Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4

Abstract

The 3-Ketosteroid-9α-Hydroxylase, also known as KshAB [androsta-1,4-diene-3,17-dione, NADH:oxygen oxidoreductase (9α-hydroxylating); EC 1.14.13.142)], is a key enzyme in the general scheme of the bacterial steroid catabolism in combination with a 3-ketosteroid-Δ-dehydrogenase activity (KstD), being both responsible of the steroid nucleus (rings A/B) breakage. KshAB initiates the opening of the steroid ring by the 9α-hydroxylation of the C9 carbon of 4-ene-3-oxosteroids (e.g. AD) or 1,4-diene-3-oxosteroids (e.g. ADD), transforming them into 9α-hydroxy-4-androsten-3,17-dione (9OHAD) or 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD), respectively. The redundancy of these enzymes in the actinobacterial genomes results in a serious difficulty for metabolic engineering this catabolic pathway to obtain intermediates of industrial interest. In this work, we have identified three homologous kshA genes and one kshB gen in different genomic regions of R. ruber strain Chol-4. We present a set of data that helps to understand their specific roles in this strain, including: i) description of the KshAB enzymes ii) construction and characterization of ΔkshB and single, double and triple ΔkshA mutants in R. ruber iii) growth studies of the above strains on different substrates and iv) genetic complementation and biotransformation assays with those strains. Our results show that KshA2 isoform is needed for the degradation of steroid substrates with short side chain, while KshA3 works on those molecules with longer side chains. KshA1 is a more versatile enzyme related to the cholic acid catabolism, although it also collaborates with KshA2 or KshA3 activities in the catabolism of steroids. Accordingly to what it is described for other Rhodococcus strains, our results also suggest that the side chain degradation is KshAB-independent.

Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms

“…Yet another type of steroid hydroxylases, Rieske monooxygenases (e.g., 3-ketosteroid 9α-hydroxylase, KSH), are involved in the so-called 9,10-seco pathway which is so far the only known aerobic route for bacterial degradation of the steroid core. The KSH enzymes were best characterized for Rhodococcus and Mycobacterium species and they represent two-component monooxygenases, composed of an oxygenase (KshA) and a flavin-dependent ferredoxin reductase (KshB) unit (Bragin et al 2013 ; Capyk et al 2009b ; Guevara et al 2017 ; Hu et al 2010 ; Penfield et al 2014 ; Petrusma et al 2011 ; Petrusma et al 2012 ; Petrusma et al 2014 ). Along with 3-ketosteroid-1(2)-dehydrogenases (KstD), KSH is responsible for the cleavage of the 9(10)-C-C bond to form 9(10)-secosteroids with an aromatized A-ring.…”

New insights into the genome of Rhodococcus ruber strain Chol-4

“…On the other hand, some of the genes detected in the central pathways are redundant in the genome of R. ruber Chol-4. Apart from certain enzymatic activities such as KstD and Ksh isoforms previously described in this strain within the steroids catabolism [19, 44, 45],there are also redundant clusters in the R. ruber genome such as 3 copies of pcaJ-pcaI involved in the catechol and protocatechuate pathways of the β-ketoadipate and leucine catabolism that encodes a succinyl-CoA: 3-ketoacid-coenzyme A transferase (EC 2.8.3.5) in NZ_ANGC02000003.1 (D092_RS10690-RS10695), NZ_ANGC02000026.1 (D092_RS24595-RS24600) and NZ_ANGC02000008.1 (D092_RS18665-RS18670) contigs with an amino acid identity of 66–73% among them and the 2 copies of the hsaEGF cluster (NZ_ANGC02000004.1 contig; Fig. 4 III) with an amino acid identity of 65–78% belonging to the 2-hydroxypentanodienoate pathway.…”

New Insights on Steroid Biotechnology

“…Other authors have attempted to construct mutant strains producing steroidal intermediates from sterols in species of the genus Rhodococcus . However, the presence of multiple steroid catabolic pathways apparently convergent and the existence of basal levels of certain isoenzymes (e.g., KstD, KshA, or KshB) have hindered the development of stable producers in these species (van der Geize et al, 2000 , 2001 , 2002a , b , 2008 ; Petrusma et al, 2011 ; Fernández de las Heras et al, 2012 ; Liu et al, 2016 ; Guevara et al, 2017a , b ). In certain members of the genus Mycobacterium such as M. smegmatis, M. tuberculosis or M. neoaurum , the deletion of a single key gene ( kshA, kshB , or kstD ) appears to be sufficient to prevent an efficient mineralization of sterols (Brzostek et al, 2005 ; Andor et al, 2006 ; Capyk et al, 2009 ; Brzezinska et al, 2013 ; Yao et al, 2014 ; Galán et al, 2017b ).…”

“…The characterization of the C-19+ gene cluster above mentioned, as well as of other steroid degradation gene clusters in bacteria, has allowed to identify multiple steroid-modifying isoenzymes with different catalytic properties (e.g., substrate specificity, cellular localization) (Knol et al, 2008 ; Petrusma et al, 2011 , 2012 ; Penfield et al, 2014 ; Guevara et al, 2017a , b ; Wang et al, 2017 ), that should be considered to design more efficient industrial bioprocesses. For example, the selective production of ADD from sterols in a single fermentation step remains a challenge at an industrial scale.…”