Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.

Microbial steroid transformations: current state and prospects

Production of recombinant proteins by yeast cells

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5'-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

Molecular Cloning of Two Human Liver 3α-Hydroxysteroid/Dihydrodiol Dehydrogenase Isoenzymes That are Identical with Chlordecone Reductase and Bile-acid Binder.

Yeast metabolic engineering – Targeting sterol metabolism and terpenoid formation

Efficient testosterone production by engineered Pichia pastoris co-expressing human 17β-hydroxysteroid dehydrogenase type 3 and Saccharomyces cerevisiae glucose 6-phosphate dehydrogenase with NADPH regeneration

There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv–GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv–GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv–GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion.

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

Human 20α-hydroxysteroid dehydrogenase (AKR1C1)-dependent biotransformation with recombinant fission yeast Schizosaccharomyces pombe.

The human sex hormone progesterone plays an essential and complex role in a number of physiological processes. Progesterone deficiency is associated with menstrual disorders and infertility as well as premature birth and abortion. For progesterone replacement therapy, the synthetic progestogen dydrogesterone is commonly used. In the body, this drug is metabolized to 20α-dihydrodydrogesterone (20α-DHD), which also shows extensive pharmacological effects and hence could act as a therapeutic agent itself. In this study, we describe an efficient biotechnological production procedure for 20α-DHD that employs the stereo- and regioselective reduction of dydrogesterone in a whole-cell biotransformation process based on recombinant fission yeast cells expressing the human enzyme AKR1C1 (20α-hydroxysteroid dehydrogenase, 20α-HSD). In a fed-batch fermentation at pilot scale (70 L) with a genetically improved production strain and under optimized reaction conditions, an average 20α-DHD production rate of 190 μM day−1 was determined for a total biotransformation time of 136 h. Combined with an effective and reliable downstream processing, a continuous production rate of 12.3 ± 1.4 g 20α-DHD per week and fermenter was achieved. We thus established an AKR-dependent whole-cell biotransformation process that can also be used for the production of other AKR1C1 substrates (as exemplarily shown by the production of 20α-dihydroprogesterone in gram scale) and is in principle suited for the production of further human AKR metabolites at industrial scale.

Biotechnological Production of 20-alpha-Dihydrodydrogesterone at Pilot Scale

Die vorliegende Erfindung betrifft ein verbessertes und effizienteres Verfahren zur Herstellung und Gewinnung von Proteinen, die mit dem menschlichen T1R2/T1R3 Rezeptor reagieren und einen susen Geschmack vermitteln.

Verfahren zur Herstellung süßer Proteine

The invention relates to an improved and more efficient method for the production and isolation of proteins that react with the human T1R2/T1R3 receptor and provide a sweet taste.

Julia Maria Naumann

Papers

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

Human 20α-hydroxysteroid dehydrogenase (AKR1C1)-dependent biotransformation with recombinant fission yeast Schizosaccharomyces pombe.

Biotechnological Production of 20-alpha-Dihydrodydrogesterone at Pilot Scale

Verfahren zur Herstellung süßer Proteine

Method for the production of sweet proteins