Patent Application: US-201314390360-A

Abstract:
the biosynthesis of fungal bicyclodiazaoctane indole alkaloids with a wide spectrum of biological activities have attracted increasing interest . their intriguing mode of assembly has long been proposed to feature a non - ribosomal peptide synthetase , a presumed intramolecular diels - alderase , a variant number of prenyltransferases , and a series of oxidases responsible for the diverse tailoring modifications of their cyclodipeptide - based structural core . until recently , the details of these biosynthetic pathways have remained largely unknown due to lack of information on the fungal derived biosynthetic gene clusters . herein , we report a comparative analysis of four natural product metabolic systems of a select group of bicyclodiazaoctane indole alkaloids including /- notoamide , paraherquamide and malbrancheamide , in which we propose an enzyme for each step in the biosynthetic pathway based on deep annotation and on - going biochemical studies .

Description:
“ sequence identity ” means that two amino acid or polynucleotide sequences are identical over a region of comparison , such as a region of at least about 250 residues or bases . optionally , the region of identity spans at least about 100 - 500 residues or bases , and spans the active domain of the polypeptide . several methods of conducting sequence alignment are known in the art and include , for example , the homology alignment algorithm ( needleman & amp ; wunsch , j . mol . biol ., 48 , 443 ( 1970 )); the local homology algorithm ( smith & amp ; waterman , adv . appl . math ., 2 , 482 ( 1981 )); and the search for similarity method ( pearson & amp ; lipman , proc . natl . acad . sci . usa , 85 , 2444 ( 1988 )). preferably , the algorithm used to determine percent sequence identity and sequence similarity is the blast algorithm ( altschul et al ., j . mol . biol ., 215 , 403 - 410 ( 1990 ); henikoff & amp ; henikoff . proc . natl . acad . sci . usa , 89 , 10915 ( 1989 ); karlin & amp ; altschul , proc . natl . acad . sci . usa , 90 , 5873 - 5787 ( 1993 )). software for performing blast analyses is publicly available through the national center for biotechnology information . other examples of alignment software , including gap , bestfit , fasta , pileup , and tfasta provided by wisconsin genetics software package ( genetics computer group , 575 science dr ., madison , wis . ), and clustalw ( thompson et al ., nuc . acids res ., 22 , 4673 - 4680 ( 1994 ); http :// www . ebi . ac . uk / tools / clustalw2 / index . html ), are known in the art . the degree of homology ( percent identity ) between a native and a mutant sequence may be determined , for example , by comparing the two sequences using computer programs commonly employed for this purpose . briefly , the gap program defines identity as the number of aligned symbols ( i . e ., nucleotides or amino acids ) which are identical , divided by the total number of symbols in the shorter of the two sequences . the default parameters for the gap program include : ( 1 ) a unary comparison matrix ( containing a value of 1 for identities and 0 for non - identities ) for nucleotides , and the weighted comparison matrix of gribskov and burgess , nucl . acids res . 14 : 6745 , 1986 , as described by schwartz and dayhoff , eds ., atlas of protein sequence and structure , national biomedical research foundation , pp . 353 - 358 , 1979 ; ( 2 ) a penalty of 3 . 0 for each gap and an additional 0 . 10 penalty for each symbol in each gap ; and ( 3 ) no penalty for end gaps . alterations of the native amino acid sequence may be accomplished by any of a number of known techniques . mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence , flanked by restriction sites enabling ligation to fragments of the native sequence . following ligation , the resulting reconstructed sequence encodes an analog having the desired amino acid insertion , substitution , or deletion . alternatively , oligonucleotide - directed site - specific mutagenesis procedures can be employed to provide an altered gene having particular codons altered according to the substitution , deletion , or insertion required . techniques for making such alterations include those disclosed by walder et al . ( gene 42 : 133 , 1986 ); bauer et al . ( gene 37 : 73 , 1985 ); craik ( biotechniques , january 1985 , 12 - 19 ); smith et al . ( genetic engineering : principles and methods , plenum press , 1981 ); and u . s . pat . nos . 