Genetic Manipulation Of The Acetate Pathway

With only a few exceptions, the transformations in any particular biosynthetic pathway are catalysed by enzymes. These proteins facilitate the chemical modification of substrates by virtue of binding properties conferred by a particular combination of functional groups in the constituent amino acids. As a result, enzymes tend to demonstrate quite remarkable specificity towards their substrates, and usually catalyse only a single transformation. This specificity means enzymes do not accept alternative substrates, or, if they do, they convert a limited range of structurally similar substrates and usually much less efficiently. Any particular organism thus synthesizes a range of secondary metabolites dictated largely by its enzyme complement and the supply of substrate molecules. Occasionally, where enzymes do possess broader substrate specificities, it is possible to manipulate an organism's secondary metabolite pattern by supplying an alternative, but acceptable, substrate. A good example of this approach is in the directed biosynthesis of modified penicillins by the use of phenylacetic acid analogues in cultures of Penicillium chryso-genum (see page 437), but its scope is generally very limited. It has also been possible, particularly with microorganisms, to select natural mutants, or to generate mutants artificially, where the new strain synthesizes modified or substantially different products. For example, mutant strains of Strep-tomyces aureofaciens synthesize tetracycline or demeclocycline rather than chlortetracycline (see page 90). Such mutants are usually deficient in a single enzyme and are thus unable to carry out a single transformation, but the broader specificity of later enzymes in the sequence means subsequent modifications may still occur. However, as exemplified throughout this book, the vast bulk of modified natural products of medicinal importance are currently obtained by chemical synthesis or semi-synthesis.

Rapid advances in genetic engineering have now opened up tremendous scope for manipulating the processes of biosynthesis by providing an organism with, or depriving it of, specific enzymes. The genes encoding a particular protein (see page 407) can now be identified, synthesized, and inserted into a suitable organism for expression; to avoid complications with the normal biosynthetic machinery, this is usually different from the source organism. Specific genes can be damaged or deleted to prevent a particular enzyme being expressed. Genes from different organisms can be combined and expressed together so that an organism synthesizes abnormal combinations of enzyme activities, allowing production of modified products. Although the general approaches for genetic manipulation are essentially the same for all types of organism and/or natural product, it has proved possible to make best progress using the simpler organisms, especially bacteria, and in particular there have been some substantial achievements in the area of acetate-derived structures. Accordingly, some results from this group of compounds are used to exemplify how genetic manipulation may provide an extra dimension in the search for new medicinal agents. However, it is important that an organism is not viewed merely as a sackful of freely diffusible and always available enzymes; biosynthetic pathways are under sophisticated controls in which there may be restricted availability or localization of enzymes and/or substrates (see the different localizations of the mevalonate and deoxyxylulose phosphate pathways to terpenoids in plants, page 172). Enzymes involved in the biosynthesis of many important secondary metabolites are often grouped together as enzyme complexes, or may form part of a multifunctional protein.

A detailed study of amino acid sequences and mechanistic similarities in various polyketide synthase (PKS) enzymes has led to two main types being distinguished. Type I enzymes consist of one or more large multifunctional proteins that possess a distinct active site for every enzyme-catalysed step. On the other hand, Type II enzymes are multienzyme complexes that carry out a single set of repeating activities. Like fatty acid synthases, PKSs catalyse the condensation of coenzyme A esters of simple carboxylic acids. However, the variability at each step in the biosynthetic pathway gives rise to much more structural diversity than encountered with fatty acids. The usual starter units employed are acetyl-CoA or propionyl-CoA, whilst malonyl-CoA or methylmalonyl-CoA are the main extender units. At each cycle of chain extension, Type I PKSs may retain the P-ketone, or modify it to a hydroxyl, methenyl, or methylene, according to the presence of ketoreductase, dehydratase, or enoylreductase activities (see page 95). The enzyme activities for each extension cycle with its subsequent modification is considered a 'module'. The linear sequence of modules in the enzyme corresponds to the generated sequence of extender units in the polyketide product. The P-ketone groups are predominantly left intact by Type II PKSs, and the highly reactive polyketide backbone undergoes further enzyme-catalysed intramolecular cyclization reactions, which are responsible for generating a range of aromatic structures (see page 61).

6-Deoxyerythronolide B synthase (DEBS) is a modular Type I PKS involved in erythromycin biosynthesis (see page 96) and its structure and function are illustrated in Figure 3.85. The enzyme contains three subunits (DEBS-1, 2, and 3), each encoded by a gene (eryA-I, II, and III). It has a linear organization of six modules, each of which contains the activities needed for one cycle of chain extension. A minimal module contains a P-ketoacyl synthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP), that together would catalyse a two-carbon chain extension. The specificity of the AT for either malonyl-CoA or an alkyl-malonyl-CoA determines which two-carbon chain extender is used. The starter unit used is similarly determined by the specificity of the AT in a loading domain in the first module. After each condensation reaction, the oxidation state of the P-carbon is determined by the presence of a P-ketoacyl reductase (KR), a KR + a dehy-dratase (DH), or a KR + DH + an enoylreductase (ER) in the appropriate module. The sequence is finally terminated by a thioesterase (TE) activity which releases the polyketide from the enzyme and allows cyclization. Thus in DEBS, module 3 lacks any P-carbon modifying domains, modules 1, 2, 5, and 6 contain KR domains and are responsible for hydroxy substituents, whereas module 4 contains the complete KR, DH, and ER set, and results in complete reduction to a methylene. Overall, the AT specificity and the catalytic domains on each module determine the structure and stereochemistry of each two-carbon extension unit, the order of the modules specifies the sequence of the units, eryAI

eryAII

eryAIII

genes proteins

AT ACP KS AT KR ACP KS AT KR ACP^f KS AT ACP KS AT DH ER KR ACP

KS AT KR ACP KS AT KR ACP TE

modules enzyme activities

O HO

SCoA

SCoA

O HO"

