CA2109221C - Production of polyhydroxyalkanoate in plants - Google Patents

Abstract

A plant which produces polyhydroxyalkanoate polymer has a recombinant genome which contains one or snore than one of the genes specifying enzymes critical to the polyhydroxyalkanoate biosynthetic pathway which occurs in certain micro-organisms such as Alcaligenes eutrophus which naturally produce same. The plant species is preferably an oil-producing plant.

Description

Wl0 92/d9747 PC.'T1~H92/00742 1 ~ ~~1~~~~
P~tOI7UCT~0Ie'9 OF POLYFiYD~tO~'~'A~.OATE 7CN PLANTS
fihis.invention relates to the production o~
polyhydroxyalkanoate in plants.
Poly-3-hydroxybutyrate (PF3B) is a linear polyester o~ p(-)-3-hydroxybutyrate. ~t was first discovered in Bacillus meqaterium in 1925.
Polyhydroxybutyrate accumulates in intracellular granules o~ a wide variety of bacteria. The .,granules appear to be membrane bound and can be stained with Hudan Hlack dye: The polymer is .10 produced under conditions of nutrient limitation and acts as a reserve of carbon and energy. The moleau~lar weight ote the polyhydroxybutyrate varies from around 50,000 to granter than 1,000,000, depending on the micro-organisms involved, the ~.5 c~nda.tions o~ growth, end the anethorl employed for extraction off' the polyhydroxyb~ztgrate.
~,blyh~~roxybutyrat~ is an ideal carbon reserve as it exists in tlhe cell in a~ highly educed state, tit is virtually insoluble); and exerts negligible 20 osmotic pressure.
Polyhydroxybutyr~te arid related golyhydroxy-alkan~bates, ~b~h as poky--~~h~droxyvalerate and .
pQly~-3-hyd~oxyoctanoate, are lbidd~gradable thermo-plastics of c~ansiderabl~ commercial importance.
25 The terms "polyhydroxyalkanoate°' and ~°~HA'° as used hereinafter i:ncZud~ polymers of ' 3--hydroxybut~~ate, p~lymers-of related ~ydx~~xyalkano~tes such as 3-hydroxyvalerate, -~~aroxyhexanoate, 3-hydroxyoctanoate, ~~ ~p~~~r~~~dec~nc~ate; and also copolymers and ~1'(> 92/ ~ 9747 PGT/G X92/007.42 ~.
mixtures of more than one of these hydroxy- .
alkanoates.
Polyhydroxyalkanoate is biodegradable and is broken down rapidly by soil micro-organisms. It is thermoplastic (it melts at ~.~0°C) and can readily be moulded into, di~rerse forms using technology well-established for the other thermoplastics materials such as high-density polyethylene which melts at around the same temperature (x.90°C). The .10 material is ideal for the production of biodegradable packaging which will degrade in .landfill sites and sewage farms. The polymer is bicicoaapata:lble, as well as biodegradable, and is well tolerated by the mammalian, including human, 25 body; its degradation product, 3-hydroxybutyrat~, is a normal mammalian metabolite. Polyhydroxy~
butyrate degrades only s~.owl~r in the body making it suitab~.e for meeli~al applications where long term degradatian is ~~~uire~~
Z~ Polyhydroxy~l~Can~ate, produced by the micrr~-organisaa Alca~.igenes eutrd_phus, is manufactured, as a copolymer of polyhydroxy-butyrate and polyhydr~xlrvalerate, by imperial Chemical industries P1GC arad sold a~nder the Trade 25 Mark SIOPOL. 'the nature o~ the polymer, for exaa~p~e the Proportions of FHB and ~HV is aeaer~aine~ by the substrate'supglied in the fermentation. It i~ nor~aally supplied in the form of pellets for therr~oprocessing: How~Ver, po,lyhy~raxy~lkanoat~ is m~re expansive tc~
mahufacture ~by exi,stingmethods than, say, pplyethxlene. It is, therefore; desirable that new; a~oxa economic ~rodu:ction of polyhydroxy-alkanoat~ be provided.

