Application of an aculeacin A acylase from Actinoplanes utahensis SW1311 in syntheses of echinocandins

FENG Guodong,XU Yongfeng,CHEN Jianfeng,ZHAO Wanzhong,WANG Xin,ZHANG Xiaosheng,JIANG Hui(.Hangzhou Huadong Medicine Group Pharmaceutical Research Institute Co.,Ltd.,Hangzhou 300,China;2.Qiushi College,Zhejiang University, Hangzhou 30058,China;3.College of Life Sciences,Zhejiang University,Hangzhou 30058,China)

SummaryEchinocandins are the first class of antifungals to target fungal cell wall.Two antifungals,including micafungin and anidulafungin,have been widely used in clinic.The synthesis of them includes an essential process: hydrolytic removal of fatty acyl side chains from FR901379 and echinocandin B(ECB)to generate cyclic hexapeptide nuclei.In this study,Actinoplanes utahensis SW1311 with acylase activity was isolated for both FR901379 and penicillin V.Then a recombinant Streptomyces coelicolor A3(2)harboring aculeacin A acylase(AAC)gene from A.utahensis SW1311 was constructed.The result showed that the AAC activity produced by the recombinant strain was 4.6-fold higher than that of A.utahensis SW1311.Additionally,the fermentation time of the recombinant strain was 30%shorter than that of 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.

Application of an aculeacin A acylase from Actinoplanes utahensis SW1311 in syntheses of echinocandins

FENG Guodong1†,XU Yongfeng1†,CHEN Jianfeng1,ZHAO Wanzhong1,WANG Xin1,ZHANG Xiaosheng2*,JIANG Hui3*(1.Hangzhou Huadong Medicine Group Pharmaceutical Research Institute Co.,Ltd.,Hangzhou 310011,China;2.Qiushi College,Zhejiang University, Hangzhou 310058,China;3.College of Life Sciences,Zhejiang University,Hangzhou 310058,China)1

SummaryEchinocandins are the first class of antifungals to target fungal cell wall.Two antifungals,including micafungin and anidulafungin,have been widely used in clinic.The synthesis of them includes an essential process: hydrolytic removal of fatty acyl side chains from FR901379 and echinocandin B(ECB)to generate cyclic hexapeptide nuclei.In this study,Actinoplanes utahensis SW1311 with acylase activity was isolated for both FR901379 and penicillin V.Then a recombinant Streptomyces coelicolor A3(2)harboring aculeacin A acylase(AAC)gene from A.utahensis SW1311 was constructed.The result showed that the AAC activity produced by the recombinant strain was 4.6-fold higher than that of A.utahensis SW1311.Additionally,the fermentation time of the recombinant strain was 30%shorter than that of 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.

micafungin;FR901379;echinocandin;antifungal;aculeacin A acylase;Actinoplanes utahensis

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,Cryptosporiopsisstrains,etal[3-16].Recently,the echinocandin B(ECB)biosynthetic gene cluster from Emericella rugulosa NRRL 11440 has been identified and characterized[17].Threesemisyntheticechinocandin 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 FR901379acylasesfromseveralStreptomyces 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 sidechain 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.

1Materialsandmethods

1.1Strains,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 mediumⅡ(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.

TherecombinantstrainsSCO-AACandS. 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 extractmalt 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.

1.2Characterization 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].

1.3Production 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 highperformanceliquidchromatography(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%NaH2PO4and 3%acetonitrile with a flow rate of 1 mL/min.

1.4Cloning 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 KODPlus-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 NdeⅠ/NotⅠDNA fragment into pTE28a vector and sequenced to confirm the fidelity.

1.5Construction of a recombinant strain sSCOAAC

Asite-specificintegrationvectorpIJ8660, 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/NotⅠDNA 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 selectedfromapramycin-resistant exconjugants and confirmed by PCR using primer pairPri53(TCC TAA GGA TCC GGC GGC TTG CGC CCG ATG CTA GTC)and PriAAC3.

2Results and discussion

2.1Acylase 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 mediumⅡand 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 mediumⅠas 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).

