rethinking production of taxolw
DESCRIPTION
BiotechnologyTRANSCRIPT
Rethinking production of TaxolW
(paclitaxel) using endophytebiotechnology§
Souvik Kusari1, Satpal Singh2, and Chelliah Jayabaskaran2
1 Institute of Environmental Research (INFU), Department of Chemistry and Chemical Biology, Chair of Environmental Chemistry
and Analytical Chemistry, TU Dortmund, Otto-Hahn-Str. 6, D-44221 Dortmund, Germany2 Department of Biochemistry, Indian Institute of Science (IISc), Bangalore 560012, Karnataka, India
Opinion
TaxolW (generic name paclitaxel) represents one of themost clinically valuable natural products known to man-kind in the recent past. More than two decades haveelapsed since the notable discovery of the first TaxolW-producing endophytic fungus, which was followed by aplethora of reports on other endophytes possessing simi-lar biosynthetic potential. However, industrial-scale Tax-olW production using fungal endophytes, althoughseemingly promising, has not seen the light of the day.In this opinion article, we embark on the current state ofknowledge on TaxolW biosynthesis focusing on the chem-ical ecology of its producers, and ask whether it is actuallypossible to produce TaxolW using endophyte biotechnol-ogy. The key problems that have prevented the exploita-tion of potent endophytic fungi by industrial bioprocessesfor sustained production of TaxolW are discussed.
TaxolW, an interesting case in point for revisiting‘endophyte biotechnology’We recently highlighted the current bottlenecks in exploit-ing a promising group of microbes called endophytic micro-organisms (or ‘endophytes’) using red biotechnology, whichare capable of producing pharmaceutically-relevant second-ary metabolites [1]. On the one hand, we are increasinglygaining a deeper understanding of how endophytes engagein bi-, tri-, and multipartite interactions with their hostplants as well as with other associated organisms (fungi,bacteria, or viruses) and endosymbionts, under the selectionpressures of various biotic (such as pathogens and feeders)and abiotic factors (such as precursors of plant/endophytesecondary metabolites and environmental conditions) inorder to produce certain ‘value-added’ natural products.On the other hand, we have failed to translate these
0167-7799/
� 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.03.011
DOI of companion article: http://dx.doi.org/10.1016/j.tibtech.2014.03.009.§This is a companion Opinion article to: Kusari, S. et al. (2014) Biotechnologicalpotential of plant-associated endophytic fungi: hope versus hype. Trends. Biotechnol.(http://dx.doi.org/10.1016/j.tibtech.2014.03.009).Corresponding authors: Kusari, S. ([email protected],[email protected]); Jayabaskaran, C. ([email protected]).Keywords: endophytic fungi; red biotechnology; plant–microbe interaction; Taxol1;endophyte biotechnology; industrial bioprocess; secondary metabolites; biosyntheticpathway; Taxol1 biosynthetic pathway; genetic engineering; co-cultivation; bio-reactor design.
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amazing discoveries into successful industrial bioprocessesfor sustained production of desirable compounds using en-dophyte biotechnology. We present the example of theblockbuster drug Taxol1 (generic name paclitaxel) to dis-cuss the trade-offs of attempting to translate potent endo-phytic Taxol1 producers into industrial microbial factories.
Chemical ecology of taxane production by yew plantsTaxol1, belonging to a class of complex diterpenoids calledtaxanes and possessing an unusual oxytane ring togetherwith a tricyclic core, is a blockbuster anticancer drug.Being unique in its mode of action of halting the prolifera-tion of cancer cells [2,3], the drug was approved by the USFDA to treat a variety of tumors including breast, ovarian,and AIDS-related Kaposi’s sarcoma, among others. It wasoriginally isolated from the bark of Pacific Yew, Taxusbrevifolia [4]. By and large, the production of Taxol1
and other taxanes is confined to a narrow taxonomic groupof higher plants belonging to the genus Taxus (familyTaxaceae, syn. Coniferales). Among the other four generaof this family, namely Amenotaxus, Autrotaxus, Pseudo-taxus, and Torreya [5], only Autrotaxus [6] and Pseudo-taxus [7] contain some simpler taxanes. Among the conifers– apart from the genus Taxus – only two other species havebeen reportedly claimed to produce taxanes. These includea close cousin of Taxus called Cephalotaxus (Cephalotax-aceae) [7] and also Podocarpus gracilor Pilger (Podocarpa-ceae) [8]. With the exception of the genus Taxus, theoccurrence of this diterpenoid in other reported gymno-sperm taxa has not been extensively studied and seemsconfined to only an exceptionally limited number of species.Interestingly, the presence of paclitaxel and other taxaneshas also been shown in an angiosperm from the familyBetulaceae, namely Corylus avellena L. [9,10]. Althoughthe molecular basis of taxane production in this angio-sperm has not received adequate attention (see [11] for arecent report on the transcriptome analysis of a Taxol1-producing endophyte harboring this plant), it might implyeither an independent evolution of Taxol1 biosynthesis inunrelated taxonomic groups or some type of evolutionaryrelatedness in the chemical ecology of these different taxa.
