The deletion m utants o f paxP a nd paxQ a re blocked for paxi lline biosynthesis and accu mulate the indole-d iterpenes paspaline and 1 3-desoxypaxilline, respectively ( McMillan et al. , 2003). This suggested that paxP a nd paxQ a re essential for paxilline biosynthesis and that paspaline and 1 3-desoxypaxilline a re the most l i kely substrates for the corresponding enzymes. To test if the block for paxilli ne biosynthesis i n L M P 1 (6.paxP) and LMQ226 (6.paxQ) mutants could be released by complementatio n , paxP and paxQ comp lementatio n constructs were prepared . These complementation constructs were also used to investigate if paspaline a nd 1 3-desoxypaxilline are the true substrates for the corresponding enzymes and also to test the participation of other proposed i ntermed iates i n paxi lline biosynthesis ( Section 3 . 3) .
3.2. 1 Preparation o f paxP and paxQ complementation constructs
To test if the paxP deletion i n LM P 1 m utant could be complemented , a paxP
complementation construct pSS 1 was prepa red . A paxP frag ment containing 583 bp of the seq uence 5' of the paxP ATG, all 1 84 9 bp of the gene seq uence and 243 bp of the 3' sequence was peR ampl ified from wild-type genomic DNA (Section 2 . 1 4 . 3) using the primers paxPEcoRIF2 and paxPEco R I R , containi ng i ntroduced EcoR I sites (Figure 3. 20A) . This product was s u bcloned into pGEM®-T Easy vector (Promega) and subseq uently seq uenced . This analysis identified one PeR-i ntrod uced error but was outside of paxP 60 bp downstrea m of the transcri ption stop site. The EcoR I frag ment containing paxP was then cloned i nto the EcoR I site of p l l99 to generate pSS 1 (Figure 3 . 20A).
In order to test if the paxQ deletion in L MQ226 mutant could be complemented , a paxQ complementation construct p SS2 was prepa red . A paxQ fragment consisti ng of 6 3 1 bp of the seq uence 5' of the paxQ ATG , all 2 087 bp of the gene seq uence and 297 bp of the 3' sequence was peR ampl ified from wi ld type genomic DNA (Section 2 . 1 4 .3) using the pri mers paxQ H i nd l l l F2 and paxQ Hind l l l R , containing i ntrod uced Hind 11 I sites (Figure 3.208).
The paxQ PCR prod uct was subcloned i nto pGEM®-T Easy vector (Promega) and su bseq uently seq uenced . Seq uence analysis confi rmed that there were no PC R-i ntrod uced errors . The Hind l l l frag ment contai ning paxQ was then ligated i nto the Hind l l l site of p l l99 to generate pSS2 (Figure 3.20B).
3.2.2 Complementatio n of I1.paxP and I1.paxQ strains
Protoplasts of LM P 1 (l1.paxP) and LMQ226 (l1.paxQ) mutants were transformed
with 5 )lg of ci rcular pSS1 (paxP) and pSS2 (paxQ) , respectively, and also with
circu lar p l l 99 vector (Section 2 . 1 3.2). The transformants were selected on RG med i u m s upplemented with 1 50 flg/ml of geneticin. Ten arbitrarily selected genetici n-resistant LMP 1 /pSS 1 and LMQ226/pSS2 transformants were colony purifi ed and screened by TLC a nalysis (Section 2.2 1 ) for thei r abil ity to make paxi l l i ne ( Figure 3. 2 1 ) .
Extracts of LMP1 and LM P 1 /p 1 l99 contained a single ind ole-d iterpene with an
R,
value s i milar to authentic paspaline ( Figure 3.2 1 A) . The presence of green bands withR,
values simi lar to a uthentic paxill i ne in seven out of ten LMP 1 /pSS 1 transformants a nalysed confi rmed that pSS1 was able to complement the paxP deletion i n LMP 1 ( F igure 3 . 2 1 A) . These transforma nts had other g reen bands simi l a r in mobility to those consistently observed i n extracts of wi ld-type P. paxilli a n d , a s shown later, correspond to other indole diterpenes i ncl ud ing p-paxitriol , PC-M6, paspaline a nd 1 3-desoxypaxi lline. The variable levels of paxi l l ine detected among the transforma nts could be due to the position of i nteg ration of the construct i nto the genome and/or d ifferences i n copy n u mber (Malonek et al. , 2005a), o r the presen ce of an incomplete promoter sequence i n pSS1 .TLC analysis of extracts of paxQ mutant a nd paxQ muta nt with pl l99 detected i ntense g reen bands with
R,
values similar to authentic 1 3-desoxypaxi lline a nd other less intense green bands corresponding to other i ndole-diterpenes (Fig u re 3 . 2 1 B). Al l the ten L MQ226/pSS2 transformants ana lysed had intense g reen bands withR,
val ues similar to authentic paxilline suggesti ng that pSS2 was able to comp lement the paxQ deletion i n LMQ226 mutant (Fig u re 3.2 1 B) .0 ... ... ... <? ... ... " ... "? � ... ... ... Gp ... C',I ... "7 ... 11: 11: 11: 11: 11: 11: 11: 11: 11: 11: ... ... ... ... ... ... ... ... ... ... ... Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. � � � � � � � � � � � � ..J ..J ..J ..J ..J ..J ..J ..J ..J ..J ..J paspaline paxilline 0 N " � Gp N N N N N N N N N N Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. .,; .,; .,; .,; .,; .,; .,; .,; .,; N N N N N N N N N N N N N N N N N N N N N N N N a a " a
�
�
a a " " a�
� � ..J � � � � � � � ..J ..J ..J ..J ..J ..J ..J ..J ..J . . . . 1 3-dp paxillineFigure 3.21 NP-TLC analysis of paxP and paxQ complementations
NP-TLC analysis of (A) paxP deletion m utant LM P 1 containing the com ple mentation co nstruct pSS 1 (lines are d rawn to show the mobility of g reen bands corresponding to authentic paxilline and paspaline) and (B) paxQ d ele tion m utant LMQ226 contain ing the complementation construct pSS2, for the presence of indole-d iterpenes (Section 2 . 2 1 ). Mycelium was g rown for 7 d ays in CDYE, supplemented with trace element mix (Section 2 . 3 .2), at 28°C with shaking at 200 rpm , freeze-dried and extracted in 2: 1 ch loroform-methanol mixture . V=p 1 l99 vecto r; P1 =pSS 1 ; P2=pSS2; 1 3-dp = 1 3-desoxypaxilline.
