Enzyme Technologies (Metagenomics, Evolution, Biocatalysis, and Biosynthesis) || Reprogramming Daptomycin and A54145 Biosynthesis to Produce Novel Lipopeptide Antibiotics

9REPROGRAMMING DAPTOMYCINAND A54145 BIOSYNTHESIS TOPRODUCE NOVEL LIPOPEPTIDEANTIBIOTICS

Richard H. Baltz, Kien T. Nguyen, and Dylan C. AlexanderCubist Pharmaceuticals, Inc., Lexington, Massachusetts

I. INTRODUCTION

Daptomycin (Fig. 1) is a cyclic acidic lipodepsipeptide antibiotic approved for thetreatment of skin and skin structure infections caused by gram-positive pathogens[1]. It has also been approved to treat bacteremia and right-sided endocarditiscaused by S. aureus , including strains resistant to methicillin (MRSA) [2]. Dap-tomycin is also efficacious in treating other infections caused by gram-positivebacteria [3], but failed to treat community-acquired pneumonia (CAP) caused byStreptococcus pneumoniae as well as current standard treatments [4]. In labora-tory studies, daptomycin has excellent antibacterial activity against pathogenicstreptococci, including S. pneumoniae [5,6], but in the presence of bovine surfac-tant daptomycin becomes sequestered and presumably not available to effectivelykill gram-positive bacteria [7]. Therefore, daptomycin is likely to be sequesteredin human surfactant in the lung. Improving antibacterial activity in the presenceof surfactant is a well-defined target for a second-generation acidic lipopeptidewith the objective of adding CAP as an indication.

A54145 is another cyclic lipodepsipeptide structurally similar to daptomycin(Fig. 1) produced by Streptomyces fradiae [8]. Notably, it has D-amino acids at

Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis,Edited by Wu-Kuang Yeh, Hsiu-Chiung Yang, and James R. McCarthyCopyright © 2010 John Wiley & Sons, Inc.

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286 REPROGRAMMING DAPTOMYCIN AND A54145 BIOSYNTHESIS

positions 2, 8, and 11 and achiral amino acids at positions 5 and 10, just as indaptomycin; therefore, it may have a three-dimensional structure similar to that ofdaptomycin [9–11]. Both daptomycin and A54145 antibacterial activities requireCa2+, and both have Ca2+-binding motifs (DXDG) at positions 7 to 10. Some ofthe natural A54145 factors have reasonably good in vitro and in vivo antibacte-rial activities, but the most active factors, which contain 3-methylglutamic acid(3mGlu) at position 12, are substantially more toxic than daptomycin [12], thusprecluding their clinical development.

Calcium-dependent antibiotic (CDA) is another cyclic lipodepsipeptide struc-turally related to daptomycin and A54145. It is produced by Streptomyces coeli-color . Unlike daptomycin and A54145, which have 13 amino acids (10 in thering and three exocyclic), CDA has a 10-amino acid ring and one exocyclicamino acid. CDA has achiral or D-amino acids in the same positions in the10-membered ring as daptomycin and A54145, so it may have a similar three-dimensional structure. The antibacterial activity of CDA factors is poor, perhapsdue to the much shorter fatty acid/amino acid side chain relative to dapto-mycin and A54145 factors [13,14]. There are a number of other cyclic peptideseven more distantly related to daptomycin that have been reviewed elsewhere[13–15].

Previous chemical modifications of daptomycin were limited to semisyntheticmodifications at the fatty acid side chain or additions to the δ-amino group ofornithine (Orn) [13,14]. Total synthesis of lipopeptides is not practical becauseof the presence of synthetically challenging unnatural amino acids. Anotherapproach to modifying daptomycin was to change the core peptide structure byreprogramming the nonribosomal peptide synthetases (NRPSs) to incorporate dif-ferent amino acids, and by deleting genes encoding auxiliary enzymes involvedin amino acid modifications to produce a variety of new cyclic depsipeptidesnot achievable by medicinal chemistry [3,9,10,13,16]. These approaches havealso been taken to reprogram A54145 biosynthesis [16–20]. In this chapter wesummarize the findings of this body of work, emphasizing the versatility, limi-tations, and “rules” governing successful engineering of lipopeptide biosynthesisto produce novel antibiotics.

II. EXPERIMENTAL DESIGN

Media and Growth Conditions The media and growth conditions for prop-agating the natural and recombinant Streptomycetes strains, and fermentationconditions to produce lipopeptides, were as described [17,20–22].

Bacterial Strains and Plasmids The parental and genetically engineeredstrains of Streptomyces roseosporus and S. fradiae, construction of recombinantplasmids, and methods to introduce plasmids into Streptomycetes strains byintergeneric conjugation and site-specific recombination have been describedelsewhere [11,17,21–24]. Key strains and plasmids are shown in Figure 2.

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RESULTS AND DISCUSSION 289

Chemical Characterization The chemical characterization of the lipopep-tides produced by recombinant Streptomycetes strains were as described[17,20,23–25].

III. RESULTS AND DISCUSSION

A. Biosynthesis of Daptomycin

The lipopeptide antibiotic A21987C1−3 factors are normally produced by S.roseosporus during fermentation. These factors differ from each other in lipidside chain (Fig. 1). Daptomycin, which has a decanoate side chain, is producedby feeding decanoic acid during fermentation [14,26]. The understanding of howdaptomycin is enzymatically assembled has benefitted greatly from the bioin-formatic and genetic analysis of the daptomycin biosynthetic gene cluster. Thedaptomycin (dpt) genes are clustered near one end of the linear chromosome ofS. roseosporus [27], and many of the genes appear to be organized in an operonthat is expressed as a very long multicistronic mRNA [28]. Transcriptome anal-ysis of low- and high-producing strains of S. roseosporus was consistent withup-regulation of one large transcript in the high producer [29]. Figure 2A showsthe general layout of the dpt genes.