From the nigh-exclusive occurrence of this class ofmolecules in yew and the evolutionary and taxonomicuniqueness of Taxaceae and more importantly Taxus,
Opinion Trends in Biotechnology June 2014, Vol. 32, No. 6
together with the fact that most of the secondary metabo-lites of plants and fungi are produced as ecosystem perfor-mance-enhancing agents [12], it is possible that before thedispersion of the progenitor members of this genus, theevolution of the biosynthetic module of this unique mole-cule would already have been well underway or evenachieved. The variety of different modifications to the basicscaffold seems to stem from the reported promiscuity ofmany of the determining enzymes, and from the richnessand independent evolution of cytochrome P450 monooxy-genases (constituting the majority of pathway genes) ingeographically isolated Taxus plants.
After the huge clinical success of Taxol1, a number ofstudies for screening the high-yielding Taxus species wereconducted at geographically and ecologically diverse sitesall over the world. The results obtained in these studiesexpanded our understanding of the role of genetic andenvironmental factors influencing the type and concentra-tion of taxanes in different Taxus species, varieties, culti-vars, organs, and tissues of various Taxus plants. Further,parameters like species, age of the plant/organ, and vari-ous biotic (pathogens, herbivores, endophytes, symbionts,etc.) and abiotic (seasonal variations, altitude of the site,soil composition and pH, heavy metals, temperature, etc.)factors were shown to play a significant role in determiningthe taxane profile of the Taxus plant tissues. The examplesof T. canadensis (from Canada) exhibiting a somewhatunique chemical composition [7] and T. mairei (from South-ern China) possessing a high content of 7-xylosyltaxanes[13] might point to such ecosystem impositions. Interest-ingly, many cultivars of T. canadensis have been reportedto have chemical profiles different to other Taxus speciesand also observed to be particularly prone to grazing byanimals [7,14]. This may indicate an intricate, yet unprov-en, connection between chemical composition (such as typeand composition of taxanes) and susceptibility to grazing ina particular ecological niche. Moreover, because most Tax-us plants are protected from insect herbivory and grazinganimals, it has been rationalized that the exposure of theplants to fungal pathogens might have necessitated theevolution of biosyntheses of taxanes. Fleshy and coloredaril of Taxus plants, strikingly, is the only aerial organlacking taxanes and might, thus, represent a purposefulexception for attracting birds for seed dispersal.
Plants respond to other ecosystem partners over time byfine-tuning their metabolism and modifying their physiol-ogy and biochemistry to establish and execute cost–benefitrelationships for optimal ecosystem performance. In thiscontext, approximately 500 types of taxanes reported fromdifferent Taxus species and varieties [7] would, from anecological standpoint, probably allow the different Taxusplants to engage in multitrophic interactions with otherecosystem partners in diverse ecological niches. One suchimportant but rather unexplored trophic dimension isprovided by their interactions with the resident endophyticfungi as well as with other ecosystem partners mediated byendophytes [15]. Reports of the Taxol1 content in differentTaxus plants and tissues correlating to the number ofendophytic fungal species highlight such an interplay[16,17]. In addition, the secondary metabolites producedby endophytic fungi have already been designated to be
directly or indirectly responsible for altering the host plantdefense chemistry, as well as for growth promotion, antag-onism towards insects, herbivores, and pathogens [12,16–18]. For instance, endophytes can enhance the availabilityof nitrogen in host plant tissues and apoplast, therebydirecting the overall flux towards increased/continuousbiosynthesis of bioactive alkaloids by the host plants([18] and references therein). A number of reports on Taxuscell cultures have shown significantly enhanced taxaneproduction upon co-culturing [12] or elicitation with anendophytic fungus, its culture, or even a purified fungalmolecule like an oligosaccharide [16,17,19].