U n l i ke the paxP complementation, the l evel of paxilline detected i n each of the LMQ226/pSS2 transforma nts was similar. Additiona l green bands similar to those consistently observed in extracts of wild-type strain were a lso observed . As wi l l be shown later, these correspond to other i nd ole-diterpenes .
3.2.3 Preparation of pSS7 with longer paxP promoter sequence
To test if the variable levels of paxi l line detected in LM P 1 /pSS 1 transformants (Section 3 . 2 .2) was due to an incomplete promoter seq uence in pSS 1 , a second paxP complementation construct, pSS7, was prepared that contai ned a longer paxP promoter seq uence ( 1 584 bp) than in pSS1 (583 bp). A 9 1 5 b p EcoR I/Smal frag ment, containing part o f t h e paxP g e n e seq uence (483 b p ) a n d 4 3 2 bp of t h e 3' untranslated region, from t h e lambda clone ",CY56 (Young et al. , 2 00 1 ) was cloned i nto the EcoRIISmal site of the pBlueScri pt 11 KS vector to generate pSS3 (Figure 3.22A). A 2950 bp Smal frag ment, containing 1 584 b p of the seq uence 5' of the paxP ATG plus the rema i ning part of the paxP gene seq uence ( 1 366 bp) , was cloned from ",CY56 i nto the Smal site of pSS3 in the correct orientation to generate pSS7 (Fig ure 3.22B) .
3.2.4 Complementation of !1paxP stra i n with pSS7
Protoplasts of paxP deletion mutant L M P 1 were co-transformed with 5 Ilg of circular p l l99 vector and pSS7 ( Section 2 . 1 3.2). Twenty six arbitrari ly selected geneticin-resistant tra n sforma nts were colony- p u rified and PC R-screened (Section 2 . 1 4.2) for integ ration of the pSS7 i nsert. Six positive transformants were fu rther screened by TLC a nalysis (Section 2 .2 1 ) for their abil ity to make paxi lline. TLC a nalysis of extracts of LMP1 and LM P 1 /p 1 199 identified green bands with
Rf
val ues si milar to authentic paspaline (Fig u re 3.23). Five out of six genetici n-resistant LM P 1 /p I l 99/pSS7 tra nsformants had green bands withRf
val ues si milar to authentic paxilline s uggesti ng that the paxP deletion i n LM P 1 mutant was complemented by pSS7 ( F ig ure 3 . 2 3 ) . These transforma nts h ad other green bands that correspond to other indole-d iterpenes . Althoug h pSS7 was able to complement L M P 1 , variable levels of paxi lline were once ag ain observed .A
amp pSS3 pSS7 pBlueScript 11 KS 2961 bp pSS3 3866 bp Pstl EcoRI EcoRV Hind III Clal San Xhol paxC paxP ACY56 paxQ 0.5 kbFigure 3.22 paxP complementation construct with different promoter
length
(A) EcoRl/Smal fragment, containing part of paxp, from ACY56 was cloned into pBlueScript 1 1 KS to g ive pSS3. (8) Smal frag ment, containing the pro moter and remaining part of paxp, from ACY56 was cloned into pSS 3 to g ive pSS7. pSS7 plasmid wa s used with pl 1 9 9 in a co-transfo rm ation of LMP1 (L'lpaxP) protoplasts.
M M
N I .... I N I N I
I"- I"- I"- I"- I"-
N 0.. """- 0.. """- 0.. """- 0.. """- 0.. """- 0.. """- � � � � � � .... .... .... .... .... 0.. 0.. 0.. 0.. 0.. 0.. 0.. 0.. :E :E :E :E :E :E :E :E ...J ...J ...J ...J ...J ...J paspaline paxilline
Figure 3.23 NP-TLC analysis of paxP com plementation with pSS7
N P-TLC analysis of paxP de letion mutant L M P 1 co nta i n ing the comp le mentation construct pSS7 (contains a longer paxP p romote r than i n the co n struct pSS 1 ) , for the p rese n ce of i n d o le-d ite rpe nes (Sectio n 2 . 2 1 ) . Mycelium was grown for 7 days in COVE, s u pplemented with trace element m i x (Section 2 . 3 . 2 ), at 2 8°C with shaking at 200 rpm , freeze-d ried and extracted in 2 : 1 chloroform-methanol mixtu re . V=p1 l99 vector; P7=pSS7.
Given the primary aim of these experi ments was to confi rm that the paxP and paxQ constructs were functional , no deta iled molecu l a r analysis was carried out to confirm whether the variable levels of paxi lline reflected copy n umber o r position o f integration .