4 , 518 , 584 and 4 , 737 , 462 , which are incorporated by reference herein . the disclosure provides an example of the comparative analysis of biosynthetic gene clusters ( mined from the whole genome ) and pathways for structurally related fungal indole alkaloids bearing the unusual bicyclo [ 2 . 2 . 2 ] diazaoctane core , including the anticancer agents (−)- notoamide a ((−)- 1 ) and (+)- notoamide a ((+)- 1 ), 35 , 36 the anthelmintic paraherquamide a ( 2 ), 37 - 39 and the calmodulin - inhibitor malbrancheamide 40 - 42 ( 3 ) ( fig1 a ) produced by aspergillus sp . mf297 - 2 , 43 aspergillus versicolor nrrl35600 , penicillium fellutanum atcc20841 , and malbranchea aurantiaca rrc1813 , respectively . these fungal natural products are assembled from an l - tryptophan , a second cyclic amino acid residue , and one or two isoprene units through biosynthetic pathways that are proposed to feature an intriguing intramolecular diels alderase ( imdase ), and a number of unique enantiomerically selective enzymes . 44 - 49 the diverse bioactivities of this natural product family suggests that elucidation of their biosynthesis could direct future structural diversification via biosynthetic engineering , thereby leading to enhanced biological activities . this comparative analysis provides significant insights into a number of intriguing biosynthetic questions : ( 1 ) which enzyme in each pathway is likely responsible for the formation of the bicyclo [ 2 . 2 . 2 ] diazaoctane core via the proposed intramolecular [ 4 + 2 ] diels - alder ( imda ) cyclization ; ( 2 ) which enzyme in the pathway of 1 and 2 installs the spiro - oxindole functionality via a putative epoxide - initiated pinacol - type rearrangement ; and ( 3 ) what genetic difference controls formation of the dioxopiperazine in 1 versus the monooxopiperazine in 2 and 3 . the most significant structural similarity between 1 - 3 is the bicyclo [ 2 . 2 . 2 ] diazaoctane core ( fig1 a ). biosynthetically , this unique structural moiety was proposed to arise from a [ 4 + 2 ] imda reaction ( fig1 b ). 44 , 46 this presumed cycloaddition reaction is also believed to catalyze the first enantiodivergent step in an otherwise common biosynthetic pathway from aspergillus sp . mf297 - 2 and a . versicolor nrrl35600 , leading to formation of (−)- 1 and (+)- 1 , respectively , together with several other enantiomeric metabolites ( fig3 ). 47 currently , it remains unknown whether a specific imdase indeed exists in these biosynthetic pathways . however , if it does exist , one would expect its encoding gene should be present in all four gene clusters . second , the spiro - oxindole is absent in 3 , suggesting the responsible enzyme is likely absent from the pathway for 3 , and present in those for 1 and 2 . third , a specific reductase responsible for reducing the tryptophan carbonyl group would be expected in the gene cluster of 2 and 3 , but not 1 . this genetic difference would account for the lack of the second amide carbonyl group in the piperazine ring of 2 and 3 . finally , the different hydroxylation status of the indole amide , distinct aromatic decoration among 1 - 3 , together with other unique structural features including the tailoring of the proline moiety and n - methylation in 2 , are also expected to be reflected at the genetic level . the following examples are provided to illustrate particular embodiments of the present invention , and are not to be construed as limiting the scope of the invention . the genomes of a . versicolor nrrl35600 , p . fellutanum atcc20841 , and m . aurantiaca rrc1813a harboring not ′, phq , and mal gene clusters , respectively were sequenced to approximately 99 , 84 , and 181 times coverage of their estimated genome size ( 35 mb ), using the illumina solexa technology ( genome analyzer iix ). first , the key biosynthetic gene note ′ ( table 1 ) encoding a non - ribosomal peptide synthetase ( nrps ) was mined from the genome sequences using the note dna sequence from the reported not gene cluster 43 as a probe for homologous genes . note ′, which shows 79 % identity and 86 % similarity to note at the amino acid ( aa ) level , was predicted to be a bimodular nrps with the a - t - c - a - t - c ( a : adenylation , t : thiolation , c : condensation ) domain organization using the pks / nrps analyzer ( http :// nrps . igs . umaryland . edu / nrps /). genome walking from note ′ toward 5 ′ and 3 ′ ends identified another nine genes ( nota ′- j ′, table 1 and fig2 ) that display high aa sequence similarity (& gt ; 70 %) with corresponding gene products of the not gene cluster . notably , the overall nucleotide identity between nota ′- j ′ ( 25 , 440 bp ) and nota - j ( 26 , 210 bp ) is 71 %, which is not surprising since both metabolic