A direct connection between Taxol1 production by Tax-us and an endophytic fungus was demonstrated in a recentexperimental setup that further corroborated these find-ings on the effect of multitrophic crosstalk on Taxol1
production [20]. The authors demonstrated that a Tax-ol1-producing Paraconiothyrium sp. induced the in plantaexpression of Taxol1 biosynthetic pathway genes of T. xmedia. Interestingly, the fungus selectively inhibitedgrowth of Taxus-specific pathogenic fungi when challengedwith them. This indicates, as concluded by the authorsthemselves, a means for the resident Taxol1-producingParaconiothyrium sp. to save its own metabolic resourcesby inducing Taxol1 production by the host to thwart itscompetitors. Utilizing the same endophyte, these authorshave further demonstrated another aspect of multitrophicinteraction by uncovering the positive synergistic effect ofendophytic fungi harbored in the same plant but notproducing Taxol1 (such as Alternaria sp. and Phomopsissp.) on the Taxol1 production of Paraconiothyrium sp. [20].Such complex interplay of ecosystem partners seems toprovide an important dimension to the production of plantnatural products such as Taxol1, which depends on thespecies involved and their genotypes, in combination withother higher-order-trophic interactions [18].
In the light of these ecological community interactionstogether with the background information on the occur-rence of hundreds of different taxane molecules – most ofthem still functionally undefined – it is compelling to drawthe conclusion that many, if not all, of these structureswould rather fulfill still uncovered ecosystem-imposedrequirements of the individual Taxus plants. This is espe-cially important within a cost–benefit context in geograph-ically distinct and ecologically imposing habitats. Thepartners involved in such multitrophic interactions could,at least theoretically, be segregated and reassembled toachieve their functional integration into a biotechnology-based toolkit to positively alter the taxane content of Taxusplants, by means of tissue or cell cultures and most deci-sively by in vitro cultured Taxol1-producing endophyticfungi. The easiest way to achieve this is, of course, findingthe inducing-organisms engaged in ecological crosstalkwith the producer or their metabolites responsible for sucha production enhancement and utilizing these in the cul-ture and associated processes. Co-culturing of the ecolog-ically interacting organisms has been demonstrated to thisend [12,16,17,20]. However, development of fully stream-lined and robust biotechnological methodologies and pro-cess technologies to achieve the desired production scalesand economics remains a daunting challenge.
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Opinion Trends in Biotechnology June 2014, Vol. 32, No. 6
TaxolW-producing endophytic fungiThe huge clinical and pharmaceutical success of Taxol1 asan anticancer drug resulted in an enormous demand for thisdrug the world over, which in turn led to its supply crisis.This especially ensued from unprecedented yew cutting,disappointingly low amounts of the drug in slow-growingyew trees, and the laborious and slow process of Taxol1
extraction, and therefore prompted the discovery of alter-native sources of this valuable compound [21]. The elabo-rated total synthesis did not appear commercially viable andsemi-synthesis from renewable yew parts such as needleswas not very productive and subject to environmental,genetic, and biological variables. Plant cell cultures of theyew finally provided some hope and have significantly con-tributed to the world’s Taxol1 supply since the 1990s [22].However, due to the low yield and cost-related issues cou-pled with factors like sensitivity to shear stress and lengthyculture duration, the search for alternative microbialsources of Taxol1 was considered imperative.