An obaect of the present invention is to provide materials and a method for the efficient production of polyhydroxyalkanoa~te.
According to the present invention there is provided a plant adapted for the production of .
polyhydroxyalkanoate comprising a recombinant genome of an oil-producing plant, which genome contains genes encoding enzymes necessary for catalysing the production of polyhydroxy- alkanoate 1p tagether with gene regulatary sequences directing expression of the said genes to target plant cell components.
. These regulatory sequences include promoter sequences directing expression of the biosynthetic 15 pathway specifically t~ the developing seed, and transit peptide sequences targeting the enzymes to appropriate subc~llular c~mpartments.
The genes encoding the enzyme or enzymes .
necessary for the catalysis of polykaydroxyalkanoate 20 production may be isolated from a micro--organism, such as Alcaligenes eutrophus, which is known to produce polyhydroxybutyrate end other polyhydroxy-alkanoates.
It is preferable, for seasons which will later ~5 b~ explained, that the plant be of a species which pr~dw~~s substantial quantities of Qil, x~at~aer than starch. Such plant species are well known and are simply referred to as ~eoil-seed" craps and include, oilseed ripe, canolai says and sunflower. Ntethods y 30 for the genetic transfo~ma ion of many oil crops are kn~wn~ for example, transformation by Agrobacterium tum~faciens methods are suitable far most: Such methods are well-described ira the literature and well-known and expensively practised 'W4 92/19747 P(.°T/G~92/0(~742 in the art. ' The biosynthesis of polyhydroxybutyrate from the substrate, acetyl-Co~4, involves three enzyme-catalysed steps, illustrated in Figure 1 herewith.
The three enzymes involved are ..
~-ketothiolase, I~P.DP linked acetoacetyl-CoA
reductase, and polyhydroxybutyrate synthase, the genes for which have been cloned from ~.lcaligenes eutrophus (Schubert et a1, 1988, ,~ Bacterial, 170).
1U When cloned into Escherichia coli the three genes are known to facilitate production of polyhydrpxyalkanoate up to 30~ of the cell weight.
Genes specafya,ng the production of alkanoates higher than the butyrate are known to exist in bacteria. Isolation of the appropriate genes allows expression of these higher polyhydroxy-alkanoates. For example, genes spec~.fying production of the polyhydroxy--octanoate and the -decanvate exist in the lbacterial species Ps~udon~ana~ oleo~rorans and ~!seudoa~onas eruginosa.
FIoweverr gene for an~log~aus polymers are widespread in bacterial species.
111 the microorganisms r~e~uired for ~~rf~rmance of this inv~~tiora a~~e publicly available from public culture collections.
bra i.~port~rat preferred feature of this invention i~ the use of an oilseed plant for a%pression of the p~lyhydrox~ralka~oate. The reason behind fur selection of oil-producing crops i~ that sucks ~alants naturally pr~duce Iaxge amounts of acetyl,;CoA substrate (under aerobic conditions) in the developing seed; which is normally used in fatty acid synthesis: Diversion of this substrate into pol~hydroxyalkana~te production will reduce '~V(192/ 19747 ~'(."T/G X392/00742 _ 5 _ ~~ ~l.)n~', :i ,.,, ,,r the amount of oil stored by the seed but will have minimal influence an other aspects of the csll~s metabolism. It is therefore possible to produce commercially viable quantities of.polyhydroxy-S alkanoate such as polyhydroxybutyrate in an .' .
oilseed.
It has been previously suggested that Alcaligenes eutrophus genes could be expressed in a starch crop but this has certain problems. :tn order to optimise polyhydroxyalkanoate production in such a crop, it would probably be necessary to down-regulate starch synthesis: However, even if .~ ~ ttais down-regulation were to be effected it would not guarantee an increased rate of acetyl--Go,~

