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(Received for publication, March 11, 1997, and in revised form, April 22, 1997)
and§¶
From the Departments of 
Chemical Engineering,
§ Chemistry, and ¶ Biochemistry, Stanford University,
Stanford, California 94305-5025
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT 
Bacterial aromatic polyketide synthases (PKSs) are a family of homologous multienzyme assemblies that catalyze the biosynthesis of numerous polyfunctional aromatic natural products. In the absence of direct insights into their structures, the use of gene fusions can be a powerful tool for understanding the structural basis for their properties. A series of truncated and hybrid proteins were constructed and analyzed within a family of PKS subunits, designated aromatases/cyclases (ARO/CYCs). When expressed alone, neither the N-terminal nor the C-terminal domain of the actinorhodin (act) or the griseusin (gris) ARO/CYC exhibited substantial aromatase activity. However, in the presence of each other, the half proteins were active. Furthermore, analysis of a set of hybrid proteins derived from the act and gris ARO/CYCs allowed us to localize the chain length dependence of this aromatase activity to their N-terminal domains. Unexpectedly, however, when the C-terminal domain of the gris ARO/CYC was expressed in a context where aromatase activity was absent, it could modulate the chain length specificity of the tetracenomycin (tcm) minimal PKS, leading to the formation of a novel 18-carbon product in addition to the expected 20-carbon one. It was also found that monodomain ARO/CYCs such as tcmN cannot substitute for the the N-terminal domain of didomain ARO/CYCs, even though they exhibit high sequence similarity with the N-terminal domain. Together, these results illustrate the utility of protein engineering approaches for dissecting the structure-function relationships of PKS subunits and for the generation of mutant alleles with novel biosynthetic properties.
INTRODUCTION
Polyketides, a large family of structurally diverse natural
products, possess a broad range of biological activities, including antibiotic and pharmacological properties. These natural products are
produced by a number of different bacteria and fungi that express
enzyme complexes known as polyketide synthases
(PKSs).1 Biosynthetic and molecular genetic
studies have demonstrated that PKSs are structurally and
mechanistically related to fatty acid synthases. Both classes of
synthases are multifunctional enzymes that catalyze repeated
decarboxylative condensations between acylthioesters (usually
acetyl, propionyl, malonyl, or methylmalonyl). Unlike typical fatty
acid synthases, PKSs introduce structural variability into the product
by varying the extent of a reductive cycle comprising of a
ketoreduction, dehydration, and enoylreduction on each 
-keto group
of the polyketide chain. Furthermore, PKSs also control chain folding
by catalyzing one or more regiospecific cyclizations in the nascent
polyketide chain (1-7).
Bacterial aromatic PKSs (2, 8) produce a broad range of polycyclic aromatic natural products such as the carbon chain precursors of doxorubicin and tetracycline. Inspired by the structural diversity and pharmaceutical relevance of these products, the molecular recognition features of PKSs have been targets of intensive analysis and manipulation via genetic engineering. The recent development of a host vector system in Streptomyces coelicolor enabled the efficient construction and expression of recombinant PKSs (9) and provided a means to decipher the function(s) and specificity of each subunit (9-18). In the process a series of heuristics have been proposed and used to engineer recombinant PKS gene clusters to generate novel polyketides in a predictive manner (17, 19). Unfortunately, even with this design capability, our understanding of the mechanisms by which aromatic PKSs control their molecular recognition features is extremely limited and remains hampered by the absence of structural information on these protein complexes. The situation could potentially be ameliorated by taking advantage of the existence of numerous allelic forms of every PKS subunit, each of which shares a high degree of sequence similarity with the others (1). Here we used sequence comparisons to design and study a series of truncated and fusion proteins.
Each aromatic PKS (for example, see Fig. 1) contains a
set of three essential subunits referred to as the minimal PKS (15). In
addition, most aromatic PKSs also include auxiliary subunits such as a
ketoreductase (KR), an aromatase/cyclase (ARO/CYC), and other cyclases.
ARO/CYCs are a particularly interesting family of subunits, since they
exhibit the following features: (a) They occur in two
architectural forms. Members of one subset, hereafter referred to as
didomain ARO/CYCs, consist of ~300 amino acid residues; whereas
members of the other subset, the monodomain ARO/CYCs, consist of ~150
residues. The N-terminal halves of didomain ARO/CYCs exhibit a high
degree of sequence similarity to the monodomain proteins (20, 21) and
to a lesser extent to their C-terminal halves (22) (Fig.
