MICROBIOLOGYTODAYVOL 31/MAY04 67

hydromorphone/hydrocodone. The biocatalyst in thisexample comprises morphine dehydrogenase (MDH)and morphinone reductase (MR) expressed in Escherichiacoli JM109. MDH and MR were isolated from Pseudo-monas putida M10, which was cultured from wasteliquors from an opiate processing plant. The products of the biotransformation are valuable pharmaceuticals,hydromorphone being some seven times more potentthan its parent compound, morphine; hydrocodone isalso used as a mild analgesic. Current methods of manu-facturing these drugs are far from satisfactory, requiringprotection and deprotection of functional groups and theuse of expensive metal catalysts. In addition, low yieldsor a mixture of products may result, depending on theroute taken.

It was possible to improve the initial whole-cellbiocatalysis in several ways: first by the incorporation of asoluble pyridine nucleotide transhydrogenase whichrestored the balance of cofactors; the use of a mutatedMDH with improved stability or its replacement withan NAD+-dependent (3–17)β-hydroxysteroid dehydro-genase which promoted cofactor recycling. The resultingwhole-cell biocatalysts were reusable and efficient, givingrise to product yields of up to 90 % in the best cases.

Small changes in chemical structure often result in adrastic alteration of a pharmaceutical property. Forexample, introduction of an hydroxyl group at the C14position of morphinan alkaloids dramatically increasestheir analgesic potency. In addition, C14-hydroxylatedmorphinans serve as intermediates in the manufacture ofnarcotic antagonists used to treat respiratory depressionfollowing opiate overdose. Such oxyfunctionalization isdifficult to achieve chemically, but there are reports ofhydroxylated compounds resulting from the incubationof morphine alkaloids with P. putida M10 or the fungus,Cylindrocarpon didymum.

� Biosensor designIncredibly perhaps in our modern society, heroin wasfirst introduced to the public by Bayer, the pharma-ceutical company, as a cough medicine in 1898. There

soon followed heroin pastilles, cough lozenges, tablets,water-soluble heroin salts and a heroin/glycerin elixir. Atthe time, such medicines met the compelling need for aneffective treatment for the symptoms of tuberculosis andpneumonia, then the leading causes of death. Heroin,which causes respiratory depression and has a sedativeaction, allowed a good night’s sleep and was seen as ablessing.

The addictive properties of heroin emerged only a yearlater when researchers reported patients developingtolerance to the drug. Over 100 years later, the fascina-tion with heroin still exists, but addiction to this andother Class A drugs stimulates the majority of crimewhich pervades our society. There is little/no productionof heroin in the UK; instead traffickers bring in smallquantities concealed about/in their person or larger bulk hidden inside vehicles, or disguised in any numberof ways as legitimate goods such as machinery or chesspieces. Current detection systems available to theCustoms Officer often require training or are cumber-some or expensive. The need for a reliable, portablesensor to detect heroin has never been greater.

Selective enrichment of bacteria by growth on heroinas sole carbon source led to the isolation of Rhodococcus sp.H1. This organism possesses an inducible heroin esterasewhich sequentially cleaves the two acetylester groupsfrom heroin to yield morphine. By coupling heroinesterase to MDH, the NADPH liberated in the presenceof heroin (or morphine) could then be coupled to a light

TOP LEFT:Fig. 1. Illustration of Papaversomniferum taken from Thomé –Flora von Deutschland, Österreichder Schweiz (1885).COURTESY KURT STÜBER(WWW.BIOLIB.DE)

ABOVE (TOP):Fig. 2. Conversion of morphine or codeine using arecombinant biocatalyst. Morphinedehydrogenase and morphinonereductase were isolated fromPseudomonas putida M10 andexpressed in Escherichia coliJM109. Whole cells were able totransform morphine/codeine intovaluable pharmaceutical products.COURTESY DEBORAH RATHBONE

ABOVE (BOTTOM):Fig. 3. Enzymic detection ofheroin. Bacterial heroin esteraseand morphine dehydrogenase arecoupled with a light output systeminvolving bacterial luciferaseallowing the detection of a fewparticles of heroin.COURTESY DEBORAH RATHBONE

MICROBIOLOGYTODAY VOL 31/MAY0466

�Alkaloids with their plethora of structuralvarieties and activities have long intriguedboth chemists and biologists. More than

10,000 are known, sourced from a variety of plants,microbes and marine organisms. The definition of an alkaloid has changed over the years as the range hasdiversified, but essentially they are complex, nitrogen-containing, usually heterocyclic molecules. Manyalkaloids are of considerable value in both human andanimal medicine. Table 1 gives the main types knownand some examples of each.

