Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
The Use of Recombinant Microorganisms for the Production of Commercial Products
Antibiotics

Since the discovery of penicillin in the late 1920s, more than 6000 antibiotics with different specificities and Mechanisms of action have been isolated from various microorganisms. Their widespread use in the Treatment of infectious diseases has helped save millions of lives. The vast majority of major antibiotics have been isolated from the Gram-positive soil bacterium Streptomyces, although they are also produced by Fungi and other Gram-positive and Gram-negative Bacteria. Globally, 100 000 t of antibiotics worth approximately 5 billion dollars are produced annually, including more than 100 million dollars spent on antibiotics added to livestock feed as supplements or growth promoters.

It is estimated that scientists discover between 100 and 200 new antibiotics each year, primarily through extensive screening programs searching thousands of different microorganisms for those that synthesize unique antibiotics. The development and clinical testing of new drugs are extremely expensive, and only those with high therapeutic value and economic viability reach the market. These account for only 1—2% of all discovered antibiotics. Recombinant DNA technology can have a major impact here. First, it can be used to design novel antibiotics with unique structures that exert more potent effects on specific microorganisms while minimizing side effects. Second, Introduction/32.html">Genetic Engineering approaches can be employed to increase antibiotic yields, thereby reducing production costs.

When developing recombinant strains of Streptomyces—the primary microorganism used for antibiotic production—It is important to ensure that the transformation and Selection of transformed Cells are not overly complex. However, unlike E. coli, Streptomyces do not exist as isolated cells but as extensive mycelia. Therefore, prior to transformation, The Cell wall must be disrupted to release individual protoplasts (Fig. 12.9). Without this, it would be impossible to distinguish transformed cells from untransformed ones, as visible colonies on solid media would arise from a group of cells rather than a single cell; consequently, colonies growing in the presence of a selective antibiotic would consist of a mixture of transformed and untransformed cells. The uptake of plasmid DNA into Streptomyces protoplasts is facilitated by polyethylene glycol. Following transformation, the protoplasts are first plated onto a solid medium to allow cell wall regeneration, and then transferred to a selective medium, typically containing either neomycin or thiostrepton, to select for transformed cells.

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Fig. 12.9. Scheme of transformation and selection of recombinant Streptomyces strains. Transformed cells are indicated by pink circles, untransformed ones by green. PEG — polyethylene glycol.

Cloning of antibiotic Biosynthesis genes

The biosynthetic pathway of a single antibiotic can consist of 10—30 enzymatic reactions, making the cloning of all its biosynthetic genes a challenging task. One approach to isolating the complete set of these genes relies on transforming one or more mutant strains unable to synthesize the antibiotic with a Gene library constructed from the chromosomal DNA of the wild-type strain. Following the introduction of the library into mutant cells, transformants capable of synthesizing the antibiotic are selected. The plasmid DNA is then isolated from the clone containing the functional, expressed antibiotic gene [i.e., the gene that restores (complements) the function lost by the mutant strain] and used as a probe to screen another wild-type chromosomal DNA library. This allows the selection of clones containing nucleotide sequences that overlap with the probe sequence. In this way, DNA segments flanking the complementing sequence are identified and subsequently cloned, reconstructing the entire antibiotic biosynthetic gene cluster. This Procedure applies when these genes are clustered at a single locus on the chromosomal DNA. If the biosynthetic genes are scattered as small clusters across different loci, at least one mutant per cluster is required to obtain the DNA clones needed to identify the remaining genes in the clusters.

This approach was successfully used to identify several genes for The biosynthesis of undecylprodigiosin from Streptomyces coelicolor A3 (Fig. 12.10). In this case, the complementation assay is based on comparing colony colors: wild-type colonies are red, whereas mutant colonies are cream-colored. Thus, complementation results in The formation of a red colony. In addition to complementation, more direct approaches can be used to identify antibiotic biosynthetic genes. For instance, genetic or biochemical experiments can be employed to identify and then isolate one or more key biosynthetic Enzymes, determine their N-terminal Amino acid sequences, and design oligonucleotide probes based on these data. This approach was used to isolate the isopenicillin N synthase gene from Penicillium chrysogenum. This enzyme catalyzes the oxidative Condensation of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N, a key intermediate in the biosynthesis of Penicillins, Cephalosporins, and cephamycins (Fig. 12.11).

