Natural echinocandins, including FR901379 and its analogues, pneumocandins, aculeacins, mulundocandins, and cryptocandin, are the first class of antifungals to target fungal cell wall^[1-3]. These natural echinocandins are cyclic hexapeptides with a long fatty acyl side chain, produced by some fungi, such as Aspergillus strains, Coleophoma strains, a Chalara strain, Glarea lozoyensis, Cryptosporiopsis strains, et al^[3-16]. Recently, the echinocandin B (ECB) biosynthetic gene cluster from Emericella rugulosa NRRL 11440 has been identified and characterized^[17]. Three semisynthetic echinocandin derivatives, including micafungin, anidulafungin, and caspofungin, have been widely used in clinic^[18]. Caspofungin is synthesized by chemical modifications of cyclic hexapeptide scaffold of pneumocandins B₀^[19]. Synthesis of micafungin and anidulafungin includes two steps: hydrolytic removal of fatty acyl side chains from FR901379 and ECB to generate cyclic hexapeptide nuclei, and addition of artificial acyl side chains into the nuclei^[20-21]. Hydrolysis of FR901379 can be catalyzed by FR901379 acylases from several Streptomyces strains^[3,22-24], several Oidiodendron strains^[23,25], and a Verticillium strain^[23]. Hydrolysis of ECB can be catalyzed by ECB deacylase from Actinoplanes utahensis NRRL 12052^[3,26-29] (Fig. 1). Recent reports on the genomic DNA of A. utahensis NRRL 12052 suggest that ECB deacylase is indeed aculeacin A acylase (AAC)^[30]. Thus, AAC and FR901379 acylases have the potential to be a green biocatalyst for pharmaceutical industry.

AAC from A. utahensis NRRL 12052 is a membrane-associated heterodimer consisting of ≈63 kDa and ≈19 kDa subunits. These two subunits and a signal peptide are translated as one peptide^[28]. It has been well characterized that AAC has a broad substrate specificity for ECB and several semisynthetic ECB derivatives^[27-28], aculeacins^[29,31-32], several penicillins^[32-33], several side chain analogues of cilofungin^[29], daptomycin and its three side-chain analogues^[34], teicoplanin^[35], pseudomycin A^[28], and capsaicins^[36]. Active AAC can be produced by heterologous expression of its gene in Streptomyces strains, such as S. lividans^{[26,28,33,37]}, S. griseus^[28,37], and S. albus^[26]. In this study, Actinoplanes utahensis SW1311 with acylase activity was isolated for both FR901379 and penicillin Ⅴ, and the AAC gene from A. utahensis SW1311 was cloned. The result showed that a recombinant Streptomyces coelicolor A3(2) harboring AAC gene from A. utahensis SW1311 produced AAC with higher activity and required shorter fermentation time compared with A.utahensis SW1311. The results not only provide a new application of AAC for micafungin synthesis, but also identify a new suitable host for AAC gene.

1 Materials and methods 1.1 Strains, growth, and culture conditions

A. utahensis SW1311 was isolated in Zhejiang Province, China. The A. utahensis SW1311 was grown on ISP2 solid medium (1% malt extract, 0.4% glucose, 0.4% yeast extract, and 2% agar, pH 7.0) at 30 ℃ for 8 days. A. utahensis SW1311 was fermented under two conditions. ConditionⅠ: A. utahensis SW1311 mycelia from ISP2 solid medium was inoculated into 30 mL of mediumⅠ(3% glucose, 0.5% peptone, 0.1% K₂HPO₄, 0.05% KCl, 0.05% MgSO₄, and 0.000 2% FeSO₄) in a 250 mL flask and grown at 30 ℃, 200 r/min for 48 h. Then 3 mL of the above culture was inoculated into 30 mL of mediumⅠin a 250 mL flask and grown at 30 ℃, 200 r/min for 72 h. ConditionⅡ: A. utahensis SW1311 mycelia from ISP2 solid medium was inoculated into 30 mL of seed mediumⅡ(1% malt extract, 2% glucose, 1% cotton seed meal, 1% yeast powder, 1% peptone, 1% soluble starch, and 0.2% CaCO₃, pH 6.5) in a 250 mL flask and grown at 30 ℃, 200 r/min for 48 h. Then 3 mL of the above culture was inoculated into 30 mL of fermentation medium Ⅱ (3% sucrose, 2% cotton seed meal, 2% yeast powder, 0.05% K₂HPO₄, 0.1% KCl, 0.1% MgSO₄, 0.2% CaCO₃, and 0.05% antifoamer, pH 6.5) in a 250 mL flask and grown at 30 ℃, 200 r/min for 72 h.