In 1993, an endophytic fungus, Taxomyces andreanae,was isolated from the bark of Taxus brevifolia and wasshown to produce Taxol1 under in vitro axenic cultureconditions [23]. This discovery was projected as the dawn ofa new era of endophyte biotechnology with billions ofdollars’ worth of global market for Taxol1 already in place,and agreements were immediately underway among lead-ing pharmaceutical companies to explore the possibility offungal Taxol1 production through industrial fermentation[21]. The inability of the fungus to show reproducible high-titer yields of Taxol1 in axenic cultures, thus not amenableto industrial scale-up, led to the disappointing failure indelivering the promises of this highly heralded discovery.However, Taxol1-producing endophytic fungi harbored inother Taxus species and even in non-Taxus plants includ-ing many angiosperms, have been regularly reported[21,24]. At the present time, around 200 endophytic fungibelonging to more than 40 fungal genera from severaldifferent orders representing mostly Ascomycota and Deu-teromycota, with only a few from Basidiomycota and Zygo-mycota, have been reported to produce Taxol1 [21,24].Many endophytic fungi are added to the list every yearunderlining the fact that only a tiny fraction of an estimat-ed one million (or more) endophytic fungal species has beencultured and screened [25]. Undeniably, none of thesediscoveries have been successfully translated into indus-trial bioprocesses so far.
TaxolW production by endophytic fungi: variable,unstable yields and ecological considerationsNumerous reports are available on the pronounced vari-ability in Taxol1 production (nanogram to milligram scaleper liter media) from various endophytic fungal isolatesacross different batch cultures. This, combined with abewildering loss of production after repeated cycles ofsubculturing [26] and the lack of a comprehensive under-standing of endophyte biology, physiology, and molecularand chemo-ecological aspects vis-a-vis their secondary me-tabolite production, has led to disagreements in the scien-tific community over the ‘ability’ of endophytes to produceTaxol1 independent of their host plant [26,27]. As many ofthese fungi might have a multinucleate hyphal phenotype,
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it is tempting to note that, if the genes encoding compo-nents of a particular secondary metabolite biosyntheticpathway are lost or not maintained in adequate copynumber due to aging after repeated subculture, a compro-mise in the target secondary metabolite biosynthesis mightbe expected. On the one hand, extra-chromosomal mainte-nance of the Taxol1 biosynthetic pathway by these fungi, ifresponsible for any production loss, could be ruled outbased on the reproducible Taxol1 pathway gene amplifica-tions from many of them even after repeated subculturing(Table 1). On the other hand, repeated subculturing, un-derstandably, might alter the growth-based requirementsof a fungus coupled to its developmental program, whichmight itself result in overall fungal physiological repro-gramming and regulatory collapse of secondary metaboliteproduction. The reported revival of Taxol1 production [28]as well as the stimulatory/inductive effect of host plantcomponents on Taxol1 production by some endophyticfungi [26] point towards a need-based Taxol1 productionscenario. All Taxol1-producing fungi are naturally resis-tant to Taxol1, which has been shown to be a strongfungicide against a plethora of fungal phytopathogens.The production of Taxol1 by endophytic fungi might thusrepresent a means to thwart attack by invading fungi tokeep plants healthy for an unhindered access to theirapoplastic space. Soliman and Raizada, working withParaconiothyrium sp. isolated from T. x media, recentlyobtained results in agreement with such a hypothesis [20].The fungus showed higher Taxol1 production upon treat-ment with the host bark extracts and more importantly,when co-cultured with one or more endophytic fungi notcapable of taxane production but isolated from the samehost plant bark. Interestingly, co-culturing with both theAlternaria sp. and Phomopsis sp. resulted in an eightfoldincrease in Taxol1 production by the fungus, which wasalmost double compared to what was obtained when co-cultured only with Alternaria sp. Such a trophic interactionbetween different fungi sharing the same ecological nichedemonstrates an ecosystem-based crosstalk for driving theTaxol1 production involving system-level coordination.
Molecular basis of TaxolW production by Taxus plantsand TaxolW-producing endophytic fungiThe molecular pathway of Taxol1 biosynthesis in differentTaxus plants has been well characterized at both nativeand recombinant levels with the discovery of close to 20different enzymatic steps spatially organized in plastids,endoplasmic reticulum, and cytosol [29–31]. However, themolecular signatures of Taxol1 biosynthesis in Taxol1-producing endophytic fungi remains largely ill-defined andan unsolved riddle. Several groups have independentlyattempted to screen many of these fungi through PCR-based approaches to seek these biosynthetic blueprintsusing primers designed from the Taxol1 biosynthesis genesequences of different Taxus plants available in thedatabases (homology-based approach; see Table 1)[21,24,26,32,33]. Indeed, reports on the PCR amplificationand cloning of many genes of this pathway from severalTaxol1-producing endophytic fungi (Table 1) facilitate adecisive re-evaluation of their ‘true’ biosynthetic potential,and in turn their potential as alternative and sustainable
Table 1. TaxolW biosynthetic pathway genes reported from endophytic fungia,b
Gene name Molecule
type
Fungus Host plant GenBank
acc. no.
