Regulatory and biosynthetic effects of the <Emphasis Type="Italic">bkd</Emphasis> gene clusters on the production of daptomycin and its analogs A21978C<Subscript>1–3</Subscript>

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus in an acidic peptide complex A21978C. In this complex, A21978C_1–3 is most abundant and contains branched-chain fatty acyl groups, while daptomycin has a straight decanoic acyl group. The branched-chain α-keto acid dehydrogenase complex (BCDH complex), encoded by bkd gene clusters in Streptomyces, is responsible for the early step of converting branched-chain amino acids into branched-chain fatty acids. In a daptomycin industrial producer S. roseosporus L30, two alleles of bkd gene clusters, bkdA1B1C1/bkdA2B2C2, and a regulatory gene bkdR located upstream of bkdA2B2C2 are identified. We show that BkdR positively regulated bkdA2B2C2 expression and was negatively auto-regulated, but is not directly involved in regulation of daptomycin gene cluster expression. However, BkdR is required for both daptomycin and A21978C_1–3 production. Furthermore, deletion of bkdA2B2C2 only led to partial reduction of A21978C_1–3 production, while the ΔbkdA1B1C1 mutant shows very weak production of A21978C_1–3, and the double bkd mutant has a similar production profile as the single ΔbkdA1B1C1 mutant, suggesting that bkdA1B1C1 gene cluster plays a dominant role in branched-chain fatty acid biosynthesis. So we reveal a unique regulatory function of BkdR and genetic engineered a bkd null strain for daptomycin production with reduced impurities.     

Heterologous production of daptomycin in Streptomyces lividans

Daptomycin and the A21978C antibiotic complex are lipopeptides produced by Streptomyces roseosporus and also in recombinant Streptomyces lividans TK23 and TK64 strains, when a 128 kbp region of cloned S. roseosporus DNA containing the daptomycin gene cluster is inserted site-specifically in the ϕC31 attB site. A21978C fermentation yields were initially much lower in S. lividans than in S. roseosporus, and detection was complicated by the production of host metabolites. However A21978C production in S. lividans was improved by deletion of genes encoding the production of actinorhodin and by medium optimization to control the chemical form of the calcium dependent antibiotic (CDA). This latter compound has not previously been chemically characterized as a S. lividans product. Adding phosphate to a defined fermentation medium resulted in formation of only the phosphorylated forms of CDA, which were well separated from A21978C on chromatographic analysis. Adjusting the level of phosphate in the medium led to an improvement in A21978C yield from 20 to 55 mg/l.     

Improvement of A21978C production in Streptomyces roseosporus by reporter-guided rpsL mutation selection

Aims:  Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram‐positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported‐guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results:  In the reporter design, dptE was chosen as the overexpressing target, and neo‐encoding neomycin phosphotransferase as the reporter. Using this reporter‐guided selection system, 20% of the selected, streptomycin‐resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10‐fold with a frequency of 1·7% A21978C overproducing strains among str^r mutants. A21978C production was increased approximately 2·2‐fold in the rpsL K43N mutant. Conclusions:  The combination of ribosome engineering and reporter‐guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study:  The strategies presented here, which integrated the advantages of both ribosome engineering and reporter‐guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites.     

Breeding of High Daptomycin-Producing Strain by Streptomycin Resistance Superposition

Daptomycin is a cyclolipopeptide antibiotic produced by Streptomyces roseosporus. It is widely used to treat drug-resistant bacterial infections; however, daptomycin yield in wild strains is very low. To improve the daptomycin production by the strain BNCC 342432, a modified method of ribosome engineering with superposition of streptomycin resistance was adopted in this study. The highest-yield mutant strain SR-2620 was obtained by increasing streptomycin resistance of BNCC 342432, and achieved daptomycin production of 38.5 mg/l in shake-flask fermentation, 1.79-fold higher than the parent strain and its heredity stability was stable. The morphological characteristics of the two strains were significantly different, and the 440^th base G of the rpsL gene in the mutant strain was deleted, which resulted in a frameshift mutation. Our results demonstrate that gradually increasing strain resistance to streptomycin was an effective breeding method to improve daptomycin yield in S. roseosporus. Open in a separate window     

A glutamic acid 3-methyltransferase encoded by an accessory gene locus important for daptomycin biosynthesis in Streptomyces roseosporus

In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.     

Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus

The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ~7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.     

Manipulation of kynurenine pathway for enhanced daptomycin production in Streptomyces roseosporus

Daptomycin is a cyclic lipopeptide natural product produced by Stretptomyces roseosporus, displaying good bactericidal activity against a wide range of gram‐positive pathogens. Daptomycin contains a 13 amino acid and kynurenine (Kyn) is essential for optimal activity of daptomycin. In this study, we characterized the Kyn pathway in S. roseosporus and investigated its role in supplying precursor for daptomycin biosynthesis. Two genes (dptJ and tdo) coding for tryptophan‐2,3‐dioxgenase existed in the chromosome. dptJ is located in the daptomycin biosynthetic gene cluster, while tdo is in other locus. Disruption of dptJ or tdo resulted in reduced yield by ～50%. The introduction of an additional copy of dptJ but not tdo led to enhanced production of daptomycin by 110%. Furthermore, disruption of kyn encoding kynureninase showed improved daptomycin productivity by 30%. Our results demonstrated that the enhancement of Kyn supply through metabolic engineering approach is an efficient way to increase daptomycin production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:847–852, 2013     

Daptomycin antibiotic production processes in fed-batch fermentation by Streptomyces roseosporus NRRL11379 with precursor effect and medium optimization

Sodium decanoate was first found to be an effective precursor for synthesis of daptomycin from Streptomyces roseosporus NRRL11379 which was increased to 71.55-fold, compared with decanoic acid. The optimal flow rate of precursor was at 600 mg/(L day) after 48 h fermentation. From protein analysis via SDS-PAGE and identification of Tandem MS/MS afterwards, it deciphered that guanosine pentaphosphate synthetase, PNPase, tripeptidylamino peptidase primarily dealing with daptomycin synthesis. By applying Taguchi’s L16 in culture optimization, the best yield was obtained from the medium with 60 g/L dextrin, 10 g/L dextrose, 1.0 g/L molasses, and 8 g/L yeast extract, respectively. The fed-batch fermentation, applied with feedback control of dextrin, stimulated the production up to 812 mg/L at 288 h. To our best knowledge, the daptomycin production in this study is significantly higher than that in previous studies and can make it more widely used in pharmaceutical industry.     

Enhancement of daptomycin production in <Emphasis Type="Italic">Streptomyces roseosporus</Emphasis> LC-51 by manipulation of cofactors concentration in the fermentation culture

The effects of eight cofactors of enzymes on daptomycin production were investigated in this work, which included nicotinic acid (VPP), riboflavin (VB₂), heme, thiamine (VB₁), biotin (VH), cyanocobalamin (VB₁₂), tetrahydrofolic acid (THF) and pyridoxal 5-phosphate (VB₆). The dry cell weight (DCW), consumption of glucose, and daptomycin production were obviously improved when proper amount of exogenous cofactors were supplemented in the medium. The effects of heme, THF, VB₁₂ and VB₆ on daptomycin production were especially notable. The daptomycin yield enhanced 363, 104, 53 and 46%, respectively, when optimized amount of these four cofactors were supplemented in the broth. Moreover, the daptomycin yield further increased to 632 mg/l, which was over 4.5-fold higher than that of the control (without cofactors), at 132 h in a 7.5-l fermenter, by supplementation all of the eight cofactors at optimized concentrations (VPP 4 mg/l, VB₂ 0.5 mg/l, heme 9 mg/l, VB₁ 0.4 mg/l, VH 0.1 mg/l, VB₁₂ 0.04 mg/l, THF 6 mg/l and VB₆ 0.4 mg/l). Further, the effects of cofactors on the corresponding key enzymes and important intracellular metabolites were studied in order to elucidate the mechanism of enhancement of daptomycin production by manipulation of cofactors concentration in the fermentation culture. It is suggested that this strategy for increasing the daptomycin production in Streptomyces roseosporus LC-51 by manipulation of cofactors concentration in the fermentation culture may provide an alternative approach to enhance the production of metabolites in other Streptomyces.     

Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster

A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu₁₂), at the expense of factors containing 3-methyl-glutamic acid (3mGlu₁₂). This provided a practical route to produce high levels of pure Glu₁₂-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.The calcium-dependent cyclic acidic lipodepsipeptide antibiotics were first reported in the 1980s and 1990s (8). These include A21978C, produced by Streptomyces roseosporus (17, 18), calcium-dependent antibiotic (CDA), produced by Streptomyces coelicolor (26), and A54145, produced by Streptomyces fradiae NRRL 18160 (11, 12, 23). A21978C (Fig. ​(Fig.1)1) has been of particular interest because the N-decanoyl lipid tail derivative of the A21978C peptide is daptomycin (8), which is approved for the treatment of complicated skin and skin structure infections caused by Gram-positive bacteria (2) and for bacteremia and right-sided endocarditis caused by Staphylococcus aureus, including strains resistant to methicillin (MRSA) (21). Daptomycin lacks efficacy in community-acquired pneumonia (CAP) infections, even though it is very active in vitro against the predominant pathogen, Streptococcus pneumoniae (8, 43). In vitro studies have shown that daptomycin becomes sequestered in bovine pulmonary surfactant, most likely in the lipid component, and has decreased antibacterial potency against Gram-positive pathogens (46); this may be a significant factor contributing to the poor clinical efficacy in CAP. Attempts to improve the efficacy of daptomycin through chemical modifications of the lipid side chain or additions to the δ-amino group of ornithine (Orn₆) (reviewed in reference 8), or by molecular engineering of peptide assembly (4, 13, 25, 37-39), have not generated a lead molecule with sufficient in vivo efficacy in a mouse pneumonia model for S. pneumoniae.Open in a separate windowFIG. 1.Structures of the lipopeptide antibiotics and NRPS protein subunit relationships. (Top) A54145 factors normally produced by S. fradiae. Note that factors A, A₁, D, and F have Glu at position 12, and factors B, B₁, C, and E have 3mGlu at position 12. (Bottom) A21978C factors normally produced by S. rosesosporus and daptomycin.A54145 factors share a number of features in common with daptomycin, but they differ at several amino acid positions (Fig. ​(Fig.1).1). The most biologically active A54145 factors against S. aureus contain four modified amino acids, l-hydroxy-Asn₂ (hAsn₂), sarcosine₅ (Sar₅), l-methoxy-Asp₉ (mOAsp₉), and l-3-methyl-Glu₁₂ (3mGlu₁₂) (14). During a standard fermentation, multiple A54145 factors are produced as the result of natural variation at position 12 (3mGlu or Glu), at position 13 (Ile or Val) and at the lipid tail attached to the peptide core. The A54145 factors A, A₁, and D (collectively designated the A-core) have the identical peptide containing Glu₁₂ and Ile₁₃ but have different lipid tails, whereas factors B, B₁, and E (the B-core) contain 3mGlu₁₂ and Ile₁₃. During fermentation of S. fradiae, factor A accumulates as a major component but plateaus early, and factor B₁ accumulates preferentially late in the fermentation (11, 12). In studies at Eli Lilly and Company, it was shown that the B-core factors were slightly more potent antibiotics, but factor B was substantially more toxic than its Glu₁₂-containing counterpart, factor A₁ (14).During the development of molecular engineering approaches to modify daptomycin biosynthesis, the genes for A54145 lipopeptide biosynthesis (lpt) were cloned and sequenced to provide nonribosomal peptide synthetase (NRPS) modules and subunits to exchange with those of daptomycin (37). Since some of the A54145 A-core factors were shown to be much less inhibited by bovine surfactant than daptomycin (40), the A54145 A-core lipopeptides should be useful starting points for both chemical and molecular engineering modification studies. We initiated a program to develop molecular genetics methods, with plasmids and host cloning strains to facilitate molecular engineering of A54145 biosynthesis in S. fradiae.In this report, we describe the engineering of a bacterial artificial chromosome (BAC) containing the A54145 biosynthesis genes by using λ-Red-mediated recombination in Escherichia coli and expression of the A54145 biosynthesis pathway in heterologous streptomycetes. The development of S. fradiae strains deleted for multiple A54145 genes and the construction of plasmid vectors with conjugation and site-specific integration functions for ectopic expression of sets of A54145 biosynthesis genes in S. fradiae and combinatorial biosynthesis (40) are discussed. This genetic system was used to generate a strain with deletion of lptI, a gene that encodes a methyltransferase involved in the biosynthesis of 3mGlu₁₂, and the mutant produced the desired A-core lipopeptides containing Glu₁₂, which are important starting materials for medicinal chemistry approaches to produce novel lipopeptides.     

