Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum

Leßmeier L, Wendisch VF. Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum. BMC Microbiology. 2015;15(1): 216.Background Methanol 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. Results The 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. Conclusion The 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

Metabolic engineering of Corynebacterium glutamicum toward the utilization of methanol

Leßmeier L. Metabolic engineering of Corynebacterium glutamicum toward the utilization of methanol. Bielefeld; 2015

Additional file 1: Table S1. of Identification of two mutations increasing the methanol tolerance of Corynebacterium glutamicum

Genome-wide comparison of mRNA levels between the strains Tol1 and C. glutamicum WT(pVWEx1) during growth in LB complex medium. Table S2. Genome-wide comparison of mRNA levels in Tol1 and C. glutamicum wild type cultivated in minimal medium with 100 mM glucose in presence or absence of methanol. Table S3. Mutations detected in strain Tol1 and a control strain in comparison to the C. glutamicum wild type genome sequence NC_006958.1. Table S4. Oligonucleotides used in this study. Figure S1. Growth in the presence of formaldehyde (A) and formaldehyde degradation (B). Figure S2. Growth of C. glutamicum wild type with propionate and methanol. Figure S3. Growth of C. glutamicum ΔramA with methanol. Figure S4. Growth of C. glutamicum Δcat with methanol. Figure S5. Biomass formation of C. glutamicum wild type, Tol1, T2840 and Δcat in minimal medium with ethanol as sole carbon source. (PDF 919 kb

Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase

Leßmeier L, Höfener M, Wendisch VF. Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase. Microbiology. 2013;159(Pt_12):2651-2662.Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the actinomycetes, is able to degrade formaldehyde but the enzyme(s) involved in this detoxification process were not known. Acetaldehyde dehydrogenase Ald, which is essential for ethanol utilization, and FadH, characterized here as NAD-linked mycothiol-dependent formaldehyde dehydrogenase, were shown to be responsible for formaldehyde oxidation since a mutant lacking ald and fadH could not oxidize formaldehyde resulting in the inability to grow when formaldehyde was added to the medium. Moreover, C. glutamicum ΔaldΔfadH did not grow with vanillate, a carbon source giving rise to intracellular formaldehyde. FadH from C. glutamicum was purified from recombinant Escherichia coli and shown to be active as a homotetramer. Mycothiol-dependent formaldehyde oxidation revealed Km values of 0.6 mM for mycothiol and 4.3 mM for formaldehyde and a Vmax of 7.7 U mg(-1). FadH from C. glutamicum also possesses zinc-dependent, but mycothiol-independent alcohol dehydrogenase activity with a preference for short chain primary alcohols such as ethanol (Km = 330 mM, Vmax = 9.6 U mg(-1)), 1-propanol (Km = 150 mM, Vmax = 5 U mg(-1)) and 1-butanol (Km = 50 mM, Vmax = 0.8 U mg(-1)). Formaldehyde detoxification system by Ald and mycothiol-dependent FadH is essential for tolerance of C. glutamicum to external stress by free formaldehyde in its habitat and for growth with natural substrates like vanillate, which are metabolized with concomitant release of formaldehyde

Regulatory associations between the metabolism of sulfur-containing amino acids and xanthan biosynthesis in Xanthomonas campestris pv. campestris B100.

Schulte F, Leßmeier L, Voß J, Ortseifen V, Vorhölter F-J, Niehaus K. Regulatory associations between the metabolism of sulfur-containing amino acids and xanthan biosynthesis in Xanthomonas campestris pv. campestris B100. FEMS microbiology letters. 2019;366(2): fnz005

Metabolic Engineering of Corynebacterium glutamicum for Alternative Carbon Source Utilization

Leßmeier L, Zahoor ul Hassan A, Lindner S, Wendisch VF. Metabolic Engineering of Corynebacterium glutamicum for Alternative Carbon Source Utilization. In: Burkovski A, ed. Corynebacterium glutamicum: From Systems Biology to Biotechnological Applications. Norfolk: Caister Academic Press; 2015: 57-70

Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate

Leßmeier L, Pfeifenschneider J, Carnicer M, Heux S, Portais J-C, Wendisch VF. Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate. Applied Microbiology and Biotechnology. 2015;99(23):10163-10176

Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris

Leßmeier L, Alkhateeb R, Schulte F, et al. Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris. Journal of Biotechnology. 2016;232:89-98.At a molecular level, the regulation of many important cellular processes is still obscure in xanthomonads, a bacterial group of outstanding relevance as world-wide plant pathogens and important for biotechnology as producers of the polysaccharide xanthan. Transcriptome analysis indicated a sucrose-dependent regulation of 18 genes in Xanthomonas campestris pv. campestris (Xcc) B100. The expression of 12 of these genes was clearly increased in the presence of sucrose. Only part of these genes was obviously involved in sucrose utilization. To identify regulatory proteins involved in transcriptional regulation, a DNA fragment-specific pull-down approach was established for Xcc. Putative promoter regions were identified and used to isolate DNA-binding proteins, which were separated by SDS PAGE and identified by MALDI-TOF mass spectrometry. This led to the identification of four transcriptional regulators, among them the global transcriptional regulator Clp and a previously identified regulator of sucrose utilization, SuxR, plus a third DNA-binding transcriptional regulator encoded by xcc-b100_2861 and recently shown to interact with a cyclic di-GMP-binding protein. The fourth regulatory protein was encoded by xcc-b100_2791. These results indicate DNA fragment-specific pull-down experiments as promising approaches to screen for specific DNA-binding regulatory proteins in Xcc. (C) 2016 Elsevier B.V. All rights reserved