Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida

Thompson, M. G.; Pearson, A. N.; Barajas, J. F.; Cruz-Morales, P.; Sedaghatian, N.; Costello, Z.; Garber, M. E.; Incha, M. R.; Valencia, L. E.; Baidoo, E.; Martin, H. G.; Mukhopadhyay, A.; Keasling, J. D.

doi:10.1101/700484

bioRxiv · 10.1101/700484

Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida

Thompson, M. G.·Pearson, A. N.·Barajas, J. F.·Cruz-Morales, P.·Sedaghatian, N.·Costello, Z.·Garber, M. E.·Incha, M. R.·Valencia, L. E.·Baidoo, E.·Martin, H. G.·Mukhopadhyay, A.·Keasling, J. D.

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Abstract

Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000x more sensitive to exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 g/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ~6 g/L. The high sensitivity and range of OplR is a powerful tool towards the development of novel routes to the biological synthesis of caprolactam. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/700484v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@ff5b8corg.highwire.dtl.DTLVardef@d365aaorg.highwire.dtl.DTLVardef@b79b18org.highwire.dtl.DTLVardef@27732f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Thompson, M. G., Pearson, A. N., Barajas, J. F., Cruz-Morales, P., Sedaghatian, N., Costello, Z., Garber, M. E., Incha, M. R., Valencia, L. E., Baidoo, E., Martin, H. G., Mukhopadhyay, A., Keasling, J. D.. 2019-07-16. Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida. https://doi.org/10.1101/700484

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