Investigaciones y proyectos sobre la realidad aumentada

An organism’s primary and secondary metabolic pathways are linked via the metabolic burden that each places on the other. Although antitumor natural products result

177 7 Anticancer Drug Discovery via Genetic Engineering

from an organisms’ secondary metabolic machinery there remains an inextricable link between natural product titers and the absolute metabolic burden with which the producing organism needs to cope. As the principal producer of anticancer natu-ral products, the actinomycetes have therefore evolved strict regulatory mechanisms crucial to the maintenance of primary metabolism but that often limit natural prod-uct titers. Indeed, these regulatory mechanisms are at the root of many obstacles encountered in the industrial production of anticancer drugs. Such obstacles have been largely addressed through a combination of approaches based on genetic engi-neering (Fig. 7.1 ). Targeted inactivation of repressor genes and overexpression of transcriptional activators both constitute strategies that have been very effective in increasing natural product titers from their wild-type strains (Fig. 7.1a ). The overexpression of structural genes has also served as a useful approach to overcome natural product-based feedback inhibition (Fig. 7.1b, c ). Additionally, gene dosage plays an important role in the improvement of production titers. Relevant genes are multiplied or overexpressed in order to increase natural product production.

Self-resistance can also contribute signi fi cantly to low titers. Increased resistance of the producer and ampli fi ed expression of structural genes can both lead to improved natural product titers (Fig. 7.1d ). Additionally, exporters, either as part of the self-defense system or simply to transport the drug outside the cell, can be overexpressed to enhance natural product synthesis (Fig. 7.1e ). Importantly, these strategies can be used individually or in combination to circumvent the problem of development- and production-limiting natural product titers.

Fig. 7.1 Important steps targeted by genetic engineering approaches to improve production titers of natural antitumor substances. ( a ) Derepression or activation of regulatory genes leads to over-expression of structural genes; ampli fi cation of genes for ( b ) precursor supply or ( c ) structural genes overcomes pathway bottlenecks; ( d ) enhancement of self-resistance increases production levels; ( e ) enhanced expression of exporter proteins and ef fl ux pumps increases the cell’s ability to produce more compound by circumventing possible cell death and/or feedback mechanisms ordi-narily used to limit compound production

7.2.1.1 Regulation

Pathway-speci fi c regulators in fl uence the transcription of structural genes and these can have either positive (activator) or negative (repressor) effects on the expression of biosynthetic genes. The identi fi cation of such elements is generally straightfor-ward as biosynthetic genes for a given natural product are typically clustered together in speci fi c regions of the chromosome. This is in contrast to pleiotropic regulators that control structural genes, pathway-speci fi c regulator genes, and others such as morphological genes or genes involved in regulating precursor and cofactor supply. Furthermore, feedback regulation mechanisms limit the amount of drug produced inside the cell. Manipulation of the regulatory system can change the production pro fi le signi fi cantly and there are clearly numerous opportunities for such engineering efforts. The often dramatic success of titer enhancement strategies has been recently reviewed (Chen et al. 2010 ) .

In the context of anticancer drug discovery and development an extensively investigated example of regulatory system manipulation involves biosynthesis of the anthracycline-type antitumor antibiotic doxorubicin (Fig. 7.2 ), the production of which is tightly regulated in Streptomyces peucetius . Overexpression of transcrip-tional activators has been shown to increase doxorubicin production up to 4.3-fold relative to the wild-type strain (Malla et al. 2010a ) . S. peucetius strains bearing plasmids with an increasingly larger number of the regulatory genes dnrN , dnrI , afsR , and metK1-sp produced increasingly greater amounts of doxorubicin.

