CN117925744B - 非核糖体肽合成酶在生产脱羧肌肽中的应用 - Google Patents
非核糖体肽合成酶在生产脱羧肌肽中的应用 Download PDFInfo
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Abstract
本发明提供了非核糖体肽合成酶在生产脱羧肌肽中的应用,属于生物技术领域。本发明首次发现非核糖体肽合成酶HiEbony具有催化组胺合成脱羧肌肽的能力,并且可用于提高脱羧肌肽的产量。所述HiEbony具有如SEQ ID NO.1所示的氨基酸序列,其编码基因核苷酸序列如SEQ ID NO.2所示。本发明提供的HiEbony在重组菌株中过表达后,摇瓶发酵能将β‑Ala和组胺转化生成脱羧肌肽,产量显著提高,且具有工业放大优势。
Description
技术领域
本发明属于生物技术领域,具体涉及非核糖体肽合成酶在生产脱羧肌肽中的应用。
背景技术
脱羧肌肽(Carcinine)的化学名称为β-丙氨酰-L-组氨,是一种由β-丙氨酸和L-组氨构成的咪唑类二肽,分子式为C8H14N4O·2HCl,相对分子质量为255.14。脱羧肌肽在常温条件下呈白色或米色粉末,无味,常温下在水中溶解度大于15g/L。早在1975年,脱羧肌肽首次于甲壳类动物体内被发现,随后又陆续发现于哺乳动物心脏中。1994年,EXSYMOL Monaco首次将脱羧肌肽运用于化妆品领域。目前,脱羧肌肽商品常以脱羧肌肽盐酸盐形式(C8H14N4O·2HCl)存在。脱羧肌肽具有稳定性好、皮透性强、作用时间长、螯合金属离子等特性,因此在抗糖化(糖逆转)、抗氧化、抗炎方面具有较广泛的应用。
目前,市面上大多数脱羧肌肽的制备方式为化学合成法,即短肽合成技术。该制备方式存在制备工艺繁琐、操作复杂、副产物多、不易纯化、生产成本高等缺点。因此,需要不断摸索更加绿色、简便、成本低的脱羧肌肽制备工艺。
微生物发酵法生产脱羧肌肽具有生产成本低、反应条件温和、原材料广泛等优点,Zhao等人[1]在文献中报道了黑腹果蝇(Drosophila melanogaster)来源的Ebony与磷酸茶氨酸转移酶(Phosphate theanine transferase,Sfp)共表达催化β-Ala和组胺反应生成脱羧肌肽。
其中Ebony是一种重要的非核糖体肽合成酶(Nonribosomal PeptideSynthetase,NRPS),实验表明Sfp对Ebony的激活至关重要,但是由于Ebony酶活有限,目前有关脱羧肌肽在大肠杆菌中生产报道的相关文献只有关于Drosophila melanogaster来源的Ebony过表达,且报道的脱羧肌肽产量不高,大大限制了放大生产。因此,亟需找到一种酶活更高的非核糖体肽合成酶用于脱羧肌肽的生产,并可应用于工业化放大。
[1]Zhao M,Song X,Liu W,et al.Whole-cell biotransformation for largescale production of carcinine in Escherichia coli[J].J Biotechnol.2022Aug 10;354:45-52.
