Abstract
BACKGROUND: Hydrocarbon-based biofuels, known as drop-in fuels, which are chemically similar to petroleum, have gained significant attention. Microorganisms that produce medium-chain alkanes hold promise for the bioproduction of drop-in fuels. Previous studies identified Klebsiella sp. NBRC100048 as having aldehyde-decarbonylating activity, enabling it to convert aldehydes into alkanes. Using a genomic fosmid library from Klebsiella sp. NBRC100048, we identified open reading frame 2991 (orf2991), which catalyzes the conversion of tetradecanal to tridecane. This gene shares high sequence similarity with the aldehyde dehydrogenase (ALDH) family in Escherichia coli. RESULTS: ALDH homolog genes from Klebsiella sp. NBRC100048 and E. coli W3110 were cloned and expressed in E. coli to assess their potential alkane-synthesizing activity. Approximately one-fifth of the tested enzymes exhibited this function, with basic local alignment search tool (BLAST) analysis classifying them under the phenylacetaldehyde dehydrogenase, succinate-semialdehyde dehydrogenase, or aldehyde dehydrogenase B families. Testing additional ALDH homologs from diverse organisms-bacteria, fungi, plants, and animals-revealed that ALDHs with alkane-synthesizing activity are widespread, occurring in Gram-positive bacteria, actinomycetes, lactic acid bacteria, and yeast species. Alkane-synthesizing activity was observed with resting cells and cell-free extracts of the E. coli transformants expressing ALDH (ORF2991) from Klebsiella sp. NBRC100048 with aldehyde as the substrate in the presence of NADH. However, under the tested conditions, the purified enzyme alone did not show detectable decarbonylase activity. These results suggest that additional cellular components, cofactors, or specific conditions may be required for the purified enzyme to exhibit the activity. CONCLUSIONS: We cloned several aldehyde dehydrogenases (ALDHs) from bacteria and yeast that have aldehyde decarbonylase activity to convert aldehydes to alkanes. Alkane-synthesizing activity was observed through the assays using resting cells and cell-free extracts of the E. coli transformants expressing ALDH. This novel function of aldehyde dehydrogenase introduces a new pathway for hydrocarbon fuel production and offers novel insights into microbial processes that may explain the natural origins of petroleum.