Consistent with this work, when the global regulatory gene afsR , an established transcriptional activator, was overexpressed in S. peucetius , doxorubicin production was enhanced up to eightfold (Maharjan et al. 2009 ) . Similar experiments with the pikromycin producer Streptomyces venezuelae and the actinorhodin producer Streptomyces lividans yielded similar results; afsR overexpression in S. venezuelae led to a ~5-fold improvement in pikromycin (Fig. 7.2 ) production relative to the wild-type strain and afsR overexpression in S. lividans led to a 1.5-fold improve-ment in actinorhodin (Fig. 7.2 ) production relative to wild-type. Similar titer improvements have been achieved in the fredericamycin producer Streptomyces griseus ATCC 49344. Overexpression of fdmR1 , which codes or a SARP family member pathway-speci fi c activator, led to a 5.6-fold improved production of fred-ericamycin A (FDM A, Fig. 7.2 ) by S. griseus . The recombinant strain produces FDM A with a titer of ~1.5 g/L suf fi cient to support future development efforts for this antitumor pentadecaketide (Chen et al. 2008a ) . These recent examples of regulatory manipulation to improve anticancer agent titers from fermentation com-pliment many earlier examples.

Just as overexpression of activators can lead to improved natural product titers so too can the repression of negative regulators. An excellent example of this comes in the form of efforts to improve the production of the antibacterial compounds platen-simycin and platencin (Fig. 7.2 ). The production of both compounds is encoded by a single gene cluster in Streptomyces platensis MA7327. Within this cluster, the ptmR1 gene was found to encode for what appears to be a member of the GntR family of transcriptional repressors. Consistent with its role as a repressor of platensimycin

179 7 Anticancer Drug Discovery via Genetic Engineering

Fig. 7.2 Clinically signi fi cant natural products whose development and application were enabled by titer improvements accomplished by altered expression of regulatory genes (doxorubicin, pikromycin, platensimycin, platencin, fredericamycin), structural genes (erythromycin A, doxoru-bicin), resistance genes (actinorhodin, C-1027) and export genes (doxorubicin) or by enhance-ments in biosynthetic precursor availability (actinorhodin, avermectin, pikromycin, FK506) during fermentation, or by a combination of these approaches

and platencin biosynthesis inactivation of ptmR1 afforded S. platensis strains that produced both natural products in titers ~100-fold superior to those of the wild-type strain (Smanski et al. 2009 ) . Clearly, repressors of biosynthetic machineries consti-tute excellent targets of regulatory modi fi cation and complement, perhaps in multiple ways, the effectiveness of targeting activators to achieve improved natural product titers (Chen et al. 2010 ) .

7.2.1.2 Precursor Supply

Bottlenecks in biosynthetic pathways restrict natural product titers and are often associated with the limitations of primary metabolism in providing key precursors necessary for secondary metabolic pathways. Such limitations can be overcome by amplifying the gene or genes that code for enzymes associated with such bottle-necks; increased enzyme levels translate to diminished bottleneck effects and hence, improved titers. Examples correlating precursor supply to improve natural product titers have focused on carbohydrate metabolism, fatty acid precursors, and intracel-lular cofactor supplies (Olano et al. 2008 ) . Such examples include heterologous overexpression of the S -adenosyl- l -methionine (SAM) synthetase metK , which improved production of a number of anticancer agents and antibiotics, such as acti-norhodin, avermectin, and pikromycin (Fig. 7.2 ), by providing SAM as a substrate or cofactor (Huh et al. 2004 ) . More recently, it has been demonstrated that titers of the immunosuppressant drug FK506 (Fig. 7.2 ) can be improved by enhancing the intracellular pool of methylmalonyl-CoA, a critical building block for FK506 production. Medium supplementation with methyl oleate and simultaneous intro-duction of the methylmalonyl-CoA mutase pathway into the FK-506 producer Streptomyces clavuligerus CKD1119 led to a threefold improvement in FK-506 titers through transient intracellular increases in acetyl-CoA, which is rapidly con-verted via the endogenous machinery to methylmalonyl-CoA (Mo et al. 2009 ) .

