Identification and expression of the 11β‐steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum

Abstract

Summary Hydroxylation of steroids has acquired special relevance for the pharmaceutical industries. Particularly, the 11β‐hydroxylation of steroids is a reaction of biotechnological importance currently carried out at industrial scale by the fungus Cochliobolus lunatus. In this work, we have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11β‐hydroxylase activity in this fungus, which is the key step for the preparative synthesis of cortisol in industry. A recombinant Corynebacterium glutamicum strain harbouring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11β‐hydroxylate several steroids as substrates. This is a new example to show that the industrial strain C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes.

Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis

“…Our results demonstrate that fortunately, the synthetic operon FAN was fully functional in M. smegmatis since only few fungal CYPs have been successfully produced in its active form in bacteria so far [ 14 , 16 , 24 , 25 , 29 ]. The detection of 14α-hydroxylated AD and ADD in the culture medium of the recombinant bacteria at high yields demonstrated that the fungal hydroxylating system has been integrated in the sterol metabolism of M. smegmatis creating a new expanded pathway.…”

“…The CYP103168 and CPR64795 encoding genes were PCR amplified as described from cDNA of C. lunatus [ 14 ]. To achieve optimal translation in the host bacterium, the PCR primers contained a Shine Dalgarno sequence (AAAGGGAG) upstream of each gene at 6 bp from the respective start codons ( Figure S1 ).…”

“…The hydroxyl group in position 11β of steroids provides important pharmacological properties [ 9 , 10 ] and from a long time this specific hydroxylation has been established as a large-scale commercial process using the fungal CYP of Cochliobolus lunatus [ 11 , 12 , 13 ]. We have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11β-hydroxylase activity in this fungus [ 14 ]. We have demonstrated that a recombinant Corynebacterium glutamicum strain harboring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11β-hydroxylate several steroids as substrates [ 14 ].…”

“…We have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11β-hydroxylase activity in this fungus [ 14 ]. We have demonstrated that a recombinant Corynebacterium glutamicum strain harboring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11β-hydroxylate several steroids as substrates [ 14 ]. In this sense, the design of alternative microbial cell factories created by recombinant DNA technologies has been explored as a challenge in the industry of steroids [ 6 , 15 , 16 ].…”

“…In this sense, the design of alternative microbial cell factories created by recombinant DNA technologies has been explored as a challenge in the industry of steroids [ 6 , 15 , 16 ]. The heterologous co-expression of hydroxylating CYPs and their natural redox partners as putative industrial biocatalyzers has been addressed in different host such as yeasts (e.g., Pichia pastoris , Saccharomyces cerevisiae , and Schizosaccharomyces pombe ), and bacteria (e.g., Escherichia coli , Bacillus subtilis , Bacillus megaterium, Corynebacterium glutamicum ) [ 10 , 14 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. All these bioconversion processes are based in whole-cell biocatalysts and require previously purified steroid synthons as substrates.…”