Fermentation
method
Refs
TS cDNA Fusarium solani Taxus celebica HM113487 SMF –
TS gDNA Taxomyces andreanae Taxus brevifolia – SMF [26]
TS gDNA Fusarium redolens Taxus baccata subsp. wallichiana – SMF [32]
TS gDNA Gibberella intermedia Taxus x media KC337345 SMF [33]
TS gDNA Mucor rouxianus Taxus chinensis – SMF [46]
TAT cDNA Ozonium sp. BT2 Taxus chinensis var. mairei AY960682 SMF –
10bH gDNA
cDNA
Ozonium sp. BT2 Taxus chinensis var. mairei AY836677
AY907826
SMF [47]
13aH cDNA Fusarium solani Taxus celebica EF626531 SMF [48]
DBAT gDNA Fusarium solani Taxus celebica GU392264 SMF –
DBAT gDNA Cladosporium cladosporoides MD2 Taxus x media EU375527 SMF [49]
DBAT gDNA Aspergillus candidus MD3 Taxus x media EU883596 SMF [50]
DBAT gDNA Fusarium redolens Taxus baccata subsp. wallichiana – SMF [32]
BAPT gDNA Taxomyces andreanae Taxus brevifolia – SMF [26]
BAPT gDNA Colletotrichum gloeosporioides Taxus x media KC337344 SMF [33]
BAPT gDNA Guignardia mangiferae Taxus x media KC337343 SMF [33]
BAPT gDNA Fusarium redolens Taxus baccata subsp. wallichiana KC924919 SMF [32]
aAbbreviations: TS, taxa-4(5),11(12)-diene synthase; TAT, taxa-4(5),11(12)-diene-5a-ol-O-acetyltranseferase; T10bH, taxane-10b-hydroxylase; T13aH, taxa-4(5),11(12)-diene-
13a-hydroxylase; DBAT, 10-deacetylbaccatin III-O-acetyltransferase; BAPT, baccatin III 13-O-(3-amino-3-phenylpropanoyl) transferase; SMF, submerged fermentation;
cDNA, complementary DNA; gDNA, genomic DNA.
bSee also [11] for some recent putative homologues of this pathway from a Penicillium aurantiogriseum NRRL 62431 strain isolated from hazelnut plant where extensive in
silico analysis of the fungal genome sequence was performed.
Opinion Trends in Biotechnology June 2014, Vol. 32, No. 6
sources of Taxol1. More than two decades have passedsince the celebrated discovery of Taxol1-producing T.andreanae and no imminent breakthrough in achievingtheir industrial and commercial utilization seems in sightdespite discovery and validation of a plethora of thesefungi. The aforementioned points attest to the mysteriouslifestyles of endophytes (alternating between endophyte-pathogen-epiphyte lifestyles), their complex and varyingphysiology under various environmental and culture con-ditions, and our inadequate knowledge about their bio-chemistry, molecular controls, and regulatory networks.The consequence of reports that a large number of unre-lated fungal taxa isolated from taxonomically and ecolog-ically diverse plant species produce Taxol1 casts doubts onwhether Taxol1 produced by endophytes is a biosyntheticproduct or an ‘adduct’ (carry-over from the host plant).However, many of these endophytic fungi that produceTaxol1 in vitro were isolated from taxonomically diversenon-Taxus species that do not contain this compound, thusnegating the notion that Taxol1 in these fungi might justbe a carry-over from the host plant tissues.