The dpt genes were cloned in cosmid and BAC vectors and sequenced [21,27].The complete dpt gene cluster present on the BAC pCV1 was expressed fromits native promoters in Streptomyces lividans after integration into the φC31 attBsite, and the recombinant produced the three major A21978C factors normallyproduced by S. roseosporus [21,30]. The dpt gene cluster encodes 12 genes thatare clearly involved in daptomycin biosynthesis. Peptide assembly is catalyzedby the giant NRPS proteins, DptA, DptBC, and DptD, encoded by the dptA,dptBC , and dptD genes (Figs. 1A and 2A; Table 1). The DptA subunit has fivemodules dedicated to the binding, activation, and coupling of the first five aminoacids, starting with Trp1 of the exocyclic tail. The Trp1 C-A-T module has threeenzymatic domains: a special condensation (C) domain, designated CIII [21] orFCL (see below), that couples long-chain fatty acids to the N-terminus of L-Trp1,an adenylation (A) domain for binding and activation of Trp; and a thiolation (T)domain or peptidyl-carrier protein (PCP) to tether the activated amino acids andgrowing peptide chains, and to facilitate cyclic peptide assembly. Module 2 hasa C-A-T-E structure (E for epimerase domain) for binding, activation, epimeriza-tion, and coupling of D-Asn2. The remaining modules in DptA (five modules),DptBC (six modules), and DptD (two modules) have the expected C-A-T or C-A-T-E structures consistent with the positions of L- and D-amino acids, exceptfor the terminal Kyn13 module of DptD, which has an additional thioesterase(TE) domain (C-A-T-TE) for ring closure and release of the completed lipopep-tides from the NRPS multienzyme. The C-domains following C-A-T-E modulesare specialized to accept D-amino acids and couple them to L-amino acids, andhave been designated CII [21] or DCL [31]. Condensation domains that coupleL-amino acids to L-amino acids have been designated LCL. In keeping with thisdescriptive nomenclature, we propose to rename CIII condensation domains for


TABLE 1 Proteins Involved in Daptomycin (Dpt) or A54145 (Lpt) Biosynthesis

Protein (aa) Protein (aa) Identity (%) Function

DptA (5812) LptA (6292) 47 NRPS (1–5)a

DptBC (7338) LptB (2148) 47 NRPS (6,7)DptBC(7338) LptC (5246) 49 NRPS (8–11)DptD (2379) LptD (2384) 53 NRPS (12,13)DptE (579) LptEF (732) 51 Acyl-CoA ligaseDptF (89) LptEF (732) 39 Acyl carrier proteinDptG (75) LptG (80) 58 UnknownDptH (271) LptH (264) 54 Editing thioesterase?DptI (328) LptI (352) 37 α-KG methyltransferaseDptJ (246) NHb — Tryptophan 2,3-dioxygenaseNH LptJ (246) — Asp oxygenaseNH LptK (262) — OH-Asp O-methyltransferaseNH LptL (319) — Asn oxygenaseDptM (319) LptM (353) 55 ABC transporter: ATP-binding proteinDptN (289) LptN (282) 56 ABC transporter: permeaseDptP (206) LptP (206) 94 Resistance to lipopeptides?

aNRPS modules (see Fig. 1).bNo homolog.

coupling fatty acids to L-amino acids as FCL. There is another type of relatedC-domain that catalyzes the coupling of β-hydroxyfatty acids [32].

There are exceptions to amino acid–coupling stereospecificities of C-domains.In both the dpt and lpt pathways, modules 6, which follows the achiral Gly5 andSar5, respectively, have DCL condensation domains, whereas modules 11, whichfollow Gly10 modules, have LCL condensation domains. The former DCL domainsmay be vestiges from an ancestral lipopeptide pathway, because CDA, friulimicin,amphomycin, laspartamicin, and glycinocins all appear to have D-amino acids atcomparable positions in the 10-membered ring [13–15].

Upstream of the NRPS genes are dptE and dptF , which encode an acyl-CoAligase and acyl carrier protein (ACP), respectively [13,21]. The DptE and DptFproteins participate in the activation and coupling of the long-chain fatty acidsto the N-terminus of L-Trp1 [33]. The dptE and dptF genes have a counterpartfused lptEF gene in the A54145 pathway (Table 1). Aside from the three D-aminoacids (D-Asn2, D-Ala8, and D-Ser11), daptomycin has three other nonproteinogenicL-amino acids (Orn6, 3mGlu12, and Kyn13).

Downstream of the NRPS genes are dptG, dptH, dptI , and dptJ . The dptGand dptH genes appear to be transcribed as part of a giant transcript, includingdptEFABCDGH [28]. The dptI and dptJ genes may also be included on thisgiant transcript [13]. The dptJ gene encodes a tryptophan-2,3-dioxygenase(TDO) involved in the conversion of Trp to Kyn. This conversion is typicallycarried out by the sequential action of TDO, kynurenine formamidase,and kynureninase. DptJ shows only 30% sequence identity (including 14%gaps) to the gene product of kynA (TDO) in S. coelicolor [13], and only


28% identity to the second TDO encoded by S. roseosporus (http://www.broad.mit.edu/annotation/genome/streptomyces_group/GenomesIndex.html) thatis likely to be involved in primary metabolism. Therefore, the dptJ gene mayhave diverged to function differently than typical TDOs. One possibility isthat it binds to the Kyn13 module as part of the NRPS multienzyme complexto provide Kyn on demand [13]. Disruption of dptJ caused a reduction indaptomycin production, confirming its role in daptomycin biosynthesis. Theresidual daptomycin biosynthesis in the dptJ mutant is probably accounted forby the expression of the second, primary metabolic TDO.

The dptI gene encodes a SAM-dependent methyltransferase that convertsα-ketoglutarate (α−KG) to 3-methyl-2-oxoglutarate, which is apparently transam-inated by a primary metabolic enzyme to give (2S,3R)-3-mGlu [34]. Deletionof dptI caused reduced overall yield and production of lipopeptides contain-ing Glu12 in place of 3mGlu12 [23]. DptI has distantly related homologs in theA54145 and CDA pathways involved in biosynthesis of 3mGlu [13,34]. LikedptI , the glmT gene from the CDA pathway expressed from the ermEp* pro-moter complemented the dptI deletion in S. roseosporus , restoring the productionof the A21978C factors containing 3mGlu12 [23]. Similarly, the dptI gene com-plemented lptI mutations in S. fradiae [17,20]. Unlike the other nonproteinogenicamino acids, Orn is likely to be provided from primary metabolism, because thereare no genes encoding its biosynthesis in the dpt gene cluster [13,14,21].