7.2.1.3 Overexpression of Structural Genes

Structural genes code for any RNA or protein product not associated with regulation and resistance. In the context of secondary metabolism, structural genes are neces-sary for providing functional platforms on which natural products are constructed. An absence or limited expression of these genes correlates to the production of interme-diates or shunt metabolites that are co-isolated with intact natural products. Such impurities can be minimized by structural gene overexpression and this strategy has also been shown to induce enhanced natural product titers. The biosynthesis of the antibiotic erythromycin A (Fig. 7.2 ) has been studied intensively in the past and serves as a model for polyketide biosynthesis in actinomycetes. A large number of natural anticancer agents are polyketides. The tailoring genes eryK (P450 hydroxy-lase) and eryG (SAM-dependent O -methyltransferase) were overexpressed in the producer strain Saccharopolyspora erythraea . Larger quantities of these enzymes

181 7 Anticancer Drug Discovery via Genetic Engineering

led to titer improvement and purity of the antibiotic erythromycin A by conversion of its intermediates (Chen et al. 2008b ) . A good example of titer improvement of antitumor substances is engineering of tailoring genes in doxorubicin production.

Glycosylation is considered to be a rate-limiting step in doxorubicin biosynthesis.

Heterologous expression of structural sugar biosynthesis and glycosyltransferase genes elevated doxorubicin levels in S. peucetius up to 5.6-fold (Malla et al. 2009 ) . Structural gene deletion or inactivation also can be used to increase natural product titers. This is particularly applicable when applied to systems in which one natural product is biosynthetically transformed to another, less useful, compound. However, by and large, structural genes have been the target of overexpression in efforts to improve anticancer natural product titers.

7.2.1.4 Overexpression of Resistance Genes

Enhanced levels of natural product resistance in the producer strain often correlate to enhanced production of compound. One example of natural product resistance involves CagA, the apoprotein of the enediyne antitumor antibiotic C-1027 (Fig. 7.2 ).

CagA binds to the toxic chromophore of C-1027, stabilizing the ordinarily labile enediyne and playing an important role in C-1027 resistance to the producer Streptomyces globisporus (Beerman et al. 2009 ; Kennedy et al. 2007 ; Liu et al.

2002 ) . Disruption of the cagA gene showed that the apoprotein is important but not essential for resistance and that other factors seem to be required for self-resistance.

Though not the sole source of resistance to C-1027, overexpression of cagA leads to increased C-1027 production (Cui et al. 2009 ) . Such a strategy to enhance titer is limited only by the resistance mechanism; overexpression of resistance genes can impact more than just one compound at a time. For instance, overexpression of streptomycin resistance ( rpsL ribosomal protein mutation) is known to increase the production of the polyketide antibiotic actinorhodin (Hesketh and Ochi 1997 ) . Additionally, multiple drug resistance mutations have been used to obtain a strain with 180-fold increased productivity. Using the same idea of enhanced resistance to multiple antibiotics a new approach called “ribosome engineering” has been devel-oped (Wang et al. 2008 ) . By screening for a Streptomyces coelicolor strain that is resistant to eight different antibiotics a strain capable of producing 1.63 g/L actinor-hodin was identi fi ed. The high degree of resistance was attributed to mutant ribo-somes which sustain a high level of protein synthesis, even at a late stage of growth when antibiotic production starts. Furthermore, the mutants showed an increased ability to accumulate ppGpp, which is an important signaling molecule for the onset of antibiotic production. This example illustrates that resistance is tied to the regula-tory network within the cell and can contribute to improved natural product titers.

7.2.1.5 Overexpression of Export Genes

Natural product biosynthesis is often subjected to negative feedback regulation and a critical component of such mechanisms is the expression of exporter genes.

Indeed, many anticancer natural products are removed from the cell by export proteins. Intracellular accumulation of products and thus inhibition of biosynthetic enzymes can be modulated by ef fl ux systems. For instance, doxorubicin produc-tion has been enhanced 2.2-fold by overexpression of the export genes drrA and drrB . These genes encode an ATP-binding cassette (ABC) transporter that pumps the antibiotic out of the cell thereby conferring resistance (Malla et al. 2010b ) . Conversely, deletion of drrA and drrB resulted in a dramatic decrease of antitumor antibiotic production in S. peucetius (Srinivasan et al. 2010 ) . For the purpose of titer improvement it is important to note that enhancement of drug export is not limited to product-speci fi c transporters. For instance, it is now well established that the nonspeci fi c multi-drug resistance (MDR) exporters can be modulated so as to increase drug production (Adrio and Demain 2006 ) .