pathways are responsible for assembling “ identical ”, yet antipodal compounds . in addition to the high sequence similarity , the genetic architecture ( i . e . order and direction of genes ) within this region is identical in the two clusters ( fig2 ). the pattern of the exon / intron arrangement in the corresponding genes is also highly similar to each other ( see supplementary information ). in contrast , the sequence similarity is reduced drastically and the gene architecture differs after notk ′/ notk ( table 1 , fig2 ), strongly suggesting the previously assigned not gene cluster ( nota - r ) probably ends at notj . at the genetic level , it is not possible to glean the key differences that account for production of antipodal notoamide metabolites , suggesting that subtle active site sequence variation in those enantiomerically selective enzymes play a critical role in the control of absolute chirality . this requires direct biochemical analysis of the key notoamide biosynthetic enzymes , including structural biology efforts , which is currently ongoing in our laboratories . second , the paraherquamide ( phq ) gene cluster ( 47 , 884 bp ) was identified from the partially assembled p . fellutanum genome by using a select group of not genes including the nrps gene note , the prenyltransferase genes notc and notf , and the p450 monooxygenase gene notg as in silico probes . 43 fifteen genes were identified that are likely involved in paraherquamide biosynthesis . the largest number of biosynthetic genes among the four studied metabolic pathways is consistent with 2 as the most complex structure compared to 1 and 3 . comparative bioinformatic analysis demonstrates that nine ( phqa , b , f , g , h , j , k , l , and m ) out of fifteen total phq genes are homologous to corresponding not genes ( table 1 ), although their homology is significantly lower than that between not and not ′ genes . notably , the bimodular phqb nrps gene is different from note in that a reductase ( r ) domain is located at its carboxy terminus instead of a condensation ( c ) domain , which is found in note and note ′. this difference is significant because the reductase ( vs condensation ) domain is presumed to account for the presence of the monooxopiperazine in 2 ( vs dioxopiperazine in 1 ) ( see below ). 50 among the remaining six cluster - specific genes , phqc shows high sequence similarity to 2 - oxoglutarate ( 2og ) and fe ( ii ) dependent oxygenases . 51 , 52 the phqd and phqe genes , which putatively encoding a pyrroline - 5 - carboxylate reductase and a short chain dehydrogenase , respectively , might be involved in the formation of the β - methyl - proline starter unit . the phqi gene that encodes the third prenyltransferase in phq is unique as it is free of introns , and therefore , distinct from the single intron - containing prenyltransferase genes phqa / notc and phqj / notf . it is worth noting that the presence of three prenyltransferase genes is inconsistent with the two isoprene groups incorporated into the structure of 2 . thus , it is of special interest to examine whether the third prenyltransferase gene is redundant or plays an alternative , and as yet unknown function in the biosynthesis of 2 . furthermore , phqn is predicted to function as a methyltransferase , likely responsible for the n - methylation in 2 . finally , the phqo p450 gene with a unique exon / intron organization pattern is hypothesized to catalyze the c14 hydroxylation of the β - methyl - proline moiety . third , the seven - gene containing mal gene cluster ( 20179 bp ) was mined from the genome of malbranchea aurantiaca rrc1813a using phqb as an in silico probe to identify the metabolic system for 3 . it has the smallest size among gene clusters of 1 - 3 , which is consistent with the simplest structure and corresponding biosynthetic pathway . the genes malb , mald , male , malf , and malg are common to the four gene clusters . thus , except for the regulatory gene of mald ( homologous to nota , nota ′ and phqg ), the remaining four biosynthetic genes ( and their homologues in not , not ′ and phq ) are possibly responsible for installing the shared structural features of 1 - 3 . this strongly suggests that the hypothetical diels alderase ( if extant ) should be represented by one of these four gene products ( see below ). interestingly , the mal genes show greater sequence similarity to phq genes than not ( or not ′) genes , perhaps indicating their closer evolutionary relationship . similar to phqb , the nrps malg harbors a reductase domain at its carboxy terminus , which is consistent with the monooxopiperazine