CN112029700B - 微生物发酵生产hip-ipa的方法、基因工程菌及应用 - Google Patents
微生物发酵生产hip-ipa的方法、基因工程菌及应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,具体涉及微生物发酵生产HIP‑IPA的方法、工程菌及应用。本发明的HIP‑IPA工程菌是将HIP‑IPA生产菌的出发菌株中的编码酰基辅酶A硫解酶的基因敲除,同时过表达编码乙酰辅酶A乙酰转移酶/硫解酶和DNA结合蛋白的基因构建获得,实现从植物甾醇出发,通过生物转化实现HIP‑IPA的生产,具有十分显著的效果。
Description
技术领域
本发明涉及基因工程技术领域,具体而言,涉及微生物发酵生产HIP-IPA的方法、工程菌及应用。
背景技术
甾体是一类具有环戊烷多氢菲环结构的化合物,通常在C-10和C-13位有甲基基团,在C-17位有烷基侧链。甾体作为一种细胞膜的组分,在生物体中具有重要的作用。一些甾体还具有激素和信号分子的作用。自上世纪50年代发现甾体药物以来,到目前为止已经鉴定了300多种甾体药物。甾体药物具有很强的抗感染、抗过敏、抗病毒和抗休克等药理作用。近年来,甾体药物在医疗领域的应用范围不断扩大,被广泛用于治疗风湿病、心血管、胶原性病症、淋巴白血病、人体器官移植、抗肿瘤、细菌性脑炎、皮肤病、内分泌失调、老年性疾病等,甾体激素药物已成为仅次于抗生素的第二大类药物。
根据《甾体化学进展》(周维善、庄治平主编,科学出版社2002年出版,ISBN 7-03-009607-X)报道,很多微生物包括诺卡氏菌(Nocardia)、分枝杆菌(Mycobacterium)、节杆菌(Arthrobocter)、和假单胞杆菌(Pseudomonas)等都可将甾体母核环戊烷多氢菲及侧链全部氧化成二氧化碳和水。代谢途径经Sih及其合作工作者(参考文献“Sih CJ, Wang KC,Tai HH. Mechanisms of steroid oxidation by microorganisms. XIII. C22 acidintermediates in the degradation of the cholesterol side chain. Biochemistry,1968, 7: 796-807”)的研究阐述如图1所示。该过程表明,在微生物降解过程中可通过控制不同酶的活力,获得如4-AD(化合物15)、9-OH-AD,HIP(化合物21)和谷内酯(化合物24)等重要的中间体。王风清等的研究表明(申请号:CN201910510202.3),在分枝杆菌中敲除编码酰基辅酶A硫解酶(Hsd4A)的基因,可用于生产22-羟基-23,24-双降胆甾-1,4-二烯-3-酮、22-羟基-23,24-双降胆甾-4-烯-3-酮和9ɑ,22-二羟基-23,24-双降胆甾-4-二烯-3-酮。美国专利US 20110191875A1报道了通过在分枝杆菌中阻断DNA结合蛋白(CxgB)的活力,抑制了植物甾醇转化生产4-雄烯二酮(4-AD)过程中副产物22-羟基-23,24-双降胆甾-1,4-二烯-3-酮和22-羟基-23,24-双降胆甾-4-烯-3-酮的产生,推测乙酰辅酶A乙酰转移酶/硫解酶(CxgA)也参与到了植物甾醇转化生产22-羟基-23,24-双降胆甾-1,4-二烯-3-酮和22-羟基-23,24-双降胆甾-4-烯-3-酮的代谢途径中。
Ⅰ
HIP-IPA,英文名为3-(1-(1-hydroxypropan-2-yl)-7a-methyl-5-oxooctahydro-1H-inden-4-yl) propanoic acid,结构式如式Ⅰ所示,可以作为合成许多甾体激素药物的中间体。Liu等通过敲除分枝杆菌中的羧酸还原酶,以植物甾醇为底物,实现了谷内酯((4aR,6aS,9aS,9bS)-6a-甲基八氢环戊二烯并[f]色烯-3,7(2H,8H)-二酮)和HIP(1,5-二氧代-7aβ-甲基-3aα-六氢茚满-4α-丙酸)的高效生产( Liu N, Feng JH, Zhang R, etal. Efficient microbial synthesis of key steroidal intermediates from bio-renewable phytosterols by genetically modified Mycobacterium fortuitum strains. Green Chem, 2019, 21: 4076-4083)。到目前为止,仍没有以植物甾醇为底物,通过生物转化生产HIP-IPA的方法。
发明内容
为解决上述现有技术中所存在的问题,本发明提供了HIP-IPA生产菌及在生产HIP-IPA中的应用,和通过微生物发酵生产HIP-IPA的方法。