Fig. 12.10. Structural formula of undecylprodigiosin.

Synthesis of Novel Antibiotics Novel antibiotics with unique properties and specificities can be obtained by genetically engineering the genes involved in the biosynthesis of already known antibiotics. One of the first experiments that yielded a novel antibiotic involved combining two slightly different antibiotic biosynthetic pathways within a single microorganism.

One of the Streptomyces Plasmids, pIJ2303, carrying a 32.5-kb fragment of S. coelicolor chromosomal DNA, contains all the genes for the enzymes responsible for the biosynthesis of the antibiotic actinorhodin, a member of the isochromanequinone family of antibiotics, from acetate (Fig. 12.12). The entire plasmid and various subclones carrying portions of the 32.5-kb fragment (e.g., pIJ2315) were introduced either into Streptomyces sp. strain AM-7161, which synthesizes the related antibiotic medermycin, or into S. violaceoruber strains B1140 or Tü22, which synthesize the related antibiotics granaticin and dihydrogranaticin. All of these antibiotics act as acid-base indicators, imparting a characteristic color to the growing culture depending on the pH of the medium (Table 12.3). In turn, the pH (and color) of the medium depends on which compound is being synthesized. Mutants of the parental S. coelicolor strain that are unable to synthesize actinorhodin are colorless. The appearance of color following the transformation of Streptomyces sp. strain AM-7161 or S. violaceoruber strains B1140 or Tü22 with a plasmid carrying all or some of the genes encoding actinorhodin biosynthetic enzymes indicates the synthesis of a novel antibiotic (Fig. 12.12, Table 12.3). Transformants of Streptomyces sp. strain AM-7161 and S. violaceoruber strain B1140 containing plasmid pIJ2303 synthesize antibiotics encoded by both the plasmid and the chromosomal DNA. However, when S. violaceoruber strain Tü22 is transformed with plasmid pIJ2303, a novel antibiotic, dihydrogranatyrhodin, is synthesized alongside actinorhodin, and when Streptomyces sp. strain AM-7161 is transformed with plasmid pIJ2315, another novel antibiotic, mederrhodin A, is synthesized.

Fig. 12.11. Biosynthesis of penicillins and cephalosporins in P. chrysogenum. Isopenicillin N synthase catalyzes the synthesis of isopenicillin N—the precursor of penicillin G, penicillin N, and cephalosporin C—from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine.

Structurally, these new antibiotics differ little from actinorhodin, medermycin, granaticin, and dihydrogranaticin, and are likely formed when an intermediate of one biosynthetic pathway serves as a substrate for an enzyme of another pathway. Once the biochemical properties of various antibiotic biosynthetic pathways are studied in detail, it will become possible to create novel, highly specific antibiotics by manipulating the genes that encode the corresponding enzymes. DEVELOPMENT OF NEW Methods FOR PRODUCING polyketide antibiotics. The term "polyketide" refers to a class of antibiotics formed by the sequential enzymatic condensation of carboxylic acids such as acetate, propionate, and butyrate. Some polyketide antibiotics are synthesized by plants and fungi, but most of them are produced by actinomycetes as secondary metabolites. Before manipulating the genes encoding the enzymes of polyketide antibiotic biosynthesis, it is necessary to elucidate the MECHANISM OF ACTION of these enzymes.

Table 12.3. Antibiotics synthesized by various Streptomyces strains, including strains transformed with plasmids pIJ2303 and pIJ23151)

1) According to Hopwood et al., Nature 314: 642—644, 1985.