production. Moreover, even if this increased production were actually achieved, it is possible that the acetyl-Cod would be rapidly utilised by respiration in the starch crop.
F'or expressir~n in higher plants the bacterial (for example Alca~:igdnes eutroph_us) genes require suitable promoter end termsnato~ seguences.
Varfoul promoters/terminat~rs are avai~.able for use. F'or constitutive expression the cauliflower mosaic virus CaMV35~ promoter and nos terminator may be ussc~. It i~ ~aowever preferred to target :,_.
synthesis of p~lyhydroxyalkanoat~ oaaly.to the developa.ng oil storage organ of the oilseed such as the embry~ of oilseed rape: 'The promoter of the rape seed storage pxot~e~.n, n~gain, could be used to obtain embryo specific expre~sxon of polyhydroxyalkanoate genes. . Expression of the polyhydroxyalkanoate genes during the precise period when lipid a,s being made will ensure effective competition by the polyhydroxyalkanoate ~N~ 92!19747 . PCT/~B92/Ofl742 _ 6 _ enxymes for available acetyl-CoA. The promoters of fatty acid synthesis genes whase expressions are switched on at this time are thus most appropriate candidates to be used as polyhydroxyalkanoate gene promoters. Examples of such promoters are those of a seed specific isoforms of rape aryl carrier protein 4ACP) or S-ketoacyl ACP reductase.
In inserting the polyhydroxyalkanoate genes into eukaryotic cells, consideration has to be given to the most appropriate subcellular compartment in which to locate the enzymes. Two factors are important: the site of production of the acetyl-CoA substrate, and the a~railabl~ space for storage of the polyhydroxyalkanoate polymer.
The acetyl-C~A required for fatty acid synthesis ~.r~, far example, developing rapeseed embryo is produced by two routes. The first, direct, route involves the activity of a pyruvate dehydrogenase enzyme located in the plastid. The sec~nd route involves the initial production of acetyl-CoA by mitochondria! pyxuvate dehydrogenase, lYsa.s to free acetate, and diffusion of the acetate int~ the pxastid where it is re-esterifi~d to CoA
by adetyl-CoA syn~ha~e. Rapeseed also produces ad~tyl-CoA i.n the cytosol, though at a lower rate than in the-plastid, via the activity of a cytosolic citrate lyase enzyme.
Considering substrate supply, the bacterial for exaanple, ,~,lcali~enes ) ~-k~tothiolase enzyme mad functioa~ in the mitochondrion; using acetyl-CoA
produ~~c~ ~,n arcs s of the re~quire~aents of respiration, or ire the cytpsol. The regulatary s~qu~ncss of the inventi~n nsay thus darent expression of the ~=~ketdthiolase gene to the WO 92/19747 PG'I'/G892/00742 - 4~ ~ ~1 f' z~ yy ;l rd FJ
mitochondrion or to the cytosol. it is however preferred to target this enzyme to the plastids, where highest rates of acetyl-CoA generation occur.
The mitochondrion lacks sufficient space for storage. of, the polyhydroxyalkanoate polymer.
Significant storage space exists in the plastids, at least in rape embryo. I~ighest storage space exists in the cytosol, the compartment normally occupied by the oil bodies.
It is not known whether the acetoacetyl-CaA
or hydroxybutyryl-CoA pathway intermediates can be .transported from p~.astid to cytosol. Certainly they would not be able try traverse.the plastid ~ envelope membrane as CoA esters. export would require that the acetoacetate or hydxoxybutyrate groups:.are recognised by the transport systems involved a.n export. of fatty acids from plastids.
These have been suggested to involve: lysis of the CoA ester, export of the free acrd, and resynthesis c~f the CoA ester in the c~tosol~ or transfer of the aryl groups to carnitir~e, and export of acyl carnitine. ~f aceto~acetyl groups array be exported from the p~~s~~.d by one of these mechanisms then it would ~e possible t~ target ~-ketothiolase to the plastid. to utilise acetyl--CoA destined for lipid synthesis, and target-acetna~~tyl~.CoA reductase and polyhydroxybutyrat~: synthase to-the cytosol to achieve polymer ~yrathesis ire thi5 more spacious coanpartznent. ~f neither acetoacetate nor 34 hydroxybutyrate groups may be exported from the pleated, polyhydroxyalka~oa~e synthesis will require that all thxee pathway enzymes are targeted to this organelle so that their are expressed in the same cell compartment W~ 92/19747 - PCT/~892/00742 ~~ g _ , ':
T~ ~~~ g~t ~he three bacterial (such as Alcaligenes eutrophus) enzymes for polyhydroxyalkanoate synthesis to the plant plastid requires the use of specific targeting regulatory elements called transit peptides.
Possible sources of plastid stroma targeting sequences are the genes fore (a) ribulose bisphosphate carboxylase/oxygenase small subunit (F~UBISCO ssu);
(b) acyl carrier protean (ACP);
(c) ~-ketoacyl ACP rsductase;
(d) enolp~rruv~ylshikiana~te-3-phosphate synthase -~ ~ ( EPSPS ) ;
(e) fr~uctase 1,6-bisphosphatase:
1g Of these th~:RUBI~CO small subunit transit .peptide has been shown to direct polypeptides to plastids in both photosynthetic and non-photosynthetzc tissues: MCP and S~-ket~aayl ACP
redu~ta~e transit peptides would a~.so operate effectively inplants such as rape embryo. The adv~ratage of using the same plastid transit peptide for all three polyhydroacyalkanoate genes is t~
e'zasure that any vat~.abilaty in the uptake of the gea~es i~ not due to the transit peptide which xs used.
Although some proteins appear to be effic~.~ntly targeted to the'pl~stid stroma by the transit peptide alba~e, ntlae proteins also require the presence of up to twenty amino acids o~ the ~9 aanino 'terminus of the mature protein. The ,.y,.;, requirement far the presence of mature sequences' apg~ar~ to depend on the size and charge ~f the pr~tein to be transported.
To obtain synthesis of p~lyhydxoxyalkanoate L..
a , v r. ~ r . o s . , . , S .'. n .., ....,........... ..,..w,..o-.... ~ ~. ... ... , !.a°. f ...a..,.
..t.>...~F'.' v....n. .n. t...m.m.5.....i....._l.~.i~ .. ...,. ..5,....... .
.. a .._o. r ....,.. .. a....._a...m ....s.~.m7, ..w>.. :,.., ,. n 'WC? 92119747 PCT/G~92/00742 polymer in plant tissues it is necessary to obtain plants expressing all three genes for the enzymes ~-ketothiolase, acetoacetyl-CoA reductase and palyhydroxybutyrate synthase. This may be achieved by using ~ne of the following strategies:
i) plants may be individually transformed with the three polyhydroxyalkanoate pathway genes.
Plants-containing individual genes are grown up in the glass-house and cross-pollinated to,,'.:
obtain hybrid plants containing two pathway genes. This procedure is then repeated to produce hybrid plants containing all three genes.
ii) Plants may be sequentially transformed with plasmids containing the individual pathway genes.
iii) Two or three pathway genes may be cotransfor~ned into the same plant by simultaneous-ir~f~ction with Agrobact~ria containing the individual genes.
iv) Plants may be transformed with plasmids , contaatning twp or three pathway genes:
A combination of these techniques may be used to obtain ~xpre~sian of aI3. ~th;ree genes in a single ~5 plant. Successive round of ~~oss--psallination are carried nut until the pror~eray are homozygous for all three genes. For metkrods ( ii ) arad ( iii ) above, .
at is adwantageo~as to insert each gene into vectors containing different selectable ~aarker genes to facilitate selection of plants containing two or three p~~IYhydroxyalkanc~ate pathway genes. ~xa~iples ~f selectable markers are genes conferring resastaa~ces to karaamyci.n; hygromy~ara, sulphonamides and bialapho~ or phasp~hinothricin.