moiety in 3 . again , the apparent redundancy of the second prenyltransferase ( 3 only contains one isoprene group ) is difficult to rationalize , but genetic disruption or rna silencing ( malb or male ) efforts are likely to shed light on the individual role of these enzymes . finally , it is evident that the flavin - dependent halogenase mala is likely involved in the introduction of one or both chlorine atoms in the biosynthesis of 3 . since the discovery of the biosynthetic gene cluster of (−)- 1 from marine aspergillus sp . mf297 - 2 , in vitro biochemical characterization of the reverse prenyltransferase notf using the nrps ( note ) product brevianamide f 53 ( 4 ) as substrate and the normal prenyltransferase notc using 6 - hydroxy - deoxybrevianamide e ( 6 ) as substrate has partially established the early steps of the notoamide pathway leading to notoamide s ( 7 ) ( fig3 ). 43 the p450 monooxygenase notg is likely catalyzing the c6 indole hydroxylation since its close homologue ftmc ( 59 %/ 72 % identity / similarity ) in fumitremorgin biosynthesis had been characterized to hydroxylate the analogous aromatic c — h bond in the indole ring of tryprostatin b , 54 , 55 which is structurally similar to deoxybrevianamide e ( 5 ). 56 as the proposed pivotal branching point in notoamide biosynthesis , 47 , 57 , 58 7 can be diverted to notoamide e ( 8 ) through an oxidative pyran ring closure putatively catalyzed by either noth p450 monooxygenase ( based on precedented examples of pyran ring formation from the epoxide intermediate generated by p450 enzymes 59 ), or the notd oxidoreductase . this step would be followed by an indole 2 , 3 - epoxidation - initiated pinacol - like rearrangement catalyzed by notb fad monooxygenase ( fmo ) leading to the formation of notoamide c ( 9 ) and notoamide d ( 10 ). 58 notably , notb ( or notb ′) is only observed in the not ( or not ′) gene cluster , consistent with the fact that this branching pathway leading to natural products 9 and 10 is only observed in notoamide biosynthesis . on the other hand , extensive precursor feeding and incorporation studies using stable isotopically labeled intermediates have supported 7 as the substrate for the hypothetical imda . 47 as a working hypothesis , a two - electron oxidation catalyzed by an oxidase would give rise to the achiral azadiene intermediate ( 11 ), which may immediately undergo a spontaneous stereoselective [ 4 + 2 ] imda cyclization in the active site of the same oxidase , yielding either (+)- notoamide t ((+)- 12 ) in aspergillus sp . mf297 - 2 or (−)- notoamide t ((−)- 12 ) in a . versicolor . the opposing conformation ( endo / exo ) assumed by achiral 11 presumably determined by the scaffolding of each putative diels - alderase might account for the enantio - divergence at this key step . the five oxidases encoded by the not gene cluster , include fmo notb and noti , p450 enzymes notg and noth , and the fad - dependent oxidoreductase notd . notb was recently identified as the notoamide e oxidase . 58 noti is highly similar to notb with 42 % protein sequence identity and 59 % similarity , and is predicted to catalyze a similar conversion from (+)- stephacidin a 60 ((+)- 13 ) to (−)- notoamide b ((−)- 14 ) via the 2 , 3 - epoxidation of (+)- 13 followed by a pinacol - type rearrangement . thus , if the putative function of notg ( see above ) is correct , noth ( or notd ) is likely the bifunctional oxidase that also functions as the imdase responsible for generation of (+)- 12 . to generate antipodal (−)- 12 , noth ′ ( or notd ′) is expected to catalyze a diels alder reaction leading to the opposite stereochemistry . currently , this hypothesis is being tested in our laboratories through in vitro characterization of noth / noth ′ ( or notd / notd ′). with comparative analysis of four gene clusters ( table 1 ), it appears that notd / notd ′ is more likely to serve as the imdase since its homologs ( phqh and malf ) are present in all clusters . this hypothesis is based on the assumption that these four biosynthetic pathways use the same type of protein scaffolding enzyme to catayze the [ 4 + 2 ] cyclo addition . however , we have recently begun to challenge this assumption ( see below ). presently , the possibility that noth / noth ′ functions as the imdase in notoamide biosynthesis cannot be excluded . once its identity is determined , the final oxidase notd ( or noth ) will likely be found to catalyze the oxidative pyran ring formation ( fig3 ). another important fact of these two related notoamide pathways is that enzymes catalyzing the biosynthetic steps after formation of 12 