Further downstream of the NRPS genes are two apparent regulatory genes,dptR1 and dptR2 [21]. Upstream of dptE are dptP, dptM , and dptN genes thatmay function in daptomycin export and/or resistance. DptM and DptN appear tobe the ATP-binding and membrane-spanning components of an ABC transporter[21] and have homologs (LptM and LptN) in the A54145 biosynthetic pathway(Table 1; [11,13]). DptP is a membrane-associated protein that has very high(94%) identity to LptP in the A54145 pathway but little similarity to other pro-teins in the databases. When a BAC vector containing dptP was inserted intoStreptomyces ambofaciens chromosome, it conferred resistance to daptomycin[54]. This finding suggests that DptP may be involved in daptomycin export orresistance in S. roseosporus . It is conceivable that DptP interacts with DptM/DptNto facilitate export of daptomycin in S. roseosporus .

The dptH gene encodes a protein that may function as an editing thioesterase[21,23], possibly functioning by cleaving stalled peptide chains at T domains, byhydrolyzing acetyl groups from acetyl-S T domains, or both [13]. Disruption ofdptH caused a 50% reduction in lipopeptide production [23], consistent with anediting function.

The dptG gene encodes a 75-amino acid protein of unknown function requiredfor optimal daptomycin production [23]. The dptG gene has homologs in othersecondary metabolite biosynthetic pathways [13]. The dptG gene is just down-stream of the NRPS genes and is cotranscribed with the NRPS genes, suggestingthat it may have some function in the NRPS multienzyme complex.


B. Biosynthesis of A54145

The A54145 biosynthetic genes were cloned in cosmids and sequenced [11]. Tofacilitate combinatorial biosynthesis and NRPS reprogramming (by domain andmodule exchanges), the A54145 biosynthetic gene cluster was cloned in a BACvector and expressed in Streptomyces ambofaciens and S. roseosporus mutantsdefective in spiramycin and daptomycin biosynthesis, respectively [17]. The het-erologous expression confirmed that the complete set of A54145 biosyntheticgenes was present on the BAC. The A54145 biosynthetic gene cluster sharessome striking similarities to that of daptomycin (Fig. 1; Table 1). The A54145gene cluster contains four NRPS genes, lptA, lptB, lptC , and lptD , which encodeNRPS proteins with five, two, four, and two modules, respectively (Figs. 1Band 2B). The lptA and lptD genes are similar to the dptA and dptD genes,encoding five and two NRPS modules, respectively, suggesting common ances-try (Fig. 1; Table 1; [13]). The lptB and lptC genes and their encoded proteins(LptB and LptC) are related to the dptBC gene and protein (DptBC) [13]. Frombiochemical studies, it appears that S. fradiae expresses only three NRPS subunits[35], suggesting that the lptB and lptC genes may encode a fused six-moduleLptBC protein by translational frameshifting [10,13].

A54145 modules 2, 8, and 11 have C-A-T-E modules, just as in the daptomycinNRPSs, suggesting that A54145 lipopeptides have the same stereochemistry asdaptomycin and CDA [13,14]. As in DptA, the LptA Trp1 C-A-T starts with a CIII

(or FCL) domain that couples long-chain fatty acids to L-amino acids. Similarly,module 13 has a TE-domain for ring closure and release of A54145 factors fromthe NRPS. The remaining modules have the expected C-A-T structures, with theone exception discussed above (Fig. 1). The A54145 NRPSs have the three typesof C-domains (FCL, LCL, and DCL) located in the same positions as observed inthe daptomycin NRPSs.

A54145 has four modified amino acids: hydroxyasparagine (hAsn3), sarcosine(Sar5), methoxyaspartic acid (mOAsp9), and 3mGlu12. The module for Sar5 hasa C-A-M-T structure which includes a methylation (M) domain that accounts forthe N-methylation of Gly to Sar. Downstream of the NRPS genes is a clusterof genes, including lptI, lptJ, lptK , and lptL, involved in the biosynthesis ofmodified amino acids. The lptI gene is a homolog of dptI and glmT geneswhich encode α-ketoglutarate methyltransferases involved in the biosynthesis of3mGlu. Deletion of lptI caused the production of A54145 factors containingGlu12 in high yields [17]. DptI and LptI proteins share only 37% identical aminoacids (Table 2), despite the fact that they both carry out the same methylation of α-KG, and as such might normally be considered as orthologous proteins. Typicalorthologous proteins in streptomycetes show 80 to 90% amino acid sequenceidentities. The drastic sequence divergence may indicate that these enzymes haveadditional nonorthologous functions, perhaps binding to the NRPS megaenzymeto provide 3mGlu upon demand for lipopeptide biosynthesis [13]. Otherwise, freecytoplasmic DptI and LptI enzymes would provide 3mGlu to the cytoplasmic poolthat could be tapped for potentially problematic protein synthesis, notwithstandingthe dilution from the key function of incorporation into lipopeptide biosynthesis.


TABLE 2 Overlap of Stop and Start Codons in NRPS Genes

Region of Stop and Start Codons Nucleotide Sequencea

dptA-dptBC TGGTGAACCGCdptBC-dpt D GGATGACGCAGlptA-lptB CAGTGAACGGTlptB-lptC TTGTGATGCTTlptC-lptD GGCTGATGCGC

aStop codons are underlined and start codons are in bold italic. In the case of LptC-LptD, twopossible start codons are shown.

A54145 factors have D-Glu in position 2, but 3mGlu is not incorporated at thisposition. Both Glu and 3mGlu can be incorporated at position 12, indicatingthat the module 12 A-domain can accept either amino acid, whereas module 2does not do so during normal fermentation. This is consistent with site-specificconversion of Glu to 3mGlu at module 12. Despite the dramatic divergence inamino acid sequences, glmT can complement a dptI defect in S. roseosporus[23], and dptI can complement an lptI defect in S. fradiae [17,20].

Three other genes in the A54145 biosynthetic gene cluster encode aminoacid–modifying functions. The lptJ gene encodes an Asp9-specific oxygenaseto produce hAsp9, and lptK encodes a SAM-dependent O-methyltransferase thatconverts hAsp9 to mOAsp9. The lptL gene encodes an Asn3-specific oxygenaseto produce hAsn3 [18].

