A concise synthesis of single‐enantiomer β‐lactams and β‐amino acids using Rhodococcus globerulus

BACKGROUND: Pharmaceutical companies continue to evaluate β‐amino acids and β‐lactams in a range of drug candidates. The development of a highly efficient and selective bioresolution of cyclic β‐lactam substrates could yield enantiopure lactams and β‐amino acids with medicinal potential. The aim of this work was to discover and develop a biocatalyst capable of selectively hydrolysing β‐lactam substrates. RESULTS: Screening of our in‐house culture collection led to the discovery of a microorganism, Rhodococcus globerulus (NCIMB 41042) with β‐lactamase activity. Whole‐cell bioresolutions of the β‐lactams 1–4 were successfully carried out and in all cases enantiomeric excesses of the residual lactam and amino acid product were found to be greater than 98%. For one example, the bioresolution was optimised to operate at 60 g L^?1 substrate concentration with a 20% wt/wt cell paste loading. CONCLUSION: A microorganism, Rhodococcus globerulus (NCIMB 41042), capable of selectively hydrolysing a range of cyclic β‐lactams, has been discovered. A scalable whole‐cell bioresolution process has been developed, leading to the synthesis of multigram quantities of enantiomerically pure β‐lactams and β‐amino acids. Copyright © 2007 Society of Chemical Industry     

Genome Mining of the Hitachimycin Biosynthetic Gene Cluster: Involvement of a Phenylalanine‐2,3‐aminomutase in Biosynthesis

Hitachimycin is a macrolactam antibiotic with (S)‐β‐phenylalanine (β‐Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β‐Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine‐2,3‐aminomutase (PAM), five polyketide synthases, four β‐amino‐acid‐carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)‐β‐Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.     

Molecular Basis for the Enantioselective Ring Opening of β‐Lactams Catalyzed by Candida antarctica Lipase B

Lipase B from Candida antarctica (CAL‐B) catalyzes the slow, but highly enantioselective (E>200), ring‐opening alcoholysis of two bicyclic and two 4‐aryl‐substituted β‐lactams. Surprisingly, the rate of the reaction varies with the nature of the alcohols and was fastest with either enantiomer of 2‐octanol. A 0.5‐g scale reaction with 2‐octanol as the nucleophile in diisopropyl ether at 60 °C yielded the unreacted β‐lactam in 39–46% yield (maximum yield is 50%) with ≥96% ee. The product β‐amino acid esters reacted further by polymerization (not isolated or characterized) or by hydrolysis due to small amounts of water in the reaction mixture yielding β‐amino acids (7–11% yield, ≥96% ee). The favored enantiomer of all four β‐lactams had similar 3‐D orientation of substituents, as did most previously reported β‐lactams and β‐lactones in similar ring‐opening reactions. Computer modeling of the ring opening of 4‐phenylazetidin‐2‐one suggests that the reaction proceeds via an unusual substrate‐assisted transition state, where the substrate alcohol bridges between the catalytic histidine and the nitrogen of the β‐lactam. Computer modeling also suggested that the molecular basis for the high enantioselectivity is a severe steric clash between Ile189 in CAL‐B and the phenyl substituent on the slow‐reacting enantiomer of the β‐lactam.     

A Unique Amino Transfer Mechanism for Constructing the β‐Amino Fatty Acid Starter Unit in the Biosynthesis of the Macrolactam Antibiotic Cremimycin

Cremimycin is a 19‐membered macrolactam glycoside antibiotic based on three distinctive substructures: 1) a β‐amino fatty acid starter moiety, 2) a bicyclic macrolactam ring, and 3) a cymarose unit. To elucidate the biosynthetic machineries responsible for these three structures, the cremimycin biosynthetic gene cluster was identified. The cmi gene cluster consists of 33 open reading frames encoding eight polyketide synthases, six deoxysugar biosynthetic enzymes, and a characteristic group of five β‐amino‐acid‐transfer enzymes. Involvement of the gene cluster in cremimycin production was confirmed by a gene knockout experiment. Further, a feeding experiment demonstrated that 3‐aminononanoate is a direct precursor of cremimycin. Two characteristic enzymes of the cremimycin‐type biosynthesis were functionally characterized in vitro. The results showed that a putative thioesterase homologue, CmiS1, catalyzes the Michael addition of glycine to the β‐position of a non‐2‐enoic acid thioester, followed by hydrolysis of the thioester to give N‐carboxymethyl‐3‐aminononanoate. Subsequently, the resultant amino acid was oxidized by a putative FAD‐dependent glycine oxidase homologue, CmiS2, to produce 3‐aminononanoate and glyoxylate. This represents a unique amino transfer mechanism for β‐amino acid biosynthesis.     