must also be enantiomerically and diastereochemically selective . specifically , in previous precursor incorporation studies of racemic 13 c - labeled (±)- 13 with aspergillus sp . mf297 - 2 and a . versicolor , 61 it was ascertained that only one enantiomer of 13 can be processed ( currently presumed by noti and noti &# 39 ;) to form downstream products . understanding the subtle differences between these two enzymes will likely provide significant insights into how related enzymes have evolved to adopt opposing enantiomeric selectivity . finally , it remains unclear which enzyme could be responsible for the final hydroxylation steps leading to notoamide a ( 1 ) and sclerotiamide 62 ( 15 ) since all five oxidative enzymes in the not (′) gene cluster has been assigned a putative function . it is possible that 1 and 15 are opportunistically produced upon the activity of unknown oxidases whose genes reside outside of the defined notoamide gene cluster . alternatively , the possibility that a not oxidase may possess bi - functionality cannot be excluded . previous feeding studies demonstrated that l - isoleucine is the precursor to the β - methyl - β - hydroxy proline moiety in 2 . 45 , 63 identification of the pyrroline - 5 - carboxylate reductase phqd and the short chain dehydrogenase phqe from phq cluster suggests a reasonable pathway from l - isoleucine to β - methyl proline ( fig4 ). similar to the partially identified biosynthesis of 4 - methyl proline in cyanobacterial nostoc sp ., 64 phqe presumably oxidizes the terminally hydroxylated l - isoleucine ( by an unknown enzyme ) to the corresponding aldehyde . spontaneous cyclization and dehydration would yield the 4 - methyl pyrolline - 5 - carboxylic acid , which is then reduced by phqd leading to the β - methyl proline precursor . the presence of a c - terminal nad ( p )- dependent reductase domain in the bimodular paraherquamide nrps ( a - t - c - a - t - r ) clearly indicates that the mechanism for dipeptide release by phqb must be different from the final condensation domain of note ( fig3 ). 50 what likely occurs is that the phqb r domain utilizes nadph for hydride transfer to reduce the thioester bond of the t domain - tethered linear dipeptide to a hemithioaminal intermediate , which spontaneously cleaves the c — s bond to release the aldehyde product . subsequently , the acid - activated aldehyde is intramolecularly trapped by the nucleophilic amine from the adjacent amino acid to form a hemiaminal intermediate , which then undergoes a spontaneous dehydration and double bond rearrangement leading to formation of the monooxopiperazine intermediate 16 ( likely existing as the enol form ) prior to all other biosynthetic steps . this hypothesis is in good agreement with previous observations 65 , 66 that the dioxopiperazine analog of preparaherquamide ( 17 ) cannot be incorporated into 2 by p . fellutanum since all substrates for downstream enzymes should bear the monooxopiperazine ring system . in this scheme ( fig4 ), formation of the diene in 16 is achieved by a reductive process , as opposed to the 2e − oxidation step proposed in the notoamide biosynthetic pathway ( fig3 ). if this is correct , in contrast to an oxidase ( noth / noth ′ or notd / notd ′) proposed to be the diels alderase in notoamide biosynthesis , the reverse prenyltransferase ( proposed to be phqj ) might act as the scaffold for an imda reaction after introduction of the reverse prenyl group to 16 . in this proposed route , the terminal double bond of the isoprene group would become the dienophile to react with the azadiene in the prenyltransferase active site , thus resulting in formation of the [ 2 . 2 . 2 ] diazaoctane intermediate 17 . following formation of 17 , the pyran ring formation is proposed to be installed by phqa prenyltransferase ( 22 % identical to notc ), phql ( 29 % identical to notg ) and phqh oxidoreductase ( 34 % identical to notd ) ( or phqm p450 enzymes ( 15 % identical to noth )). the fmo phqk ( 32 % identical to noti ) is likely responsible for generation of the spiro - oxindole , and the n - methylation is likely mediated by the phqn methyltransferase leading to the isolable natural product paraherquamide f 38 , 67 ( 18 ). however , the order of these biosynthetic steps cannot be predicted without further in vivo genetic studies and / or in vitro biochemical analysis . in late - stage paraherquamide biosynthesis , the third p450 monooxygenase phqo is probably responsible for the c14 hydroxylation , transforming 18 to paraherquamide g 38 , 67 ( 19 ), and paraherquamide e 38 , 67 ( 20 ) to the final product 2 . however , expansion from the 6 - membered ring pyran ( in 18 and 19 ) to the 7 - membered dioxepin ring ( in 2 and 20 ) represents a poorly understood but intriguing process . possibly , phqc that encodes a 2og - fe ( ii )- oxygenase is involved in this ring expansion , which is consistent with previous reports showing this class of enzyme functioning as an expandase . 