It was shown recently that oxidation of asparagine by AsnO to produce hAsnduring CDA biosynthesis occurs with free Asn as substrate [36]. This observa-tion suggests that the same mechanism may apply during A54145 biosynthesis.However, the AsnO and LptL oxygenases are only 47% identical in amino acidsequence [13]. As with the DptI and LptI genes, this low sequence identity isnot consistent with orthologous functions and suggests that each protein mayhave a second function, perhaps binding to the NRPS megaenzyme to providehAsn on demand at module 3. It is noteworthy that A54145 has a D-Asn residueat position 11 which is not hydroxylated, supporting the notion that oxidationoccurs site-specifically at module 3.

C. Genetic Engineering of S. roseosporus and S. fradiae

Ectopic Expression Systems Since S. roseosporus appears to express the threegiant NRPS and accessory proteins from a single very long transcript [28], withoverlapping translational stop and start codons (Table 2), this transcriptionalorganization poses challenges for genetic engineering. There are two possiblesolutions to engineering the daptomycin gene cluster. The first approach isto delete the complete pathway from S. roseosporus and engineer the clonedpathway on the BAC vector in E. coli , leaving all genes in the naturaltranscriptional organization, and then reintroduce the engineered pathway intoS. roseosporus at an ectopic chromosomal locus. Alternatively, the engineered


daptomycin biosynthetic pathway might be expressed in a heterologous host.Both of these approaches have been enabled by cloning the complete daptomycinbiosynthetic gene cluster on a BAC vector that has transfer functions forconjugation from E. coli into Streptomycetes; the vector also encodes functionsto catalyze the site-specific integration into the bacteriophage φC31 attB sitein streptomycete chromosomes, and daptomycin has been produced by thismechanism in S. lividans [21,30].

A second approach is to delete one or more genes in the daptomycin biosyn-thetic pathway in frame to avoid generating a polar mutation that disrupts thecoding sequences downstream in the transcript, and to express one or more com-plementing genes from ectopic chromosomal loci under the transcriptional controlof a strong promoter. This approach has been accomplished by using two differentplasmids that insert site-specifically into different chromosome sites (φC31 attBand IS117 attB ), and by expressing the cloned genes from the strong constitutiveermEp* promoter [21,24,28]. By using this system, the three giant daptomycinNRPS genes (or engineered versions of these or related genes) were expressedfrom three different chromosomal sites. Recombinants expressing the three NRPSsubunits from three different chromosomal sites produced A21978C factors atabout 100% of control [21,28]. Figure 2a shows the features of the S. roseosporusstrains containing different deletions, and the plasmid vectors used for ectopictrans-complementation with homologous, heterologous, and engineered NRPSgenes.

A similar ectopic trans-complementation system was devised for combina-torial biosynthesis in S. fradiae [17]. In this case, genes were expressed fromthe native site and two bacteriophage attachment sites (φC31 attB and φBT1attB ). It was shown that the complete set of A54145 biosynthetic genes could bedeleted from the native locus and expressed from a BAC vector very efficiently(>100% of control) by inserting them at either the φC31 or φBT1 attB site.In addition, the A54145 genes were expressed in strains of S. roseosporus andS. ambofaciens at efficiencies of 23% and 88% of the S. fradiae control [17].This relatively efficient heterologous expression established several cloning hostoptions for engineering the A54145 biosynthetic pathway.

The ectopic trans-complementation system in S. fradiae was tested further byexpressing the three NRPS genes from three different sites, and the recombinantproduced A54145 factors at 85% of control. In this case, transcription of the lptBCand lptD genes was expressed at ectopic loci from the ermEp* promoter, andthe lptA gene was expressed from its native promoter [17]. This ectopic trans-complementation established the use of both φC31 and φBT1 att/int systemsin S. fradiae to generate combinatorial libraries of novel lipopeptides related toA54145.

Subunit Exchanges The NRPS genes that catalyze the formation of daptomycin,A54145, and CDA peptides appear to have evolved from a common ancientancestral pathway [13]. Of particular note is that they all encode the enzymaticmachinery to produce cyclic peptides containing the rare amino acid, 3mGlu, at


the same position of the ring (Fig. 1; [13]). Also, all respective pathways havedimodular NRPS terminal subunits that incorporate 3mGlu at postition 12 andKyn, Ile, or Trp at position 13. Inspection of the N-terminal amino acid sequencesof the three NRPSs suggested that they have related interpeptide docking domainsthat might form single-coiled coil structures with DptBC [13].

The dptD gene was deleted in S. roseosporus , and the lptD or cdaPS3genes were inserted at the φC31 attB site. Recombinants produced the expectedhybrid lipopeptides CB-182,098 containing Trp13 or CB-182,107 containingIle13 (Table 3) at 50% and 25% of control, respectively (Table 4; [22,28]).Interestingly, when the CdaPS3 subunit was expressed under the control of theermEp* promoter in the S. roseosporus parent strain expressing the completedaptomycin pathway, the recombinant produced more than 50% of lipopeptidescontaining Trp13, and the overall yield of total lipopeptides was greater thanthat of the control [22]. This indicated that the heterologous CdaPS3 expressedfrom the ermEp* promoter competed well with the native DptD, suggestingthat the protein–protein interactions between related NRPS multienzymes arestrongly selected to maintain functionality, even in heterologous contexts. Thedata also support the conclusion that the TEs from LptD and CdaPS3 haverelaxed substrate specificities and can cyclize and release lipopeptides distantlyrelated to the native substrates. This promiscuity in docking, peptide coupling,and cyclization may be part of the natural evolutionary process for testing newcombinations of genes and proteins to generate novel lipopeptides.

Module and Domain Exchanges The successful subunit exchanges for DptDprovided a system to explore the efficiency and flexibility of module fusions atT-C linkers between C-A-T and C-A-T-TE modules in DptD. The system wasfirst tested by making homologous dptD C-A-T::C-A-T-TE fusions, inserting arestriction enzyme cleavage site in the T-C linker region to facilitate subsequentfusions. The flexibility of the linker was tested by generating three differentdouble-amino acid substitutions, a four-amino acid insertion, and a four-aminoacid deletion in the T-C linker. The reconstituted dptD genes were cloned intopHM11a and expressed from the ermEp* promoter at the IS117 attB site, and allrecombinants produced the same high levels of A21978C1–3 factors [37]. Thisresult indicated that the amino acid sequence and total length of the T-C linker(at least within ±4 amino acids) is not critical for efficient coupling of L-3mGluto L-Kyn.

