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Cytochrome P450 reductases from poppy plants

a technology of cytochrome p450 and reductases, which is applied in the field of poppy alkaloids production, can solve the problems of limiting the activity rate of cytochrome p450, poor transferability of reductases from diverged species, and not being able to establish the exact nature of “bottlenecks”

Inactive Publication Date: 2006-08-17
JOHNSON & JOHNSON RES PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Benefits of technology

The patent text describes the identification and isolation of a polynucleotide that encodes a cytochrome P-450 reductase enzyme from an alkaloid poppy plant. This enzyme is involved in the production of morphine and other alkaloids in the plant. The invention allows for the control of the total alkaloid content of a plant by alleviating bottlenecks in the production process. The polynucleotide can be expressed in plants and the enzyme can be directed to specific cellular compartments or the extracellular environment. The invention also provides a recombinant DNA construct for expressing the polynucleotide and a method for preparing variants of the cytochrome P-450 reductase enzyme.

Problems solved by technology

There are also thought to be a number of rate limiting steps (“bottlenecks”) where limitations in the availability of either substrates, co-factors or certain enzymes, determine which particular branch of the synthetic pathway is favoured and therefore the ultimate “mix” of alkaloids and the type of alkaloid which is predominantly in the plant.
This imbalance may be a regulatory step for the reduction and therefore be rate-limiting of the cytochrome P450 activity.
Although there is some promiscuity within species, available data suggests that there is poor transferability of reductases from diverged species.
Notwithstanding this body of work, to date it has not been possible to establish the exact nature of the “bottlenecks” in the alkaloid metabolism pathway or to identify the key enzymes which may be responsible and which could be used to manipulate alkaloid metabolism in the opium poppy in order to achieve higher yields of alkaloids generally, and specific alkaloids in particular.
In fact, it is possible that any increase in the yield of straw may result in the relative content of alkaloid decreasing through dilution.

Method used

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Examples

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example 1

Enzyme Purification and Amino Acid Sequencing

[0085] Plant cell cultures. Cell suspension cultures of P. somniferum and E. californica were routinely grown in 1-litre conical flasks containing 400 mL of Linsmaier-Skoog medium (17) over 7 days at 23° C. on a gyratory shaker (100rpm) in diffuse light (750 lux). Elicitation of E. californica cell suspension cultures was achieved by the aseptic addition of methyl jasmonate to a final concentration of 100 μM to the medium (18).

[0086] Purification and sequence analysis. Cells were harvested from seven-day-old suspension cultures of P. somniferum by vacuum filtration, immediately shock frozen and stored at −20° C. All of the following operations were carried out at 4° C. 500 g frozen tissue were then homogenised with a mortar and pestle in 1 litre 0.1 M tricine / NaOH, pH 7.5 containing 15 mM thioglycolic acid. Cell debris was removed by centrifugation at 10,000×g, 30 min. The supernatant was filtered through four layers of cheesecloth and ...

example 2

Generation of Partial cDNAs From P. somniferum and E. californica

[0091] Optimised PCR primers were then designed based on highly homologous sites on both the amino acid and nucleotide levels in the plant cytochrome P-450 reductase sequence comparison (FIG. 3). The precise sequence of the primers used for the first round of PCR was:

5′-CA ITI CII CCT CCT TTC CC-3′  and    T3′-ACC TAC TTC TTA CGI CAA GG-5′.                C      TGC

[0092] Polymerase chain reaction (PCR) generated partial cDNAs encoding cytochrome P-450 reductases from P. somniferum and E. californica were produced by PCR using cDNA produced by reverse transcription of total RNA isolated from 3 to 5-day-old suspension cultured cells. DNA amplification was performed under the following conditions: 5 cycles of 94° C., 30 sec; 45° C., 1 min; 72° C., 1 min; 25 cycles of 94° C., 30 sec; 55° C., 30 sec, 72° C., 1 min. At the end of 30 cycles, the reaction mixtures were incubated for an additional 5 min at 72° C. prior to c...

example 3

cDNA Isolation and Heterologous Expression of Cytochrome P-450 Reductase in Saccharomyces cerevisiae

[0099] cDNA clones encoding the Papaver and Eschscholzia cytochrome P-450 reductases were isolated by screening of cDNA libraries prepared in either 1-ZAP II or Uni-ZAP XR (Stratagene) using the partial clones generated by PCR as hybridization probe. The clones that yielded positive results through a third screening were converted to pBluescript KS (+) by excision. After determination of the nucleotide sequence on both strands, the full length reading frame, free of the 5′- and 3′-flanking sequences, was generated by PCR using either Taq DNA polymerase (Perkin Elmer) and was subcloned into pGEM-T (Promega) or Pfu DNA polymerase and was subcloned into pCR-Script SK (+) (Stratagene).

[0100] The P. somniferum cytochrome P-450 reductase cDNA in pGEM-T, designated pGEM-T / PsoCPR, was digested with the restriction endonucleases Not I and Hin dIII and the 2096 bp fragment was ligated into No...

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Abstract

The present invention relates to production of alkaloids from poppy plants and in particular togenes encoding enzymes in the alkaloid pathway, to proteins encoded by the gens, to plants transformed or transfected with the genes and to methods of altering alkaloid content or blend of poppy plants.

Description

TECHNICAL FIELD [0001] The present invention relates to production of alkaloids from poppy plants and in particular to genes encoding enzymes in the alkaloid pathway, to proteins encoded by the genes, to plants transformed or transfected with the genes and to methods of altering alkaloid content or blend of poppy plants. INTRODUCTION [0002] The opium poppy Papaver somniferum is grown under strict government control, for the production of medically useful alkaloids such as morphine and codeine. The alkaloid content of poppy straw (includes threshed poppy capsules) is the most important parameter in the efficiency of opium alkaloid production. There have been numerous attempts to increase the yield of alkaloid per ton of poppy material. The vast majority of approaches focus on improving agricultural practices and on established methods of conventional breeding in the attempt to increase cultivation efficiencies and to modifying the genotype of the opium poppy plants. [0003] In additio...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01H1/00C07H21/04C12N9/06C12N15/82C12N5/04C12N9/02C12N15/53
CPCC12N9/0042C12N15/8205C12N15/8243
Inventor KUTCHAN, TONIZENK, MEINHARTATKINS, DAVIDFIST, ANTHONY
Owner JOHNSON & JOHNSON RES PTY LTD
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