发明内容
针对以上问题,本发明提供了非核糖体肽合成酶在生产脱羧肌肽中的应用。本发明首次发现非核糖体肽合成酶HiEbony具有催化组胺合成脱羧肌肽的能力,并且可用于提高脱羧肌肽的产量。所述HiEbony具有如SEQ ID NO.1所示的氨基酸序列,其编码基因核苷酸序列如SEQ ID NO.2所示。本发明提供的HiEbony在重组菌株中过表达后,摇瓶发酵能将β-Ala和组胺转化生成脱羧肌肽,产量显著提高,且具有工业放大优势。
为了实现上述发明目的,本发明的技术方案如下:
一方面,本发明提供了一种非核糖体肽合成酶或其编码基因在生产脱羧肌肽中的应用,所述非核糖体肽合成酶为HiEbony或其突变体,所述HiEbony的氨基酸序列为SEQ IDNO.1。
SEQ ID NO.1的序列如下:
MGSLPQLSIVKGVQRELIPKKLHKLFESNLESCKNKTALSFIENINLDPTEI
DYTTLNENANQIARLIISQAKQHELKPNQDGDWIVSVCMQPTEKLVTILLAIW
KSGAAYLPMDPSFPANRMQHIRKEAQPFLIICDDDVDGQQFEPTITLTVEECFD
KRLEFANSNIDSSELFNSTENDLAIVLYTSGSTGVPKGVRLPHAVILNRLQWQ
WNQFPYSATEKTGCFKTALTFVDSVSEIWGPLLNGMRLIIVPKAITKDPEQLVD
VLDLYKIERLVLVPTLLRSLLMYLSMTTKPNALKGLKSWICSGEPLALSLAKEF
YHYFEEGKQCLYNFYGSTEVMGDVTYFACESSKQLNSLERIPIGYPVDNTVVY
LLDAELRPVKTGEIGEIYVSGLNLADGYVNGRDPHRFLKNPLAVDTDYSRLYQ
TGDFGSLNKGVIMFEGRTDSQIKIRGHRVDLSEVEKILLTIDAIDKAIVLCYNA
GEIDQALLAFITMKRGERCYCGLDIESLLKSKLTDYMIPHVIVMESIPLLVNGK
VDRQYLLKTYAESNNNNNSTEIEVDYAAVPENLMNVARDLFEIVGQVIGRSAR
TKLSVSSNFYEMGGNSLNSIYTVTQLRERGYFISITEFIAAKNFGEILHQISKTEI
TLQEDSHDKYLKMQATPLTIDDQQATIDIITSSFYGKADLEHWLIDEIHPEDYA
NILKEIWEVLVEKDLSFMIRDENGRPIGVALNFDARDEPEVNIESKLQVIFEFLE
YLEGPIRDNQLPPGANQILHSFMMGTVEDLNAQENVAVINFMEDQVLSLAKR
KNFAGIFTTNTNPLTQQLGTNVYNYEILLDYQVNQYVYSDNYKPFGAAPDSQ
RAIVHWKDIRDK*。
具体地,所述编码基因的核苷酸序列包括SEQ ID NO.2或与SEQ ID NO.2具有75%以上同源性的核苷酸序列。
SEQ ID NO.2的序列如下:
ATGGGCAGCCTGCCGCAGCTGAGCATTGTGAAAGGCGTGCAGCGCGA
ACTGATTCCGAAAAAACTGCATAAACTGTTTGAAAGCAACCTGGAAAGCT
GCAAAAACAAAACCGCGCTGAGCTTTATTGAAAACATTAACCTGGATCCGA
CCGAAATTGATTATACCACCCTGAACGAAAACGCGAACCAAATCGCCCGCC
TGATTATCAGCCAAGCGAAACAGCATGAACTGAAACCGAACCAAGATGGC
GATTGGATTGTGAGCGTGTGCATGCAGCCGACCGAAAAACTGGTGACCATT
CTGCTGGCGATTTGGAAAAGCGGCGCGGCGTATCTGCCGATGGATCCGAGC
TTTCCGGCGAACCGCATGCAGCATATTCGCAAAGAAGCGCAGCCGTTTCTG