O HO"

SCoA

O HOHO-

O HOHO

SCoA

co2h modules enzyme activities

o y oh deoxyerythronolide o y oh deoxyerythronolide

ACP: acyl carrier protein AT: acyltransferase DH: dehydratase ER: enoyl reductase KR: P-ketoacyl reductase KS: P-ketoacyl synthase TE: thioesterase

DEBS 1

DEBS 2

DEBS 3

Module 1

Module 3

Module 5

release load

Module 2

Module 4

Module 6

SCoA

load

Module 1

Module 2

at acp ks at kr acp ks at kr acp TE DEBS 1 plus TE domain Module 1

"CC

each module programmes the nature of the extender unit added (via AT), and the oxidation state of the p-carbon in the preceding unit (via KR, DH, ER)

module 2 9

loading load

Module 2

, release at acp ks at kr acp ks at kr acp ks at acp TE

truncated DEBS plus TE domain ohVY

AT ACP ks at kr acp ks at kr acp ks at acp ks at dh er kr acp "^>|~|<s at kr acp ks at kr acp te~y loading domain from avermectin PKS specifies isobutyryl-CoA

load

Module 2

Module 4

Module 6

at acp ks at kr acp ks at kr acp ks at acp ks at dh er kr acp

ks at kr acp ks AT kr acp te

acyltransferase domain from rapamycin PKS specifies malonyl-CoA

module 6

at acp ks at kr acp ks at kr acp ks at acp ks at dh er kr acp ^>f~ks at m acp ks at kr acp tlt^>

toh deletion of ketoreductase domain prevents p-carbon processing h O*

and the number of modules determines the size of the polyketide chain. The vast structural diversity of natural polyketides arises from combinatorial possibilities of arranging modules containing the various catalytic domains, the sequence and number of modules, and the post-PKS enzymes which subsequently modify the first-formed product, e.g. 6-deoxyerythronolide B ^ erythromycin (see page 96). Genetic engineering now offers vast opportunities for rational modification of the resultant polyketide structure.

A few representative examples of successful experiments leading to engineered polyketides are shown in Figure 3.86. Reducing the size of the gene sequence so that it encodes fewer modules results in the formation of smaller polyketides, characterized by the corresponding loss of extender units; in these examples the gene encoding the chain terminating thioesterase also has to be attached to complete the biosynthetic sequence. Replacing the loading domain of DEBS with that from another PKS, e.g. that producing avermectin (see page 97), alters the specificity of the enzyme for the starter unit. The loading module of the avermectin-producing PKS actually has a much broader specificity than that for DEBS; Figure 3.86 shows the utilization of isobutyryl-CoA as features in the natural biosynthesis of avermectin B1fc. Other examples include the replacement of an AT domain (in DEBS specifying a methylmalonyl extender) with a malonyl-specific AT domain from the rapamycin-producing PKS (see page 103), and deletion of a KR domain, thus stopping any P-carbon processing for that module with consequent retention of a carbonyl group. Not all experiments in gene modification are successful, and even when they are yields can be disappointingly lower than in the natural system. There is always a fundamental requirement that enzymes catalysing steps after the point of modification need to have sufficiently broad substrate specificities to accept and process the abnormal compounds being synthesized; this becomes more unlikely where two or more genetic changes have been made. Nevertheless, multiple modifications have been successful, and it has also been possible to exploit changes in a combinatorial fashion using different expression vectors for the individual subunits, thus creating a library of polyketides, which may then be screened for potential biological activity.

Non-ribosomal peptide synthases (see page 421) are also modular and lend themselves to similar genetic manipulation as the Type I PKSs. The production of modified aromatic polyketides by genetically engineered Type II PKSs is not quite so 'obvious' as with the modular Type I enzymes, but significant progress has been made in many systems. Each Type II PKS contains a minimal set of three protein subunits, two P-ketoacyl synthase (KS) subunits and an ACP to which the growing chain is attached. Additional subunits, including KRs, cyclases (CYC), and aromatases (ARO), are responsible for modification of the nascent chain to form the final cyclized structure. Novel polyke-tides have been generated by manipulating Type

II PKSs, exchanging KS, CYC, and ARO sub-units among different systems. However, because of the highly reactive nature of poly-^-keto chains, the cyclizations that occur with the modified gene product frequently vary from those in the original compound. Compared with Type I PKSs, the formation of new products with predictable molecular structure has proven less controllable.

The polyketide synthases responsible for chain extension of cinnamoyl-CoA starter units leading to flavonoids and stilbenes, and of anthraniloyl-CoA leading to quinoline and acridine alkaloids (see page 377) do not fall into either of the above categories and have now been termed Type

III PKSs. These enzymes differ from the other examples in that they are homodimeric proteins, they utilize coenzyme A esters rather than acyl carrier proteins, and they employ a single active site to perform a series of decarboxylation, condensation, cyclization, and aromatization reactions.

Was this article helpful?

0 0
Reducing Blood Pressure Naturally

Reducing Blood Pressure Naturally

Do You Suffer From High Blood Pressure? Do You Feel Like This Silent Killer Might Be Stalking You? Have you been diagnosed or pre-hypertension and hypertension? Then JOIN THE CROWD Nearly 1 in 3 adults in the United States suffer from High Blood Pressure and only 1 in 3 adults are actually aware that they have it.

Get My Free Ebook


Post a comment