Heterologous production of daptomycin in Streptomyces lividans
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- Penn, J., Li, X., Whiting, A. et al. J IND MICROBIOL BIOTECHNOL (2006) 33: 121. doi:10.1007/s10295-005-0033-8
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.
KeywordsDaptomycinA21978CCalcium-dependent antibiotic (CDA)ActinorhodinStreptomyces lividans
We describe improvement of the yield of the A21978C lipopeptides in S. lividans through minimizing expression of the host background metabolites. This was done using both genetic and medium optimization approaches. The first heterologous production experiments for the A21978C lipopeptide in S. lividans used TK64 and TK23 strains with an intact actinorhodin pathway [15–17]. The actinorhodins are colored polyketides produced in copious quantities by S. coelicolor and some strains of S. lividans under many fermentation conditions, and interfere with the detection and purification of other secondary metabolites from the fermentation. As a consequence, medium development to enhance expression of the heterologous NRPS pathway in S. lividans was restricted to formulations that minimize the production of actinorhodin. However, S. lividans is also capable of producing large amounts of other metabolites that, in comparison, dwarfed the A21978C lipopeptides produced. It was therefore desirable to minimize the production of host metabolites in order to achieve cleaner chromatographic profiles for easier detection and purification of new compounds produced by recombinant strains.
Materials and methods
Strains and plasmids
Strains and plasmids
Streptomyces lividans 66
TK64 (pStreptoBac V)
TK23 (pStreptoBac V)
TK23-521 (pStreptoBac V)
BAC vector with oriT, Amr, att/intϕC31
pStreptoBac V::128 kbp dpt region
pStreptoBac V::90 kbp dpt region
Transformation of S. lividans
Construction of Act− S. lividans
Culture media and growth conditions
Although a number of different media were initially explored, two were examined in more detail for their ability to support production of the A21978C lipopeptides in S. lividans. Both media also support good production of the A21978C lipopeptides in S. roseosporus. Medium A was a complex medium consisting of 1% glucose (BDH), 2% soluble starch (Sigma), 0.5% yeast extract (Difco), 0.5% casein (Sigma) and 4.6% MOPS (Sigma), adjusted to pH 7 and autoclaved. Medium B was a defined medium consisting of 2% glycerol, 0.25% sucrose, 1.2% L-proline, 1.5% MOPS, 0.056% K2HPO4, 0.05% NaCl, 1% Tween 80, 0.5% trace salts solution (per liter: 1 ml 1 M H2SO4, 1.722 g ZnSO47H2O, 1.112 g FeSO47H2O, 0.223 g MnSO44H2O, 0.062 g H3BO3, 0.125 g CuSO45H2O, 0.048 g Na2MoO42H2O, 0.048 g CoCl26H2O, 0.083 g KI), 0.2% vitamin mixture solution (per liter: 0.025 g thiamine, 0.025 g riboflavin, 0.025 g pantothenate, 0.025 g nicotinic acid, 0.025 g pyridoxine, 0.025 g thioctic acid, 0.0025 g folic acid, 0.0025 g cyanocobalamin, 0.0025 g p-aminobenzoic acid, 0.05 ml vitamin K1, 2 ml Tween 80), 0.02% 10 mg/l CaCl22H20, 0.02% 10 mg/l MgSO47H2O, 0.00055% FeSO47H2O, adjusted to pH 7 and filter sterilised.
Fermentations were initiated by inoculation of an enriched oatmeal slant (medium A9 ) containing 100 mg/l apramycin with approximately 0.25 ml of biomass from a cryovial stored at −135°C. After 7–10 days incubation at 28°C, a mixed mycelial and spore suspension was generated by the addition of 4 ml 0.1% Tween 80, and 2 ml were inoculated into 40 ml of seed medium A345  containing 25 mg/l apramycin in a baffled flask to initiate the seed stage. Seed flasks were shaken at 240 rpm and 30°C for 24–28 h before a 5% transfer to production flasks containing 50 ml of medium A or B without apramycin (to facilitate detection of A21978C production through its antibacterial activity; data not reported here). Replicate flasks were sampled from day 2 until day 6 of the production fermentation by aseptically removing approximately 1 ml broth and centrifuging it for 10 min at 10,000 rpm.
