Identification of Absidia orchidis steroid 11ß-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone.

Hydrocortisone is an effective anti-inflammatory drug and also an important intermediate for synthesis of other steroid drugs. The filamentous fungus Absidia orchidis is renowned for biotransformation of acetylated cortexolone through 11ß-hydroxylation to produce hydrocortisone. However, due to the presence of 11α-hydroxylase in A. orchidis, the 11α-OH by-product epi-hydrocortisone is always produced in a 1:1 M ratio with hydrocortisone. In order to decrease epi-hydrocortisone production, Saccharomyces cerevisiae was engineered in this work as an alternative way to produce hydrocortisone through biotransformation. Through transcriptomic analysis coupled with genetic verification in S. cerevisiae, the A. orchidis steroid 11ß-hydroxylation system was characterized, including a cytochrome P450 enzyme CYP5311B2 and its associated redox partners cytochrome P450 reductase and cytochrome b5. CYP5311B2 produces a mix of stereoisomers containing 11ß- and 11α-hydroxylation derivatives in a 4:1 M ratio. This fungal steroid 11ß-hydroxylation system was reconstituted in S. cerevisiae for hydrocortisone production, resulting in a productivity of 22 mg/L·d. Protein engineering of CYP5311B2 generated a R126D/Y398F variant, which had 3 times higher hydrocortisone productivity compared to the wild type. Elimination of C20-hydroxylation by-products and optimization of the expression of A. orchidis 11ß-hydroxylation system genes further increased hydrocortisone productivity by 238% to 223 mg/L·d. In addition, a novel steroid transporter ClCDR4 gene was identified from Cochliobolus lunatus, overexpression of which further increased hydrocortisone productivity to 268 mg/L·d in S. cerevisiae. Through increasing cell mass, 1060 mg/L hydrocortisone was obtained in 48 h and the highest productivity reached 667 mg/L·d. This is the highest hydrocortisone titer reported for yeast biotransformation system so far.

Application of α- and ß-naphthoflavones as monooxygenase inhibitors of Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651 in transformation of 17α-methyltestosterone.

In this work, 17α-methyltestosterone was effectively hydroxylated by Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651. A. coerulea KCh 93 afforded 6ß-, 12ß-, 7α-, 11α-, 15α-hydroxy derivatives with 44%, 29%, 6%, 5% and 9% yields, respectively. S. racemosum KCh 105 afforded 7α-, 15α- and 11α-hydroxy derivatives with yields of 45%, 19% and 17%, respectively. Chaetomium sp. KCh 6651 afforded 15α-, 11α-, 7α-, 6ß-, 9α-, 14α-hydroxy and 6ß,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6ß,14α-dihydroxy derivatives were determined as new compounds. Effect of various sources of nitrogen and carbon in the media on biotransformations were tested, however did not affect the degree of substrate conversion or the composition of the products formed. The addition of α- or ß-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.

Cloning and identification of a novel steroid 11α-hydroxylase gene from Absidia coerulea.

Steroid 11-hydroxylation by filamentous fungi is a major route for industrial scale production of key intermediates in the synthesis of steroid drugs. Although it is well established that enzymes involved in fungal hydroxylation of steroids are cytochrome P450s (CYP), few fungal steroid hydroxylase genes have been identified. In this study, we identified a novel 11α-hydroxylase gene CYP5311B1 from Absidia coerulea AS3.65 by a combination of transcriptome sequencing, real-time qRT-PCR and heterologous expression in Pichia pastoris. The full-length open reading frame (ORF) of CYP5311B1 is predicted to encode a CYP protein of 527 amino acids whose expression in Pichia cells was confirmed by western blot. In addition, the major hydroxylation product was characterized by HPLC and 2D NMR. CYP5311B1 was highly induced by steroid substrate at the transcriptional level. The cloning and identification of an 11α-hydroxylase gene from A. coerulea should aid in a better understanding of the structural basis underlying regio- and stereoselectivity, and substrate specificity of fungal steroid 11α-hydroxylases, thus facilitating the engineering of more efficient steroid hydroxylases for industrial applications.

Two Novel Fungal Phenolic UDP Glycosyltransferases from Absidia coerulea and Rhizopus japonicus.

