Complete Genome Sequence of Nocardia iowensis DSM 45197T (= NRRL 5646T).

We report the complete genome sequence and predicted functional profile of Nocardia iowensis DSM 45197T. N. iowensis DSM 45197T is a spore-forming, mesophilic, Gram-positive bacterium that was isolated from garden soil in Osceola, Iowa, USA. This organism has been exploited for its production of glycocinnamolyspermidine antibiotics and biotransformation of xenobiotic substances. Other significant features of N. iowensis DSM 45197T include the first fully characterized carboxylic acid reductase (CAR) and the first bacterial nitric oxide synthase system. The genome sequence of N. iowensis DSM 45197T can facilitate further understanding of its function, as well as the pathogenesis of Nocardia spp. N. iowensis DSM 45197T has a genome size of 8.95 Mbp; about 46% of the coding sequences have no known homologues and were labeled hypothetical proteins. This finding implies further potential for biomedical and biotechnological research applications.

Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase.

Nocardia strain NRRL 5646, isolated from a garden soil sample in Osceola, Iowa, USA, was initially of interest as an antibiotic producer. It contained biocatalytically important enzymes and represented the first described nitric oxide synthase enzyme system in bacteria. The present polyphasic taxonomic study was undertaken to differentiate strain NRRL 5646(T) from related species of the genus Nocardia. Chemotaxonomic analyses included determinations of the fatty acid methyl ester profile (C(16 : 1)omega6c/C(16 : 1)omega7c, C(16 : 0), C(18 : 1)omega9c and C(18 : 0) 10-methyl as major components), quinone [cyclo MK-8(H(4)) as the major component], polar lipid (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major components) and mycolic acid. These results supported its placement within the genus Nocardia. Biochemical testing and 16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses differentiated strain NRRL 5646(T) from recognized Nocardia species. Previous studies have demonstrated that other genetic sequences (carboxylic acid reductase, Nocardia phosphopantetheinyl transferase and GTP cyclohydrolase I) from strain NRRL 5646(T) can also be used to substantiate its uniqueness. The level of 16S rRNA gene sequence similarity between strain NRRL 5646(T) and the type strains of Nocardia tenerifensis and Nocardia brasiliensis was 98.8 %. However, strain NRRL 5646(T) could be clearly distinguished from these Nocardia species based on DNA-DNA hybridization data. Consequently, strain NRRL 5646(T) is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed. The type strain is NRRL 5646(T) (=UI 122540(T)=NRRL B-24671(T)=DSM 45197(T)).

Aldehyde oxidoreductase as a biocatalyst: Reductions of vanillic acid.

Aldehyde oxidoreductase (carboxylic acid reductase) catalyzes the Mg(2+), ATP and NADPH dependent reduction of carboxylic acids to their corresponding aldehydes. The identification of the gene from Nocardia sp. NRRL 5646 and its expression in E. coli BL21-CodonPlus(®)(DE3)-RP/pHAT305 provided an avenue to develop a biocatalyst for reduction of carboxylic acids. In addition to aromatic acids, the recombinant carboxylic acid reductase also accepts several aliphatic mono, di and tri carboxylic acids as substrates. A recently identified Nocardia sp., phosphopantetheinyl transferase gene (npt) enhanced the activity of carboxylic acid reductase. Coexpression of car and npt in E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.83 resulted in a purified recombinant carboxylic acid reductase with improved specific activity of 2.2U/mg protein. The utility of the recombinant carboxylic acid reductase as a biocatalyst has been demonstrated using vanillic acid as substrate. E. coli BL21-CodonPlus(®)(DE3)-RP/pHAT305 expressing Car reduced 50% of vanillic acid to vanillin in 10h. E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.83 resting cells expressing Car and Npt reduced 90% of vanillic acid to vanillin in 6h. Enhanced, in vivo cofactor NADPH regeneration by glucose dehydrogenase (gdh) was accomplished using E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.85, that carried car, npt, and gdh. Resting cell reactions using E. coli BL21-CodonPlus(®)(DE3)-RP/pPV2.85 with in situ product removal by XAD-2 resin efficiently reduced 5g/L of vanillic and benzoic acids within 2h.

Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus.

The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform steroid biotransformations under single phase, pulse feed conditions. The steroids 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17beta-hydroxyandrost-4-en-3-one (testosterone) (5), 3beta-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17alpha,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17alpha,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2beta, 6beta/7beta and 15beta/16beta hydroxylations. P. chrysosporium was more prone functionalize steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.

Bioconversion of Stemodia maritima diterpenes and derivatives by Cunninghamella echinulata var. elegans and Phanerochaete chrysosporium.

Stemodane and stemarane diterpenes isolated from the plant Stemodia maritima and their dimethylcarbamate derivatives were fed to growing cultures of the fungi Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. C. echinulata transformed stemodin (1) to its 7alpha-hydroxy- (2), 7beta-hydroxy- (3) and 3beta-hydroxy- (4) analogues. 2alpha-(N,N-Dimethylcarbamoxy)-13-hydroxystemodane (6) gave 2alpha-(N,N-dimethylcarbamoxy)-6alpha,13-dihydroxystemodane (7) and 2alpha-(N,N-dimethylcarbamoxy)-7alpha,13-dihydroxystemodane (8). Stemodinone (9) yielded 14-hydroxy-(10) and 7beta-hydroxy- (11) congeners along with 1, 2 and 3. Stemarin (13) was converted to the hitherto unreported 6alpha,13-dihydroxystemaran-19-oic acid (18). 19-(N,N-Dimethylcarbamoxy)-13-hydroxystemarane (14) yielded 13-hydroxystemaran-19-oic acid (17) along with the two metabolites: 19-(N,N-dimethylcarbamoxy)-2beta,13-dihydroxystemarane (15) and 19-(N,N-dimethylcarbamoxy)-2beta,8,13-trihydroxystemarane (16). P. chrysosporium converted 1 into 3, 4 and 2alpha,11beta,13-trihydroxystemodane (5). The dimethylcarbamate (6) was not transformed by this microorganism. Stemodinone (9) was hydroxylated at C-19 to give 12. Both stemarin (13) and its dimethylcarbamate (14) were recovered unchanged after incubation with Phanerochaete.

Stemodane and stemarane diterpenoid hydroxylation by Mucor plumbeus and Whetzelinia sclerotiorum.

Incubation of stemodin (1) with Mucor plumbeus ATCC 4740 resulted in the formation of 2alpha,6beta,13-trihydroxystemodane (2), 2alpha,3beta,13-trihydroxystemodane (3), 2alpha,11beta,13-trihydroxystemodane (4) and 2alpha,13,14-trihydroxystemodane (5), while stemodinone (7) afforded 6alpha,13-dihydroxystemodan-2-one (8) and 6alpha,12alpha,13-trihydroxystemodan-2-one (9). Metabolites obtained from the bioconversion of stemarin (11) were 8,13,19-trihydroxystemarane (12) and 2alpha,13,19-trihydroxystemarane (13). 19-N,N-Dimethylcarbamoxy-13-hydroxystemarane (14) was not transformed by the fungus. Stemodin (1) was incubated with Whetzelinia sclerotiorum ATCC 18687 to produce 2alpha,7beta,13-trihydroxystemodane (6) and 2alpha,11beta,13-trihydroxystemodane (4). Stemodinone (7) was converted to 7beta,13-dihydroxystemodan-2-one (10). Compounds 2, 4, 9, 10, 12 and 13 have not been previously reported.