Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum

Abstract

BackgroundMethanol is present in most ecosystems and may also occur in industrial applications, e.g. as an impurity of carbon sources such as technical glycerol. Methanol often inhibits growth of bacteria, thus, methanol tolerance may limit fermentative production processes.ResultsThe methanol tolerance of the amino acid producing soil bacterium Corynebacterium glutamicum was improved by experimental evolution in the presence of methanol. The resulting strain Tol1 exhibited significantly increased growth rates in the presence of up to 1 M methanol. However, neither transcriptional changes nor increased enzyme activities of the linear methanol oxidation pathway were observed, which was in accordance with the finding that tolerance to the downstream metabolites formaldehyde and formate was not improved. Genome sequence analysis of strain Tol1 revealed two point mutations potentially relevant to enhanced methanol tolerance: one leading to the amino acid exchange A165T of O-acetylhomoserine sulfhydrolase MetY and the other leading to shortened CoA transferase Cat (Q342*). Introduction of either mutation into the genome of C. glutamicum wild type increased methanol tolerance and introduction of both mutations into C. glutamicum was sufficient to achieve methanol tolerance almost indistinguishable from that of strain Tol1.ConclusionThe methanol tolerance of C. glutamicum can be increased by two point mutations leading to amino acid exchange of O-acetylhomoserine sulfhydrolase MetY and shortened CoA transferase Cat. Introduction of these mutations into producer strains may be helpful when using carbon sources containing methanol as component or impurity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0558-6) contains supplementary material, which is available to authorized users.

Evolutionary engineering of Corynebacterium glutamicum

“…In the methanol study, tolerance was increased by using media with 50 mM methanol. [24] The evolved strain showed a higher growth rate under methanol concentrations between 500 mM and 2000 mM. Sequencing identified 29 mutations and combined engineering of two of them, metY (A165T) and cat(Q342*), increased the methanol tolerance of the parental strain to that of the evolved strain.…”

“…After 206 days, the evolved strain was able to grow, but the reported growth rate of 0.03 h −1 is still far away from industrial applications. Interestingly, the authors describe the metY (G1256A) mutation; another mutation in the same gene ( metY (A165T)) was also found in the methanol tolerance study . An elegant approach to realize methanol‐essential growth was recently reported for E. coli .…”

“…The increased glucose consumption rate probably resulted from a higher ptsI expression, which codes for a component of the phosphotransferase system (PTS). In the methanol study, tolerance was increased by using media with 50 m m methanol . The evolved strain showed a higher growth rate under methanol concentrations between 500 m m and 2000 m m .…”

“…Two different studies applied rbALE to increase tolerance for inhibitors present in corn stover hydrolysate [23] and for methanol, which is an impurity found in certain glycerol waste streams. [24] A fixed amount of inhibitor was added, and cells were selected for growth. The corn stover evolved strain showed a higher degradation rate of the inhibitors furfural, HMF, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, and acetic acid.…”

“…Overview of different objectives in C. glutamicum ALE studies. ALE has successfully been applied to increase the growth rate, substrate utilization, stress tolerance, and small molecule production . Growth coupling strategies have been used to increase precursor supply and small molecule production …”