Proteomic analysis of formaldehyde toxicity and engineered formaldehyde utilization in Methylobacterium extorquens
EMSL Project ID
49856
Abstract
Methylotrophy involves the production of formaldehyde, a highly toxic metabolite, as an intermediate. It is critical to understand how cells sense and respond to formaldehyde levels, as well as the constraints it places upon the natural and engineered systems. Here we propose to use proteomics to examine the broad cellular response to these two related challenges in the model organism, Methylobacterium extorquens. The Marx lab has discovered a novel regulatory protein, EfgA, conserved across methylotrophs. EfgA directly binds formaldehyde and interacts with the ribosome to slow or shut down translation; this is the only example we are aware of in which a protein directly senses metabolites to control ribosomal activity. We have also discovered EfgB, a putative cyclase that produces cGMP, a secondary messenger with very little known role in bacteria. EfgB appears to be involved in the response to protein damage. Through quantitative analyses of the proteome in different mutants and growth conditions we seek to elucidate the broader cellular response that ensues when cells face formaldehyde toxicity. Furthermore, we have succeeded in engineering/evolving M. extorquens to grow on methanol using the foreign ribulose monophosphate (RuMP) formaldehyde assimilation pathway. Although the evolved isolates are still slower than wild-type (WT), this is the first success in achieving methanol-dependent growth via the RuMP pathway in a heterologous host1,2. Currently, we are further optimizing RuMP-dependent methanol growth via a library of 104 gene-edited variants (see below) to combinatorially alter promoter strengths throughout the pathway. Analysis of the proteome in the evolved and highly-fit edited isolates will provide insight into the cellular response and remaining challenges to effective utilization of this pathway.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2017-10-01
End Date
2018-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)