6-deoxyerythronolide B production through chromosomal localization of the deoxyerythronolide B synthase genes in E. coli

Chromosomal engineering was used to localize the deoxyerythronolide B synthase (DEBS) genes and propionyl-CoA carboxylase (PCC) genes to the BAP1 Escherichia coli chromosome creating the new strain YW9. YW9 then featured a plasmid-free heterologous pathway for the production of the polyketide product 6-deoxyerythronolide B (6dEB, a precursor to the antibiotic erythromycin) highlighted by the successful chromosomal integration of five genes total and three DEBS genes each approximately 10 kb in length. The new strain was tested for small-scale 6dEB biosynthesis and compared to 6dEB production from plasmid-derived gene expression at 22, 30, and 37 degrees C. YW9 produced 6dEB at each temperature tested; whereas, the current plasmid-based system could only produce 6dEB at 22 and 30 degrees C. As determined by MS analysis, average production levels for YW9 were 0.47 (22 degrees C), 0.52 (30 degrees C), and 0.11 (37 degrees C)mg/L.     

Engineered biosynthesis of a novel amidated polyketide, using the malonamyl-specific initiation module from the oxytetracycline polyketide synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster

A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.     

Biochemical analysis of the substrate specificity of the beta-ketoacyl-acyl carrier protein synthase domain of module 2 of the erythromycin polyketide synthase

The beta-ketoacyl-acyl carrier protein synthase (KS) domain of the modular 6-deoxyerythronolide B synthase (DEBS) catalyzes the fundamental chain building reaction of polyketide biosynthesis. The KS-catalyzed reaction involves two discrete steps consisting of formation of an acyl-enzyme intermediate generated from the incoming acylthioester substrate and an active site cysteine residue, and the conversion of this intermediate to the beta-ketoacyl-acyl carrier protein product by a decarboxylative condensation with a paired methylmalonyl-SACP. We have determined the rate constants for the individual biochemical steps by a combination of protein acylation and transthioesterification experiments. The first-order rate constant (k(2)) for formation of the acyl-enzyme intermediate from [1-(14)C]-(2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (2) and recombinant DEBS module 2 is 5.8 +/- 2.6 min(-)(1), with a dissociation constant (K(S)) of 3.5 +/- 2.8 mM. The acyl-enzyme adduct was formed at a near-stoichiometric ratio of approximately 0.8:1. Transthioesterification between unlabeled diketide-SNAC 2 and N-[1-(14)C-acetyl]cysteamine gave a k(exch) of 0.15 +/- 0.06 min(-)(1), with a K(m) for HSNAC of 5.7 +/- 4.9 mM and a K(m) for 2 of 5.3 +/- 0.9 mM. Under the conditions that were used, k(exch) was equal to k(-)(2), the first-order rate constant for reversal of the acyl-enzyme-forming reaction. Since the rate of the decarboxylative condensation is much greater that the rate of reversion to the starting material (k(3) > k(-)(2)), formation of the acyl-enzyme adduct is effectively irreversible, thereby establishing that the observed value of the specificity constant (k(cat)/K(m)) is solely a reflection of the intrinsic substrate specificity of the KS-catalyzed acyl-enzyme-forming reaction. These findings were also extended to a panel of diketide- and triketide-SNAC analogues, revealing that some substrate analogues that are not converted to product by DEBS module 2 form dead-end acyl-enzyme intermediates.     

Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry

Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.     

6-MSAS-like polyketide synthase genes occur in lichenized ascomycetes

Lichenized and non-lichenized filamentous ascomycetes produce a great variety of polyketide secondary metabolites. Some polyketide synthase (PKS) genes from non-lichenized fungi have been characterized, but the function of PKS genes from lichenized species remains unknown. Phylogenetic analysis of keto synthase (KS) domains allows prediction of the presence or absence of particular domains in the PKS gene. In the current study we screened genomic DNA from lichenized fungi for the presence of non-reducing and 6-methylsalicylic acid synthase (6-MSAS)-type PKS genes. We developed new degenerate primers in the acyl transferase (AT) region to amplify a PKS fragment spanning most of the KS region, the entire linker between KS and AT, and half of the AT region. Phylogenetic analysis shows that lichenized taxa possess PKS genes of the 6-MSAS-type. The extended alignment confirms overall phylogenetic relationships between fungal non-reducing, 6-MSAS-type and bacterial type I PKS genes.     

Chemobiosynthesis of novel 6-deoxyerythronolide B analogues by mutation of the loading module of 6-deoxyerythronolide B synthase 1

Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler "monoketide" acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1(o) for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.     

Intermodular communication in polyketide synthases: comparing the role of protein-protein interactions to those in other multidomain proteins

Although the role of protein-protein interactions in transducing signals within biological systems has been extensively explored, their relevance to the channeling of intermediates in metabolism is not widely appreciated. Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) are two related families of modular megasynthases that channel covalently bound intermediates from one active site to the next. Recent biochemical studies have highlighted the importance of protein-protein interactions in these chain transfer processes. The information available on this subject is reviewed, and its possible mechanistic implications are placed in context by comparisons with selected well-studied multicomponent protein systems.     