In the present study, two novel phenolic UDP glycosyltransferases (P-UGTs), UGT58A1 and UGT59A1, which can transfer sugar moieties from active donors to phenolic acceptors to generate corresponding glycosides, were identified in the fungal kingdom. UGT58A1 (from Absidia coerulea) and UGT59A1 (from Rhizopus japonicas) share a low degree of homology with known UGTs from animals, plants, bacteria, and viruses. These two P-UGTs are membrane-bound proteins with an N-terminal signal peptide and a transmembrane domain at the C terminus. Recombinant UGT58A1 and UGT59A1 are able to regioselectively and stereoselectively glycosylate a variety of phenolic aglycones to generate the corresponding glycosides. Phylogenetic analysis revealed the novelty of UGT58A1 and UGT59A1 in primary sequences in that they are distantly related to other UGTs and form a totally new evolutionary branch. Moreover, UGT58A1 and UGT59A1 represent the first members of the UGT58 and UGT59 families, respectively. Homology modeling and mutational analysis implied the sugar donor binding sites and key catalytic sites, which provided insights into the catalytic mechanism of UGT58A1. These results not only provide an efficient enzymatic tool for the synthesis of bioactive glycosides but also create a starting point for the identification of P-UGTs from fungi at the molecular level.IMPORTANCE Thus far, there have been many reports on the glycosylation of phenolics by fungal cells. However, no P-UGTs have ever been identified in fungi. Our study identified fungal P-UGTs at the molecular level and confirmed the existence of the UGT58 and UGT59 families. The novel sequence information on UGT58A1 and UGT59A1 shed light on the exciting and new P-UGTs hiding in the fungal kingdom, which would lead to the characterization of novel P-UGTs from fungi. Molecular identification of fungal P-UGTs not only is theoretically significant for a better understanding of the evolution of UGT families but also can be applied as a powerful tool in the glycodiversification of bioactive natural products for drug discovery.

Hydroxylation of DHEA and its analogues by Absidia coerulea AM93. Can an inducible microbial hydroxylase catalyze 7α- and 7ß-hydroxylation of 5-ene and 5α-dihydro C19-steroids?

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and 5α-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis of the hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to the mixture of allyl 7-hydroxy derivatives, while EpiA and 5α-androstan-3,17-dione were converted mainly to 7α- and 7ß-alcohols accompanied by 9α- and 11α-hydroxy derivatives. On the basis of (i) time course analysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells at different pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between the C-H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, it is postulated that the same enzyme can catalyze the oxidation of C7-Hα as well as C7-Hß bonds in 5-ene and 5α-dihydro C19-steroids. Correlations observed between the structure of the substrate and the regioselectivity of hydroxylation suggest that 7ß-hydroxylation may occur in the normal binding enzyme-substrate complex, while 7α-hydroxylation-in the reverse inverted binding complex.

A comparative characterization of indigenous keratinase enzymes from district Khairpur, Sindh, Pakistan.

To isolate and characterize keratinolytic fungi and bacteria from indigenous soils, a total of 80 samples were collected from Ghari Mori District. Khairpur, and these organisms were isolated using standard microbiological technique. The isolated keratinolytic microorganisms comprised: Absidia sp., Chrysosporium asperatum, Chrysosporium keratinophilum, Entomophthora coronata, Bacillus subtilis and Staphylococcus aureus and their keratinolytic properties were distinguished from the production of keratinase by measurement of zone of hydrolysis on skimmed milk agar (p<0.05). C.keratinophylum and B. subtilis produced largest zone among all the isolated species. The crude keratinase revealed that the optimum time for production of the enzyme was seven days, optimum temperature 30°C and optimum pH 9 for C.keratinophylum but for B. subtilis, the optimum time was three days, optimum temperature 37°C and optimum pH 7. The enzyme activity of C. keratinophylum and B. subtilis were determined to be 220 U/ml and 260 U/ml respectively (P<0.05).

Efficient hydrolytic reaction of an acetate ester with fungal lipase in a liquid-liquid interface bioreactor (L-L IBR) using CaCO3-coated ballooned microsphere.

In a liquid-liquid interface bioreactor using a CaCO3-coated ballooned microsphere, 2-ethylhexyl acetate was efficiently hydrolyzed to 2-ethyl-1-hexanol with Absidia coerulea NBRC 4423 compared with using talc-coated or non-coated ballooned microsphere. It was assumed that CaCO3 brought about stabilization of lipase by Ca²âº and maintenance of medium pH.