Next, the system was tested by making C-A-T::C-A-T-TE fusions by ligat-ing the 3mGlu12 module from dptD to the Ile13 or Trp13 modules from lptDand cdaPS3 , respectively. The recombinants produced A21978C1–3 derivativescontaining Ile13 or Trp13 in high yields (79 and 123% of control, respectively;Table 4). The improvements in lipopeptide yields over those obtained withsubunit exchanges may be due primarily to conservation of the homologousprotein–protein docking relationship between DptBC and DptD. In addition, thisexperiment further demonstrated that the TE-domains from LptD and CdaPS3can effectively cyclize and release hybrid lipopeptides that differ substantiallyfrom their native substrates.

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TABLE 4 Efficiency of Production of Lipopeptides Following Subunit Exchanges,Module Exchanges, or Domain Fusions in dptD Expressed from an Ectopic Locus

Subunit D Structurea Lipopetide Product Relative Yield (%) Ref.

= CamGluT-CaKynTTe A21978C1 –3 100 [22]# CamGluT-CaKynTTe A21978C1 –3 96 [22]{}CAmGluT-CATrpTTe A21978C1 –3 (Trp13) 50 [22]{}CAmGluT-CAIleTTe A21978C1 –3 (Ile13) 25 [22]# CamGluT::CaKynTTe A21978C1 –3 100 [37]# CamGluT: CaKynTTe A21978C1 –3 99 [37]# CamGluT:::CaKynTTe A21978C1 –3 118 [37]#CamGluT::CATrpTTe A21978C1 –3 (Trp13) 123 [37]#CamGluT::CAIleTTe A21978C1 –3 (Ile13) 79 [37]#CamGluT::CAAsnT:: Te A21978C1 –3 (Asn13) 0 [37]#CamGluT::CAAsn::TTe A21978C1 –3 (Asn13) ∼43 [37]

a=, homologous protein–protein docking expressed from normal dpt gene cluster; #, homologousprotein–protein docking expressed from ectopic chromosomal locus; {}, heterologous protein–proteindocking from ectopic locus; -, normal interpeptide linker; ::, mutant interpeptide linker in frame;:::, mutant linker with four-amino acid insertion; :, mutant linker with four-amino acid deletion.Heterologous domains are shown in bold italic.

Since few C-A-T-TE modules were available to explore amino acid substitu-tions at position 13 in the daptomycin core peptide, the trans-complementationsystem in S. roseosporus was used to generate hybrid C-A-T-TE modules retain-ing the homologous DptD TE for ring closure. There are three possible ways togenerate hybrid C-A-T-TE modules with heterologous A-domains, and all requiredouble fusions: (1) insert an A domain (C-A-T-C::A::TTe); (2) insert a C-A di-domain (C-A-T::C-A::T-TE); or insert a C-A-T tri-domain (C-A-T::C-A-T ::TE).Since fusions at the T-C linker worked effectively in other experiments, and sinceit is important to keep homologous C-A di-domains together for mechanistic rea-sons (see below), only the C-A and C-A-T exchanges were explored [37]. TheC-A and C-A-T domains from the LptC C-AD-Asn11-T-E module were exchangedfor the C-A and C-A-T domains from the C-A Kyn13-T-TE module of DptD. Therecombinant expressing the C-A-T::C-A::T-TE double fusion under the controlof the ermEp* promoter at the IS117 attB site produced A21978C1–3 deriva-tives containing L-Asn13 at about 43% of control, but the strain expressing theC-A-T::C-A-T ::TE double fusion produced undetectable levels of lipopeptides(Table 4).

This work supports the conclusion that it is important to maintain ahomologous interaction between native T and TE domains, as strongly impliedfrom recent structural studies [38–40]. The T-domain from the C-AKyn13-T-TEmodule normally interacts with the upstream C- and A-domains as well as the TEdomain, whereas the T-domain from the C-AD-Asn11-T-E module normally inter-acts with the upstream C- and A-domains and the downstream E- and C-domains.Thus, their functions are very different, and apparently not interchangeable. Sincethere are at least three different types of T-domains from C-A-T, C-A-T-E, and


TABLE 5 Production Levels of Novel Lipopeptides by Recombinants ContainingModule or Multidomain Exchanges in DptBC

Lipopeptide YieldDptBC Structurea (% of Control)

CaOrn6T-CaAsp7T-CaD−Ala8TE-CaAsp9T-CaGlyT-CaD−Ser11TE 100CaOrn6T-CaAsp7T-CaD−Ala8TE-CaAsp9T-CaGlyT::CAD-Ala8T::E 50CaOrn6T-CaAsp7T-CaD−Ala8TE-CaAsp9T-CaGlyT::CAD-Asn11T::E 17CaOrn6T-CaAsp7T-CaD−Ala8TE-CaAsp9T-CaGlyT::CAD-Asn11TE* 10CaOrn6T-CaAsp7T-CaD−Ala8TE-CaAsp9T-CaGlyT::CAD-Asn11TE 9CaOrn6T-CaAsp7T::CAD-Ser11T::E-CaAsp9T-CaGlyT-CaD−Ser11TE 18CaOrn6T-CaAsp7T::CAD-Asn11T::E-CaAsp9T-CaGlyT-CaD−Ser11TE 10CaOrn6T-CaAsp7T::CAD-Asn11TE*::CaAsp9T-CaGlyT-CaD−Ser11TE 4

aThe fusion sites in the T-C, T-E, and E-C linkers are shown as (::), and the heterologous domains arein bold italic. The CAD-Asn11TE* module has a hybrid LptC/DptBC interpeptide docking sequence[13,24]. The CAD-Asn11TE module has the native LptC interpeptide docking sequence [13,25].

C-A-T-TE modules, we will refer to these domains as TC (T followed by a C), TE

(T followed by an E), and TTE (T followed by a TE) in subsequent discussions.In S. fradiae, the strain deleted for lptD gene could not be complemented

by dptD under the control of the ermEp* promoter [19]. However, a C-A-T::CAKyn::T-TE double fusion in lptD was expressed in a mutant S. fradiae strain(�lptD �lptI::tsr), and the recombinant produced an A54145 analog containingGlu12-Kyn13 (Table 2).