Polyketide antibiotics are synthesized via the same pathway as long-chain Fatty acids. With each condensation cycle, a β-keto group is added to the growing carbon chain. The process consists of a series of repeating steps, including reduction of the keto group, dehydration, and reduction of the β-enoyl groups in the growing polyketide chain. There are two classes of polyketide synthases—enzyme complexes responsible for the synthesis of polyketide antibiotics (Fig. 12.13). The first class consists of synthases that catalyze the biosynthesis of aromatic Polyketides; each synthase is a single polypeptide with a single Active Site that sequentially catalyzes the biosynthetic reactions (Fig. 12.13, A). The second class includes synthases made up of several Polypeptides (A–E in Fig. 12.13, B); each has its own active site and possesses a specific enzymatic activity that catalyzes a particular biosynthetic reaction.

If each enzymatically active domain of a multifunctional polyketide synthase subunit catalyzes a specific reaction, then the loss of any single activity will affect only one biosynthetic step, and modifying a domain with a known function will lead to predictable changes in The Structure of the synthesized antibiotic. For example, by studying in detail the genetic and BIOCHEMICAL COMPONENTS OF erythromycin biosynthesis in Saccharopolyspora erythraea cells, it was possible to introduce specific changes into the genes associated with the biosynthesis of this antibiotic and to synthesize erythromycin derivatives with altered properties. First, the Primary Structure of a 56-kb S. erythraea DNA fragment containing the ery gene cluster was determined, and then the erythromycin polyketide synthase was modified in two different ways. To achieve this, they either 1) deleted the DNA region encoding β-ketoreductase, or 2) introduced a change into the DNA region encoding enoylreductase. Deletion of the β-ketoreductase gene led to the accumulation of an intermediate in which a carbonyl group, rather than a hydroxyl group, was attached to the C-5 atom of the ring (Fig. 12.14), while a mutation in the enoylreductase gene resulted in the formation of a double bond between the C-6 and C-7 atoms (Fig. 12.14). These experiments demonstrate that once a gene cluster encoding the biosynthetic enzymes of a specific polyketide antibiotic is identified and characterized, introducing specific changes into these genes allows for the targeted Modification of the antibiotic's structure. Furthermore, by cutting and splicing various DNA regions, polyketide synthase domains can be rearranged to produce novel polyketide antibiotics.

Fig. 12.12. Structural formulas of various isochromanequinone antibiotics synthesized by Streptomyces strains. Wild-type S. coelicolor and plasmid pIJ2303 encode actinorhodin; Streptomyces sp. synthesizes medermycin, and S. violaceoruber produces granaticin and dihydrogranaticin. The synthesized hybrid antibiotics are mederrhodin A and dihydrogranatyrhodin.

All aromatic polyketide gene clusters contain three genes encoding the so-called minimal polyketide synthase. This enzyme complex includes a ketosynthase (with an acyltransferase domain), a chain length factor, and an acyl carrier protein. The minimal polyketide synthase is responsible for synthesizing the aromatic polyketide backbone, while its modifications are carried out by Other Enzymes acting in concert with it. The genes encoding all these enzymes are typically organized into a single cluster (Fig. 12.15). Each gene cluster encodes the synthesis of a specific antibiotic. By exchanging genes between clusters, two novel aromatic polyketide antibiotics were synthesized (Fig. 12.16), further illustrating the potential of genetic engineering.

Fig. 12.13. Schematic representation of the structure of an aromatic polyketide synthase, in which the active site resides within a single polypeptide (A), and a polyketide synthase that is a complex of several polypeptides with different active sites (B). Both types of enzymes contain multiple domains (A–E), each possessing its own enzymatic activity.

Fig. 12.14. Genetically engineered erythromycin derivatives. A. A mutation in the enoyl reductase gene results in a product with a double bond between the C-6 and C-7 atoms (colored circle). B. Deletion of the β-ketoreductase gene is accompanied by the formation of a derivative with a C-5 carbonyl group instead of a hydroxyl group (colored circle). (After Katz, Donadio, Annu. Rev. Microbiol. 47: 875—912, 1993.)

