Identification of Transcriptional Regulators for a Novel Glyoxylate Production Pathway in Methylobacterium extorquens AM1 (LB255)

Abstract

Methylobacterium extorquens AM1 is of interest as a platform for production of biofuels and biodegradable plastics from methanol. Biofuels such as 1‐butanol can be derived from intermediates in the Ethylmalonyl‐CoA pathway; however, sequestration of these intermediates for biofuel production limits the cell’s ability to produce glyoxylate, a metabolite required to power carbon assimilation. A second glyoxylate production pathway, the Oxalyl‐CoA Reductase pathway, exists but is mainly operational during oxalate growth. Use of this pathway may compensate for loss of glyoxylate during 1‐butanol production but an understanding of pathway regulation is needed to enhance cell engineering efforts. Expression studies showed upregulation of this pathway by exogenous oxalate and glyoxylate and QscR was identified as a potential transcriptional activator using transposon mutagenesis. Subsequent studies showed direct binding to the Oxalyl‐CoA Reductase pathway promoter by QscR. Interestingly, growth with succinate prevented upregulation in vivo and neither oxalate nor glyoxylate affected binding in vitro, consistent with the presence of an additional regulator. Future studies will use transposon mutagenesis to identify mutations that cause an increase or decrease in fluorescence from an Oxalyl‐CoA Reductase pathway promoter fusion identifying any additional transcriptional regulators. These studies will aid our ability to engineer M. extorquens to use the alternative glyoxylate production pathway to supply cellular glyoxylate, freeing the Ethylmalonyl‐CoA pathway for biofuel and bioplastic production.Grant Funding Source: Supported by California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Award and Louis Stokes Alliances for Minority Participation (LSAMP)