The recombinant strain sSCO-AAC and S. coelicolor A3(2) were grown on MS solid medium (2% mannitol, 2% soybean powder, and 2% agar) at 30 ℃ for 4 days. sSCO-AAC and S. coelicolor A3(2) mycelia from MS solid medium were inoculated into 30 mL of 2 ×YT medium (1.6% tryptone, 1% yeast extract, and 0.5% NaCl) in a 250 mL flask and grown at 30 ℃, 200 r/min for 36 h. Then 3 mL of the above culture was inoculated into 30 mL of YEME (yeast extract malt extract) medium (34% sucrose, 1% glucose, 0.5% tryptone, 0.3% yeast extract, 0.3% malt extract, and 5 mmol/L MgCl₂) in a 250 mL flask and grown at 30 ℃, 200 r/min for 48 h.

1.2 Characterization of the acylase activity by using penicillin Ⅴ as a substrate

The mycelia and the supernatant of A. utahensis SW1311 culture, sSCO-AAC culture, and S. coelicolor A3(2) culture were separated by centrifugation under 8 000 g for 10 min at 4 ℃, respectively. Then 0.5 mL of the supernatant was added into 0.1 mL of 428 mmol/L penicillin Ⅴ in 100 mmol/L potassium phosphate buffer (pH 8.0). The reaction mixture was incubated at 45 ℃ for 30 min and stopped by addition of 0.4 mL acetate. Then 0.75 mL of the above-obtained mixture was added into 0.25 mL of 5 g/L p-dimethylaminobenzaldehyde (PDAB) solution and then centrifuged to remove the precipitate. The released 6-aminopenicillanic acid (6-APA) was monitored by ultraviolet (UV) absorbance detection of the final mixture at 415 nm^[32-33,38].

1.3 Production of FR901379 nucleus

FR901379 (140 mg, 50% purity) was added into 30 mL of A. utahensis SW1311 culture or sSCO-AAC culture and incubated at 30 ℃ for various time. The above reaction mixture was centrifuged to remove the precipitate and then filtered with 0.22 μm film. The FR901379 nucleus production was determined using high performance liquid chromatography (HPLC) system (Agilent 1100 Series; Agilent Technologies, USA) equipped with a YMC-Pack ODS-AM column (250 mm × 4.6 mm, YMC, Kyoto, Japan). The column temperature was maintained at 25 ℃ and the UV detector was set at 210 nm. The mobile phase contained 0.5% NaH₂PO₄ and 3% acetonitrile with a flow rate of 1 mL/min.

1.4 Cloning of AAC gene from A. utahensis SW1311

AAC gene was amplified by polymerase chain reaction (PCR) from the genomic DNA of A. utahensis SW1311 by using primer pair PriAAC5 (G CGC CAT ATG ACG TCC TCG TAC ATG CG) and PriAAC3 (G CGC AAG CTT TCA GCG TCC CCG CTG TGC CA). The PCR mixture (50 μL) contained ≈200 ng of genomic DNA, 0.3 μmol/L each of two primers, 0.2 mmol/L each of dNTPs, 1.5 mmol/L MgSO₄, 5μL of 10×PCR buffer for KOD-Plus-Neo (Toyobo, Japan), and 1 unit of KOD Plus-Neo DNA polymerase (Toyobo, Japan). The following PCR condition was repeated for 30 cycles: 98 ℃ for 10 s, 60 ℃ for 30 s, and 68 ℃ for 90 s. Then the PCR product was cloned as an Nde Ⅰ/Not Ⅰ DNA fragment into pTE28a vector and sequenced to confirm the fidelity.

1.5 Construction of a recombinant strain sSCO AAC

A site-specific integration vector pIJ8660, containing egfp, aac(3) Ⅳ, int ФC31, and attP, was used to construct an integrative recombinant plasmid^[39]. The egfp was replaced with ermEp* promoter and a multiple cloning site to generate plasmid pSN1. AAC gene from pET28a was cloned as an NdeⅠ/NotⅠ DNA fragment into the same sites of pSN1 to produce plasmid pIJ-AAC. The pIJ-AAC was introduced into E. coli ET12567 (pUZ8002) for E. coli-S. coelicolor A3(2) conjugation using standard procedures^[40]. The recombinant strain sSCO-AAC was selected from apramycin-resistant exconjugants and confirmed by PCR using primer pair Pri53 (TCC TAA GGA TCC GGC GGC TTG CGC CCG ATG CTA GTC) and PriAAC3.