3). (b) Didomain ARO/CYCs catalyze the
aromatization of the first six-membered carbocyclic ring derived from
polyketide backbones that have undergone a C-9 reduction (Fig.
2) (16). (c) Monodomain ARO/CYCs alter the
folding pattern of the nascent polyketide backbone by catalyzing a
C-9/C-14 aldol condensation instead of the more typical C-7/C-12
condensation (18, 23). Additionally, they have also been implicated to
possess aromatase activity (18). (d) Didomain ARO/CYCs
exhibit a hierarchy with respect to their substrate specificity for
polyketides of different chain lengths (17). For example, an ARO/CYC
from a decaketide synthase (e.g. the griseusin
(gris) PKS) catalyzes aromatization of octaketide,
nonaketide, or decaketide substrates, whereas an octaketide ARO/CYC
(e.g. the actinorhodin (act) ARO/CYC) only recognizes octaketide substrates.
In an attempt to gain insights into the structural basis for these multifarious properties of the ARO/CYC family of proteins, truncated and fusion proteins were constructed and analyzed.
EXPERIMENTAL PROCEDURES
DNA manipulations were performed using standard in vitro techniques with Escherichia coli XL1-Blue as a host organism. Expression plasmids were passaged through E. coli ET12567 (dam, dcm hsdS Cmr) (24) to generate unmethylated DNA for introduction into S. coelicolor CH999 by transformation (9). E. coli strains were grown under standard conditions (25), and S. coelicolor strains were grown on R2YE agar plates (26).
Construction of Genes Encoding Hybrid ARO/CYCsPlasmids
pRZ50, pRZ51, pRZ52, pRZ54, pRZ56, pRZ60, pRZ64, and pRZ67 (see Tables
I, II, and III) are derivatives of pSEK21 (15). Plasmids pRZ53, pRZ55,
pRZ57, pRZ61, pRZ65, pRZ71, pRZ73, pRZ74, and pRZ77-80 (see Tables I,
II, and III) are derivatives of pSEK23 (15). The 5
and 3 halves of
the act and gris ARO/CYC genes were amplified
separately via polymerase chain reaction. Based on sequence
similarities (Fig. 2), the N-terminal domain for the act
ARO/CYC was defined to consist of residues 1-145; whereas the
C-terminal domain was defined to consist of residues 146-317.
Likewise, the N-terminal domain of the gris ARO/CYC
consisted of residues 1-148, and the C-terminal domain consisted of
residues 149-320. The monodomain portion of tcmN was
defined as residues 1-169, as in an earlier study (21). All segments
were amplified with the following designed sequences (5 to 3): a
PstI restriction site, a ribosome binding site (AGGAGGA), an
NdeI restriction site overlapping the start codon, the
natural coding sequence, a stop codon, and an EcoRI
restriction site. 5 segments also included an NdeI
restriction site immediately upstream of the stop codon. Thus, each
fragment represented a gene by itself. In addition, wild-type or hybrid
genes could be constructed by fusing different 5 and 3 fragments
together at the NdeI site. In turn, these genes were
inserted into either pSEK21 (which carries the act minimal PKS and the
act ketoreductase) or pSEK23 (which carries the
tcm minimal PKS and the act ketoreductase)
immediately downstream of the minimal PKS gene set as described
previously (15).
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Strains of S. coelicolor CH999
containing plasmids pRZ50-57, pRZ61, pRZ64, pRZ65, pRZ67, pRZ71,
pRZ73, pRZ74, and pRZ77-80 were grown on R2YE agar plates (26) for 7 days, after which the agar was finely chopped and extracted twice with
ethyl acetate/1% acetic acid. The organic extract was concentrated by
evaporation and flashed through a silica gel (Baker) chromatography
column in ethyl acetate/1% acetic acid. The predominant brown fraction was further purified via high performance liquid chromatography (HPLC)
using a 20-60% acetonitrile/water/1% acetic acid gradient on a
preparative reverse phase (C-18) column (Beckman). Absorbance was
monitored at 280 and 410 nm. The yields of all products were similar to
those reported earlier (9, 12-15). Structures of mutactin (compound
1; Ref. 27), SEK34 (compound 2; Ref. 16), RM20
(compound 3; Ref. 9), RM20b (compound 4; Ref. 12), RM20c
(compound 5; Ref. 12), and SEK43 (compound 6;
Ref. 17) (Fig. 4) were confirmed by NMR spectroscopy.