The most important clue to this apparent paradox of‘endophytes’ producing Taxol1 with their endogenous bio-synthetic capabilities might lie in the very origin of Taxol1
biosynthesis in these unique microorganisms. Horizontalgene transfer (HGT) is often hypothesized to explain relat-ed genes being present in taxonomically distant organisms.However, HGT cannot account for co-opting such a complexpathway orchestrated in different subcellular compart-ments with genes possibly scattered over different plantchromosomes. As discussed above, even the maintenanceof this pathway as an extra-chromosomal module wouldnot appear to be true. The reported differences in the plantand endophytic fungal Taxol1 biosynthetic genes, TS (tax-adiene synthase) and BAPT [baccatin III 13-O-(3-amino-3-phenylpropanoyl) transferase], further refute the HGT
theory [32]. Because TS catalyzes the so-called committedstep of this diterpenoid-derived pathway in plants(Figure 1), the molecular basis and evolutionary originof this biosynthetic step could account for the generallyaccepted and experimentally observed phenomenon ofTaxol1 production by endophytic fungi only upon specificinduction. Indeed, the reported positive selection pressure-mediated changes in TS [34,35] might point towards thepossibility of gain-of-function gene transfer events at anearly stage from a fungus to plant even involving co-evolution, and also among different fungi with diversityarising from gene duplication and divergence under differ-ent habitat-imposed selection pressures.
Whether the Taxol1 biosynthetic pathway is conservedamong plants and endophytic fungi especially with regardto gene clustering and regulation is an interesting andimportant question. If this was true, much of our currentunderstanding about this pathway from plants would beapplicable to Taxol1-producing endophytic fungi for anysort of genetic manipulation and regulation. Reports of thediscovery of several plant–Taxol1 biosynthetic genes inmany Taxol1-producing endophytic fungi even hint in parttowards an overall related pathway in these fungi (Table1). However, as is known for the biosynthesis of manycomplex secondary metabolites (for instance, gibberellins),the plant and fungal pathways might differ substantiallyand might even represent convergent or parallel evolution[11,36,37]. Perhaps not surprisingly then, the Taxol1 bio-synthetic pathway in plants is known to have many enzy-matic steps, which divert the flux away from Taxol1 [38].By contrast, there have been contradicting reports on theindependent Taxol1 biosynthetic capacity of endophytesfor which the ‘presumed’ Taxol1-producing endophyteswere examined in detail [27]. The search for the possiblegenetic signatures of this pathway using even state-of-the-art tools and techniques, however, led to the conclusion
307
MVA/MEP Pathway
Paclitaxel
OPP
HH
OHH
H
H
HO
HO
HO
HO
AcOAcO
AcOOH
OH
O
O
O
O
O
O
O
OAcO
HO
O
O
NH
OHOH
OH
AcO
O
O
OO
O
O
H
OH
OHAcO
AcO
OH
OH
OCcA
NH2
NH2
NH2
NH2
O
O
OHHO
O
O
O
O
OHH
HH
CAc
CAc
H
OPP
OPP
OPP
Isopenteyldiphosphate
Taxa-4(5),11(12)-diene
Taxa-4(5),11(12)-diene-5α-ol
Taxa-4(5),11(12)-diene-5α-acetate
Taxa-4(5),11(12)-diene-10β-ol-5α-acetate
Farnesyldiphosphate
Isopenteyldiphosphate
Geranylgeranyldiphosphate
10-Deacetylbacca�n III Bacca�n III
β-Phenylalanine CoA
β-Phenylalanine
3’-N-debenzoyl-2’-deoxypaclitaxel
α-Phenylalanine
GGPPS
T10βH
TATDBTNBT
PAM
*
T5αH
DBAT
BAPT
TS
+
‡
TRENDS in Biotechnology
Figure 1. Prevalent consensus biosynthetic route for Taxol1 in Taxus species. Abbreviations: MVA, mevalonic acid; MEP, 2-C-methyl-D-erythritol-4-phosphate; GGPPS,
geranylgeranyldiphosphate synthase; TS, taxa-4(5),11(12)-diene synthase that catalyzes the committed step of this pathway; T5aH, taxa-4(5),11(12)-diene-5a-hydroxylase;
TAT, taxa-4(5),11(12)-diene-5a-ol-O-acetyltranseferase; T10bH, taxane-10b-hydroxylase; z, ‘oxytane ring’ formation and branch migration enzymes including taxane 2a-O-
benzoyltransferase (T2BT or DBBT = debenzoyltaxane-20-a-O-benzoyltransferase) as well as C-13 hydroxylation and steps taking pathway flux towards non-Taxol1-type
molecules; DBAT, 10-deacetylbaccatin III-O-acetyltransferase; BAPT, baccatin III 13-O-(3-amino-3-phenylpropanoyl) transferase; DBTNBT, 30-N-debenzoyl-20-deoxytaxol-N-
benzoyltransferase which follows hydroxylation in the side chain by an unknown enzyme; PAM, phenylalanineaminomutase; *, b-phenylalanine coenzyme A ligase.
