Identification of gene expression profiles in the actinomycete Gordonia neofelifaecis grown with different steroids.

Gordonia neofelifaecis NRRL B-59395 was initially isolated from the fresh feces of a clouded leopard based on its ability to degrade cholesterol. The transcriptome profiles of G. neofelifaecis NRRL B-59395 grown with cholesterol, androstenedione (AD), and pyruvic acid were compared by RNA-Seq. The sterol catabolic genes are highly conserved in G. neofelifaecis, Rhodococcus jostii RHA1, and Mycobacterium tuberculosis. The RNA-Seq results indicated that the genes involved in the sterol side chain cleavage were exclusively induced by cholesterol, while the genes involved in the degradation of rings A/B and C/D were up-regulated by both cholesterol and AD. It appears that the induction mechanisms for the genes responsible for side chain cleavage and those for degradation of rings are different. There are approximately 21 genes encoding transporter proteins that are differentially expressed in cholesterol or AD compared with pyruvic acid. The genes camABCD and camM encode two systems that take up cholate, and they have been shown to be cholesterol- and AD-inducible. The potential biological functions of other differentially expressed genes are also discussed. These results will promote the functional characterization of the sterol catabolic genes and also provide important clues in understanding the mechanisms of their gene expression, and they may help us understand the mechanism underlying microbial cholesterol catabolism.

[Accumulation of 9α-hydroxy-4-androstene-3,17-dione by co-expressing kshA and kshB encoding component of 3-ketosteroid-9α-hydroxylase in Mycobacterium sp. NRRL B-3805].

9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.