Patent Application: US-73763509-A

Abstract:
an isolated nucleic acid molecule cloned from artemisia annua encodes an alcohol dehydrogenase . artemisia annua adh1 enzymatically oxidizes artemisinic alcohol to artemisinic aldehyde . the nucleic acid molecule , and the enzyme encoded thereby , may be used in processes to produce artemisinic aldehyde , dihydroartemsinic aldehyde , artemisinic acid and / or dihydroartemisinic acid in a host cell . artemisinic aldehyde , dihydroartemisinic aldehyde , artemisinic acid and / or dihydroartemisinic acid can be chemically converted to the antimalarial compound artemisinin .

Description:
artemisinic acid was isolated from dichloromethane extracts of a . annua flower buds and leaves ( teoh , polichuk , reed , nowak , & amp ; covello 2006 ) and was used to synthesize artemisinic aldehyde according to the method described by chang et al . 2000 , the disclosure of which is incorporated herein by reference . dihydroartemisinic aldehyde was synthesized from the isolated dihydroartemisinic acid ( see above ). the acid was converted to methyl dihydroartemisinate with excess diazomethane in diethyl ether at 0 ° c . for 5 minutes . the ether and diazomethane were removed under a stream of nitrogen and the methyl ester was reduced to ( 11r )- dihydroartemisinic alcohol with excess 1 . 5 m diisobutyl aluminum hydride in toluene at room temperature for 10 min under nitrogen . with subsequent extraction , oxidation to the aldehyde with pyridinium chlorochromate ( corey & amp ; suggs 1975 ) and purification by hplc the ( 11r )- dihydroartemisinic aldehyde was produced at an overall yield of 48 % with & gt ; 90 % purity according to gc analysis . artemisia annua l . seeds were obtained from elixir farm botanicals , brixey , mo ., usa and from pedro melillo de magalhães , state university of campinas , brazil ( line 2 / 39 ). seeds were germinated and grown in soil in a controlled environment chamber with 16 hour / 25 ° c . days and 8 hour / 20 ° c . nights . plants that had reached the height of approximately 1 . 2 m ( about 3 months ) were transferred to flowering chamber with 12 hour / 25 c days and 12 hour / 20 c nights for the elixir line and 8 hour / 25 ° c . days and 16 hour / 20 ° c . nights for line 2 / 39 . flower buds that developed after 19 - 21 days in the flowering chamber were harvested for total rna isolation . total rna was extracted and isolated from glandular trichomes and flower buds using a modified method described by logeman , et al . 1987 . cdna synthesis from 1 . 5 micrograms of total rna and construction of the trichome and flower bud cdna library were carried out with creator ™ smart ™ cdna library construction kit ( clontech ). a total of 6 , 239 clones and 2 , 208 clones for trichome and flower bud libraries , respectively were randomly picked and their dna sequences determined . sequencing was performed on an ab13700 dna sequencer using bigdye terminator cycle sequencing kit ( applied biosystems ) and the m13 reverse primer . dna sequence traces were interpreted and vector and low quality sequences were eliminated using phred ( ewing et al . 1998 ) and lucy ( chou & amp ; holmes 2001 ). clustering of the resulting est dataset was done using stackpack ( miller et al . 1999 ) and sequence similarity was identified by blast ( altschul et al . 1990 ). the gene corresponding to an artemisia annua cdna clone was identified by sequence similarity searches as showing similarity to other nucleotide sequences encoding alcohol dehydrogenases was designated artemisia annua adh1 . the nucleotide sequence of the open reading frame of adh1 is given by seq id no : 1 . the gateway vector pdest17 had the adh1 gene inserted and was subsequently transformed into a competent expression e . coli strain rosetta ( de3 ). these cells were grown overnight in 3 ml of lb broth amended with 100 μg / ml ampicillin . an aliquot of 1 ml overnight culture was sub cultured to 100 ml of fresh lb broth also amended with 100 μg / ml of ampicillin . the culture was incubated at 37 ° c . until the optical density reached 0 . 8 at 600 nm . a final concentration of 1 mm iptg was then added to the culture and the flask was incubated at 30 ° c . for another 4 hours . cells were harvested by centrifugation at 10 , 000 rcf for 10 minutes . pelleted cells were resuspended in 20 mm sodium phosphate ph 7 . 4 and 500 mm nacl . cell walls were disrupted by sonication on ice at intervals of 0 . 7 seconds for 1 minute , followed by a 1 minute cool down this was repeated three times . the sonicated cells were centrifuged at 10 , 000 rcf for 10 minutes ; the supernatant which contained the soluble adh1 was concentrated and desalted by spin dialysis ( using amicon ultra - 15 devices ; millipore , mass . ), and stored in a new tube at 4 ° c . this method of recombination and expression was also used for a control plasmid pdest17 - gus . enzyme assay was prepared to determine the specific activity of adh1 . the assay contained a final concentration of 1 mm nicotinamide adenine dinucleotide ( nad + ), 0 . 1 μg / μl artemisinic alcohol or dihydroartemisinic alcohol ( aaoh or dhaaoh ) and approximately 90 μg of crude protein . the assays were incubated at 30 ° c . for 30 minutes at 500 rpm , followed by an ethyl acetate extraction . the upper organic phase was transferred to a gc vial and the sample was run on an agilent 6890n — network gc system coupled to an agilent 5973 network mass selective detector . the column present in the gc was an innowax column ( 30 m , i . d . 0 . 250 mm , film 0 . 25 μm ), with a 50 : 1 split injector , the temperature gradient began at 125 ° c . and increased at 5 ° c ./ minute to a maximum temperature of 300 ° c . enzyme assays were prepared to determine whether adh1 acted specifically on artemisinic alcohol or if there was also activity observed with dihydroartemisinic alcohol as a substrate . these substrates were tested with cells that were expressing the adh1 gene as well as a control gus gene . it was observed that adh1 acts on aaoh and not dhaaoh ( fig4 ). fig4 a shows gc / ms analysis of crude adh1 incubated with artemisinic alcohol ( retention time = 20 . 55 min ), and the presence of product artemisinic aldehyde ( 14 . 37 min ). peaks were confirmed by mass spectrometry . fig4 b shows gc / ms analysis of crude adh1 incubated with dihydroartemisinic alcohol ( 19 . 05 min ), and no observed product . no product was observed with extracts of e . coli transformed with the control plasmid pdest17 - gus . references : the disclosures of the following references are incorporated herein by reference in their entirety . altschul , s . f ., gish , w ., miller , w ., myers , e . w ., & amp ; 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