Multiple arrows imply more than one biosynthetic step. The Taxol1 biosynthetic pathway is proposed to have about 20 different enzymatic steps in Taxus plants.
Opinion Trends in Biotechnology June 2014, Vol. 32, No. 6
that no such pathway exists in these fungi. Interestingly,more recent findings based on genome mining [11] revealedthat such a scenario does not seem to be true.
Concluding remarks and future perspectivesGenetic engineering of endophytic fungi known to produceTaxol1, both by gene overexpression and random muta-genesis coupled with genome shuffling, have beenattempted in only a very limited number of fungal isolates.In Ozonium sp. EFY-21 isolated from T. chinensis var.mairei, overexpression of Taxus TS gene under a fungalspecific promoter resulted in about fivefold increase inTaxol1 production as compared to control [39]. Multiplemutagenesis of Nodulisporum sylviforme provided thestrain NCEU-1 from which three hereditarily stablestrains were obtained by mutagenesis. Protoplasts (roundfungal cells generated from spores and lacking the cellwall) generated from these and fused randomly finally ledto three strains that showed an increase in Taxol1 yield by
308
31, 64, and 45% over the control, respectively [40]. Thereported Taxol1 pathway metabolic engineeringapproaches in Escherichia coli [41–43] and Saccharomycescerevisiae [44,45] have mostly focused on taxadiene engi-neering. Reported attempts to engineer the Taxol1 biosyn-thetic pathway beyond taxadiene encountered metabolicbottlenecks as observed by total absence or insignificantyields of any intermediate beyond taxadiene. This wasshown for seven consecutive gene transfers in S. cereviseae,which is the highest number of steps (for the Taxol1
pathway) engineered in a heterologous host so far [45].Notwithstanding these unsuccessful endeavors, however,the several hundred milligrams per liter (reaching �1 g/l)yields of taxadiene obtained in few such attempts togetherwith reports of biotransformation of intermediate taxanesby several microbial enzymes provide some strategiesworth exploring to realize sustained Taxol1 supply usingendophyte (and related microbial) biotechnology. Thiswould be especially interesting when supplemented with
Box 1. Sustained TaxolW supply using endophytes
Genetic engineering approaches can be used to engineer the Taxol1
biosynthetic pathway (Figure IA). This includes overexpression of the
important or all of the Taxol1 biosynthetic pathway genes and their
promoter modulation, including other complimentary genetic engi-
neering approaches such as epigenetic engineering involving tran-
scription factors or chromatin modifier elements, and gene silencing.
Taxol1-producing endophytes and other heterologous hosts including
simpler organisms such as yeast and bacteria could serve as ideal
candidates for such an attempt. Taking advantage of inter-organismal
effects could also lead to greater Taxol1/taxane yields (Figure IB). This
strategy may involve co-cultivation of two or more organisms for
elicitation of Taxol1 biosynthesis, Taxol1 pathway-intermediate bio-
transformation, or utilization of metabolic dead ends/high yield Taxol1
pathway-intermediates from one organism by another organism as the
exogenously supplied substrates, and even combinatorial Taxol1/
taxane biosynthesis by different organisms. Various molecular-,
biotechnological-, and bioprocess-related methodologies, tools, tech-
niques, and optimization strategies, both stand-alone or in combina-
tion, are known or proposed to affect the Taxol1/taxane yields of a
given endophyte or a recombinant heterologous host (Figure IC). These
would seem to prove especially relevant when used in combination
with the genetic engineering and inter-organismal combinatorial
approaches. For example, a heterologous host engineered for the
Taxol1 biosynthetic pathway gene(s) could be optimized for an
inducible Taxol1 production, a greater cellular release of Taxol1,
two-phase growth and production cycles, and silencing or modulation
of its own Taxol1 pathway negative feedback, flux-diversionary, or
dead-end metabolite metabolic steps.