基于AlphaFold 2和分子对接探讨非还原型聚酮合酶的碳甲基化程序

【目的】探讨非还原型聚酮合酶(non-reducing polyketide synthase, NR-Pks)的碳甲基化程序差异的原因。【方法】以红色红曲菌(Monascus ruber) M7中红曲色素和桔霉素的NR-Pks为研究对象，采用生物信息学方法和AlphaFold 2软件，分析了这两种NR-Pks及其各结构域的序列和结构差异。再基于分子对接等技术，比较了它们的碳甲基转移酶结构域(C-methyltransferase domain,CMeT)分别与其他结构域及其中间产物的结合特征。【结果】两种NR-Pks各结构域的序列和结构相似性高，但其整体结构差异大，表明碳甲基化差异可能源于结构域互作差异。进一步分析发现，桔霉素Pks的CMeT比红曲色素Pks的更容易结合携带底物的酰基载体蛋白结构域(acylcarrier protein,ACP)，使其中间产物更容易受到CMeT催化。CMeT和β-酮酰基合成酶结构域(β-ketosynthase domain, KS)相比，与甲基受体底物的结合自由能更低。【结论】NR-Pks中的CMeT能通过与KS竞争，从而影响其产物的碳甲基化程度。...     

Inter-domain movements in polyketide synthases: a molecular dynamics study

Insights into the structure and dynamics of modular polyketide synthases (PKS) are essential for understanding the mechanistic details of the biosynthesis of a large number of pharmaceutically important secondary metabolites. The crystal structures of the KS-AT di-domain from erythromycin synthase have revealed the relative orientation of various catalytic domains in a minimal PKS module. However, the relatively large distance between catalytic centers of KS and AT domains in the static structure has posed certain intriguing questions regarding mechanistic details of substrate transfer during polyketide biosynthesis. In order to investigate the role of inter-domain movements in substrate channeling, we have carried out a series of explicit solvent MD simulations for time periods ranging from 10 to 15 ns on the KS-AT di-domain and its sub-fragments. Analyses of these MD trajectories have revealed that both the catalytic domains and the structured inter-domain linker region remain close to their starting structures. Inter-domain movements at KS-linker and linker-AT interfaces occur around hinge regions which connect the structured linker region to the catalytic domains. The KS-linker interface was found to be more flexible compared to the linker-AT interface. However, inter-domain movements observed during the timescale of our simulations do not significantly reduce the distance between catalytic centers of KS and AT domains for facilitating substrate channeling. Based on these studies and prediction of intrinsic disorder we propose that the intrinsically unstructured linker stretch preceding the ACP domain might be facilitating movement of ACP domains to various catalytic centers.     

Benzophenone synthase and chalcone synthase from Hypericum androsaemum cell cultures: cDNA cloning,functional expression,and site-directed mutagenesis of two polyketide synthases

Benzophenone derivatives, such as polyprenylated benzoylphloroglucinols and xanthones, are biologically active secondary metabolites. The formation of their C13 skeleton is catalyzed by benzophenone synthase (BPS; EC 2.3.1.151) that has been cloned from cell cultures of Hypericum androsaemum. BPS is a novel member of the superfamily of plant polyketide synthases (PKSs), also termed type III PKSs, with 53-63% amino acid sequence identity. Heterologously expressed BPS was a homodimer with a subunit molecular mass of 42.8 kDa. Its preferred starter substrate was benzoyl-CoA that was stepwise condensed with three malonyl-CoAs to give 2,4,6-trihydroxybenzophenone. BPS did not accept activated cinnamic acids as starter molecules. In contrast, recombinant chalcone synthase (CHS; EC 2.3.1.74) from the same cell cultures preferentially used 4-coumaroyl-CoA and also converted CoA esters of benzoic acids. The enzyme shared 60.1% amino acid sequence identity with BPS. In a phylogenetic tree, the two PKSs occurred in different clusters. One cluster was formed by CHSs including the one from H. androsaemum. BPS grouped together with the PKSs that functionally differ from CHS. Site-directed mutagenesis of amino acids shaping the initiation/elongation cavity of CHS yielded a triple mutant (L263M/F265Y/S338G) that preferred benzoyl-CoA over 4-coumaroyl-CoA.     

植物类型III聚酮合酶超家族晶体结构与功能

植物类型Ⅲ聚酮化合物合酶(PKS)催化合成多种植物次生代谢产物的基本分子骨架,参与植物体许多重要生物学功能的行使,一直是研究蛋白结构与功能关系、基于结构进行分子改造的重要模式分子家族。目前在蛋白质数据库(PDB)中有超过80个不同种属来源的类型Ⅲ PKS的三维结构被报道,其中包括了研究最为透彻的查尔酮合酶在内的7种酶的晶体结构,这些结构的发表对于阐明该类酶复杂多变的底物专一性、链延伸和不同的环化反应机制奠定了结构基础。三维空间结构解析以及基于定点突变的结构功能分析是进行酶工程、基因工程的基础。以下系统综述了植物类型Ⅲ PKS超家族晶体结构和功能的研究进展。     

Reconstitution of the FK228 biosynthetic pathway reveals cross talk between modular polyketide synthases and fatty acid synthase

Functional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide in Chromobacterium violaceum strain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway in Escherichia coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.     

Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein.

We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.     

Identification of csypyrone B2 and B3 as the minor products of Aspergillus oryzae type III polyketide synthase CsyB

Since our first report on the identification of the fungal type III polyketide synthase (PKS) genes csyA～D in Aspergillus oryzae RIB40, type III PKS homologues have also been found in other fungal species. We previously reported the isolation and structural determination of csypyrone B1 as the main product of CsyB when inductively expressed in Aspergillus oryzae. Herein we report the isolation and identification of the two minor products of the csyB transformant in addition to csypyrone B1 as 4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid and 5-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)pentanoic acid. These compounds were named csypyrone B2 and B3, respectively, and both are homologues of main product csypyrone B1 with different side chain lengths. This result suggests that the carbon skeleton of the csypyrone B precursor is constructed by the condensation of fatty acyl-CoA and acetylmalonyl-CoA followed by pyrone formation. The alkyl side chain of the precursor may be oxidatively cleaved by enzyme(s) in the host fungus to give variations of csypyrone B with propanoic acid, butyric acid, or pentanoic acid side chains.     

Precursor-directed biosynthesis of 6-deoxyerythronolide B analogues is improved by removal of the initial catalytic sites of the polyketide synthase

Precursor-directed biosynthesis has been shown to be a powerful tool for the production of polyketide analogues that would be difficult or cost prohibitive to produce from medicinal chemistry efforts alone. It has been most extensively demonstrated using a KS1 null mutation (KS1⁰) to block the first round of condensation in the biosynthesis of the erythromycin polyketide synthase (DEBS) for the production of analogues of its aglycone, 6-deoxyerythronolide B (6-dEB). Here we show that removing the DEBS loading domain and first module (mod1Δ), rather than using the KS1⁰ system, can lead to an increase in the utilization of some chemical precursors and production of 6-dEB analogues (R-6dEB) in both Streptomyces coelicolor and Saccharopolyspora erythraea. While the difference in utilization of the precursor was diketide specific, in strains fed (2R*, 3S*)-5-fluoro-3-hydroxy-2-methylpentanoate N-propionylcysteamine thioester, twofold increases in both utilization of the diketide and 15-fluoro-6dEB (15F-6dEB) production were observed in S. coelicolor, and S. erythraea exhibited a tenfold increase in production of 15-fluoro-erythromycin when utilizing the mod1Δ rather than the KS1⁰ system.     

Erythromycin biosynthesis. The 4-pro-S hydride of NADPH is utilized for ketoreduction by both module 5 and module 6 of the 6-deoxyerythronolide B synthase.

Incubation of chirally deuterated NADPH with 6-deoxyerythronolide B synthase (DEBS) modules 5 and module 6 and analysis of the derived triketide lactones established that the two ketoreductase domains, KR5 and KR6, are both specific for the 4-pro-S hydride of the nicotinamide cofactor.     

Expression and characterization of polyketide synthase module involved in the late step of cephabacin biosynthesis from Lysobacter lactamgenus

The cephabacins produced by Lysobacter lactamgenus are beta-lactam antibiotics composed of a cephem nucleus, an acetate residue, and an oligopeptide side chain. In order to understand the precise implication of the polyketide synthase (PKS) module in the biosynthesis of cephabacin, the genes for its core domains, beta-ketoacyl synthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), were amplified and cloned into the pET-32b(+) expression vector. The sfp gene encoding a protein that can modify apo-ACP to its active holo-form was also amplified. The recombinant KS, AT, apo-ACP, and Sfp overproduced in the form of His6-tagged fusion proteins in E. coli BL21(DE3) were purified by nickel-affinity chromatography. Formation of stable peptidyl-S-KS was observed by in vitro acylation of the KS domain with the substrate [L-Ala-L-Ala-LAla- L-3H-Arg] tetrapeptide-S-N-acetylcysteamine, which is the evidence for the selective recognition of tetrapeptide produced by nonribosomal peptide synthetase (NRPS) in the NRPS/ PKS hybrid. In order to confirm whether malonyl CoA is the extender unit for acetylation of the peptidyl moiety, the AT domain, ACP domain, and Sfp protein were treated with 14C-malonyl-CoA. The results clearly show that the AT domain is able to recognize the extender unit and decarboxylatively acetylated for the elongation of the tetrapeptide. However, the transfer of the activated acetyl group to the ACP domain was not observed, probably attributed to the improper capability of Sfp to activate apo-ACP to the holo-ACP form.