ATTATTTGCGATGACGATGTGGATGGTCAGCAGTTTGAACCGACCATTACCC
TGACCGTGGAAGAATGCTTTGATAAACGCCTGGAATTTGCGAACAGCAACA
TTGATAGCAGCGAACTGTTTAACAGCACCGAAAACGATCTGGCGATTGTTC
TGTATACGAGCGGCAGCACCGGCGTGCCGAAAGGCGTGCGCCTGCCGCAT
GCGGTGATTCTGAACCGCCTGCAGTGGCAGTGGAATCAGTTTCCGTATAGC
GCGACCGAAAAAACCGGCTGCTTTAAAACCGCGCTGACCTTTGTGGATAG
CGTGAGCGAAATTTGGGGCCCGCTGCTGAACGGCATGCGCCTGATTATTGT
GCCAAAAGCGATTACCAAAGATCCGGAACAGCTGGTGGATGTGCTGGATCT
GTATAAAATTGAACGCCTGGTGCTGGTGCCGACCCTGCTGCGCAGCCTGCT
GATGTATCTGAGCATGACCACCAAACCGAACGCGCTGAAAGGCCTGAAAA
GCTGGATTTGCAGCGGCGAACCGCTGGCGCTGAGTTTAGCCAAAGAGTTTT
ATCATTATTTTGAAGAAGGCAAACAGTGCCTGTATAACTTTTATGGCAGCAC
CGAAGTGATGGGCGATGTGACCTATTTTGCGTGCGAAAGCAGCAAACAGC
TGAACAGCCTGGAACGCATTCCGATTGGCTATCCGGTGGATAACACCGTGG
TGTATCTGCTGGATGCGGAACTGCGCCCGGTGAAAACCGGCGAAATTGGC
GAAATTTATGTGAGCGGCCTGAACCTGGCGGATGGCTATGTGAACGGCCGC
GATCCGCATCGCTTTCTGAAAAACCCGCTGGCGGTGGATACCGATTATAGCC
GCCTGTATCAGACCGGCGATTTTGGCAGCCTGAACAAAGGCGTGATTATGT
TTGAAGGCCGCACCGATAGTCAGATTAAAATTCGCGGCCATCGCGTGGATC
TGAGCGAAGTGGAAAAAATTCTGCTGACGATTGACGCGATTGATAAAGCGA
TTGTGTTATGCTATAACGCGGGCGAAATTGATCAAGCGCTGCTGGCGTTTAT
TACCATGAAACGCGGCGAACGCTGCTATTGCGGCCTGGATATTGAAAGCCT
GCTGAAAAGCAAACTGACCGATTATATGATTCCGCATGTGATTGTGATGGA
AAGCATTCCGCTGCTGGTGAACGGCAAAGTGGATCGTCAGTATCTGCTGAA
AACCTATGCGGAAAGCAACAATAACAATAACAGCACGGAAATTGAAGTGG
ATTATGCGGCGGTGCCGGAAAACCTGATGAACGTGGCGCGCGATCTGTTTG
AAATTGTGGGCCAAGTGATTGGCCGCAGCGCGCGCACCAAACTGAGCGTG
AGCAGCAACTTTTATGAAATGGGCGGCAACAGCCTGAACAGCATTTATACC
GTGACGCAGCTGCGCGAACGCGGCTATTTTATTAGCATTACCGAATTTATTG
CGGCGAAAAACTTTGGCGAAATTCTGCATCAGATTAGCAAAACCGAAATTA
CCCTGCAAGAAGATAGCCATGATAAATATCTGAAAATGCAAGCGACCCCGC
TGACCATTGATGATCAGCAAGCGACCATTGATATTATTACGAGCAGCTTTTA
TGGCAAAGCGGATCTGGAACATTGGCTGATTGATGAAATTCATCCGGAAGA