WO 92/19?~4? PCT/GH92100?42 The invention will now be described by way o~f example only with reference to the accompanying drawings, in which: ' Figure 1 shows the pathway for polyhydroxybutyrate production in Alcaligenes .
eutrophus;
Figure 2 is a physical map of the 5.2kb Smal-EcoRI fragment of Alcaligenes eutrophus DNA;
Figure 3 is a map of the plant expression vector p~7Rli;
Figure 9 shows the Southern blot analysis of , the three gCR products produced during the making o-f the ssu transit peptide-ketothiolase construct;
Figure 5 is a graph of S-~reto~thio~.ase enzyme s activities in tobacco leaves;
Figure 6 is a graph of NADP acetoacetyl CoA
reductase enzyme adtivities in tobacco leaves.
EiF~,E
A 5.2kb Smal--EcbRI fragment which codes for 2D all three polyhydroxya7.ka~no~te ( PF3A) biosynthetic genes had previously been isolated from Al,calic,~enes eutrophus (Schubert a al, 198, ~ Bacteriol, 17Q).
This fragment cloned into the vector p~C9 (New England Siolabs) t~gether with a 2.3kb F~tI sub fragment Toned into ~lue~cr~pt RS- (Stratagene) ~aer~ provided by D'r Stei.nbuchel of the university of Got~ingen, G~~~any.' A r~estricti.on map of the fragment is shown xn Figure 2. The positirans of the restri.cti~n sites and the positions of the 3Q genes far ~-ketothiola'se, a~e~to~cetyl CoA
reductase; and polyhydroxybutyrate tPHH) synth~se are shown.
The ex~aressic~ra vector chosen to gain constitutive expression of P~1~ biosynthetic genes W~ 92/19747 - 9'C.T/GH9Z/0~742 'j -~ ~ ~ ~ ~' '~
in tobacco and oilseed rape plants was pJRli. This vector contains the cauliflower mosaic virus CaMV35S promoter and the nos terminator, separated by a multiple cloning site to allow the insertion of the PHA genes. The vector also contains the kanamycin resistance n~II gene as a selectable marker. Figure 3 is a map of the plant expression vector pJRla. Vector p~3RlRi was also wtilised;
this vector contains the expression cassette in the opposite orientation.
All routine molecular bialogical techniques were these of Sambraok et al (1989, A laboratory w- manual, Second edition). Oligonucleotides were all synthesised on an Applied Biosystems 380H DIVA
synthesiser. PCR machines used were Techne PHC-1 Programmable Dri-Hlocks. Taq polymerase was . ;;::r~:
obtained ~rom Perkin-Elmer/Cetus. Restrictian enzymes and ~ther modifying enzymes were obtained ~rpm lNew England Bialabsr Gibco/DRL, Northumbria Hiologicals Dimited and Pharaaacia. ~er~uencing kits were obtained from Cambridge Hiosciences (~Qq~enase) and Promeg~ (Tac~track). All radio-isotopes were supplied by Amersham International.
3.. Construction of vectors to ain constitutive c tosolid ex ressxon o~ Pte; ath~ra eases.
1.~.: ~-ketotha.ola~e the ~-k~tbthiolase g~r~e was isolated as a le3kb Pstl-PIeT fragment groan the 2.3kb Pstl , 0 f ra~~nent cif pKS-: ~ 2 . 3P7 > ~'hi s fragment was blunt-ended ~ ~rith Klenow and ~ra~ ir~~er~ed auto the dephosphorylated Smal site of pJRZi. The resulting p~asraid vas dea~ated pJRliTa R~c~amkainant plasmads were identified by colony hy~ra.disatian using the 9~0 92!19747 PCTlGH92/00742 _ 12 ~..3kb insert fragment as a probe. Restriction mapping of recombinants revealed those containing a single ~-ketothiolase insert in the sense orientation. The orientation of the insert was confirmed by sequencing using a primer that hybridised to the 3' end of the CaMV355 promoter.
1, . ~ . ~lcetyoacetyl-Cod reductase The acetoacetyl-CoA reductase gene was isolated as a 0.9kb Aval;t-XmnII fragment fram pKS. ..
::2.3P7. This fragment was inserted into pJRIi as described for pJRIiT. However, the orientation of the insert fragment in recombinant plasmids could not be confirmed by restriction mapping due to the unavailability of suitable restriction enzyme sites. Therefore faur recombinants were sequenced using the CaMV35S 3' primer and, of these, one'was found to contain a sense insert. This plasmid was denoted p~Rli~R.
r ~ w ~~~ ~y~thase Z0 The PHA s~ntha~e g~~e was isolated from piiS-::2.3P7 as a $stBl-Stul~fragm~nt. This fragment was blunt--ended and ' inserted iaato p,7RIi as described f~~ pJRIaT and pJRIiR. The identity of recombinant (pJAr3.S, plae~aids cantain~ng a singly insert in the dense orientatibn was confirmed by restriction bopping and by ~equenc~.ng with the CaMV35S 3~ pra:mer.