2 Results and discussion 2.1 Acylase activity of A. utahensis SW1311 culture

Penicillin Ⅴ was used as a substrate to characterize the acylase activity of A. utahensis SW1311, which was isolated from Zhejiang, China, rapidly and economically as described previously^[32-33,38]. A. utahensis SW1311 was grown under various conditions, including different media, growth time, fermentation temperature, etc. The mycelia and the supernatant of A. utahensis SW1311 culture were separated by centrifugation. Penicillin Ⅴ was incubated with the supernatant, and the released 6-aminopenicillanic acid (6-APA) was monitored with fluorescamine (Fig. 1). By using seed medium Ⅱ and fermentation mediumⅡas seed medium and fermentation medium respectively, an unexpected degradation of ECB nucleus was detected when ECB was incubated with the culture supernatant. By using mediumⅠas both seed medium and fermentation medium, the unexpected degradation of ECB nucleus was mild (data not shown). Thus, the acylase activity was quantified under the latter fermentation condition. The result showed the acylase activity achieved the highest level (0.444 unit/mL) when A. utahensis SW1311 was grown in mediumⅠas seed medium at 30 ℃ for 48 h and then fermented in medium Ⅰas fermentation medium at 30 ℃ for 72 h. When the growth temperature changed from 30 ℃ to 28 ℃ and 32 ℃, the acylase activity decreased by 60% and 40%, respectively (Table 1).

2.2 Production of FR901379 nucleus catalyzed by A. utahensis SW1311 culture

FR901379 was used as a substrate to test whether the acylase activity of A. utahensis SW1311 could be applied to biosynthesize FR901379 nucleus. FR901379 was purified by 50% from the culture of a FR901379 producing strain Coleophoma empetri^[15-16]. A. utahensis SW1311 culture grown in medium Ⅰ as fermentation medium at 30 ℃ for 72 h was added with various concentrations of FR901379 and then incubated at 30 ℃. The production of FR901379 nucleus was monitored by HPLC periodically (Fig. 2). When the final concentration of FR901379 was 2.00 g/L or 3.00 g/L, the highest conversion yield (about 85%) was achieved and the incubation time was 16 h. When the final concentration of FR901379 increased to 4.00 g/L and 5.00 g/L, the highest conversion yield both decreased by 81.9% and 73.6% respectively, and also required longer incubation time (22 h and 24 h) (Table 2).

2.3 Cloning of AAC gene from A. utahensis SW1311

A pair of PCR primers, based on AAC gene from A. utahensis NRRL 12052 (GenBank accession number JRTT00000000)^[30], were designed to clone AAC gene from A. utahensis SW1311. AAC gene was amplified by PCR from the genomic DNA of A. utahensis SW1311 and sequenced. Protein sequence alignment showed that AAC from A. utahensis SW1311 was 100%/97% identical with AACs from A. utahensis NRRL 12052 and another A. utahensis strain (GenBank accession number D10610)^[37], respectively (Fig. 3).

2.4 Construction of a recombinant strain sSCO AAC

AAC gene was cloned into a modified integration vector pIJ8660^[39] to construct a plasmid pIJ-AAC, in which AAC gene was under the control of ermEp*, a strong promoter in Streptomyces. The pIJ-AAC was introduced into S. coelicolor A3(2) to make AAC gene overexpression recombinant strain sSCO-AAC. sSCO AAC was confirmed by apramycin resistance and PCR using specific primers for amplification of the ermEp*-AAC fusion gene.

2.5 Acylase activity of sSCO-AAC culture

The acylase activity of the recombinant strain sSCO-AAC was characterized by using Penicillin Ⅴ as a substrate. The acylase activity reached the highest level (2.060 unit/mL) when sSCO-AAC was grown in 2×YT medium as seed medium at 30 ℃ for 36 h and then fermented in YEME medium as fermentation medium at 30 ℃ for 48 h. The acylase activity was not detected by using S. coelicolor A3(2) culture as a control, indicating that the acylase activity came from the heterologous production of AAC in S. coelicolor A3(2) (Table 1). Thus, the acylase activity of sSCO AAC was 4.6-fold higher than that of A. utahensis SW1311. The total fermentation time of sSCO-AAC was 36 h, which was 30% shorter than that of A. utahensis SW1311.

2.6 Production of FR901379 nucleus catalyzed by the sSCO-AAC culture

sSCO-AAC culture grown in 2×YT medium as fermentation medium at 30 ℃ for 48 h was added 2.00 g/L of FR901379 and then incubated at 30 ℃. After incubation for 3 h, the conversion yield reached 88.1% (Fig. 2,Table 2). Thus, the required incubation time for sSCO-AAC culture was less than 1/5 of that for A. utahensis SW1311 culture. Additionally, the total area of other peaks in Fig. 2D is smaller than that in Fig. 2C, suggesting that the by-products of the reaction mixture catalyzed by sSCO-AAC culture are less than those of A. utahensis SW1311 culture.

3 Conclusion

The AAC produced by A. utahensis SW1311 can catalyze the hydrolysis of FR901379. Heterologous production of this AAC in a recombinant S. coelicolor A3(2) resulted in higher yield of AAC and shorter fermentation time compared with that of A. utahensis SW1311. Therefore, this recombinant S. coelicolor A3(2) has the potential to be applied in the industrial production of micafungin. These results here not only expand the application of AAC for micafungin production, but also identify S. coelicolor A3(2) as a suitable host for AAC expression.

Acknowledgements: We thank Dr. WANG Yueyue from School of Life Sciences, Fudan University for helpful discussions during the preparation of the manuscript.