sequence analysis capabilities to identify underlying genomic mutations in the isolated production strains.