Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

• Published in: Applied microbiology and biotechnology Vol. 96; no. 1; pp. 283 - 297
• Main Authors: Lu, Jingnan, Brigham, Christopher J., Gai, Claudia S., Sinskey, Anthony J.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01-10-2012; Springer; Springer Nature B.V
• Subjects: Alcohol; Alcohols; Amino acids; Analysis; Biodiesel fuels; Bioenergy and Biofuels; Biofuels; Biomedical and Life Sciences; Biosynthesis; Biotechnology; Branched chain amino acids; Butanols - metabolism; Butanols - toxicity; Carbon sequestration; Carbon sinks; Cultivation; Cupriavidus necator - genetics; Cupriavidus necator - growth & development; Cupriavidus necator - metabolism; Dehydrogenases; Enzymes; Ethanol; Gene expression; Genes; Gram-negative bacteria; Hydroxybutyrates - metabolism; Life Sciences; Metabolic Engineering; Metabolic Networks and Pathways - genetics; Microbial Genetics and Genomics; Microbiology; Nutrients; Pentanols - metabolism; Plasmids; Polyesters - metabolism; Polyhydroxybutyrate; Ralstonia eutropha; Studies; Toxicity; 3-Methyl-1-butanol; Biofuel; Branched-chain amino acid; Isobutanol; Branched-chain alcohol
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-012-4320-9

Wild-type
Ralstonia eutropha
H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of
R. eutropha
with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered
R. eutropha
. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type
R. eutropha
. After the elimination of genes encoding three potential carbon sinks (
ilvE
,
bkdAB
, and
aceE
), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the
R. eutropha
mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that
R. eutropha
carbon flux can be redirected from PHB to branched-chain alcohols and that engineered
R. eutropha
can be cultivated over prolonged periods of time for product biosynthesis.