具体而言,本发明采用以下技术方案:
本发明的第一方面提供了一种HIP-IPA生产的基因工程菌,其特征在于,所述基因工程菌是将HIP-IPA生产菌的出发菌中的编码酰基辅酶A硫解酶的基因的表达量降低、失活或敲除,同时过表达编码乙酰辅酶A乙酰转移酶/硫解酶和DNA结合蛋白的基因构建获得。
所述HIP-IPA生产菌的出发菌是放线菌和假单胞菌,其中所述放线菌包括红球菌、诺卡式菌、分枝杆菌、链霉菌和节杆菌。优选地,所述的HIP-IPA生产菌的出发菌是分枝杆菌,优选地,其中敲除了FadD3(acyl-CoA 合成酶)基因和羧酸还原酶基因作为出发菌。在一个具体实施方式,所述出发菌在野生分枝杆菌Mycobacterium fortuitum:ATCC 6841基础上敲除了FadD3(acyl-CoA 合成酶)基因和羧酸还原酶基因获得的(申请号:CN201711051707 .5)。本发明最优选是在该出发菌的基础上进行分子改造获得的。
优选地,所述酰基辅酶A硫解酶的编码基因具有SEQ ID NO:1所示核苷酸序列。其中,使该基因的表达量降低、失活或敲除可以采用已知的技术实现,例如基因编辑等各种可行的方法。
优选地,所述乙酰辅酶A乙酰转移酶/硫解酶具有SEQ ID NO:2所示的氨基酸序列或者与SEQ ID NO:2所示的氨基酸序列具有至少80%的同源性的氨基酸序列。所述乙酰辅酶A乙酰转移酶/硫解酶基因来源于放线菌和假单胞菌,优选地,所述放线菌包括红球菌、诺卡式菌、分枝杆菌、链霉菌和节杆菌。
更佳地,所述乙酰辅酶A乙酰转移酶/硫解酶基因来源于分枝杆菌。优选地,所述DNA结合蛋白具有SEQ ID NO:3所示的氨基酸序列或者与SEQ ID NO:3所示的氨基酸序列具有至少80%的同源性的氨基酸序列。
所述DNA结合蛋白基因来源于放线菌和假单胞菌,优选地,所述放线菌包括红球菌、诺卡式菌、分枝杆菌、链霉菌和节杆菌。更佳地,所述DNA结合蛋白基因来源于分枝杆菌。
本发明的第二方面提供了所述HIP-IPA生产工程菌在发酵生产HIP-IPA中的应用。优选地,所述应用是利用所述的基因工程菌,以甾醇为发酵原料进行发酵以获得HIP-IPA。
本发明的第三方面提供了一种通过微生物发酵生产HIP-IPA的方法,包括上述的HIP-IPA生产工程菌发酵以生产HIP-IPA。在本发明的方法中,所述发酵优选在25-45℃下进行,更优选在25-37℃下进行。发酵时的pH优选为7-8。发酵优选进行3-12天,例如进行3-10天,更优选4-10天。根据具体的应用和条件,可以根据要求进行调整所述HIP-IPA生产菌的发酵接种量,例如可以为:HIP-IPA生产菌种子液的OD600nm(600nm下的OD值)为1-20(例如10)、体积为发酵用培养液的1%-20%。例如,当OD值低时,可以接种较大体积的种子液;当OD值高时,可以接种较小体积的种子液。
本发明与现有技术相比具有以下优点和积极效果:实现了从植物甾醇出发,通过生物转化实现HIP-IPA的生产,进一步地,本发明能以高底物投料浓度,获得高收率的目标产物,摩尔收率不低于60%。
附图说明
图1为甾醇在微生物中代谢途径。
图2所示为hsd4A基因敲除PCR验证结果,其中泳道1-6为验证菌株,泳道1-5为基因敲除未成功的菌株,泳道6为基因敲除成功菌株,M为分子量标记。
图3所示实施例4的发酵液萃取样品的气相色谱图,其中a为出发菌株发酵液萃取样品,b为酰基辅酶A硫解酶基因缺失菌株发酵液萃取样品,c为酰基辅酶A硫解酶基因缺失、乙酰辅酶A乙酰转移酶/硫解酶和DNA结合蛋白编码基因过表达菌株发酵液萃取样品,d为HIP-IPA标准品。
图4所示是实施例4制得的HIP-IPA的1H-NMR图。
具体实施方式
为更好的理解本发明的内容,下面结合具体实施例作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限制本发明的范围,未进行具体说明的操作步骤均为常规的操作。
实施例1:基因敲除质粒的构建
酰基辅酶A硫解酶的编码基因(hsd4A)如SEQ ID NO:1所示,按照以下的方法构建敲除hsd4A的质粒。
以分枝杆菌(HIP生产菌,申请号:CN201711051707.5)的基因组为模板进行PCR扩增。PCR体系和引物如下。
PCR体系:
5×Phusion GC Buffer 10 μl
2mM dNTPs 5 μl
Primer F 1 μl
Primer R 1 μl
Template DNA 50-100 ng
DMSO 1.5 μl
Phusion 0.5 μl
ddH2O 至 50 μl
PCR程序:98℃ 3 min;98℃ 10 s变性,58℃ 20 s退火,72℃ 30 s延伸,30个循环;72℃ 10 min。其中所用引物序列为:
上游片段引物:
Up-F 5' acgttgttgccattgctgcagACTTCTTCTGCTCCTCGGTGC 3'
Up-R 5' CAGCTGAGCGTTGACAGCTTagtactATCTCGTCGAGGACGTCGGAGG 3'
下游片段引物:
Down-F 5'TCCGACGTCCTCGACGAGATagtactAAGCTGTCAACGCTCAGCTGTTC 3'
Down-R 5'GTACCGCGGCCGCTTAATTAAGCCCTGCAGGAACACGGAGAAC 3'
将扩增出的上、下游片段与pGOAL19中的片段(该片段的选取和与该片段的连接参照文献“Parish T, Stoker NG. Use of a flexible cassette method to generate adouble unmarked MycoIPActerium tuberculosis tlyA plcABC mutant by genereplacement. Microbiology, 2000, 146: 1969-75”所述的方法进行)以及p2NIL载体相连接。将连接产物转化到DH5α感受态细胞中,涂Kan、Hyg双抗LB(胰蛋白胨:10g/L,酵母提取物:5g/L,氯化钠:10g/L,Kan:50μg/ml,Hyg:50μg/ml,琼脂:1.5%)平板,同时加IPTG与X-Gal,37℃过夜培养。挑蓝色单菌落培养后提质粒进行PacI单酶切验证后,再测序进一步确证是否构建成功。
实施例2:敲除菌株的筛选
将构建好的基因敲除质粒电转入分枝杆菌(HIP生产菌,申请号:CN201711051707.5)感受态细胞中,涂Kan抗性LB平板(胰蛋白胨:10g/L,酵母提取物:5g/L,氯化钠:10g/L,Kan:50μg/ml,琼脂:1.5%)并加IPTG和X-gal,进行第一次筛选。从中挑取蓝色单菌落至蔗糖板(胰蛋白胨:10g/L,酵母提取物:5g/L,蔗糖:10g/L,琼脂:1.5%) (加IPTG和X-gal),进行第二次筛选。在蔗糖板上挑白色菌落至液体LB培养基,30℃培养约36小时后,提基因组,以目的基因的Up-F、Down-R为引物进行PCR验证。若基因敲除成功,则PCR产物应约为1900 bp的单一片段。图2显示成功获得了hsd4A基因敲除的分枝杆菌菌株。
实施例3:基因表达菌株的构建
1、表达质粒构建
应用引物cxgAB-F和cxgAB-R扩增来自Mycobacterium sp. NRRL B-3805基因组中的乙酰辅酶A乙酰转移酶/硫解酶基因(cxgA)和DNA结合蛋白基因(cxgB),获得带有与质粒pMV261的15bp同源臂的cxgAB片段后,与表达质粒pMV261经EcoRI和HindIII酶切纯化获得单片段连接,获得重组表达质粒261-cxgAB。
PCR体系:
5×Phusion GC Buffer 10 μl
2mM dNTPs 5 μl
Primer F 1 μl
Primer R 1 μl
Template DNA 50-100 ng
DMSO 1.5 μl
Phusion 0.5 μl
ddH2O 至50 μl
PCR程序:98℃ 3 min;98℃ 10 s变性,58℃ 20 s退火,72℃ 30 s延伸,30个循环;72℃ 10 min。其中所用引物序列为:
cxgAB-F: 5' GCGGATCCAGCTGCAGAATTCATGGGTTTGCGTGGTGACG 3'
cxgAB-R: 5' TACGTCGACATCGATAAGCTTCTATTCGGCGGCGGTGTAGTG 3'
将连接产物转化到DH5α感受态细胞中,涂Kan抗性LB平板(胰蛋白胨:10g/L,酵母提取物:5g/L,氯化钠:10g/L,Kan:50μg/ml,琼脂:1.5%),37℃过夜培养。挑单菌落培养后提质粒进行酶切验证后,再测序进一步确证是否构建成功。
2、分枝杆菌表达菌的构建
通过电转化将构建好的重组表达质粒261-cxgAB导入上述hsd4A基因敲除成功的分枝杆菌感受态细胞中,经Kan抗性筛选得到带有质粒261-cxgAB的重组菌株。
实施例4:发酵生产HIP-IPA
种子培养基:葡萄糖:6 g/L;酵母粉:15 g/L;NaNO3:5.4 g/L;甘油:2 g/L;NH4H2PO4:0.6 g/L;pH: 7.5;115°C灭菌30分钟
发酵培养基:NaNO3:6.37 g/L;KH2PO4:1.05 g/L;Na2HPO4:2.14 g/L;MgSO4:0.82g/L;KCl:0.21 g/L;CaCl2:0.1 g/L;玉米浆干粉:14.23 g/L;植物甾醇:20 g/L;大豆油:12%;pH: 7.8;121°C灭菌30分钟。
发酵培养步骤:
1、30℃下LB平板(胰蛋白胨:10g/L,酵母提取物:5g/L,氯化钠:10g/L,琼脂:15 g/L)培养72小时以活化菌种;
2、从活化平盘接种至种子培养基中,180 rpm,30°C培养3天;
3、将种子液在无菌条件下取样进行取样镜检,镜检无染菌方可接种;按10%的接种量接种至3 L发酵培养基中;
4、500 rpm,30°C发酵培养,通气比为0.5 vvm,培养24小时后提高温度至42°C并保持30分钟,再降低发酵温度至30°C继续发酵,整个发酵过程pH在7-8之间,不需要控制;发酵前期因为是有油体系,所以底物植物甾醇会有结块现象,因此前期最好不要取样,在体系变均匀后可以定时取样进行检测。具体而言,每8小时取样50ml,盐酸调节pH为3,加入二氯甲烷萃取得到粗提物,TLC分析植物甾醇的残留量。当植物甾醇转化为HIP-IPA的反应转化率达到95%时,终止发酵。发酵后的整个体系灭菌后静置直至油水分离,待油水分离后取水相,盐酸调节pH为3,二氯甲烷萃取水相直至萃取完全,记录整个萃取相体积,通过制作标准曲线进行定量,得到整个发酵收率,气相色谱结果如图3所示。
制得的样品HIP-IPA的核磁数据,核磁谱图如图4所示:
1H NMR (400MHz ,CHLOROFORM-d) δ = 3.68 (dd, J = 3.0, 10.4 Hz, 1 H),3.43 (dd, J = 6.6, 10.5 Hz, 1 H), 2.60 - 2.46 (m, 2 H), 2.45 - 2.28 (m, 3 H),2.22 - 2.14 (m, 1 H), 2.06 - 1.94 (m, 1 H), 1.88 - 1.70 (m, 3 H), 1.69 - 1.47(m, 4 H), 1.44 - 1.31 (m, 2 H), 1.09 (d, J = 6.6 Hz, 3 H), 1.04 (s, 3 H)。
发酵罐验证结果如下表所示:
从上表可知,本发明构建的工程菌能以高底物投料浓度,获得高收率的目标产物,摩尔收率达63%,真正实现了从植物甾醇出发,通过生物转化实现HIP-IPA的生产。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 微生物发酵生产HIP-IPA的方法、基因工程菌及应用
<130> 202001015
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 924
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<213> Mycobacterium fortuitum ATCC6841
<400> 1
atgaccactg acgacgctca gatcgatctg tccggaaagg tggccgtggt caccggtgcc 60
gccgcgggcc tcggcacgtg ccgaagccat cggtctggcg aaggccggcg ccaccgtggt 120
ggtcaacgac atggccggcg cgctggacgc ctccgacgtc ctcgacgaga tcgcggccgc 180
aggctccaag ggtgtcgccg tggccgggga catcagtgcg cgcacgaccg ccgacgagct 240
ggtcgccacc gccgacggtc tgggcggtct ggacatcgtc gtcaacaacg cgggcatcac 300
ccgtgaccgg atcctgttca acatgagcga cgaagagtgg gacgccgtca tcgcagtgca 360
cctgcgcggc cacttcctgc tcacccgcaa cgccgccgtg tattggcgta acaaggccaa 420
ggcgggggac gggaccgtct acggccggat catcaacacg tcgtcggagg ccggtctgtc 480
aggtccggtc ggacagccca actacggagc cgccaaggcc ggtatcaccg cgctgacggt 540
gtcggcggcc cgggccctgg aacgcttcgg cgtgcgggcc aacgcgatcg ccccgcgtgc 600
ccgcaccgcc atgaccgccg gcgtcttcgg ggatgctccc gaaaacgccc gacggtcaga 660
tcgacccgct gtcgaccgat cacgtcgtca ccctcgtgcg attcttggcc gccccggcgt 720
ccgaagctgt caacgctcag ctgttcatcg tctatggtcc aactgtcacg ttggtcgcgc 780