TTATGCGAACATTCTGAAAGAAATTTGGGAAGTGCTGGTGGAAAAGGATCT
GAGCTTTATGATTCGCGATGAAAACGGCCGCCCGATTGGCGTGGCGCTGAA
CTTTGATGCGCGCGATGAACCGGAAGTGAACATTGAAAGCAAACTGCAAG
TGATTTTTGAATTTCTGGAATATCTGGAAGGCCCGATTCGCGATAATCAGCT
GCCGCCGGGCGCGAATCAGATCCTGCATAGCTTTATGATGGGCACCGTGGA
GGATCTGAACGCGCAAGAAAACGTGGCGGTGATTAACTTTATGGAAGATCA
AGTGCTGAGTCTGGCGAAACGCAAAAACTTTGCGGGCATTTTTACCACCA
ACACCAACCCGCTGACGCAGCAGCTGGGCACCAACGTGTATAACTATGAAA
TTCTGCTGGATTATCAAGTGAATCAGTATGTGTATAGCGATAACTATAAACCG
TTTGGCGCGGCGCCGGATAGTCAGCGCGCGATTGTGCATTGGAAAGATATT
CGCGATAAATAA。
具体地,所述应用通过利用HiEbony上调的重组微生物发酵生产脱羧肌肽。
具体地,所述应用通过利用HiEbony上调的重组微生物的全细胞催化底物生产脱羧肌肽。
优选地,所述全细胞包括但不限于培养液、细胞裂解液、细胞裂解液的上清部分、细胞裂解液的沉淀部分。
优选地,所述底物包括β-丙氨酸、组胺或组氨酸中的任意一种或多种。
进一步优选地,所述底物为β-丙氨酸和组胺或β-丙氨酸和组氨酸。
具体地,所述应用为在提高脱羧肌肽产量中的应用。
另一方面,本发明提供了一种重组质粒,所述重组质粒包含上述HiEbony编码基因。
优选地,所述重组质粒为pEZ07-sfp-HiEbony。
又一方面,本发明提供了上述重组质粒的构建方法,包括以下步骤:
S1、扩增HiEbony基因片段,获得目的基因;
S2、将目的基因、含sfp基因的片段与酶切回收的载体片段进行克隆构建;
S3、转化细胞,筛选阳性克隆,提取质粒验证,得到含有HiEbony的表达质粒。
具体地,步骤S1以合成基因为模板,利用引物对扩增HiEbony基因片段。
优选地,所述的引物对为pHW232-F/pHW232-R,所述pHW232-F具有如SEQ ID NO.3所示的核苷酸序列,所述pHW232-R具有如SEQ ID NO.4所示的核苷酸序列。
具体地,步骤S1中所述的HiEbony基因核苷酸序列如SEQ ID NO.2所示,所述HiEbony基因的氨基酸序列如SEQ ID NO.1所示。
具体地,步骤S2中所述的载体为pEZ07载体。
又一方面,本发明提供了一种重组微生物,所述重组微生物为HiEbony上调的重组微生物。
具体地,所述重组微生物中HiEbony的表达量或活性提高;或HiEbony的编码基因增加、拷贝数或活力提高。
具体地,所述重组微生物包括但不限于大肠杆菌、芽孢杆菌、谷氨酸棒杆菌、沙门氏菌或酵母菌。
又一方面,本发明提供了一种脱羧肌肽的生产方法,所述生产方法包括利用上述的重组微生物发酵生产脱羧肌肽或利用上述的重组微生物的全细胞催化底物生产脱羧肌肽。
本发明的积极和有益效果在于
(1)目前并没有关于HiEbony可以合成脱羧肌肽的相关现有技术的公开,本专利是关于HiEbony合成脱羧肌肽的技术首次公开。
(2)本发明提供的HiEbony在重组菌株中过表达后,摇瓶发酵能将β-Ala和组胺转化生成脱羧肌肽,产量显著提高,且具有工业放大优势。
附图说明
图1为HPLC测定发酵液中的脱羧肌肽。
图2为NRPS的表达质粒在野生型宿主中的表达。
图3为NRPS的表达质粒在改造后的宿主中的表达。
图4为验证HiEbony的效果。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实验方法1在大肠杆菌中基因敲除的方法