2: C~nstruct~~n d~ vectors fr~r bonstitutive lastid tar eted ex re~sa~~n of ~H~ athr~a enx es .
~rar~spor~ into pla~t,xds of the nnmponen~.
polyp,eptides for each c~f the. PHH pathway enzymes can be achieved by ~dditi.on of a transit peptide sequence to the 5' end of the gene sequence.
The first gene ~o be tailored was W~ 92/~ 9747 P~.'f/GB92/0~742 13 ~~~~~~~il ketothiolase. A technique involving polymerase ' chain reaction (PC~t) was employed in order to join the pea ~tUB~SCt7 small subunit transit peptide sequence in frame with the ketothiolase gene.
Linking the transit peptide to the ketothiolase gene involved three experiments. The first experiment added a small portion of the ,~5' end of the ketothiolase gene onto the 3' end of the transit peptide sequence. The seGOnd experiment added a small portion of the 3° end of the transit peptide onto the 5' end of ketothiolase gene. The third experiment utilised the overhangs produced in .the preceding exp~riments~te~ extend across the junction and praduce full length transit peptide 1,5 linked in frame with the ketothiolase gene.
Four PCR primers were designed:-1. 5! end of the transit Peptide allowing extension award its 3' end:
,TGG CTS' CTA TG1~. TAT CCT CTT CAG CT

2. 3' end o~ transit peptide linked to 5' end of ke~othiolase gene allowing extensa.c~n toward 5' end of transit pep~idea ACG .~.TG A.CA ~.CG TC~: GTC ATG C~sC TTT ACT CTT
CCA CCs TTG CTT GT
TPK~

3. 3° end of 'transit pepti;de,linked to 5' end of ketothaol~se gene all~win~ extension toward the 3~ end of the keteathi~las~ gene:
~0 PTT ,ACA AGC ACT GGT GGA A,GA GTA ~G TGC
~ATG ACS" GAC GTT GTC ATC GT
TFKT

