Abstract
The biochemistry and genetics of the bacterial steroid catabolism have been extensively studied during the last years and their findings have been essential to the development of biotechnological applications. For instance, metabolic engineering of the steroid-eater strains has allowed to obtain intermediaries of industrial value. However, there are still some drawbacks that must be overcome, such as the redundancy of the steroid catabolism genes in the genome and a better knowledge of its genetic regulation. KshABs and KstDs are key enzymes involved in the aerobic breakage of the steroid nucleus. Rhodococcus ruber Chol-4 contains three kshAs genes, a single kshB gene and three kstDs genes within its genome. In the present work, the growth of R. ruber ΔkshA strains was evaluated on different steroids substrates; the promoter regions of these genes were analyzed; and their expression was followed by qRT-PCR in both wild type and ksh mutants. Additionally, the transcription level of the kstDs genes was studied in the ksh mutants. The results show that KshA2B and KshA1B are involved in AD metabolism, while KshA3B and KshA1B contribute to the cholesterol metabolism in R. ruber. In the kshA single mutants, expression of the remaining kshA and kstD genes is re-organized to survive on the steroid substrate. These data give insight into the fine regulation of steroid genes when several isoforms are present.
Highlights
Sterols and related steroids are widespread in nature due to their important physiological activities that vary from their structural role in the eukaryotic membrane to their action as hormones
The biochemistry and genetics of the bacterial steroid catabolism have been extensively studied during the last years and their findings have been essential to the development of biotechnological applications
We used the synthetic ribosomal binding site (RBS) AAGGAGG that has been proved to be effective in the Gram-positive Streptomyces [41] and the strongest RBS found for effective expression of genes in actinobacteria: (AA)GGA(G)G(AA)A(AAA)CAT-ATG [42]
Summary
Sterols and related steroids are widespread in nature due to their important physiological activities that vary from their structural role in the eukaryotic membrane to their action as hormones. These molecules are highly resistant to degradation due to their hydrophobic nature and reduced presence of functional groups; it is possible to find bacteria that can degrade steroid compounds [1]. Different pathways for aerobic or anaerobic steroid degradation may co-exist in the same organism, the role and participation of steroid genes involved in cholesterol catabolism have been clarified by biochemical and genetic studies [1,3,4,5,7,8,9,10]. The genome search for catabolic steroid genes encoding clusters has resulted in important advances in metabolic engineering and sterol bioconversion of both
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