本发明采用Datsenko的RED同源重组方法在大肠杆菌中进行基因敲除(Datsenko2000.Proc Natl Acad Sci USA,97(12):6640-6645),相应的基因敲除引物见Baba2006.Mol Syst Biol,2(1)0008。
实验方法2摇瓶发酵验证重组菌株生产脱羧肌肽的方法
1、试剂
(1)LB培养基:每升培养基中含酵母粉5g,氯化钠10g,蛋白胨10g,去离子水定容至1L(著[美]J.莎姆布鲁克.黄培堂译,分子克隆指南2002,1595)。
上述溶液进行高压蒸汽灭菌,灭菌温度121℃,时间20-30min。
(2)发酵培养基:每升培养基中葡萄糖30g,5N-5倍的盐溶液200mL,TM3溶液1mL,柠檬酸铁10mg,七水硫酸镁246mg,氯化钙111mg,硫胺素1μg,以无菌去离子水定容至1L。
其中5N-5倍的盐溶液为十二水合磷酸氢二钠75.6g,磷酸二氢钾每升15g,氯化钠2.5g,氯化铵25g,以离子水定容至1L;TM3溶液为四水氯化锌2.0g,六水氯化钙2.0g,两水钼酸钠2.0g,五水硫酸铜1.9g,硼酸0.5g,盐酸100mL,去离子水定容至1L。
上述溶液进行高压蒸汽灭菌,灭菌温度121℃,时间20-30min。同时准备空摇瓶,每瓶按照称取0.4g碳酸钙,发酵时碳酸钙最终浓度为20g/L。
2、仪器:恒温摇床、恒温培养箱。
3、摇瓶发酵方法:
(1)接种重组菌株至含有抗生素的3mL的LB培养基中,置37℃摇床250rpm培养;
(2)取培养16h后的种子液200μL转移至2mL含有抗生素的LB液体培养基,于37℃摇床250rpm培养4h;
(3)将2mL二级种子全部转入装有18mL发酵培养基的摇瓶中,置37℃摇床中,250rpm培养4h。
(4)添加IPTG使其终浓度1mM,同时添加1g/Lβ-Ala+2g/L组胺和/或2g/LL-组氨酸,调节摇床温度至34℃,继续培养20h左右,取0.2mL发酵液和0.8mL水混匀离心后(12000rpm,1min),取上清样品过0.22μm的水系滤膜后,进行HPLC检测,检测方法详见实验方法3。
实验方法3HPLC测定发酵液中的脱羧肌肽
HPLC的参数如下:
色谱柱:SB-AQ 4.6*150*5μm;
流动相A:磷酸二氢钾和辛烷磺酸钠混合液(称取磷酸二氢钾6.8g,辛烷磺酸钠2.16g加水至1000g,溶解,用磷酸调节PH到3.0;用0.22nm水相滤膜过滤,超声备用);
流动相B:乙腈;
流动相A:流动相B=89:11;
柱温:30℃;
初始流速:1.0mL/min;
检测波长:200nm;
进样量:5μL;
检测时间:15min。
HPLC图谱如图1所示,脱羧肌肽的出峰时间为12min。
实施例1NRPS的表达质粒构建
本实验室通过对文献中Drosophila melanogaster来源的NRPS在NCBI上进行氨基酸BLAST,通过序列比对,选取与文献报道的NRPS氨基酸序列同源性较低的NRPS(见表1),以下基因均在苏州金唯智生物科技有限公司将序列针对大肠杆菌密码子优化后进行基因合成。
表1不同来源的NRPS
来源 | 基因库编号 | 基因 | 同源性 |
Drosophila melanogaster | SGD:S000005036 | DmEbony | 100% |
Hermetia illucens | CAD7085107.1 | HiEbony | 58.70% |
Teleopsis dalmanni | NP_001020145.1 | TdEbony | 71.70% |
Zeugodacus cucurbitae | XP_011180928.2 | ZcEbony | 73.30% |
Topomyia yanbarensis | XP_058813264.1 | TyEbony | 57.66% |
Leptinotarsa decemlineata | ATB56363.1 | LdEbony | 53.36% |