The structure determination of RZ53 (compound 7, 15 mg/liter
in CH999/pRZ53 cultures) is described here.
RESULTS
To evaluate the function of the N- and C-terminal halves of the didomain ARO/CYCs and to test whether they do indeed fold into separate domains, recombinant gene clusters were constructed in which the putative domains of the act and gris ARO/CYCs were expressed as separate polypeptides in conjunction with either the act (octaketide producer) or tcm (decaketide producer) minimal PKS and the act KR (Table I). Earlier studies have established that the presence of SEK34 (2) or SEK43 (6) are indicative of the existence of first ring aromatase activity in a 16- or 20-carbon polyketide producing strain respectively (16, 17). Neither the N-terminal (CH999/pRZ50, pRZ77, and pRZ78) nor the C-terminal (CH999/pRZ51, pRZ73, and pRZ74) domains of the act or gris ARO/CYC showed substantial aromatase activity when expressed alone. The extract from CH999/pRZ50 (N-terminal domain of act ARO/CYC) exhibited an HPLC peak characteristic for SEK34, but the product yields were inadequate for NMR analysis. An in vitro assay with 14C-labeled malonyl CoA of this strain also supported the production of SEK34.2 (Unexpectedly, the gris C-terminal domain alone led to the formation of a novel product, RZ53 (7) (see below).) However, when the N- and C-terminal domains were co-expressed as separate polypeptides (CH999/pRZ64, pRZ79, and pRZ80), SEK34 (2) was produced by CH999/pRZ64 and pRZ79, whereas SEK43 (6) was produced by CH999/pRZ80, suggesting that the domains can associate productively with themselves and/or the PKS complex. It should be noted that the biosynthesis of comparable amounts of mutactin by CH999/pRZ64 and RM20b by CH999/pRZ80 implies that domain disconnection leads to only partial aromatase activity in these two strains.
Dissecting Chain Length Hierarchy Using Hybrid Didomain ARO/CYCsAs summarized above, didomain ARO/CYCs exhibit an interesting hierarchy with respect to their recognition of polyketide chain length. To dissect the structural basis for this property, a set of recombinant PKS gene clusters containing hybrid genes derived from the act and gris ARO/CYCs was constructed. The results described in Table II show that ARO/CYC proteins containing the N-terminal domain of the gris subunit (CH999/pRZ54, pRZ55, and pRZ61) catalyzed the aromatization of both octaketide and decaketide substrates. In contrast, those containing the N-terminal domain of the act subunit (CH999/pRZ52, pRZ53, and pRZ71) recognized only octaketide subunits. Thus, chain length recognition appears to reside exclusively within the N-terminal domain of didomain ARO/CYCs.
Replacement of the N-terminal Domain of Didomain ARO/CYCs with a Monodomain ARO/CYCAs mentioned above, the N-terminal domains of the didomain ARO/CYCs exhibit significant sequence similarity to the monodomain ARO/CYCs, which suggested that the monodomain ARO/CYC may be able to replace the N-terminal domain. To investigate this hypothesis, hybrid proteins where the monodomain ARO/CYC portion of the tcmN protein was inserted in place of the N-terminal domain of the act and gris ARO/CYCs were constructed. As summarized in Table III, none of these hybrids displayed functional didomain ARO/CYC activity, indicating that the monodomain ARO/CYCs cannot substitute for the N-terminal domain of the didomain ARO/CYCs.
Didomain ARO/CYC Influence on Polyketide Chain LengthDuring the course of the above studies it was observed that, although the C-terminal domain of the gris ARO/CYC could not catalyze first ring aromatization by itself, its co-expression with the tcm minimal PKS and the act ketoreductase (CH999/pRZ74) led to the production of a relatively abundant new metabolite that was not formed in the presence of the gris N-terminal domain alone (CH999/pRZ78) or the act N- or C-terminal domains alone (CH999/pRZ50 and pRZ51, respectively). Indeed, production of this metabolite was also observed in strains containing the act/gris and tcm/gris hybrid proteins (CH999/pRZ53 and pRZ65, respectively). Likewise, because this metabolite was also produced by CH999/pRZ80, which contains partial aromatase activity, but not in CH999/pRZ61, which exhibits full aromatase activity, its production is the result of the presence of the gris C-terminal domain in a context, where it is unable to participate in first ring aromatase activity. (Upon more sensitive analysis, this new metabolite was also observed in strains CH999/pRZ71 and pRZ57 (>1 wt% of total polyketide products); however, its production levels in these strains were more than an order of magnitude lower than the production levels in their counterpart strains, CH999/pRZ53 and pRZ65 (~10 wt% of total polyketide products), respectively.)