Exchanges of C-A-T tri-domains and C-A-T-E modules were carried out atpositions 8 and 11 in DptBC at interdomain T-E and E-C linkers (includingsome at interpeptide docking sites) using λ Red-mediated recombination on BACclones containing dpt genes in E. coli . Recombinant BACs were introduced intoa S. roseosporus strain deleted for the dpt pathway, and transconjugants pro-duced the predicted A21978C1–3 derivatives [24]. The hybrid lipopeptide yieldswere highest with the C-A-T exchanges (10 to 50% of control; Table 5). In thesecases the homologous interactions of E-domains with downstream DCL-domainswithin the DptBC protein or between DptBC and DptD were conserved. All con-structions also exchanged TE-domains that normally interact with downstreamE-domains, in addition to interacting with upstream A- and C-domains and down-stream C-domains. The exchange of the complete C-AD-Asn11-T-E module for theC-AD–Ser11-T-E module yielded a recombinant that produced hybrid lipopeptidesat 9% of control yields, thus indicating that the LptC C-terminal docking pep-tide interacted successfully with the N-terminal docking peptide of DptD. Thesetri-domain and module exchanges were coupled with subunit exchanges and dele-tion of the dptI gene to generate combinatorial libraries of lipopeptides (22–24;Table 3).

In S. fradiae, λ Red-mediated recombination was used to exchangesingle or multiple modules, or multidomains in plasmid pDA2048 con-taining lptBCD expressed from ermEp* or plasmid pDA2054 containinglptEFABCDGHJKLMNP genes expressed from the natural promoter(s) ([16,17];


Fig. 2b), using splicing sites located in the interdomain regions [20] similar tothose used in the dpt cluster [24,37]. Exchanges at position 8 eliminated thestop codon of lptB , generating fused lptBC genes. C-A-T tri-domain exchangesat positions 8 or 11 (both C-A-T-E modules as in dptBC ) in the fused lptBCgene were carried out in plasmid pDA2048 (Fig. 2B) and introduced intostrains DA740 (�lptBCD) and DA901 (�lptBC �lptI::tsr) at the φC31 attBsite, generating novel lipopeptides containing the desired changes coupledwith 3mGlu12 or Glu12. In all cases, the source of C-A-T tri-domains wasfrom C-A-T-E modules from dptBC (D-Ala8 and D-Ser11) or lptC (D-Asn11).Exchanges at positions 2, or 2 and 3 in lptA, or 2 to 8 in lptA and fused lptBCwere made in plasmid pDA2054 and introduced into strain DA1187 at the φC31attB , thus producing novel lipopeptides containing Glu12. The same changesat positions 2, 2 and 3, and 2 to 8 were coupled with 3mGlu12 by introducingplasmid pKN55 containing the dptIJ genes into the φBT1 attB site. Novellipopeptides with one to three amino acid substitutions were produced in yieldsof 3 to 48 mg/L (versus 300 mg/L in the control), the highest being produced bythe recombinant containing the C-ASer11-T tri-domain from dptBC exchangedfor the C-AAsn11-T tri-domain in lptC . In this case, the construction conservedthe TE configuration, and the native docking interaction between the module11 E-domain of lptC and the DCL of module 12 in lptD . The truly hybridlipopeptide containing seven contiguous amino acids (positions 2 to 8) from thedpt pathway, and the other six from the lpt pathway produced about 1 mg/L [20].

Disruption of Genes Involved in Amino Acid Modifications As discussed above,disruption of the dptI and lptI methyltransferase genes caused the production oflipopeptides containing Glu12. The S. roseosporus strain blocked in dptI producedA21978C factors containing Glu12 at about 50% of control total [23], whereas theS. fradiae mutant blocked in lptI function produced about 140% of control totalA54145 factors, all containing Glu12 [17]. A54145 factors have three additionalmodified amino acids: hAsn3, Sar5, and mOAsp9 (Fig. 1). The genes encoding theenzymes involved in the biosynthesis of hAsn and mOAsp have been disruptedindividually and in combinations [18]. Disruption of the lptL gene encodingan asparagine oxygenase caused the production of lipopeptides containing Asn3instead of hAsn3. Disruption of the lptK encoding a hAsp O-methyltransferasecaused the production of lipopeptides containing hAsp9 instead of mOAsp9, anddisruption of lptJ encoding an aspartic acid oxygenase produced Asp9. Onemutant containing a deletion of methyltransferase (M) domain of the CAGly5MTmodule in lptA produced a derivative of A54145 containing Gly substituted forSar. These experiments indicate that the NRPS multienzymes, including specificA domains involved in incorporation of modified amino acids, are capable ofprocessing noncognate amino acids related to the natural substrates to assemblerelated lipopeptides. The antibacterial properties of lipopeptides containing oneor more amino acid modifications are summarized in Table 3.

Initiation of Biosynthesis by Multiple Related Fatty Acids The biosynthesis ofA21978C and A54145 is initiated by the coupling of preferred fatty acids to

LESSONS LEARNED AND CONCLUSIONS 301

the N-terminus of Trp1. During A21978C biosynthesis, anteiso-undecanoyl, iso-dodecanoyl, and anteiso-tridecanoyl are preferred substrates for incorporationinitiated by the DptE and DptF acyl CoA ligase and ACP enzymes, whereasduring A54145 biosynthesis the shorter n-decanoyl, iso-decanoyl, and anteiso-undecanoyl are the preferred substrates for incorporation initiated by the fusedLptEF enzyme (Fig. 1; Table 1; [13,14,17,30]). It remains to be seen if thespecificity resides strictly in the acyl-CoA ligase and ACPs (DptE and DptF orLptEF), or if the FCL condensation domain of the Trp1 modules plays some role.The fatty acid preferences can be overridden by feeding high levels of otherfatty acids with varying chain lengths [13,14,26,41]. Therefore, all of the aminoacid modifications generated by genetic engineering are naturally coupled withmultiple lipidations, thus expanding the numbers of novel lipopeptides generatedby combinatorial biosynthesis. In some cases, lengthening the lipid side chaincan improve antibacterial activity. For example, the compounds related to CB-182,106 (with Val13 substituted for Kyn13; Table 3), but with iso-dodecanoyl oranteiso-tridecanoyl lipid side chains, had fourfold lower MICs against S. aureus,Enterococcus faecalis , and Enterococcus faeceum than that of CB-182,106 [22].