ccccccaccg cggagaagca cttcaccgcg aactccgacg cgtgggatcc ggcagacctc 840
agcggggcac tgcacgatta ctttgctgat cgtgacgcgg cgcgtggatt ctcggccacc 900
gagctgatgg cgtcacgaga ctga 924
<210> 2
<211> 401
<212> PRT
<213> Mycobacterium neoaurum NRRL B-3805
<400> 2
MGLRGDAAIV GFHELPATRK PTGTAEFTIE QWARLAAAAV ADAGLSVQQV DGLVTCGVME 60
SQLFVPSTVA EYLGLAVNFA EIVDLGGASG AAMVWRAAAA IELGLCQAVL CAIPANYLTP 120
MSAERPYDPG DALYYGASSF RYGSPQAEFE IPYGYLGQNG PYAQVAQMYS AAYGYDETAM 180
AKIVVDQRVN ANHTPGAVFR DKPVTIADVL DSPIIASPLH MLEIVMPCMG GSAVLVTNAE 240
LARAGRHRPV WIKGFGERVP YKSPVYAADP LQTPMVKVAE SAFGMAGLTP ADMDMVSIYD 300
CYTITALLTL EDAGFCAKGT GMRFVTDHDL TFRGDFPMNT AGGQLGYGQP GNAGGMHHVC 360
DATRQLMGRA GATQVADCHR AFVSGNGGVL SEQEALVLEG D 401
<210> 3
<211> 138
<212> PRT
<213> Mycobacterium neoaurum NRRL B-3805
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MTESSARPVP LPTPTSAPFW DGLRRHEVWV QFSPSSDAYV FYPRILAPGT LADDLSWRQI 60
SGDATLVSFA VAQRPVAPQF ADAVPHLLGV VQWTEGPRLA TEIVGVDPAR LRIGMAMTPV 120
FTEPDGADIT LLHYTAAE 138
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<211> 42
<212> DNA
<213> 人工序列:Up-F
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<212> DNA
<213> 人工序列:Up-R
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<212> DNA
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<213> 人工序列:cxgAB-F
<400> 8
gcggatccag ctgcagaatt catgggtttg cgtggtgacg 40
<210> 9
<211> 42
<212> DNA
<213> 人工序列:cxgAB-R
<400> 9
tacgtcgaca tcgataagct tctattcggc ggcggtgtag tg 42
Claims (6)
2.如权利要求1所述HIP-IPA生产的基因工程菌在发酵生产HIP-IPA中的应用。
3.如权利要求2所述的应用,其特征在于,利用所述的基因工程菌,以甾醇为发酵原料进行发酵以获得HIP-IPA。
4.一种通过微生物发酵生产HIP-IPA的方法,其特征在于,包括将如权利要求1所述的HIP-IPA生产的基因工程菌,以甾醇为发酵原料进行发酵获得HIP-IPA的步骤,以及收集HIP-IPA的步骤。
5.如权利要求4所述的方法,其特征在于,所述发酵是在25-45℃下,发酵液的pH为7-8的条件下进行。
6.如权利要求5所述的方法,其特征在于,所述发酵是在25-37℃,发酵液的pH为7-8的条件下进行。
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CN101918436A (zh) * | 2007-11-16 | 2010-12-15 | 维莱尼姆公司 | 制造雄烯二酮的组合物和方法 |
CN109722455A (zh) * | 2017-10-31 | 2019-05-07 | 中国科学院天津工业生物技术研究所 | 微生物发酵生产谷内酯的方法、工程菌及应用 |
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