Fig.1Hydrolysis of FR901379,echinocandin B(ECB),and penicillin V catalyzed by FR901379 acylase and aculeacin A acylase(AAC)

Table 1Acylase activity of A.utahensis SW1311 culture,sSCO-AAC culture,and S.coelicolor A3(2)culture by using penicillin V as a substrate

2.2Production 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 mediumⅠas 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).

2.3Cloning 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).

2.4Construction of a recombinant strain sSCOAAC

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.sSCOAAC was confirmed by apramycin resistance and PCR using specific primers for amplification of the ermEp*-AAC fusion gene.

2.5Acylase activity of sSCO-AAC culture

Fig.2HPLC analysis of the FR901379 nucleus production catalyzed by cultures

Table 2Production of FR901379 nucleus catalyzed by A.utahensis SW1311 culture,sSCO-AAC culture,and S.coelicolor A3(2)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 sSCOAAC 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.

Fig.3Protein sequence alignment of two peptides encoded by aculeacin A acylase(AAC)genes from A.utahensis SW1311 and another A.utahensis strain(GenBank accession number D10610)[37]

2.6Production 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.

3Conclusion

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 expandtheapplicationofAACformicafungin production,but also identify S.coelicolor A3(2)as a suitable host for AAC expression.

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

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犹他游动放线菌SW1311中阿库来菌素A酰基酶在棘白菌素类抗真菌药物合成中的应用(英文).

浙
江大学学报（农业与生命科学版）,2017,43(4):416-424

冯国栋1†，许永锋1†，陈建烽1，赵万忠1，王欣1，张小晟2*，江辉3*（1.华东医药集团新药研究院有限公司，杭州310011；2.浙江大学求是学院，杭州310058；3.浙江大学生命科学学院，杭州310058）

棘白菌素类抗真菌药物米卡芬净和阿尼芬净的合成工艺中包括一个关键步骤：水解除去FR901379分子和棘白菌素B（echinocandin B,ECB）分子的脂肪酸侧链，形成环状的6元多肽核心。FR901379和ECB的水解可以分别被FR901379酰基酶和阿库来菌素A酰基酶（aculeacin A acylase,AAC）催化，因此，FR901379酰基酶和AAC的发掘、表征和生产在米卡芬净和阿尼芬净的工业生产上具有重要的应用价值。本研究首先筛选到了犹他游动放线菌SW1311，发现该菌株发酵液具有酰基酶活性，并摸索了不同发酵条件对酰基酶活性的影响。然后将犹他游动放线菌SW1311中的AAC基因克隆到改造过的质粒载体pIJ8660中，并将该质粒转化到天蓝色链霉菌（Streptomyces coelicolor）A3(2)中对AAC基因进行高表达，得到重组菌株sSCO-AAC。最后将sSCO-AAC生产的AAC活性和所需培养时间与犹他游动放线菌SW1311进行比较，表征了sSCO-AAC的发酵液水解FR901379的反应。结果表明，犹他游动放线菌SW1311中的酰基酶具有水解FR901379和青霉素V的酰基活性。用重组菌株sSCO-AAC生产的AAC活性比犹他游动放线菌SW1311的高4.6倍，且该重组菌株所需的培养时间比犹他游动放线菌SW1311缩短了30%。该结果不仅将ACC的应用范围从阿尼芬净合成拓宽到了米卡芬净合成，而且还揭示出天蓝色链霉菌A3(2)可以作为一个良好的AAC表达菌株。本研究对阿尼芬净和米卡芬净的工业化生产具有潜在的应用价值。

米卡芬净；FR901379；棘白菌素；抗真菌药物；阿库来菌素A酰基酶；犹他游动放线菌

Q 81Document codeA

10.3785/j.issn.1008-9209.2016.12.061

Projects supported by Zhejiang Provincial Natural Science Foundation of China(No.LR16H300001)and National Natural Science Foundation of China(No.31670008).

*Corresponding authors:JIANG Hui(http://orcid.org/0000-0002-4534-1322),E-mail:jianghuisioc@zju.edu.cn;ZHANG Xiaosheng(http://orcid. org/0000-0002-4379-6497),E-mail:sid@zju.edu.cn

FENG Guodong(http://orcid.org/0000-0002-3177-9032),E-mail:guodong_feng@hotmail.com.†These authors contributed equally.

2016-12-06;Accepted:2017-03-09