Improving antibiotic production

Genetic engineering can be used not only to create new antibiotics but also to increase the efficiency of synthesizing existing ones. A limiting factor in the industrial production of antibiotics using Streptomyces spp. is often The amount of oxygen available to the cells. Due to the poor solubility of oxygen in Water and the high density of the Streptomyces culture, oxygen is often insufficient, which slows cell growth and reduces antibiotic yield. To solve this problem, one can, first, modify the design of the bioreactors in which the Streptomyces culture is grown, and second, use Genetic engineering METHODS to develop Streptomyces strains that utilize the available oxygen more efficiently. These two approaches are not mutually exclusive.

Fig. 12.15. Biosynthetic gene clusters of the aromatic polyketide antibiotics actinorhodin (act), tetracenomycin (tcm), frenolicin (fren), and griseusin (gris). Each cluster contains genes encoding a minimal polyketide synthase, which is responsible for the Synthesis of the polyketide backbone. Enzymes encoded by other genes catalyze its modification reactions. The tapered end of each gene indicates the direction of its METABOLISM/31.html">Transcription.

Fig. 12.16. Engineered biosynthetic pathways for the polyketide antibiotics SEK43 and SEK26. Designations: act — actinorhodin, tcm — tetracenomycin, fren — frenolicin, gris — griseusin, min PKS — minimal polyketide synthase, KR — β-ketoreductase, ARO — aromatase, CYC — cyclase.

One strategy used by some aerobic microorganisms to survive under oxygen-limiting conditions is the synthesis of a Hemoglobin-like product capable of binding oxygen and delivering it to cells. For example, the aerobic bacterium Vitreoscilla sp. synthesizes a homodimeric heme-containing protein functionally similar to eukaryotic hemoglobin. The Vitreoscilla "hemoglobin" gene was isolated, cloned into a Streptomyces plasmid vector, and introduced into the cells of this microorganism. Upon expression, Vitreoscilla hemoglobin accounted for approximately 0.1% of the total cellular protein of S. coelicolor, even when expression was driven by the native promoter of the Vitreoscilla hemoglobin gene rather than a Streptomyces promoter. Transformed S. coelicolor cells grown at low dissolved oxygen levels (approximately 5% of air saturation) synthesized 10 times more actinorhodin per gram of dry cell weight and exhibited a higher growth rate than untransformed cells. This approach can also be used to supply oxygen to other microorganisms growing under oxygen-deficient conditions.

The Starting Material for the Chemical synthesis of certain cephalosporins—antibiotics with minor side effects and activity against a wide range of bacteria—is 7-aminocephalosporanic acid (7-ACA), which in turn is synthesized from the antibiotic cephalosporin C (Fig. 12.11). Unfortunately, no natural microorganisms capable of synthesizing 7-ACA have been identified to date. A novel biosynthetic pathway for 7-ACA was engineered by introducing specific genes into a plasmid of the fungus Acremonium chrysogenum, which normally synthesizes only cephalosporin C. One of these genes was a cDNA from the fungus Fusarium solani encoding D-Amino Acid Oxidase, while the other was derived from the genomic DNA of Pseudomonas diminuta and encoded cephalosporin acylase. In the plasmid, these genes were placed under the control of the promoter of

A. chrysogenum. In the first step of the new biosynthetic pathway, cephalosporin C is converted into 7-β-(5-carboxy-5-oxopentanamido)cephalosporanic acid (keto-AD-7-ACA) by D-amino acid oxidase (Fig. 12.17). Part of this product reacts with hydrogen peroxide, one of the by-products, to form 7-β-(4-carboxybutanamido)cephalosporanic acid (GL-7-ACA). Cephalosporin C, keto-AD-7-ACA, and GL-7-ACA can all be hydrolyzed by cephalosporin acylase to yield 7-ACA; however, only 5% of cephalosporin C is directly hydrolyzed to 7-ACA. Therefore, both enzymes are required to produce 7-ACA in high yield.

Fig. 12.17. Genetically engineered biosynthetic pathway of 7-aminocephalosporanic acid (7-ACA) from cephalosporin C. The D-amino acid oxidase gene was isolated from the fungus F. solani, and the cephalosporin acylase gene from the bacterium P. diminuta.