Analysis by HPLC, HPLC-MS and NMR
Broth supernatants were analyzed by HPLC at ambient temperature using a Waters Alliance 2690 HPLC system and 996 PDA detector with a 4.6×50 mm Symmetry C8 3.5 μm column and a Phenomenex Security Guard C8 cartridge. The mobile phase, buffered with 0.10% trifluoracetic acid, was initially held at 10% acetonitrile(aq) for 2.5 min, followed by a linear gradient over 6 min to 100% acetonitrile and re-equilibration after a further 3.5 min; the flow rate was 1.5 ml/min. Up to 50 μl of the supernatant was injected to monitor the production of the native A21987C lipopeptides; their concentrations were determined by comparison to reference daptomycin purified from S. roseosporus fermentations and provided by Cubist Pharmaceuticals, Inc., Manufacturing Dept. Preparative HPLC was performed on a radially compressed cartridge column consisting of two 40×100 mm Waters Nova-Pak C18 60 Å 6 μm units and a 40×10 mm Guard-Pak with identical packing.
Mass spectrometric (MS) data were obtained by LC-MS analysis on a Finnigan SSQ710C system using electrospray ionization in positive ion mode, with a scan range of 200–2,000 daltons and 2 s scans. The LC method was run at ambient temperature on a Waters Symmetry C8 column (2.1×50 mm 3.5 μm particle size). The initial conditions of 90% water, 10% acetonitrile and 0.01% formic acid were maintained for 0.5 min, followed by a linear gradient to 100% acetonitrile and 0.01% formic acid over 6 min, and this composition was held for 3.5 min before re-equilibration; the flow rate was 0.35 ml/min. The electrospray capillary voltage was 21.2 V with a collisional induced dissociation offset of 0 or −10 V. The capillary temperature was maintained at 250°C. 1 H and 13 C spectra were recorded in DMSO-d6 at 308 K using a Bruker ACF400 spectrometer at 400 and 100 MHz, respectively.
Results and discussion
Streptomyces lividans TK64 harbors str-6, a Lys88 to Glu mutation in ribosomal protein S12 (encoded by rpsL) that not only confers resistance to streptomycin, but also has been implicated in enhancing the production of actinorhodin in S. lividans TK24 . As the high level of actinorhodin production might be related to the rpsL mutation present in the host, pCV1 was also introduced into S. lividans TK23, which lacks the str-6 marker  but the same result was obtained, production of both A21978C and high levels of actinorhodin. Furthermore, similarly high levels of actinorhodin were also produced by control strains of both TK64 and TK23 carrying only the BAC vector even though neither strain could produce the A21978C lipopeptides.
It was difficult to accurately quantify the A21978C lipopeptides produced in crude broth by these strains due to co-chromotography with host peaks. A total maximum yield of the three main A21978C factors was estimated at around 20 mg/l. These were produced early in the fermentation along with numerous other host metabolites. One series of host metabolites caused particular problems for A21978C analysis. These had UV/visible spectra with λmax at 222, 281 and 345 nm. LC-MS analysis suggested molecular weights of 1,494, 1,500 and 1,514. These are similar, but not identical to, the non-phosphorylated members of the CDA complex (1,494 corresponds to CDA4a; 1,500 and 1,514 have not previously been reported; the UV-visible maxima correspond to members of the complex containing a Z-2,3-dehydrotryptophan residue) . For further characterization of these compounds, a number of other S. lividans strains containing smaller DNA fragments cloned from S. roseosporus that were known not to produce A21978C  were examined. One of these, CBUK136744, containing an 90 kbp DNA insert, was an effective producer of the putative CDA factors and the absence of A21978C production yielded a simpler chromatographic profile, facilitating purification. A 5l fermentation was conducted in multiple shake flasks and the component produced in the highest abundance (that with putative molecular weight 1,514) was purified by preparative HPLC. NMR spectroscopic analysis clearly indicated the presence of two tryptophan residues, and supported the presence of one hydroxyphenylglycine residue, one serine and at least two aspartate residues and methyl groups consistent with the presence of 3-methyl-glutamic acid and a short alkyl chain. These features are as expected for a member of the CDA complex. The compound was not characterized further.
In conclusion, we demonstrated that daptomycin can be produced in S. lividans when the daptomycin gene cluster is inserted site-specifically in the chromosomal ϕC31 attB site. The yields were improved to 55 mg/l, about one-third the amount produced by wild type S. roseosporus, by adjusting the level of phosphate in the medium, and compound isolation was facilitated by deleting the act gene cluster. Surprisingly, the conditions that favored the expression of the heterologous daptomycin gene cluster also enhanced the expression of the otherwise cryptic CDA pathway in all the modified TK23 and TK64 S. lividans strains studied. Since both lipopeptide pathways compete for some of the same precursors, deletion of the CDA pathway should facilitate further improvement in daptomycin yields, and product isolation. The cloning of complete antibiotic or other secondary metabolite pathways on BAC vectors, coupled with the stable insertion into the chromosome of robust streptomycetes, such as S. lividans in the present work, should serve as a particularly useful approach to express biosynthetic gene clusters from slow-growing actinomycetes not amenable to facile scale-up in large scale fermentation.