In Vitro Reconstitution of a PKS Pathway for the Biosynthesis of Galbonolides in Streptomyces sp. LZ35

The galbonolides are 14‐membered macrolide antibiotics with a macrocyclic backbone similar to that of erythromycins. Galbonolides exhibit broad‐spectrum antifungal activities. Retro‐biosynthetic analysis suggests that the backbone of galbonolides is assembled by a type I modular polyketide synthase (PKS). Unexpectedly, the galbonolide biosynthetic gene cluster, gbn, in Streptomyces sp. LZ35 encodes a hybrid fatty acid synthase (FAS)‐PKS pathway. In vitro reconstitution revealed the functions of GbnA (an AT‐ACP didomain protein), GbnC (a FabH‐like enzyme), and GbnB (a novel multidomain PKS module without AT and ACP domains) responsible for assembling the backbone of galbonolides, respectively. To our knowledge, this study is the first biochemical characterization of a hybrid FAS‐PKS pathway for the biosynthesis of 14‐membered macrolides. The identification of this pathway provides insights into the evolution of PKSs and could facilitate the design of modular pools for synthetic biology.     

The Phormidolide Biosynthetic Gene Cluster: A trans‐AT PKS Pathway Encoding a Toxic Macrocyclic Polyketide

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16‐membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans‐type acyltransferase (trans‐AT) ORFs along with KS‐AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans‐AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Highly Efficient Suzuki Coupling Reaction of α‐Chloroalkylidene‐β‐lactones and β‐Lactams with Organoboronic Acids

The activation of C Cl bond of (Z)‐α‐chloroalkylidene‐β‐lactones and (E)‐α‐chloroalkylidene‐β‐lactams via the Suzuki cross‐coupling reaction is reported in this paper. Alkyl, heteroaromatic, substituted phenyl‐ and alkenylboronic acids can be coupled with a wide variety of α‐chloroalkylidene‐β‐lactones and β‐lactams in excellent yields within a short period of time. The cross‐coupling reaction of optically active substrates leads to the optically active compounds without racemization of the corresponding chiral center.     

Biosynthesis of Phenylnannolone A,a Multidrug Resistance Reversal Agent from the Halotolerant Myxobacterium Nannocystis pusilla B150

The myxobacterial strain Nannocystis pusilla B150 synthesizes the structurally new polyketides phenylnannolone A–C. Apart from some common volatiles and siderophores, these are the first natural products from the genus Nannocystis. Phenylnannolone A shows inhibitory activity towards the ABCB1 gene product P‐glycoprotein and reverses daunorubicin resistance in cancer cells. To decipher the biochemical reactions leading to the formation of phenylnannolone A, the putative biosynthetic genes were identified (phn1, phn2). Phn2 is a polyketide synthase (PKS) with an NRPS‐like loading module, and its domain order is consistent with the phenylnannolone A structure. The functionality and substrate selectivity of the loading module were determined by means of a γ‐¹⁸O₄‐ATP pyrophosphate exchange and a phosphopantetheine ejection assay. A specific activation of cinnamic acid by the AMP‐ligase was detected. Phn1 is a putative butyryl‐CoA carboxylase (BCC), providing ethylmalonyl‐CoA for the formation of the ethyl‐substituted part of phenylnannolone A. Phn1 is the first BCC found in biosynthetic genes for an ethyl‐substituted natural compound. Biosynthesis of phenylnannolone A, putatively encoded by phn1 and phn2, thus utilizes the first biosynthetic machinery in which both a BCC and a PKS are involved.     

