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

Abstract

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.

Figure 1

Expression profiles of selected putative CYP genes of C. lunatus exposed to various steroids: deoxycorticosterone (DOC), androstenedione (AD), progesterone (PROG), cholic acid (ChoAc) and β‐sitosterol (Β‐SITO) and C corresponds to the negative control without DNA, NoIND corresponds to the no induced condition (fungal cultures not exposed to steroid, but exposed to the solvent Tyloxapol at same concentration) and gDNA corresponds to the genomic DNA as a positive control. The glyceraldehyde 3‐phosphate dehydrogenase gene has been used as housekeeping, an internal control. A. sqRT‐PCR for CYP transcript determination. B. Fold change of CYP130168 mRNAs determined by qRT‐PCR. Error bars represent the standard deviation of three independent experiments.

Figure 2

Schematic representation of the genes contained in the FAN operon and FIN operons. The sequences of the intergenic regions (R1‐R3) are indicated in the table. The sequences of the restriction sites are in cursive, and the corresponding restriction enzymes are annotated. The RBS sequences are indicated in bold.

Figure 3

A. Deoxycorticosterone (DOC) biotransformation by C. glutamicum (pXKFAN) and C. glutamicum (pXKFIN). Mass spectra obtained from ion 331 (characteristic ion of DOC) (blue line) and mass spectra obtained from ion 347 (characteristic ion of corticosterone (CORT)) (red line). B. Deoxycorticosterone biotransformation by C. lunatus. Mass spectra obtained from ion 331 (characteristic ion of DOC) (blue line) and mass spectra obtained from ion 347 (characteristic ion of corticosterone (CORT)) (red line). All the experiments were repeated three times and a representative one was chosen.

Figure 4

Cortexolone biotransformation by C. glutamicum (pXKFAN). Mass spectra obtained from ion 347 (characteristic ion of cortexolone (Reichstein's Substance S, RSS) (blue line) and mass spectra obtained from ion 363 (characteristic ion of hydrocortisone (HC)) (red line).