Systematic gene disruption and chemical compleme ntation studies have shown that at least five enzymes cata lyse the formatio n of paxi lline i n P. paxilli
(McMillan et al. , 2003; Young et al. , 2 00 1 ) . Deletion mutants of paxG (encodes a GGPP synth ase), paxM (encodes a FAD-dependent monooxygenase) (McMilla n and Scott, unpubli s hed results) a nd paxC (encodes a prenyltransferase) (Young et al. , u n published resu lts) are u nable to synthesise any detectable i ndole-d iterpenes. H owever, deletion muta nts of paxP and paxQ
(both encode cytochrome P450 monooxygenases) accumul ate the i ndole diterpenes paspaline a nd 1 3-desoxypaxi lline, respectively (McMillan et al. ,
2003) . These studies led Parker a nd Scott to propose that PaxG , PaxM and PaxC are req uired for bi osynthesi s of the first stable indole-d iterpene product paspaline, a nd PaxP and PaxQ for the fu rther tra nsformatio n s to paxilline ( Parker a nd Scott, 2004) . Recently, Monahan and Scott identified two additional pax genes with i n the cl u ster, paxA and paxB (putative membrane proteins), deletions of which are also u nable to synthesise a ny d etectable i ndole-d iterpenes (Monahan and S cott, unpublished results). Taken together, these results suggest that at least five gene prod ucts ( PaxG , PaxM , PaxC, PaxA and PaxB) are needed for paspaline biosynthesis. In order to d etermine how many of these genes a re req u i red for paspaline biosynthesis a series of constructs were p repared containing diffe rent combinations of these g enes and transformed i n to paxilli ne-negative deletion derivatives.
3. 1 .1 Preparation and functional analysis of pSS8
A Hind l l l restriction fragment (- 1 1 kb) from the lambda clone, ACY55,
contai ning the five pax genes viz. paxG, paxA , paxM, paxB a nd paxC was
cloned i nto the Hind l l l site of the geneticin-resistant vector p l l 99 to generate pSS8 ( F ig ure 3 . 1 ) . This frag ment was missing the last six amino acids and the stop codon of PaxC . Hence, the functionality of the genes at either e nd of the
restriction frag ment, i .e. paxG and paxC, was checked by tra nsforming pSS8
into the P. paxilli deletion muta nts LMG23 (.6.paxGA) and ABC83 (.6.paxC)
(Section 2 . 1 3 . 2 ) . Subsequent to the generation of the LMG23 mutant Monahan
Hind 11 I 0.5 kb pax1 22 ggpps1 6 ggpps31 � � � paxG paxA t..CY55 pax M Hind 11 I p1l99-3 p1l99-4 �
Figure 3.1 pSS8 (paxGAMBC) construct
pax214 pax127 � � paxB Spel nptll CYP-6 � paxC Hind 11 I
The construct pSS8 was made by cloning the Hind 1 1 I fragment containing paxG,
paxA, paxM, paxB and paxC from the lambda clone, t..CY55, into the Hind l l l site of p1 l99.
Restriction sites used for clon ing and positions of primers used for screening transformants are shown in g reen and pink, respectively.
s howed that the paxG deletion also i ncluded 370 bp of the 3' cod ing seq uence of paxA (unpublished results) .
Protopl asts of LMG23 and ABCS3 were transformed with 5 f.lg of ci rcular p l l 99 or pSSB. The tra nsformants were selected on RG med i u m supplemented with 1 50 f.lg/ml of genetici n . Four to seven stable tra nsformants were colony p urified and screened by TLC analysis (Section 2 .2 1 ) for thei r abil ity to synthesise paxi lline or other indole-diterpenes, which stain g reen with Ehrlich's reagent (Fig u re 3.2). N o indole-diterpenes were detected i n extracts of the paxGA
mutant or paxGA mutant with p l 199 (Figure 3.2A) . However, all the fou r LMG23 tra nsforma nts containing pSSS, LMG2 3 . PS- 1 to 4 , were able to complement the paxGA deleti o n . These transformants had i ntense g reen bands with
R,
val ues similar to a uthentic paxilline. In addition to paxilline, other g reen bands, similar to those consistently observed i n extracts of wi ld-type P. paxilli, were also observed. As wi l l be shown later, these correspond to other i ndole diterpenes incl ud i ng �-paxitriol , PC-M6, paspaline and 1 3-desoxypaxi lline. These compounds h ave been proposed to be paxi l l i ne i ntermedi ates ( M u nday F inch et al. , 1 996).Extracts of paxC mutant or paxC mutant with p l l 99 did not accu mul ate any detecta ble i ndole-diterpenes (Figure 3.2B). Two out of fou r ABCS3 tran sformants conta i n i ng pSSS, ABCS3. PS- 1 and ABCS3. PS-2 , were able to complement the paxC deletion. Since the transforma nts were not screened for the presence of i ntact copies of the integ rating construct, and were a rbitrarily selected as genetici n-resistant colonies, the i ndole-diterpene negative phenotype of the other two ABCS3 transformants , ABC S 3 . PS-3 and ABCS3. PS- 4, could be due to the absence of intact copies of pSSS. U n l ike the paxGA
complementatio n , paxC complementing transformants d i d not accumulate any indole-diterpene i ntermediates, but just the fi nal product paxilline. These resu lts confirmed that paxG and paxC present at either end of the Hind 1 1 I restrictio n fragment were fu nctiona l .