In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl‐CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl‐CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self‐malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self‐acylation. This lack of self‐malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein–protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.     

Palladium(II)‐Catalyzed 1,4‐Addition of Arylboronic Acids to β‐Arylenones for Enantioselective Synthesis of 4‐Aryl‐4H‐chromenes

The enantioselective 1,4‐addition of arylboronic acids to β‐arylenones to give β‐diaryl ketones was carried out at 0–25 °C in the presence of a dicationic palladium(II) catalyst, [Pd(S,S‐chiraphos)(PhCN)₂](SbF₆)₂. Addition of a silver salt such as silver tetrafluoroborate [AgBF₄] or silver hexafluoroantimonate [AgSbF₆] (5–10 mol %) was effective to achieve high enantioselectivities at low temperatures (92–99 % ee) and to reduce the catalyst loading to 0.05 mol %. The protocol provided a simple access to 4‐aryl‐4H‐chromenes. Optically active chromenes were synthesized with up to 99 % ee via dehydration of the 1,4‐adducts between arylboronic acids and β‐(2‐hydroxyaryl)‐α,β‐unsaturated ketones.     

Biosynthetic Code for Divergolide Assembly in a Bacterial Mangrove Endophyte

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3‐amino‐5‐hydroxybenzoate and the rare extender units ethylmalonyl‐CoA and isobutylmalonyl‐CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer–Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.     

Crystal Structures of BapA Complexes with β‐Lactam‐Derived Inhibitors Illustrate Substrate Specificity and Enantioselectivity of β‐Aminopeptidases

β‐Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of β‐amino acids, which serve as building blocks for the design of non‐natural peptidomimetics. We have identified the β‐lactam antibiotic ampicillin and the ampicillin‐derived penicilloic acid as novel inhibitors of the β‐aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K_i values of 0.69 and 0.74 mM , respectively). We report high‐resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4‐(2‐aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA‐catalyzed reactions with β‐peptide substrates.     

Bifunctionality of ActIV as a Cyclase‐Thioesterase Revealed by in Vitro Reconstitution of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

Type II polyketide synthases iteratively generate a nascent polyketide thioester of the acyl carrier protein (ACP); this is structurally modified to produce an ACP‐free intermediate towards the final metabolite. However, the timing of ACP off‐loading is not well defined because of the lack of an apparent thioesterase (TE) among relevant biosynthetic enzymes. Here, ActIV, which had been assigned as a second ring cyclase (CYC) in actinorhodin (ACT) biosynthesis, was shown to possess TE activity in vitro with a model substrate, anthraquinone‐2‐carboxylic acid‐N‐acetylcysteamine. In order to investigate its function further, the ACT biosynthetic pathway in Streptomyces coelicolor A3(2) was reconstituted in vitro in a stepwise fashion up to (S)‐DNPA, and the product of ActIV reaction was characterized as an ACP‐free bicyclic intermediate. These findings indicate that ActIV is a bifunctional CYC‐TE and provide clear evidence for the release timing of the intermediate from the ACP anchor.     

The AT2 Domain of KirCI Loads Malonyl Extender Units to the ACPs of the Kirromycin PKS

The antibiotic kirromycin is assembled by a hybrid modular polyketide synthases (PKSs)/nonribosomal peptide synthetases (NRPSs). Five of six PKSs of this complex assembly line do not have acyltransferase (AT) and have to recruit this activity from discrete AT enzymes. Here, we show that KirCI is a discrete AT which is involved in kirromycin production and displays a rarely found three‐domain architecture (AT₁‐AT₂‐ER). We demonstrate that the second AT domain, KirCI‐AT₂, but not KirCI‐AT₁, is the malonyl‐CoA‐specific AT which utilizes this precursor for loading the acyl carrier proteins (ACPs) of the trans‐AT PKS in vitro. In the kirromycin biosynthetic pathway, ACP5 is exclusively loaded with ethylmalonate by the enzyme KirCII and is not recognized as a substrate by KirCI. Interestingly, the excised KirCI‐AT₂ can also transfer malonate to ACP5 and thus has a relaxed ACP‐specificity compared to the entire KirCI protein. The ability of KirCI‐AT₂ to load different ACPs provides opportunities for AT engineering as a potential strategy for polyketide diversification.     