Other reviews provide a deeper study of the bio-synthesis and microbial transformation of the vast arrayof alkaloids; here, we concentrate on the microbialmetabolism and transformation of three of the morenotorious alkaloids: morphine, heroin and cocaine, andoutline some of the applications that have arisen from our work.

� Sources of narcoticsOpium and morphinan alkaloids. The Greek word opos,meaning milky juice of plants, effectively describes thewhite latex material that exudes from a cut unripe seedcapsule of Papaver somniferum, the opium poppy (Fig. 1).Opium is the condensed form of this latex. It is not amodern substance, being mentioned in cuneiformwriting on Sumerian clay tablets from 4000 BC. Apapyrus dating back some 3,500 years also suggests its use ‘to prevent excessive crying of children’! The main

constituents of opium include alkaloids such asmorphine, codeine and thebaine, which are extracted and used directly (or their derivatives) in modern-day medicine, providing a range of analgesic (pain-relieving), antitussive (cough suppressing) or narcotic-antagonist properties. Morphine is the main componentof opium, determines psychotropic action and is aneffective pain reliever. Diamorphine (heroin) is a semi-synthetic opiate prepared by the chemical acetylation ofmorphine and finds notoriety as a drug of abuse.

Tropane alkaloids. Cocaine is extracted from the leavesof the coca plant, Erythroxylum coca. Traditionally, leavesof E. coca were chewed by Andean peasants and workers toreduce hunger pain, giving the strength and enduranceto work for many hours at high altitudes. In moderntimes, E. coca is no longer simply a minor crop used bypeasants in far-off lands: the cocaine trade has become ahuge industry.

Cocaine was first isolated in pure alkaloid form in1860 by the German chemist, Dr Albert Neimann, fromleaves brought to Europe. Over the next 20 years or so, it was used extensively by doctors as a stimulant, a local anaesthetic, and, bizarrely, as a cure for morphinedependence. It could be found in over-the-countertonics, toothache cures, in medicines and as chocolate-covered tablets until 1916. The popular drink, Coca-Cola, initially developed as a non-alcoholic tonic andcure-all, contained cocaine until as recently as 1904 ataround 60 mg per serving.

� Drugs as a food sourceBacteria are extremely versatile organisms, able to use awhole range of compounds as sources of nitrogen and/orcarbon for growth. During the course of research in our laboratory, bacteria were isolated from a range ofenvironmental sources which were capable of growing on morphine, heroin or cocaine. By elucidating themetabolic pathways by which bacteria degrade complexmolecules like the morphine alkaloids, we have been ableto isolate some novel enzymes with some interesting anduseful applications.

� Biocatalysis – microbial factoriesIn the chemical industry a variety of catalysts are neededfor well-defined chemical transformations. Often thedesired product is available only at low concentrations oras a mixture with unwanted by-products. Costly down-stream purification processes might then make thechemical synthesis economically non-viable or of lowyield. An alternative is the use of enzymes or whole-cell biocatalysts. Because of their high stereo- or regio-selectivity, these biocatalysts enable highly specifictransformations under moderate reaction conditions.

This is best demonstrated in Fig. 2 which shows abiological system able to convert morphine/codeine to

Microbial narcoticsDeborah A. Rathbone and Neil C. Bruce

The microbialmetabolism andtransformation ofalkaloid narcoticssuch as heroin,morphine andcocaine areattracting theattention ofresearchers. Theresults offer someinterestingapplications, asDeborah Rathboneand Neil Brucedescribe.

Table 1. Types of alkaloid

Alkaloid group Examples Action

Indole Strychnine Poison; bird/mammal/insect control agentVinblastine Anticancer agentPhysostigmine Cholinesterase inhibitor

Isoquinoline Sanguinarine Antimicrobial agentNaloxone Narcotic antagonistMorphine, codeine AnalgesicsPholcodine Antitussive

Pyridine Nicotine Natural insecticidePyrrolizidine Heliotrine Hepatotoxic agent; teratogenQuinoline Quinine Cardiac therapySteroidal α-Tomatine Natural antifungal agent

Jervine Cardiac therapy; hypertension treatment; teratogen Tropane Cocaine Local anaesthetic; tonic; drug of abuse

Atropine Cardiac stimulant Scopolamine Anticholinergic agent; briefly used as a truth drugHyoscyamine Treatment of Parkinson’s disease, GI tract disorders, rhinitis