Antibacterial Properties of Novel Lipopeptides The amino acid modificationsand substitutions and antibacterial properties of a representative group of novellipopeptides produced by combinatorial biosynthsesis in S. roseosporus or S.fradiae are shown in Table 3. Antibacterial activities against S. aureus weredetermined with and without 1% bovine surfactant to identify candidates to treatS. pneumoniae pulmonary infections, because daptomycin activity is stronglyinhibited by pulmonary surfactant [7]. In vitro antibacterial activities in the pres-ence or absence of surfactant displayed by engineered daptomycin and A54145derivatives varied by over 100-fold. None of the daptomycin derivatives wereparticularly promising in the surfactant inhibition test, the best being the deriva-tive containing a Val substitution for Kyn13. However, several of the A54145derivatives had promising activities. The three most active derivatives of A54145(CB-182,561, CB-182,390, and CB-183,298), each with distinctly different pep-tide core modifications, had good MICs in the presence of 1% bovine surfactant(2 μg/mL) and were 32-fold more active than daptomycin in the presence ofsurfactant. These data indicate that molecular engineering and combinatorialbiosynthesis can successfully modify the fundamental properties of complexlipopeptide antibiotics.

IV. LESSONS LEARNED AND CONCLUSIONS

Ectopic Expression of NRPS Genes The native loci for the daptomycin andA54145 gene clusters are located near one end of their respective linear chromo-somes [27,42], whereas the φC31 attB and φBT1 sites are more central in the S.coelicolor genome [43]. The φBT1 attB site lies about 1 Mb to the right of oriC ,and φC31 attB lies about 90 kb to the left of oriC . Therefore, in S. fradiae thethree-site pathway reconstitution works well even though the individual enzymes


are expressed from three different loci, each likely to be separated by megabasestretches of DNA. In addition, the NRPS genes expressed from the ectopic lociwere transcriptionally driven by the strong constitutive ermEp* promoter. Sim-ilar results were obtained in ectopic trans-complementation experiments in S.roseosporus , using ermEp* to drive transcription of NRPS genes from φC31attB and IS117 attB sites [28]. Therefore, early expression of some NRPS genesdriven by a constitutively expressed promoter does not have detrimental effectson product yield in most cases. The use of a single constitutively expressedpromoter simplifies the design and emphasizes the general utility of ermEp*[44,45]

Adenylation Domains A-domains are critical to NRPS function and geneticengineering applications because they bind and activate specific amino acids.Each amino acid binding pocket has a well-defined 10-amino acid binding “code”[46,47]. In the daptomycin and A54145 pathways, A-domains are localized inmodules as C-A-T, C-A-T-E, C-A-M-T, or C-A-T-TE. In some cases, A-domainscan accept noncognate but related amino acids. For example, LptD module 13can bind and activate Ile (the primary substrate) or Val, and the ratios of fac-tors containing Ile or Val can be modulated by supplementing the fermentationmedium with Ile or Val [17,41]. In the work presented in this chapter, it is clearthat the A-domains for 3mGlu, hAsn, and mOAsp will also process Glu, Asn,and Asp (or hAsp), respectively, in mutants blocking the modifications. That begsthe question of how certain NRPS modules exclude the incorporation of modi-fied amino acids at other positions. For example, A54145 has D-Glu2, Asp7, andD-Asn11 residues, but these amino acids are not modified. One possibility is thatthe amino acid modifications take place on or in the vicinity of specific modules,generating substrates in situ at the appropriate A-domains [13]. This concept isdiscussed in more detail below.

Condensation Domains There are three types of C domains encountered in dap-tomycin and A54145 NRPS genes: FCL for coupling long-chain fatty acids toL-amino acids; LCL for coupling L-amino acids to L-amino acids; and DCL forcoupling D-amino acids to L-amino acids. In vitro enzyme studies have shownthat the C domain of tyrocidine module 2 has an acceptor position for the down-stream T-bound amino acid nucleophile that discriminates against D-isomers anddifferences in the side chain, but the donor position had low specificity for theside chain of the upstream T-bound peptide electrophile [48]. More recently,structural studies on SurA-C (C-A-T-TE) showed that the C and A domains areclosely engaged and the C-A linker makes multiple contacts with the catalyticplatform [40,49]. In contrast, the A-T linker lacks any contacts with the catalyticplatform. This reinforces the notion that it is prudent, and perhaps necessary, tomaintain natural C-A di-domain relationships when engineering NRPSs. Choos-ing the appropriate type of C-domain (FCL,LCL, or DCL) is also critical forsuccessful engineering. In the studies described in this chapter, A-domains havebeen transplanted successfully in NRPSs as C-A di-domains, C-A-T modules,C-A-T tri-domains, and C-A-T-E modules.

LESSONS LEARNED AND CONCLUSIONS 303

Thiolation Domains T-domains are highly flexible, dynamic proteins that carryout multiple tasks [39,40,50,51]. From a functional perspective, there are threetypes of T-domains in the Dpt and Lpt NRPS proteins. TC-domains are found in17 C-A-T modules in the Dpt and Lpt NRPSs. TC-domains have a core functionof interacting with the upstream C- and A-domains, and downstream C-domainsduring peptide assembly. TE-domains are present in six C-A-T-E modules in Dptand Lpt NRPSs. In addition to the core function, TE-domains interact with theadjacent E-domains during epimerization. TTE-domains (one each in DptD andLptD) have the upstream core functions and they interact with TE-domains duringcyclyzation and release of the finished lipopeptides. All of the successful geneticengineering constructs described in this chapter maintained or reconstituted mod-ules containing T-domains to conserve the functional relationships as C-A-TC-C,C-A-TE-E-C, or C-A-TTE-TE. The most telling experiment directly addressingT-domain specificity was one where parts from a C-A-TE-E (front end) and aC-A-TTE-TE (back end) were assembled as C-A-TE::TE or C-A::TTE-TE . Thefirst construct with the wrong T-domain (TE) failed to yield product, whereas theconstruct that maintained the native TTE-TE relationship worked effectively.

Epimerase Domains E-domains are important to set the stereochemistry of dap-tomycin and A54145. The E-domains in the Dpt and Lpt NRPS multienzymesare located at positions 2, 8, and 11. In all six cases they are embedded in C-A-TE-E modules followed by modules in the same or downstream protein withDCL-domains. Because of the upstream TE relationship, and the downstream DCL

constraints, all constructions were carried out to maintain the functional interac-tions by exchanging complete C-A-TE-E modules or C-A-TE tri-domains. Bothtypes of exchange worked, but C-A-TE exchanges resulted in higher lipopeptideyields. C-A-TE exchanges maintain the functional interaction of the heterologousC-A di-domain, and the native interaction between the E- and the downstreamDCL-domains, while exchanging functionally equivalent TE-domains.