4. 3' er~d of ket~thiolase gene:
ACC CCT ACC TTA TTT GCG CSC GAC T

a For the fist experiment template DNA was pSM64 (transit peptide sequence) and the primers were TP1 and TPKB with an annealing temperature of 65°C. The derived PCR products were run out on an agarose gel and the band corresponding to 199bp cut out and electroeluted from the gel.
In the second experiment template DNA was pKS::2.3P7, the primers involved were TPKT and K1 and the annealing temperature 68°C. The products of the PCR reaction were again run out on a gel and the required I.207kb band isolated and electroeluted from the gel slice.
The third experiment utilised the~~DNA isolated from the previous experiment as template and the primers TP1 and Kl. The annealing temperature was 65°C and although this PCR experiment was very inefficient some full length product (1.352kb) was formed.
A small portion of each of the three PCR
products was run out on an agarose gel. Southern blot analysis using three of the oligos as probes (TP1, K1 and TPKT) was carried out. Results are given in Figure 4 and show that the product of the third reaction contained the 5' end of the transit peptide, the overlap of 3' transit peptide and 5' ketothiolase gene, and the 3' end of the ketothiolase gene.
It was necessary to check the sequence of this product as it is known that PCR can incorporate base mismatches. The PCR product was blunt-ended and cloned into Smal cut and phosphatased pUClB.
Six clones were identified which contained the PCR
product. The clones were sequenced using the universal and reverse primers (Sequenase~'kit and Trademark ' WO 92/19747 PCT/GB92/00742 Taqtrack~kit). Clones with completely correct ' sequence through the transit peptide and the 5' end of the ketothiolase gene up to a TthIIIl restriction site within the gene were identified.
From one of these clones a TthIIIl-Kpnl fragment , was excised. The Kpnl site was cutback to give a blunt end, and a TthILIl-Smal fragment of Alcaligenes eutrophus DNA from pKS-::2.3P7 corresponding to the major portion of the ketothiolase gene was inserted. Positive clones were sequenced across the joins. The transit peptide-ketothiolase fragment was excised and inserted into pJRlRi.
For the transit peptide-reductase construct PCR was also utilised. This required only one PCR
experiment as a Dde I site (unique in the transit peptide and,reductase sequence ) was present close to the 5' end of the gene. The PCR experiment required two primers:-I. Sequence homologous to the 5' end of the transit peptide which would allow extension toward the 3' end. A Cla I site was incorporated into the sequence 5' to the transit peptide sequence.
AGC ATC GAT GGA TGG CTT CTA Z'GA TAT CCT CTT
CAG CT
CLATP
2. Sequence homologous to just past the Dde I
site in the reductase gene,li,nked in frame with 3' transit peptide sequence to allow extension toward the 5' transit peptide.
ATG CGC TGA GTC ATG CAC TTT ACT CTT CCA CCA
TTG CTT GTA AT
TPDDER
Trademark W~ 92/19747 ' P~.'T/GB92/00742 16 _ After PCR with these two primers and transit' peptide DNA as teanplate the 195 by product was identified on agarose gels and isolated by electroelution. DdeI XmnT reductase gene was isolated and ligated to Ddel cut PCR product.
, After agarose gel electrpphoresis the 1.063 kb band was isolated, out with CIaI and ligated into Clal EcoR~ Bluescript gK(-). Positives are being characterised.
3. Transformation of plants ~itla the P~tB genes 3.1. ~rgrobacterium transformations .
Cesium-pure p3RIiT, pJRIiR, pJRIiE and pJRIi were individually transformed into ~c~robacterium tume~aciens strain LBA4Q04 by direct uptakes as follows> LB (lOmls) was inoculated with A
tumefaciens strain LBA4404. The culture was shake-incubated at 28°C for appraxianately 16 hours until tie opt~ical'density (t~D) at 660 nm was 0.5. The :cells were recovered by c~ntrifuc~at~.on (3000 rpm So~cvall RT6000B, 6 mina. 4°C)~ They were r~suspended a.~ 250N1 of ice-cold 20mr~i CaCl2. The cell suspension was then dispensed into pre-chilled Ep~endorf tubes an 0.1m1 aliquots. APProxi:mat~ly l,ug of caesium-pure pla~znid DN~r was added to each tube. The cells 'were ti~en hea~c--shocked by ~rasping in liquid nitrogen ~oll~wedl by incubation at 37°C
fdr 5 minutes. La 'medium ( l~nl ) was added and 'the cells were allowed ~o recover by incubation (shaken) at 28°C for 3-4 hours: The sell pellets were obtained by centrifugation (11,5008; 30 seconds; 20P~) and xesuspended in O.Lml LB.
Rec9mbinant cells were selected on L8 (agar-salidified) co~taining,kanamyoin (50,ug/ml).
streptomycin (500pg/ml) and rifampicin (~:OOpgfml) ''1y~ 92J19'l~7 '; i~r~d~
P~LT/oG 892104742 - 1! - ~:~,~e~Fwrfr following incubation at 28°C. Mini-prep DNA of the resultant A~grobacterium strains was then isolated and analysed by restriction enzyme digestion to ensure that na re-arrangements had occurred.
3.2. Plant Transformations Tobacco leaf pieces and oilseed rape petioles were inoculated a.ndividually with strains LBA4404/JRIi, LBA4404/p~RIiT, LBA4404/p~RI3R and LBA4404/p~RxiS. Plants were cultured in a growth raom with a temperature of 25°C and a photaperiod of 16 hours.
Brassica napes cv. Westar seedlings were sterilised in l0% sodium hypochlorite and washed in sterile water before germin2~tian on MS medium (Imperial)(containing 3% sucrose and 0.7% phytagar (Gibco). The cotyledons were excised from 5 day old seedlings and the petioles ~f which were placed in MS msdi~m as above but supplemented w~,th 4.5,~g/m1 benzy7.aroinapurine (B~,P): The cotyledons were cultured in this medium for 24 hours after which their petioles were dipped in an ~.grobacterium solution. The Agra~aacterium culture hid green grown n~rernight in LB medium containing kanaanycin ( 50pag/m~l ) fallowing which the Agrobactex~iu~n ells had been pel~eted and washed in liquid MS ~ncdium and di~.uted to OD~~O 0.1. the inoculated petioles were returned to the MS medium containing 4.5,~g/m2 BAP and 'incubated in tha culture room for 2 days. The cotyledons were then transferred to'MS medium supplemented with BAP
( 4 . 5,uc~/ml ) . carbenicillin (Du~hefa ) ( 500,ug/ml ) and kanamycin (l5;ug/ml): The cotyledons were subcultured an this medium every 2 weeks until the produ~tian of green callus arad e~rentually shoots.