A
... � '? "00 l1li l1li l1li > 11. 11. 11. 11. .., ,.; ,.; ,.; ,.; ,.; N N N N N N <:) <:)
�
<:)�
<:) � � � � .J .J .J .J .J .J paxillineB
... I � '? "l1li l1li l1li l1li > 11. 11. 11. 11.
.., ,.; ,.; ,.; ,.; ,.;
l1li l1li l1li l1li ID l1li
U U U U U U
III � III III III III
'" '" '" '"
paxilline
F i g u re 3 . 2 N ormal P hase ( N P)-TLC a na lysis of paxG and paxC complementations
N P-T LC a n a l ys i s of (A) pax GA d e l et i o n m u ta nt L MG23 and
(8)
paxC deletion mutant ABC83, con t a i n i n g t h e c o m p l e m e n t a t i o n c o n st r u c t p S S 8 (paxGA MBC ) . Myceli u m was g rown fo r 7 days i n C O V E , s u p p l e m e n t e d w i t h t r a c e e l e m e n t m i x ( Section 2 . 3 . 2 ) , at 28°C with shaking at 2 0 0 rp m , f reeze-d r i e d a n d e x t racted i n 2 : 1 c h l o rofo r m m eth a n o l m ixtu re (Section 2 . 2 1 ) . V= p 1 l 9 9 vector; P8=pSS8.3 . 1 .2 pSS8 transformation of pax deletion m utants LM662 a nd CY2
In order to defi ne the mi nimum n u mber of g enes required for paspaline biosynthesis pSS8 was introd uced into two d ifferent genetic backgrounds. The first was LM662 (You ng et al. , 200 1 ) that lacks the entire pax cl uster i.e. paxG, paxA , paxM, paxB, paxC, paxP and paxQ. The other backg round was C Y2 (Young et al. , 1 998) that lacks the enti re pax cluster plus considerable fla n ki ng seq uences . The reason for selecti ng two d ifferent pax negative backgrounds was to check if there were any other pax genes o utside the reg ion d efi ned by the LM662 deletio n . Protoplasts of LM662 and CY2 were transformed with 5 j.lg of ci rcular p l l99 or pSS8 (Section 2 . 1 3.2) and geneticin-resista nt transforma nts selected on RG med i u m su pplemented with 1 50 J..lg/ml of genetici n .
Twelve geneticin-resista nt LM662/pSS8 transforman ts were arbitrarily selected and screened for i ntegration of the pSS8 insert by PCR ampl ification (Sectio n 2 . 1 4 . 2) with primer sets d esigned to ampl ify each end o f the insert (Fig u re 3 . 3A) . Seven out of twelve tra nsforma nts were found to have both prod u cts . The absence of PCR prod ucts with pax primers was not due to D NA templ ate quality as confirmed by the presence of prod u cts in all templates when ampl ified with primers to tub2. These positive transformants were analysed fu rther by Southern blotting and hybridisation ( Secti on 2 . 1 0) using a
e
2PJ labelled Hind 1 1 I fragment from pSS8 as the probe (Fig ure 3. 38). This a nalysis showed that all seven transformants contai ned at l east one complete copy of the Hind 11 I frag ment. The copy number was calculated by compari ng the sig nal obtained with wild-type. 8y this comparison, the additional weaker h ybridising ba nds found i n some tra nsformants cou ld be due to the i ntegration of partia l cop ies of the i nsert.Extracts of the seven tra nsforma nts that contai ned the pSS8 insert were then analysed by TLC (Section 2 . 2 1 ) for i ndole-diterpenes ( Fig ure 3 .4A) . A g reen band with an
R,
value si milar to authentic paspaline was visible for three of the seven transformants viz. LM662. P8-3 , LM662 . P8-9 a nd LM662 . P8-1 2 . These transformants had at least th ree cop ies of the Hind 1 1 I fragment contai ningpaxGAMBC (Fig u re 3.38). Extracts of all seven L M662/pSS8 transfo rmants
ggpps18131 pax21 4/cVP.o N .. ..