Thioesterase Domains The TE domains are essential for the cyclization andrelease of completed lipopeptides. The subunit exchange and module exchangeexperiments with DptD, LptD, and CdaPS3 demonstrated that the TE domainsfrom LptD and CdaPS3 work very efficiently with heterologous substrates closelyrelated to daptomycin. In addition, DptD TE carried out functional ring closureof a number of engineered lipopeptides related to daptomycin, including onewith the noncognate substrate for ring closure, L-Asn13 [37]. In addition, theLptD TE catalyzed ring closure with the noncognate terminal Kyn13 [19]. Thesein vivo studies are generally consistent with in vitro chemoenzymatic studiescarried out with cloned T-TE di-domains [52,53]. The substrate promiscuity ofthe TE-domains may represent an important component in natural evolution oflipopeptides, and facilitates the genetic engineering to produce new lipopeptides.

Interpeptide Linkers Certain interpeptide linkers that connect the individualdomains within modules or that connect modules can be used effectively to


engineer NRPSs. The studies described in this chapter demonstrate that wholemodules can be exchanged by making fusions at intermodule T-C linkers, andrecombinants produced a high yield of hybrid lipopeptides. For example, the T-Clinker in DptD is flexible and can be modified by double amino acid substitutions,and deletions or additions of four amino acids without harming productivity.This flexibility opens up the possibility of engineering the nucleotide sequenceof the linker to insert restriction enzyme cleavage sites that facilitate modulefusions. Successful C-A-T-E module exchanges have been made at intermoduleE-C linkers, C-A di-domain exchanges have been made at A-T linkers, and C-A-Ttri-domain exchanges have been made at T-E linkers.

Amino Acid Modifications The methyltransferases (DptI, LptI, and GlmT) thatparticipate in the formation of 3mGlu during lipopeptide biosynthesis have acommon substrate, α-KG, which is methylated to (3R)-3-methyl-2-oxoglutarate,a substrate for transamination by enzymes from primary metabolism to form(2S,3R)-3-methyl glutamate (3mGlu) [34]. If the methyltransferases simply cat-alyze the methylations in the cytoplasm, the respective dptI, lptI , and glmT genesshould be orthologs, and their enzyme products should show high amino acidsequence conservation, similar to that of other orthologous proteins in strepto-mycetes (e.g., ≥80% amino acid identities). However, in pairwise comparisons,DptI, LptI, and GlmT display only 35 to 37% sequence identities (13; Table 3).This percent sequence identity is substantially lower than the sequence identitiesobserved in the nonorthologous but related NRPS genes (Table 1). For instance,DptD and LptD share 53% identical amino acids, even though they catalyzethe incorporation of different amino acids at position 13. DptP and LptP, on theother hand, may be orthologous, because they display 94% sequence identity. It isapparent that DptI, LptI, and GlmT are not typical orthologous proteins, althoughthey have undoubtedly derived from a common ancestral gene: their genes andproteins are more closely related to each other than to other methyltransferasegenes and proteins. This situation poses a conundrum. If these genes are paralogs,they must have nonorthologous functions in addition to the methylation of α-KG.

One possible paralogous function would be binding to and interacting with spe-cific NRPS modules that incorporate 3mGlu into the growing lipopeptide chainsduring peptide assembly [13]. This model addresses two questions: (1) How canthe production of 3mGlu be excluded from normal cytoplasmic metabolism anddirected into lipopeptide assembly exclusively, thus avoiding dilution and pos-sible toxic incorporation into proteins; and (2) Why is 3mGlu incorporated atposition 12 but not at position 2 during A54145 biosynthesis? Both 3mGlu mod-ules in DptD and LptD can incorporate Glu at varying efficiencies if 3mGlu is notavailable, so the discrimination against 3mGlu at position 2 is probably not dueto stringent amino acid substrate specificity. The same argument can be made forthe incorporation of hAsn at position 3 but not position 11 in A54145 assembly.In this case, the amino acid–binding pockets in the respective A-domains havethe nearly identical binding codes (DLTKVGDVN for hAsn3 and DLTKVGDVSfor D-Asn11; [11]). Similarly, the mOAsp9 binding pocket is identical to the three

REFERENCES 305

Asp binding pockets in the CDA pathway, and differs from the binding pocketsof the Asp7 module in A54145 and the Asp7 and Asp9 modules in the dapto-mycin NRPS by a single conserved Ile versus Leu or Val at position 299 [11].Disruption of any of the genes encoding enzymes that modify Asn or Asp in S.fradiae led to productive incorporation of unmodified or partially modified aminoacids, consistent with the notion that the respective binding pockets do not dis-criminate for or against the use of modified amino acids, yet the production ofA54145 factors containing Asn3 or Asp9 is not observed in the wild-type strain.Therefore, the production of modified amino acids in situ at the A domains is anattractive model that can be tested experimentally.

Combinatorial Biosynthesis The daptomycin and A54145 biosynthetic pathwaymanipulations demonstrate that combinatorial biosynthesis is feasible and canyield in practical titers interesting new structures not obtainable by medicinalchemistry. The NRPS multienzymes can be altered by a number of types ofgenetic manipulations as described here, amino acid modification patterns can bealtered by gene disruptions, and lipid side chains can be varied naturally or byfeeding alternative substrates. The tools for facile engineering in E. coli , partic-ularly the use of λ Red-mediated recombination, for conjugal transfer from E.coli to Streptomyces , for site-specific insertion into streptomycete chromosomes,and for the expression of different genes or sets of genes from ectopic loci underthe control of the ermEp* promoter worked well for these two pathways, andshould be generally applicable to other complex secondary metabolite pathways.The “rules” for successful engineering deduced from structural studies carriedout primarily in the Walsh and Marahiel laboratories and from the outcomes ofthe genetic engineering experiments described here should be applicable to otherNRPS pathways.

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Enzyme Technologies (Metagenomics, Evolution, Biocatalysis, and Biosynthesis) || Reprogramming Daptomycin and A54145 Biosynthesis to Produce Novel Lipopeptide Antibiotics