The structure of this new metabolite, designated RZ53 (7) (Fig. 4), was solved using a combination of NMR (Table IV), mass spectroscopy, and isotope labeling analysis. The chemical shifts were remarkably similar to those of corresponding atoms in RM20 (3) (9) except for the absence of a methylene and a carbonyl signal. Sodium [1,2-13C]acetate feeding experiments confirmed that the carbon chain of RZ53 was derived from nine acetate units. The coupling constants calculated from the 13C NMR spectrum of the enriched RZ53 sample also facilitated peak assignment. High resolution fast atom bombardment gave a molecular weight of 282.0897, which is consistent with C17H14O4 MW (282.0892). Thus, RZ53, a novel "unnatural" natural product, is the first known nonaketide to be produced by the tcm PKS.
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DISCUSSION
Without any direct insight into their structures, some cognizance into the structural basis of several properties of the didomain ARO/CYC has been acquired via domain truncation and replacement. Our studies demonstrate that the N-terminal domain is responsible for the variability observed in the polyketide chain length recognition by the ARO/CYC. This variability could either arise due to differences in the substrate binding pockets of individual subunits, or it may reflect differential affinities for the minimal PKS subunits themselves. Given the availability of cell-free systems for polyketide biosynthesis (28, 29), further in vitro analysis could shed light on this question.
In the course of these studies, we also obtained structural insights into yet another interesting attribute of these enzymes, their ability to influence the chain length specificity of the minimal PKS within a window of one acetate unit. Specifically, we have established that it is the C-terminal domain of the didomain ARO/CYC that is responsible for this trait. This is not the first report of auxilary subunits influencing the chain length specificity of the minimal PKS. Recent studies of the frenolicin (fren) PKS showed that both the act KR and tcmN can modulate the relative distribution of 16-carbon and 18-carbon polyketide products (19). A similar phenomenon has also been reported in the case of the whiE PKS (30), which produces 22-carbon and 24-carbon backbones. However, until now it has been assumed that these observations are peculiar to PKSs that exhibit relaxed chain length specificity in nature, because at least the frenolicin producer, S. roseofulvus, is known to produce both 16- and 18-carbon natural products with very similar structures (31, 32). In contrast, neither the naturally occurring PKS from S. glaucescens nor any engineered PKS containing the core tcm subunits has yielded an 18-carbon product thus far. Our new findings therefore provide the clearest evidence that certain auxiliary PKS subunits can not only control the folding, reduction, and aromatization of the nascent chain but can also secondarily influence the chain length of the final product by plus or minus one acetate unit.
The hypothesized didomain nature of the act and gris ARO/CYCs (22) was verified by expressing the proposed domains as separate proteins and demonstrating that they can associate productively to manifest the properties attributed to an intact didomain ARO/CYC. However, because neither domain exhibited significant aromatase activity when expressed alone, it appears that both domains must work in concert to aromatize the first ring. Furthermore, fusion proteins between the monodomain ARO/CYC portion of tcmN and the C-terminal domains of the act or the gris ARO/CYC disclosed that although there is good sequence homology between monodomain ARO/CYCs and N-terminal domains, monodomain ARO/CYCs are incapable of executing the role(s) of the N-terminal domain in the aromatization of the reduced first ring.
In summary, the utility of deletion and domain swapping analysis between different alleles of the aromatic PKS subunits has been demonstrated. In conjunction with in vitro studies, higher resolution gene shuffling should yield further insights into the mechanistic basis for ARO/CYC function and specificity and may even give rise to additional ARO/CYC subunits with novel properties.
FOOTNOTES
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X63449[GenBank] (actinorhodin (act) sequence), X77865[GenBank] (griseusin (gris) sequence), and M80674[GenBank] (tetracenomycin (tcm) sequence).
To whom correspondence should be addressed.
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