Production of carbon-13-labeled cadaverine by engineered Corynebacterium glutamicum using carbon-13-labeled methanol as co-substrate.
Methanol, a one-carbon compound, can be utilized by a variety of bacteria and other organisms as carbon and energy source and is regarded as a promising substrate for biotechnological production. In this study, a strain of non-methylotrophic Corynebacterium glutamicum, which was able to produce the polyamide building block cadaverine as non-native product, was engineered for co-utilization of methanol. Expression of the gene encoding NAD+-dependent methanol dehydrogenase (Mdh) from the natural methylotroph Bacillus methanolicus increased methanol oxidation. Deletion of the endogenous aldehyde dehydrogenase genes ald and fadH prevented methanol oxidation to carbon dioxide and formaldehyde detoxification via the linear formaldehyde dissimilation pathway. Heterologous expression of genes for the key enzymes hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase of the ribulose monophosphate (RuMP) pathway in this strain restored growth in the presence of methanol or formaldehyde, which suggested efficient formaldehyde detoxification involving RuMP key enzymes. While growth with methanol as sole carbon source was not observed, the fate of 13C-methanol added as co-substrate to sugars was followed and the isotopologue distribution indicated incorporation into central metabolites and in vivo activity of the RuMP pathway. In addition, 13C-label from methanol was traced to the secreted product cadaverine. Thus, this synthetic biology approach led to a C. glutamicum strain that converted the non-natural carbon substrate methanol at least partially to the non-native product cadaverine.
Genetics of Prokaryotes, Faculty of Biology and Center for Biotechnology, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
Lennart
L
Leßmeier
Genetics of Prokaryotes, Faculty of Biology and Center for Biotechnology, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany.
Johannes
J
Pfeifenschneider
LISBP-INSA de Toulouse, 135 avenue de Rangueil, 31077, Toulouse CEDEX 04, France.
Marc
M
Carnicer
LISBP-INSA de Toulouse, 135 avenue de Rangueil, 31077, Toulouse CEDEX 04, France.
Stephanie
S
Heux
LISBP-INSA de Toulouse, 135 avenue de Rangueil, 31077, Toulouse CEDEX 04, France.
Jean-Charles
JC
Portais
Genetics of Prokaryotes, Faculty of Biology and Center for Biotechnology, Bielefeld University, Universitätsstraße 25, 33615, Bielefeld, Germany. volker.wendisch@uni-bielefeld.de.
Volker F
http://orcid.org/0000-0003-3473-0012
VF
Wendisch
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D002103:Cadaverine / Q000378:metabolism*; D002247:Carbon Isotopes / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D007553:Isotope Labeling; D060847:Metabolic Engineering; D000432:Methanol / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002247:Carbon Isotopes; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D000446:Aldehyde-Lyases; C021355:hexose phosphate synthetase; D019747:Aldose-Ketose Isomerases; C023918:phospho-3-hexuloisomerase; D002103:Cadaverine; D000432:Methanol
13C-labeling; Bacillus methanolicus; Cadaverine; Corynebacterium glutamicum; Diaminopentane; Methanol; Methylotrophy