� �
" .. .. IL IL IL CL IL CL N N N N N N N N N N N .. .. .. .. .. .. ..� �
�
� �
� � - _ ... - ... -- - - - - -- - - - - ... ..., .. .. .. .. .. -- - � .- - " .. .. IL IL IL CL CL N N N N N .. .. ... .. ... .. .. ... ... OD .. OD .. OD � � � � � � � 23.1 9.4 -1.87 5.5 -2.33 -0.3 4.4 2.3 2.0Figure 3.3 PCR screening and Southern analysis of LM662 transformants containing pSSB (paxGAMBC)
(A) PCR screening for pSS8 (paxGAMBC) in LM662 background. Each transformant was screened with one primer set at either end of the construct (ggpps 1 6/31 and pax2 1 4/CYP- 6). The transformants (labelled black) that gave a PCR product for both sets of primers and also LM662 and LM662.v. 1 (negative control and vector only control, respectively) were screened further by Southern ana lysis as shown in (8). See Fig ure 3 . 1 for primer positions. tub2 was used as a control (amplified with primer set Etub2/3). (8) Southern a nalysis of LM662/paxGAMBC tra nsfo rmants. Hind l l l -d igested genomic DNA fro m LM662/paxGAMBC transformants hybridised with the [32P]-labelled Hind 1 1 I fragment from the pSS8 construct. The numbers on the left correspond to the sizes of ),D NA/Hind 11 I frag ments and that o n the right correspond to the expected size of the restriction fragment that hybrid ised to the probe.
All fragment sizes are shown in kb. P8=pSS8; V=p1l99 vector.
Standard PCR components (Section 2 . 1 4.2) were used with the following thermocycle con
d itions: 1 cycle of 94°C for 2 min; 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for
either 2 min (for ggpps1 6/3 1 ) or 3 min (for pax2 1 4/CYP-6) or 1 m in (for Etub2/3); 1 cycle of
A
13-dp paspaline paxillineB
I.M662. P8-1 1 1662.P8-9 LM6 6 2 . P8-6 I.M662.P84 1662.P8·J I.M662.V.1 1..'\1662 wlld-I}'pe 0.0 ... co co � ...J 5.0 .... � ... co co � '1 ,. 'of tI! 00 00 00 00 a, Cl. a, Cl. ... N N N co co co co co co co co � � � � ...J 1 0.0 1 5.0 20.0Retention time (min)
0 .... "7 "7 00 00 Cl. Cl. N N co .., co .., � � pas paline 25.0 N .... 00 " Cl.
�
N co .., :E � .� 30.0 F i g u re 3 .4 N P-TLC a n d Reverse P h a se ( RP)-H P L C a na l ysis of LM662/pSS8 transformantsLM662 transformants containing the construct pSS8 (paxGA MBC) were analysed by (A) NP-TLC a nd
(8)
R P-H P L C for the prese n ce of indole d iterpe nes (Section 2 .2 1 ) . Myce l i u m wa s g rown for 7 d a ys i n C OVE, supplemented with trace element mix (Section 2 . 3 .2), at 2 8°C with shak ing at 200 rpm , freeze-d ried and extracted in 2 : 1 chloroform-methanol mixture. V=p 1 199 vector; P8=pSS8; 1 3-d p= 1 3-desoxypaxi lline.were fu rther a nalysed by H PLC (Sectio n 2 . 2 1 ) . This analysis s howed that all transformants contai ned a metabolite that had the same retention time
(Rt
=2 0 . 5 min) as a uthentic paspaline (Figure 3.48) showi ng a n absorption maxi mum at 2 3 0 nm and a n absorption minimum at 280 n m cha racteristic of an indole moiety. The low levels of paspal i ne detected compared to wild-type probably reflects the ectopic integ ration of pSS8. The hig hest levels of paspaline coi n ci ded with transformants containing multiple copies of pSS8.
Ten a rbitrari ly selected genetici n-resistant CY2/pSS8 transformants were screened for i nteg ration of the pSS8 i nsert by PCR amplifi cation (Section 2 . 1 4 . 2 ) with pri mer sets desig ned to amplify each end of the insert (Figure 3 . 5A) . Seven out of ten transformants gave both products . Al l templates gave a prod uct when ampl ified with pri mers to tub2 confirming that the absence of prod ucts with the pax pri mers was n ot d ue to template q uality . These positive transforma nts were fu rther analysed by Southern blotti ng a nd hybridisatio n ( Section 2 . 1 0) using a
e
2P]-labelled Hind I I I frag ment from p S S 8 as the probe ( Fi g u re 3 . 5 8). This analysis showed that at least one copy of the Hind l l l frag ment was present i n all transfo rmants . The additional weaker hybridising bands observed in some tra nsforma nts could be due to the integ ration of incomplete copies of the insert.TLC analysis of extracts of the seven positive tra nsforma nts (see above) showed a g reen band with an
R,
val ue similar to authenti c paspaline i n all transforma nts except CY2 . P8- 1 ( F i g u re 3.6A). This result was fu rther confirmed by H PLC analysis (Fig ure 3.68). The i nabi lity of the transformant CY2 . P8-1 to synthesise paspaline, despite containing the five pax genes , cou ld be due to the position of i nteg ration of the construct i n the genome (Malonek et al. ,2005a) .
It was confi rmed that the i ntrod uction of pSS8 i nto LM662 and CY2 restored to these strains the abil ity to synthesise paspaline. The fact that both LM662/pSS8 and CY2/pSS8 transformants synthesised paspal ine suggested that either genetic backg round was su itable for fu rther comp lementation