68 finally , the biosynthetic genes , including phqi as well as phqm ( or phqh , the one uninvolved in the pyran ring formation ), do not have a clearly prescribed role and appear to be redundant . except for using l - proline instead of β - methyl proline as the starter unit , the biosynthetic route through premalbrancheamide ( 21 ) ( fig5 ) is proposed to parallel that of paraherquamide biosynthesis through 17 ( fig4 ). mediated by nrps malg ( a - t - c - a - t - r , 37 % identical to phqb ) and prenyltransferase male ( 36 %/ 34 % identical to notf / phqj ), 21 is produced with its structure slightly different from 17 in lacking the c1 methyl group . subsequently , the halogenase mala presumably chlorinates the c9 position ( malbrancheamide numbering ) first to afford the isolable natural product malbrancheamide b ( 22 ), which could be further chlorinated by mala at c8 leading to the final product malbrancheamide ( 3 ). this putative pathway is partially supported by the previous feeding study showing that the 13 c labeled 21 can be incorporated into 22 by m . aurantiaca . 69 lack of observed 13 c labeled 3 from the fermentation broth was interpreted to suggest that the second chlorination might be too slow to incorporate detectable levels of 13 c material from 22 to 3 . notably , the order of these two chlorinations seems unexchangeable since the c8 - monochloro regioisomer of 22 ( c9 - monochlorinated ) was not detected as a natural product despite considerable effort . 42 it is also possible that the dichloro species , malbrancheamide , arises from a pre - halogenated tryptophan - based assembly . blast ( http :// blast . ncbi . nlm . nih . gov /) sequence analysis revealed significant homology of mala to the family of flavin - dependent tryptophan halogenases . 70 - 73 this result suggests two alternative malbrancheamide biosynthetic pathways . first , mala could chlorinate tryptophan at c4 and c5 ( tryptophan numbering ) sequentially prior to being loaded onto the second t domain of malg . then , both monochlorinated and dichlorinated tryptophan could be processed by subsequent assembly enzymes , thereby respectively leading to 22 and 3 in parallel . second , mala might only monochlorinate the c4 position of tryptophan , resulting in 22 . then , 22 is converted into 3 by either mala or another unidentified halogenase that resides outside mal . to test these hypotheses , it would be the best to conduct in vitro functional analysis of purified mala against selected substrates such as l - tryptophan and 22 . alternatively , whether or not the 13 c labeled 22 can be incorporated into 3 in an in vivo precursor feeding study would also provide useful information about the timing of the two chlorination steps in malbrancheamide biosynthesis . according to the proposed malbrancheamide biosynthetic pathway ( fig5 ), only three enzymes are required to assemble the final product 3 . inactivation of these seemingly redundant genes including malb , malc , and malf ( table 1 ) is currently underway . interestingly , the malc short chain dehydrogenase related to phqe , which is presumed to participate in preparation of β - methyl proline starter unit in paraherquamide biosynthesis ( see above ), is present in the mal gene cluster although apparently unnecessary for malbrancheamide biosynthesis . this implies that malc , together with other redundant genes , might be residuals from ancestral or a horizontally transferred gene cluster ( e . g . one analogous to phq ). the evolving biosynthetic gene cluster not only recruits new genes , but also eliminates or retains unused genes when facing a diverse living environment and selection pressure during its evolutionary history . 24 recently , a novel malbrancheamide - type natural product named spiromalbramide ( 23 ) ( fig5 ) was isolated from a marine invertebrate - derived malbranchea graminicola fungal strain . 