Asymmetric Aza‐Morita–Baylis–Hillman‐Type Reactions: The Highly Enantioselective Reaction between Unmodified α,β‐ Unsaturated Aldehydes and N‐Acylimines by Organo‐co‐catalysis

The highly enantioselective organo‐co‐catalytic aza‐Morita–Baylis–Hillman (MBH)‐type reaction between N‐carbamate‐protected imines and α,β‐unsaturated aldehydes has been developed. The organic co‐catalytic system of proline and 1,4‐diazabicyclo[2.2.2]octane (DABCO) enables the asymmetric synthesis of the corresponding N‐Boc‐ and N‐Cbz‐protected β‐amino‐α‐alkylidene‐aldehydes in good to high yields and up to 99% ee. In the case of aza‐MBH‐type addition of enals to phenylprop‐2‐ene‐1‐imines, the co‐catalytic reaction exhibits excellent 1,2‐selectivity. The organo‐co‐catalytic aza‐MBH‐type reaction can also be performed by the direct highly enantioselective addition of α,β‐unsaturated aldehydes to bench‐stable N‐carbamate‐protected α‐amidosulfones to give the corresponding β‐amino‐α‐alkylidene‐aldehydes with up to 99% ee. The organo‐co‐catalytic aza‐MBH‐type reaction is also an expeditious entry to nearly enantiomerically pure β‐amino‐α‐alkylidene‐amino acids and β‐amino‐α‐alkylidene‐lactams (99% ee). The mechanism and stereochemistry of the chiral amine and DABCO co‐catalyzed aza‐MBH‐type reaction are also discussed.     

β‐Aminopeptidase‐Catalyzed Biotransformations of β2‐Dipeptides: Kinetic Resolution and Enzymatic Coupling

We have previously shown that the β‐aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non‐natural β³‐peptides and β³‐amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N‐terminal β²‐amino acid residues as substrates under aqueous conditions. The suitability of a β²‐peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C_α substituent of the N‐terminal β²‐amino acid. BapA was shown to convert a diastereomeric mixture of the β²‐peptide H‐β²hPhe‐β²hAla‐OH, but did not act on diastereomerically pure β²,β³‐dipeptides containing an N‐terminal β²‐homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA‐catalyzed transformation of the diastereomeric mixture of H‐β²hPhe‐β²hAla‐OH proceeded along two highly S‐enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis‐to‐hydrolysis ratio (v_S/v_H) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N‐terminal β‐amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of β²‐homophenylalanine was detected (ee>99 % for H‐(S)‐β²‐hPhe‐OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure β²‐amino acids and the enantioselective coupling of N‐terminal β²‐amino acids to peptides.     

Identification of the Biosynthetic Gene Cluster for Himeic Acid A: A Ubiquitin‐Activating Enzyme (E1) Inhibitor in Aspergillus japonicus MF275

Himeic acid A, which is produced by the marine fungus Aspergillus japonicus MF275, is a specific inhibitor of the ubiquitin‐activating enzyme E1 in the ubiquitin–proteasome system. To elucidate the mechanism of himeic acid biosynthesis, feeding experiments with labeled precursors have been performed. The long fatty acyl side chain attached to the pyrone ring is of polyketide origin, whereas the amide substituent is derived from leucine. These results suggest that a polyketide synthase–nonribosomal peptide synthase (PKS‐NRPS) is involved in himeic acid biosynthesis. A candidate gene cluster was selected from the results of genome sequencing analysis. Disruption of the PKS‐NRPS gene by Agrobacterium‐mediated transformation confirms that HimA PKS‐NRPS is involved in himeic acid biosynthesis. Thus, the him biosynthetic gene cluster for himeic acid in A. japonicus MF275 has been identified.     

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D ‐ravidosamine and D ‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D ‐ravidosamine and 5‐membered D ‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.