The action mechanism of daptomycin

Daptomycin is a lipopeptide antibiotic produced by the soil bacterium Streptomyces roseosporus that is clinically used to treat severe infections with Gram-positive bacteria. In this review, we discuss the mode of action of this important antibiotic. Although daptomycin is structurally related to amphomycin and similar lipopeptides that inhibit peptidoglycan biosynthesis, experimental studies have not produced clear evidence that daptomycin shares their action mechanism. Instead, the best characterized effect of daptomycin is the permeabilization and depolarization of the bacterial cell membrane. This activity, which can account for daptomycin’s bactericidal effect, correlates with the level of phosphatidylglycerol (PG) in the membrane. Accordingly, reduced synthesis of PG or its increased conversion to lysyl-PG promotes bacterial resistance to daptomycin. While other resistance mechanisms suggest that daptomycin may indeed directly interfere with cell wall synthesis or cell division, such effects still await direct experimental confirmation. Daptomycin’s complex structure and biosynthesis have hampered the analysis of its structure activity relationships. Novel methods of total synthesis, including a recent one that is carried out entirely on a solid phase, will enable a more thorough and systematic exploration of the sequence space.     

Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

Cerulenin resistance in a cerulenin-producing fungus. Isolation of cerulenin insensitive fatty acid synthetase

Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens, is a potent inhibitor of fatty acid synthetase systems. This antibiotic specifically blocks the activity of β-ketoacyl thioester synthetase (condensing enzyme). The mechanism of the resistance of C. caerulens to cerulenin was investigated. The rate of growth in medium containing up to 100 gmg/ml cerulenin was as rapid as that in cerulenin-free medium. At a cerulenin concentration of 300 μg/ml, the rate of growth was still more than half that of the control. The addition of cerulenin (200 μg/ml) to a culture of growing cells has almost no effect on the incorporation of [${}^{14}\text{C}$]acetate into cellular lipids. Fatty acid synthetase was purified from C. caerulens to homogeneity. Properties of this fatty acid synthetase were almost the same as those of yeast fatty acid synthetase except for the sensitivity to cerulenin. C. caerulens synthetase is much less sensitive to cerulenin than fatty acid synthetases from other sources. These findings suggested that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus.     

Novel Strategy of Using Methyl Esters as Slow Release Methanol Source during Lipase Expression by mut+ Pichia pastoris X33

One of the major issues with heterologous production of proteins in Pichia pastoris X33 under AOX1 promoter is repeated methanol induction. To obviate repeated methanol induction, methyl esters were used as a slow release source of methanol in lipase expressing mut⁺ recombinant. Experimental design was based on the strategy that in presence of lipase, methyl esters can be hydrolysed to release their products as methanol and fatty acid. Hence, upon break down of methyl esters by lipase, first methanol will be used as a carbon source and inducer. Then P. pastoris can switch over to fatty acid as a carbon source for multiplication and biomass maintenance till further induction by methyl esters. We validated this strategy using recombinant P. pastoris expressing Lip A, Lip C from Trichosporon asahii and Lip11 from Yarrowia lipolytica. We found that the optimum lipase yield under repeated methanol induction after 120 h was 32866 U/L, 28271 U/L and 21978 U/L for Lip C, Lip A and Lip 11 respectively. In addition, we found that a single dose of methyl ester supported higher production than repeated methanol induction. Among various methyl esters tested, methyl oleate (0.5%) caused 1.2 fold higher yield for LipA and LipC and 1.4 fold for Lip11 after 120 h of induction. Sequential utilization of methanol and oleic acid by P. pastoris was observed and was supported by differential peroxisome proliferation studies by transmission electron microscopy. Our study identifies a novel strategy of using methyl esters as slow release methanol source during lipase expression.