74 this new derivative contains the spiro - oxindole moiety that is found in notoamides and paraherquamides , but is absent from malbrancheamides . based on the comparative analysis of not , not ′, phq , and mal gene cluster , we are now capable of predicting that an fmo gene homologous to noti , noti ′ or phqk should reside in the uncharacterized biosynthetic gene cluster of 23 . so far , the solexa genome sequencing of m . graminicola has been completed . this prediction will be tested in the near future as soon as the biosynthetic gene cluster is mined and annotated from genome sequences . in principle , the shared genes from different clusters are responsible for assembling the common structural core among similar natural products . the cluster - specific gene products are presumed to modify these structures by a series of variant tailoring steps , thereby leading to structural diversification . however , it is noteworthy that the redundant genes and multifunctional genes could complicate comparative analysis of gene clusters . therefore , conclusions can only be unambiguously drawn after genetic and / or biochemical confirmation of enzymatic activities . following these simple but logical principles , we performed a comparative analysis wasperformed for four related gene clusters including not , not ′, phq , and mal , based on the proposed complete biosynthetic pathways for (+)/(−)- notoamides , paraherquamides , and malbrancheamides with a biosynthetic enzyme assigned for each individual step ( fig3 - 5 ). for example , the function of the not - specific gene notb can be readily connected to the pathway specific transformation from notoamide e ( 8 ) to notoamide c ( 9 ) and d ( 10 ). this was recently confirmed by in vitro characterization of notb fmo enzyme . 58 furthermore , detailed comparative analysis resulted in nomination of the oxidases noth and noth ′ ( or notd and notd ′), and the prenyltransferases phqj and male as putative diels - alderases to catalyze the distinctive imda reactions for these pathways . next , comparative functional analysis of these enzymes in vitro will enable us to test this long standing hypothesis regarding the existence of a diels - alderase in the biosynthesis of fungal indole alkaloids with the bicyclo [ 2 . 2 . 2 ] diazaoctane core . it is striking that nature has conscripted two evolutionarily related gene cluster paradigms , to construct the novel bicyclo [ 2 . 2 . 2 ] diazaoctane ring system by vastly different mechanistic protocols ( fig6 ). in one instance , for the notoamides , the net transformation from the nrps - loaded dipeptide to the bicyclo [ 2 . 2 . 2 ] diazaoctane core , a net two - electron oxidation is required to reach the key , putative azadiene species required for the proposed imda construction . in the other , the paraherquamide and malbrancheamide systems , the nrps - loaded dipeptide substrate is cleaved in a net two - electron reduction , that we speculate cyclizes and dehydrates to the related ( reduced ) azadiene species for the homologous imda construction . this insight was most readily presented to us , by the analysis of the respective gene cluster annotations , and has provided a very satisfying level of corroboration with labeled precursor incorporation experiments that at first , seemed incongruous . we expect that the tremendous insights that the bioinformatics analyses have provided in these systems , will render understanding the possible biogenesis of these and related natural products more efficient , congruent and intellectually satisfying . the foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting . since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art , the invention should be construed broadly to include all variations within the scope of the appended claims and equivalents thereof . 1 . j . w .- h . li and j . c . vederas , science , 2009 , 325 , 161 . 2 . d . j . newman and g . m . cragg , j . nat . prod ., 2007 , 70 , 461 . 3 . r . li and c . a . townsend , metab . eng ., 2006 , 8 , 240 . 4 . r . h . baltz , j . ind . microbiol . biotechnol ., 1998 , 20 , 360 . 5 . s . baba , y . abe , t . suzuki , c . ono , k . iwamoto , t . nihira and m . hosobuchi , appl . 6 . j .- h . noh , s .- h . kim , h .- n . lee , s . y . lee and e .- s . kim , appl . microbiol . biotechnol ., 2010 , 86 , 1145 . 8 . d . e . cane , c . t . walsh and c . khosla , science , 1998 , 282 , 63 . 10 . c . sanchez , l . zhu , a . f . brana , a . p . salas , j . rohr , c . mendez and j . a . salas , proc . natl . acad . sci . u . s . a ., 2005 , 102 , 461 . 11 . j . pollier , t . moses and a . goossens , nat . prod . rep ., 2011 , 28 , 1897 . 12 . j . l . que and w . b . tolman , nature , 2008 , 455 , 333 . 13 . a . l . goff , v . artero , b . jousselme , p . d . tran , n . guillet , r . métayé , a . fihri , s . palacin and m . fontecave , science , 2009 , 326 , 1384 . 14 . k . t . watts , b . n . mijts and c . schmidt - 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