Abstract
Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate–pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism.
| Original language | English |
|---|---|
| Article number | kuae024 |
| Journal | Journal of Industrial Microbiology and Biotechnology |
| Volume | 51 |
| DOIs | |
| State | Published - 2024 |
Funding
The work was authored under Triad National Security, LLC (\u201CTriad\u201D) Contract No. 89233218CNA000001 with the U.S. Department of Energy. This work was also partially authored by Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC, for the U.S. DOE under contract DE-AC05-00OR22725. This work was authored in part by Alliance for Sustainable Energy, LLC, the manager, and operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE-AC36-08GO28308. The authors thank Lorena Fernandez Cabezon and Taek Soon Lee at the Joint Bioenergy Institute for building the C. glutamicum plasmid pLFC007. The authors are grateful to William Henson, Christopher Johnson and Gregg Beckham from National Renewable Energy Laboratory for providing strain (RH189) and gene deletion plasmids (pRH80, pRH122) and for providing critical comments on the manuscript. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facilities operated for the U.S. Department of Energy (DOE) Office of Science. The work was supported through the Agile BioFoundry, which is funded by the US Department of Energy, Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office (BETO) under contract NL0032182.
| Funders | Funder number |
|---|---|
| Office of Science | |
| Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office | |
| Bioenergy Technologies Office | NL0032182 |
| Bioenergy Technologies Office | |
| U.S. Department of Energy | DE-AC05-00OR22725 |
| U.S. Department of Energy | |
| National Renewable Energy Laboratory | DE-AC36-08GO28308 |
| National Renewable Energy Laboratory |
Keywords
- Biomanufacturing
- Biosensor
- Chorismate
- Flow cytometry
- High-throughput screening
- Non-model organism
- Promoter optimization
- Tool transfer
- cis
- cis-muconic acid