5 to 100 linked genes (or more, depending on the complexity of the molecule) that together regulate expression and encode the biosynthetic enzymes responsible for natural product assembly and structural modification. In addition, natural product gene clusters include one or more genes that encode enzymes for cellular self-protection and extracellular drug transport. Typically, a complete pathway includes a cluster of genes encompassing 20-150 kilobases (kb). In actinomycetes, such as Streptomyces, the typical genome is about 8 megabases [12,13]. For combinatorial biology to successfully generate novel natural products, it must be possible to transfer efficiently rather large segments of DNA (>40 kb) from natural product-generating donor microorganisms into an appropriate engineered expression host. That is an essential aspect of Cubist's technological proficiency [7].

B. Combinatorial Biology: General Features of a New Drug Discovery Technology

Combinatorial biology is a proprietary leading edge drug discovery technology that was developed recently from advances made in molecular genetic manipulation and enzymology of natural product biosynthesis in actinomycetes, fungi, and other microbial systems [14]. This technology involves transfer of metabolic pathways from natural secondary metabolite-producing microorganisms (e.g., Streptomyces, Micromonspora, Actinomodura) to an engineered host (e.g., S. lividans) that allows control over timing and level of expression of natural product biosynthetic genes [7].

There are several ways that new natural products are created by combinatorial biology approaches. (1) It is now possible to transfer entire metabolic pathways from a donor strain to an engineered host using diverse molecular genetic tools (e.g., mobilizable vectors) [14]. This process allows a single pathway to be isolated and manipulated genetically to create libraries of recombinants capable of producing modified forms of a particular natural product. (2) Transfer of metabolic pathways from a natural donor strain to an engineered host can lead to expression of ''silent'' pathways that are not normally expressed under the culture conditions used to grow the donor strain [14]. Thus combinatorial biology can harness previously undetected metabolic pathways that result in discovery of new natural products. (3) Cubist has developed proprietary technology that involves efficient reassembly or ''shuffling'' of natural product biosynthetic pathway genes in a process of ''combinatorial pathway'' construction [7].

C. Combinatorial Biology Technology

The starting point for library construction is high molecular weight genomic DNA isolated from cultured donor strains or directly from the environment. Once geno-

mic DNA is isolated and suitably prepared, it is used to create two basic types of libraries. The first type of library, the natural pathway library (Fig. 3), provides metabolic pathways wherein the genes that comprise the pathway have their native linear configuration. This allows production of the same or similar molecules prescribed in the original donor strain [7]. Natural pathway library clones provide rapid access to the desired biosynthetic gene cluster and an immediate strategy for construction of ''biased'' combinatorial libraries and for screening such libraries using an engineered Streptomyces expression host in conjunction with the macrodroplet screening system (Fig. 3). The second type of library involves prior enrichment for DNA that contains natural product biosynthetic genes, followed by ''shuffling'' of the genes associated with donor biosynthetic pathways in a process that mimics natural processes of genetic recombination [7]. In both library construction approaches, DNA specifying production of natural products is introduced into an expression vector and transferred through conjugation or transformation into an appropriate host that provides effective production of natural products. The recombinant microorganisms created in this way can be screened directly by encapsulation in a gel macrodroplet that contains a target organism or reporter-based assay system. The integration of combinatorial biology expression libraries and HTS relieves a significant bottleneck in the natural product drug discovery process (Figs. 3, 4).

In addition to the examples of combinatorial biology library formats described above, other approaches for the discovery of novel natural products are available. An important example involves generation of ''biased'' combinatorial libraries (Fig. 5) [8]. In this library format, a selected group of microorganisms is chosen for their specific ability to produce an important class of natural products. Through genetic manipulation, a library of recombinant microorganisms is generated that produce variously modified forms of the particular natural product chem-otype. This approach is the recombinant version of enzymatic biotransformation and akin to medicinal chemistry on a known pharmacophore. However, since libraries are efficiently generated and screened in an engineered, characterized expression host, new natural products may be discovered more quickly and subjected to immediate fermentation scale-up once a promising lead has been discovered (Fig. 5).

D. Vector Systems for Expression of Natural Products

Combinatorial biology technology development programs involve construction of advanced molecular and genetic tools that provide increasingly efficient access to natural products for different donor and expression host systems. Toward this, two basic types of vector expression systems that accommodate a broad size range of DNA fragments to be cloned, mobilized, and expressed have been developed for several systems. One type of vector expression system includes shuttle

Figure 3 Natural pathway library construction.

Figure 4 Combinatorial pathway library construction.

Figure 4 Combinatorial pathway library construction.

Unique random CB clones In each strain

Unique random CB clones In each strain

Figure 5 Combinatorial pathway biased library for lead optimization and ''biological medicinal chemistry.''

Host's chromosome

Natural pathway' ( '

clone expressing V — J

"backbone" structu --—---------------------

Figure 5 Combinatorial pathway biased library for lead optimization and ''biological medicinal chemistry.''

cosmids. These allow efficient cloning of up to 50-kb fragments of DNA from a diverse range of microbes. These vectors have the capability to be mobilized (via interspecies conjugation), from E. coli directly into a broad range of potential expression hosts, and many are designed for efficient integration within the host chromosome. This strategy is being pursued in order to enhance long-term genetic stability of the cloned biosynthetic pathways. This capability is particularly important when high-level production of a combinatorial biology-derived natural product is required.

A second type of vector expression system derived from bacterial artificial chromosome (BAC)-based plasmids is a key combinatorial biology genetic tool [15]. These vectors are designed for cloning of DNA fragments greter than 120 kb. This feature is important because many complex natural product biosynthetic pathways are large clusters of genes that cannot be accommodated on cosmids due to the size limitation imposed by lambda packaging. Here again, flexible features are being built in to these vectors to allow facile mobilization, gene expression, and chromosomal integration.

E. Bacterial Expression Hosts

A critical aspect in development of the genetic toolbox for combinatorial biology involves the use of engineered bacterial host systems for stable expression of donor DNA containing genes for natural product biosynthesis. Engineered host systems have been developed (or are under development) for Streptomyces liv-idans, Streptomyces fradiae, and Myxococcus xanthus. For Streptomyces, a series of advanced-engineered strains are being developed in which indigenous metabolic pathways (e.g., actinorhodin, undecylprodigiosin for S. lividans) have been disabled or deleted from the genome. This simplifies the potential baseline product profile and removes competing endogenous pathways that may drain metabolic flux potential away from the desired heterologous system. Further modifications to these expression hosts will include genetic manipulation of regulatory genes involved in antibiotic production to increase yields from the heterologous pathways [16-18].

Was this article helpful?

0 0

Post a comment