Miscellaneous Thiocolchicoside Analgesic; myorelaxantCaffeine Stimulant

RO

O

O

NCH3

RO

O

O

NCH3

RO

O

HO

NCH3

NADP+ NADPH NADH NAD+

Morphinone R = HCodeinone R = CH3

Morphinedehydrogenase

Morphinonereductase

Morphine R = HCodeine R = CH3

Hydromorphone R = HHydrocodone R = CH3

NCH3

O

HOCH3COO

O

CH3COO

NCH3 NCH3

O

HO

HO ONADP+ NADPH

FMNH2 FMN

RCHO RCOOH+ O2 + H2O

Heroin esterase

Luciferase

FMN :NADPH oxidoreductase

Morphinedehydrogenase

output system (Fig. 3). A lab prototype device was able to detect the presence of one or two particles of heroin,and correlated well with authentic samples of drug-containing urine and seized street samples (Fig. 4).

� TherapeuticsA strain of Rhodococcus MB1 was isolated from rhizo-sphere soil surrounding the cocaine-producing plantErythroxylum coca. Thebacterium contained anesterase which was able to cleave cocaine intoecgonine methyl ester andbenzoate (Fig. 5).

Whilst antibody-basedtherapies are suitable forthe more extended periodsof time required for cocaineaddiction rehabilitation, amore rapid detoxification is vital in an emergencysituation such as an over-dose. Since cocaine esteraseexhibits the fastest report-ed hydrolysis of cocaineinto non-psychoactive

MICROBIOLOGYTODAY VOL 31/MAY0468

metabolites, it has avaluable potential thera-peutic application as anintravenous treatment forcocaine overdose.

The crystal structure of cocaine esterase hasrecently been solved (Fig.6); it is the first structure of a cocaine-degradingenzyme to be reported. A thorough understandingof the structure–functionrelationship of this enzyme,

which such data allows, provides vital informationrequired for the generation of more efficient cocaineantibody catalysts.

� SummaryBy exploiting the metabolic versatility of microbes, it ispossible to isolate novel enzymes whose properties makethem attractive for a variety of applications such asbiocatalysis, biosensor design and therapeutics to name but a few. The rapid advancement of recombinanttechnologies has already allowed for their improvementand points the way towards a new generation of valuableand sustainable resources.

� Dr Deborah A Rathbone is a Garfield WestonPostdoctoral Research Associate at CNAP,Department of Biology (Area 8), University of York,PO Box 373, York, YO10 5YW, UK.Tel. 01904 328784; Fax 01904 328786email: dr16@york.ac.uk� Professor Neil C. Bruce is the Chair inBiotechnology at CNAP.Tel. 01904 328777; Fax 01904 328801email ncb5@york.ac.uk

Further readingBoonstra, B., Rathbone,D.A. & Bruce, N.C. (2001).Engineering novel biocatalyticroutes for production ofsemisynthetic opiate drugs.Biomol Eng 18, 41–47.

Larson, N., Turner, J.,Stevens, J., Rosser, S.,Basran, A., Lerner, R.,Bruce, N. & Wilson, I.(2002). Crystal structure of abacterial cocaine esterase. NatStruct Biol 9, 17–21.

Lister, D.L., Kanungo, G.,Rathbone, D.A. & Bruce,N.C. (1999). Transformationsof codeine to importantsemisynthetic opiatederivatives by Pseudomonasputida M10. FEMS MicrobiolLett 181, 137–144.

Rathbone, D.A. & Bruce,N.C. (2002). Microbialtransformations of alkaloids.Curr Opin Microbiol 5, 274–281.

Rathbone, D.A., Lister, D.L. & Bruce, N.C. (2002).Biotransformation of alkaloids.In The Alkaloids: Chemistry andBiology, pp. 1–82. Edited byG.A. Cordell. Amsterdam:Elsevier.

TOP RIGHT: Fig. 4. Authentic street samples of heroin compared withpharmaceutical-grade heroin andmorphine.

LOWER RIGHT:Fig. 5. The enzymic conversionof cocaine to non-psychoactivemetabolites.

BOTTOM RIGHT:Fig. 6. The structure of cocaineesterase from Rhodococcus sp.MB1. Structure made with ProteinData Bank coordinate file 1JU3using PyMOL.

ALL COURTESY DEBORAH RATHBONEN

O

H2O

Cocaine

Cocaineesterase

CH3COOCH3

H

C

O

Ecgonine methylester Benzoic acid

COOCH3

CH3

NH

OH

HO C

O