New dioscin-glycosidase hydrolyzing multi-glycosides of dioscin from Absidia strain.

A novel dioscin-glycosidase that specifically hydrolyzes multi-glycosides such as 3-O-alpha-L-(1 --> 4)-rhamnoside, 3-O-alpha-L-(1 --> 2)-rhamnoside, 3-O-alpha-L-(1 --> 4)-arabinoside and beta-D-glucoside on diosgenin was isolated from Absidia sp.d38 strain; and it was purified and characterized. The molecular weight of the new dioscin-glycosidase is about 55 kDa in SDS-PAGE. The the dioscin-glycosidase gradually hydrolyzes either 3-O-alpha-L-(1 --> 4)-Rha or 3-O-alpha-L-(1 --> 2)-Rha of dioscin to 3-O-alpha-L-Rha-beta-D-Glc-diosgenin; further rapidly hydrolyzes the other alpha-L-Rha of 3-O-alpha-L-Rha-beta-D-Glc-diosgenin to main intermediate products 3-O-beta-D-glc-diosgenin; and subsequently hydrolyzes intermediate products to final product of aglycone; the new enzyme also gradually hydrolyzes 3-O-alpha-L-(1 --> 4)-arabinoside, 3-O-alpha-L-(1 --> 2)-rhamnoside and beta-D-glucoside of [3-O-alpha-L-(1 --> 4)-Ara, 3-O-alpha-L-(1 --> 2)-Rha]-beta-D-Glc-diosgenin into final product diosgenin, exhibiting significant differences from previously reported glycosidases. The optimal temperature of the new dioscin-glycosidase is 40 degrees C and the optimal pH is 5.0. The activity of the new dioscin-glycosidase was not affected by the Na+, K+ and Mg2+ ions; it was significantly inhibited by the Cu2+ and Hg2+ ions; and it was slightly affected by the Ca2+ ions.

[Purification and properties of isoflavone-glucosidase].

A high activity isoflavone-glucosidase, which hydrolysis glycosides, was obtainde using liquid fermentation from Absidia sp. R strain. The isoflavone-glucosidase was purified 11 folds with yielding rate of 10.9% after ammonium sulfate precipitation and DEAE-Cellocuse (DE-52) ion exchange chromatography. SDS-PAGE results showed that the molecular weight is 53kD. And the optimum temperature, the optimum pH, Km and pI of the enzyme are 50 deegrees C, 5.0, 1.3 x 10(-2) mol/L and 3.2, respectively. The isoflavone-glucosidase is also rather stable under 60 degrees C and in pH range from 5.0 to 7.0. The enzyme can be activated by Co2+ and Ca2+, and be inhibited by Ag+ and Cu2+.

Purification and characterization of ginsenoside-alpha-L-rhamnosidase.

In this paper the ginsenoside-alpha-(1-->2)-L-rhamnosidase from microorganisms was purified and characterized. The enzyme hydrolyzed the 6-C, alpha-(1-->2)-L-rhamnoside of 20(S) and 20(R)-ginsenoside Rg2 to produce the 20(S) and 20(R)-ginsenoside Rh1, but hardly hydrolyzed the alpha-rhamnoside of pNPR. The enzyme molecular weight was about 53 kDa. The optimum temperature of enzyme reaction was 40 degrees C, and the optimum pH was 5.

Shrimp chitin as substrate for fungal chitin deacetylase.

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5-10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2x10(5) in chitin to 1.2 x 10(4) in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.

Nitrogen bridgehead compounds, facile synthesis of bioactive cyanopyrimido[1,2-a]pyrimidinones.

Synthesis of some new cyanopyrimido[1,2-a]pyrimidinones 5-22 have been achieved via interaction of 2-amino-6-anisyl-5-cyano-4(3H)-pyrimidinone (1) with some heterocycles having a vicinal chloroester, chlorocyano or mercaptocyano group, dimethyl acetylenedicarboxylate, active methylene compounds, ethyl 2-acetyl-3-anisylpropenoate, ethyl 3-aryl-3-cyanopropenoates, ethyl 2-cyano-3-ethoxyacrylate and some enones or enals. Some of the isolated products were subjected to biological screening tests.