vvo ~zi i 97a~ P~i~~9z>oo7az - a.a -Shoots were excised and cultured on MS containing carbenicillin (500pg/ml) and kanamycin (l5,ug/ml) until they were transferred to the glasshouse.
Nicotiana tabacum cv SRI seeds were sterilised as described above and germinated on MS medium (containing 3~ sucrose and 0.8~ bactoagar). The shoot tips from these seedlings were then micropropagated on this media to provide plants for transformation studies. Leaf pieces from these 20 plants were dipped in an Agrobacterium solution (prepared as described above) and were then cultured on MS medium containing 3% sucrose, 0.8~
bactoagar, l,ug/m1 RAP amd 0 ~ l,ug/a~l NAA, for 2 days.
. The leaf p~,eces were then cultured on the same ~.8 meda.a supplemented with carbenicillin (500,ug/ml) and kanamycin (100,~g/ml) for 5 weeks. Regenerated shoots vJere excised and cultured on MS containing 3% sucrose; 0:8~ laa~~oagar; 200,~g/ml carbenicillin and lOO,ug/ml k~n~mycxn for 2 passages of 5 weeks 20 before transfer to the glasshouse.
Kanamycxn~-resistant bobacco and rape plants were obtained for khose transformed individually with JRIi, JRIiT,'JRIiR end JRIiS.
~ , 3 s ~otr~r~sfo~~a~i~ns 25 Rape cotyledpns and ~c~bacco leaf pieces were also a:n~cul~~eci with z~axtures of l~grohact~rium straa:ns.' These ina~ulat~~ns wire performed as described previously except that 3~:1 mixtures of d~.lut~d Aq_roba~texium culture, of the wane ogtical 30 porosity, were grepared i~raed3;ately priest to ~. inoculation:
~ o Piio~he~aical assessment of plaxats aggression of Alcaligenes eutrophus p~tA
pathway enzymes in plant tissues ways detected by 'W~ 92r'~9747 ~ PC'I'/GB92/00742 - 19 - G, ~y~~t.~~~
enzyme activity assays. The presence of the enzyme polypeptides was also detected by Western blot -analysis»
For the latter analyses rabbit polyclonal antibodies were raised to the purified S-ketothiolase and MADp acetoacetyl CoA reductase enzymes from Alcaligenes eut~ophus» macteria were '..
pelleted, washed, and crude extracts prepared as described by Haywood and Large (1901, Fsiochem ~J, 199, 107--201) . ~-keto~h~.olase A was purified by chromatography on hydroxylapatxte, followed by anion exchange chromatography on FELC mono Q, . f~llowed by gel filtration on Superdex E--200 (Pharmacia), wing modifida~aons of method described lby Haywood et al (1980, FEMS Microbiology ~.etter~, S2, 91-9~). 1~ADP acetoa~etyl-CoA
reductase was purified using the same techniques, with an additional affinaty chromatography step on 2',5' ASP s~pharose (Pharmacia). Purafi.ed proteins ZO were subjected to sodium dod~cyl sulphate polyacrylaanzde gel electrophoresis (SDS PAGE) according tea the method of Laemmli (1970; Nature, 222, 680-685 ) 0 The final ~3-keto~hiolase p~Ep~sation showed a sa.ngle coomassie blue stained band at 4lkd. ' ~hie final ~reduct~se preparation choked a major band et 26kd» 3mg o~ purified ketotha:~lase end 2mg of purifx~d reducta~e wire .
subjected to preparative SDS PAGE» The bands c~rresporading to the two enzymes were ~l~ctroeluted fr~m'the gels and injected into rabbits to raise polyclonal anta~bod~.es ~ Eera, from primary and secondary bleec%s following injection were sh~awn to contain ant~;bs~dies spe~ifie for their taxget enzymes via Western blot analyses crf crude W~ 92/1974? PCT/GB92/00742 AlcalicLenes extracts.
Crude extracts of tobacco leaves were prepared by grinding leaf tissue in 50mM potassium phosphate buffer pH7.0 containing 2mM dithiothreitol. After centrifugation at 30,0008, enzyme assays for ketothialase and acetaacetyl CoA reductase were conducted on aliquots of the supernatants by the methods described by Haywood et al (1988, FE1~S
Microbiology Letters, 52, 91-96; 52, 259-264). PHB
synthase assays were conducted on aliquots of the 30,0008 supernatants and aliquots of the pellets, ..resuspended in extraction buffer, by the methad of . H~ywood et a1 (1989, FEri~S Microbiology Letters, 57, 1~6?.
For Western blot analysis, aliquots of the 30,0008 supernatants were subjected to SDS PAGE and electrophoreti~~lly transferred to nitrocellulose filters:, Filters were then rinsed in TBS (50mrt Tris-~ICl pH7.9, 150mM Na~CI~ and incubated in TeS
plus 5~ bovine serum albumin. P~~teins reacting with anti-ketothiolase or anti-reductase serum were detected by incubating the falters in 200m1 THS
containing 2m1 of the relevant ssxum for 1~-2h.
Hound first antibody was subsequently detected 2~ using 84a anti--rabbit IgG alkaline ghosphatase.
conjugate and nitroblue tetra~ola:u~ alkaline phosph.a~as~ colour dwelopr~~nt reagent ( aioRad Laboratoxies).
lriibial biochemical analyses wire carried out on su3~~ultured t~obacca pl~r~ts g=owing in tissue 'culture. Eigh een kanamycin. resistant plants transformed with ~lRla. ketothiolase wire subjebted to enzyme'analy~is and results compared with untransfor:med control plants. Leaves of the same W~ 92/19767 PL~'1G~92/Of1742 ~,~.~~~~~
size were extracted.
.Figure 5 shows the ~-ketothiolase enzyme activities in the tobacco leaves. The identification numbers of individual plants are S shown on the x axis. Plants to the left of the dotted line are untransformed control plants.
Plants to the right of the line are transformed with JRli ketothiolase. ;
A low level of ketothiolase activity was detected in untransformed control plants. Dearly all of the JFtli ketothiolase transformed plants had ketothiolase activity higher than control. The , highest activity was 34 nmol/min/ang protein, 2.8 times higher than the h3.ghest control plant. zn t~lestern blots the anti-~ketotha:olase antibody detected a polypeptide at 9lkd in untransformed' control tobacco plants ~ possibly corresponding to the endogenous ketothiolase enzyme activity. t~ha.le a ~lkd polypeptide '~a~ also detected in extracts of ,lRli ketothiolo~se transformed plants, the Western blots could not quantitata,vely distinguish transformed from untransfAr~ned plants.
Figure 6 shows the NhDP acetaacetyl Cod reductase enzyme ~dtiviti.es in lea~res of the tissue culture grown tobacco Plants. The identification aaumbers of individual plants are shown on the x axis: Plants to the left ~f the dotted Iine are untransformed control plants: Plants to the right of the line are tr~nsfor~ued'with p~Rli reductase.
p, low level ~f ac~to~cetyl Cod reductase activity eras detected in unt.ran~f~rm~d control plants: Dearly alb the 21 ~Rli reducta~e transformed plants had reductase activity higher ~.han control. The highest act~.vity was 30 VY() 92/19747 ~ ~ _ pC.T/~~92/0074Z

