Process for the overexpression of dehydrogenases

1. A process for selective introduction of a double bond into ring A of a steroid skeleton by overexpression of 3-keto steroid-Δ 1 -dehydrogenases , wherein

a) a Δ 1 -dehydrogenase gene encoding a polypeptide comprising Seq. ID No. 11 is isolated from Bacillus sphaericus , cloned and amplified,

b) promoter and terminator elements of the 3-keto steroid-Δ 1 -dehydrogenase gene or other promoter and terminator elements are isolated from the same or another bacterium, cloned and amplified,

c) expression plasmids are constructed in which the 3-keto steroid-Δ 1 -dehydrogenase gene from a), is flanked by promoter and terminator sequences of the 3-keto steroid-Δ 1 -dehydrogenase gene or by other promoter and terminator elements from b), is contained,

d) B. sphaericus, B. subtilis , or E. coli host bacteria are transformed with the expression plasmid that is produced under c), and

e) the thus produced bacteria are cultivated, and the selective dehydrogenation at 1-position in the steroid skeleton of hydrocortisone (F), hydrocortisone-17-acetate (MAF), hydrocortisone-21-acetate (EAF), 4-androstene-3-17-dione(AD), fluocortolone A acetate (FCAA) or 11β,17α-Dihydroxy-6α,9α-difluoro-16α-methylprogesterone (DDFMP) is performed with these cultures, whereby

i) a high substrate concentration at unaltered operating times is used, and

ii) no disruptive secondary zones are produced.

2. The process according to claim 1 , wherein said promoter comprises Seq. ID No. 9 or a sequence having at least 90% homology to Seq. ID No.9.

3. The process according to claim 1 , wherein said selective dehydrogenation at the 1-position in the steroid skeleton forms betamethasone, clobetasone, clocortolone, Δ 1 -11β,17α-dihydroxy-6α,9α-difluoro-16α-methylprogesterone, deflazacort, dexamethasone, diflocortolone, fluocinolone acetonide, fluocortolone, hydroxy acid or prednisolone and derivatives thereof.

4. The process according to claim 1 , wherein said promoter or terminator is a constitutive promoters that is p(veg), a promoter of bacteriophages Φ29 or SPO1, an inducible promoters that is p(aprE) or p(sacB) from Bacillus subtilis , a hybrid promoter that is a ladI-controlled SPO1-promoter, a terminator of Eseherichia coli that is t(rrnB) or of Bacillus subtilis that is t(senS) or t(senN).

5. The process of claim 1 , wherein said terminator is the terminator of the 3-Keto steroid-Δ 1 -dehydrogenase gene from Bacillus sphaericus comprising SEQ ID NO: 10.

6. The process of claim 1 , wherein said host cell is B. sphaericus.