(A)TSGGPPS T5αH TAT T10βH DBAT PAM etc.
Metabolic engineering of Taxol® biosynthe�c pathway
Heterologous hosts
Flux balance (pathway and protein engineering)
Enzyme engineering
Co-culture and culture op�miza�on
Bioreactor design
Removal of metabolic bo�lenecks/dead ends
Strain improvement (gene overexpression,mutagenesis, genome shuffling. gene silencing,engineering, and physiochemical op�miza�on
for increased product excre�on)
Superior Taxol® yields ???
Endophy�c fungi
(B)
(C)
d i
TRENDS in Biotechnology
Figure I. Possible strategies for achieving sustained Taxol1 supply using endophytes. (A) Genetic engineering approaches involving the Taxol1 biosynthetic pathway.
(B) Inter-organismal contribution towards greater Taxol1/taxane yields. (C) Bioprocessing-based strategies for increasing Taxol1 yield.
Opinion Trends in Biotechnology June 2014, Vol. 32, No. 6
309
Box 2. Outstanding questions
� Is the Taxol1 biosynthesis in endophytic fungi similar to that of
Taxus plants and what is its origin?
� Why do endophytic fungi tend to lose their Taxol1 production
potential upon extensive propagation?
� Do these endophytic fungi produce Taxol1 in planta and, if so,
how does that differ from their in vitro production, especially with
regard to its biosynthetic regulation?
� What is the molecular basis of the reported elicitation of
endophyte Taxol1 biosynthesis by host plant components?
� Do the host plant Taxol1 pathway intermediates play a part in
fungal Taxol1 biosynthesis?
� How much is the storage, release, and compartmentalization of
Taxol1 biosynthesis in fungi affected by metabolic flux dynamics
connecting primary and secondary metabolism or vice versa?
Opinion Trends in Biotechnology June 2014, Vol. 32, No. 6
contributions from heterologous hosts and other optimiza-tion methodologies and tools such as intracellular com-partment optimization, storage and efflux modulation, andcontrol of pathway regulatory elements (Box 1). However,delineation of the molecular mechanisms of Taxol1 bio-synthesis and regulation thereof remains a prerequisite forall such endeavors. Most notably, as seen from the Taxol1
biosynthetic pathway of Taxus sp., there seems to be anobvious hurdle in engineering such a lengthy and complexpathway in its entirety in heterologous hosts. Transforma-tion of genes of the entire pathway is a challenge and moreimportantly, regulation of Taxol1 production encompass-ing epigenetic modulation and signaling crosstalk itselfremains a poorly understood topic. Taxol1-producing en-dophytic fungi, therefore, still present a viable and long-term target, despite many unanswered questions (Box 2).
AcknowledgmentsResearch in the laboratory of S.K. (INFU, TU Dortmund) is supported inpart by the International Bureau (IB) of the German Federal Ministry ofEducation and Research (BMBF/DLR), Germany, the Ministry ofInnovation, Science, Research, and Technology of the State of NorthRhine-Westphalia, Germany, the German Academic Exchange Service(DAAD; ‘Welcome to Africa’ initiative), and the German ResearchFoundation (Deutsche Forschungsgemeinschaft, DFG). S.K. is a VisitingResearcher at the Department of Plant Sciences, University of Oxford,South Parks Road, Oxford OX1 15 3RB, United Kingdom. S.K. gratefullyacknowledges M. Spiteller for approving and authorizing, Gail M.Preston for hosting, and TU Dortmund for supporting his stay at theUniversity of Oxford. Research in the C.J. laboratory (IISc Bangalore) issupported by grants from the Department of Biotechnology (DBT), Indiaand the Council of Scientific & Industrial Research (CSIR), India. S.S.thanks CSIR and DBT for fellowships. We thank Bhagat Singh forassistance in preparation of Figure 1. We apologize to the numerousinvestigators whose publications could not be cited here owing to spaceconstraints.
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