nmol/min/mg protein, 4 fold higher than the highest control plant. In Western blots the anti-reductase antibody did not detect any polypeptide with a m.w.
of 26kd in extracts of untransformed control glants. ,~, 26kd polypeptide was however detected in extracts of the JRIi reductase transformed plantso Expression of the bacterial reductase gene in tobacco leaves was therefore demonstrated.

Claims (10)

CLAIMS:

1. A plant cell for the production of polyhydroxyalkanoate comprising a recombinant genome of an oil-producing plant, wherein the genome contains genes encoding enzymes necessary for catalyzing the production of polyhydroxyalkanoate together with gene regulatory sequences directing expression of the said genes to target plant cell components.

2. A plant cell according to claim 1 wherein the genes encoding the enzymes necessary for the catalysis of polyhydroxyalkanoate production are isolated from a micro-organism.

3. A plant cell according to claim 2 wherein the micro-organism is Alcaligenes eutrophus.

4. A plant cell according to claim 1 in which the oil-producing plant is selected from the group consisting of oilseed rape, canola, soya and sunflower.

5. A plant cell as claimed in claim 4 wherein the oil-producing plant is Brassica napus.

6. A plant cell as claimed in claim 4, wherein the plant cell is an oil storage organ cell to which the gene regulatory sequences direct the expression of the polyhydroxyalkanoate genes.

7. A plant cell as claimed in claim 6, wherein the plant cell is a developing oil storage organ cell to which expression is directed.

8. A plant cell as claimed in claim 6, wherein the plant cell is an embryo cell to which expression is directed.

9. A plant cell as claimed in claim 1, in which the said gene regulatory sequences direct expression of the polyhydroxyalkanoate genes to the cytosol or to the mitochondrion or to the plastid.

10. A plant cell as claimed in any one of claims 1 to 9, in which the enzymes comprise .beta.-ketothiolase, acetoacetyl-CoA reductase and polyhydroxybutyrate synthase.