Novel acetone metabolism in a propane-utilizing bacterium, Gordonia sp. strain TY-5

Abstract

In the propane-utilizing bacterium Gordonia sp. strain TY-5, propane was shown to be oxidized to 2-propanol and then further oxidized to acetone. In this study, the subsequent metabolism of acetone was studied. Acetone-induced proteins were found in extracts of cells induced by acetone, and a gene cluster designated acmAB was cloned on the basis of the N-terminal amino acid sequences of acetone-induced proteins. The acmA and acmB genes encode a Baeyer-Villiger monooxygenase (BVMO) and esterase, respectively. The BVMO encoded by acmA was purified from acetone-induced cells of Gordonia sp. strain TY-5 and characterized. The BVMO exhibited NADPH-dependent oxidation activity for linear ketones (C3 to C10) and cyclic ketones (C4 to C8). Escherichia coli expressing the acmA gene oxidized acetone to methyl acetate, and E. coli expressing the acmB gene hydrolyzed methyl acetate. Northern blot analyses revealed that polycistronic transcription of the acmAB gene cluster was induced by propane, 2-propanol, and acetone. These results indicate that the acmAB gene products play an important role in the metabolism of acetone derived from propane oxidation and clarify the propane metabolism pathway of strain TY-5 (propane --> 2-propanol --> acetone --> methyl acetate --> acetic acid + methanol). This paper provides the first evidence for BVMO-dependent acetone metabolism.

FIG. 1.

Two-dimensional protein analysis of cell extract from Gordonia sp. strain TY-5 cells grown on citrate (left panel) and induced by acetone (right panel). The acetone-induced proteins (A and B) are indicated by arrows.

FIG. 2.

The 5,374-bp SacII-PstI DNA fragment of Gordonia sp. strain TY-5 harboring the acmA and acmB genes. The dashed part of orf2 was not cloned in this study.

FIG. 3.

Whole-cell reactions with Gordonia sp. strain TY-5 cells grown on 2-propanol under aerobic (A) and anaerobic (B) conditions. 2-Propanol-grown cells were washed to remove 2-propanol and suspended in a reaction mixture containing 2-propanol (1.96 mM). The concentrations (Conc.) of 2-propanol (circles) and acetone (squares) were determined by gas chromatography.

FIG. 4.

Stoichiometric production of methyl acetate from acetone with an E. coli transformant overexpressing the acmA gene. The initial concentration (Conc.) of acetone was 13.4 mM. The concentrations of acetone (squares) and methyl acetate (circles) were determined by gas chromatography.

FIG. 5.

Northern blot analysis of total RNA from Gordonia sp. strain TY-5. (A) Expression of acmAB and orf1 was detected by using the indicated fragments as hybridization probes. Total RNA was prepared from cells grown on sodium citrate (lanes C) and acetone (lanes A), and 10 μg of RNA was loaded into each lane. (B) Expression of acmAB was detected by hybridization with the acmA fragment. Total RNA was prepared from cells grown on sodium citrate (lane C), propane (lane P), 2-propanol (lane 2-P), and acetone (lane A). Ten micrograms of total RNA was loaded into each lane.

FIG. 6.

Propane metabolism in Gordonia sp. strain TY-5. The genes involved in each step of the reaction are indicated in the boxes.