本实验室针对检索出的6个不同来源的NRPS,分别设计引物通过无缝克隆构建到低拷贝载体pEZ07(载体pEZ07同中国专利申请号:201510093004.3中)上,获得表达质粒pHW231-pHW236(质粒信息见表2),质粒构建相关引物见表3。
表2质粒信息表
质粒 | 质粒信息 |
pHW231 | pEZ07-sfp-DmEbony |
pHW232 | pEZ07-sfp-HiEbony |
pHW233 | pEZ07-sfp-TdEbony |
pHW234 | pEZ07-sfp-ZcEbony |
pHW235 | pEZ07-sfp-TyEbony |
pHW236 | pEZ07-sfp-LdEbony |
表3质粒构建引物
注:表中,F和R为扩增引物,其中:F表示正向引物,R表示反向引物。
以pHW232(即pEZ07-sfp-HiEbony)构建为例介绍表达质粒构建过程,具体步骤如下:
以枯草芽孢杆菌BS168(Bacillus subtilis subsp.Subtilis str.168)基因组为模板,利用引物对sfp-F/sfp-R扩增sfp基因片段,得到大小为717bp的片段且电泳无杂带。以合成基因为模板,利用引物对pHW232-F/pHW232-R扩增HiEbony基因片段,得到大小为2668bp的片段且电泳无杂带。直接进行过柱回收纯化,使用捷瑞胶回收纯化试剂盒(购自上海捷瑞生物工程技术有限公司,货号GK2043),得到的纯化片段与pEZ07载体(NcoI/XhoI酶切)进行EZ克隆构建,使用GBclonart无缝克隆试剂盒(购自苏州神洲基因有限公司,货号GB2001-48)。
所述的EZ克隆构建包括以下步骤:
重组克隆反应液于45℃水浴锅温浴30min,然后转移到冰上放置5min,加入TG1化转感受态细胞,混匀放置5min,42℃热击2min,冰浴2min后加入800μL复苏培养基LB,复苏培养1h后,离心(8000rpm,1min),涂布含100mg/L壮观霉素的LB平板,次日挑取克隆培养过夜,提取质粒进行酶切验证,最终构建得到质粒pEZ07-sfp-HiEbony,编号pHW232。其中,HiEbony的核苷酸序列如SEQ ID NO.2所示,HiEbony的氨基酸序列如SEQ ID NO.1所示。
实施例2NRPS的筛选
1、NRPS的表达质粒在野生型宿主中的表达
将实施例1中构建得到的NRPS表达质粒pHW231-pHW236分别转化至宿主大肠杆菌W3110(ATCC 27325),分别得到含不同表达质粒的NRPS重组菌株。将以上NRPS重组菌株按照实验方法2所述的方法进行摇瓶发酵,并采用实验方法3中所述的HPLC方法测定各发酵液中的脱羧肌肽含量。测定结果如图2所示。
2、NRPS的表达质粒在改造后的宿主中的表达
按照实验方法1所述的基因敲除方法在大肠杆菌W3110(ATCC 27325)上进行pepD基因和dpp基因的敲除,构建得到敲除了脱羧肌肽降解基因和摄取基因的宿主SHK25(W3110ΔpepDΔdpp)。
将实施例1中构建得到的NRPS表达质粒pHW231-pHW236分别转化至宿主SHK25(W3110ΔpepDΔdpp)中,分别得到含不同表达质粒的NRPS重组菌株。将以上NRPS重组菌株按照实验方法2所述的方法进行摇瓶发酵,并采用实验方法3中所述的HPLC方法测定各发酵液中的脱羧肌肽含量。测定结果如图3所示。
图2-图3显示,无论在野生型W3110宿主细胞还是改造后的SHK25宿主细胞中,HiEbony的过表达都能显著积累脱羧肌肽,比文献中记载的DmEbony过表达产量高1.4倍左右。
实施例3验证HiEbony的效果
为了近一步确认基因HiEbony能够催化组胺生成脱羧肌肽,本发明在摇瓶发酵时添加底物β-丙氨酸和组氨酸,同时在表达质粒pHW231与pHW232上串入基因PdHDC(组氨酸脱羧酶,催化底物组氨酸转化为组胺),分别构建pHW244(pEZ07-sfp-DmEbony-PdHDC)与pHW245(pEZ07-sfp-HiEbony-PdHDC),转化宿主细胞SHK25中,得到重组菌株,按照实验方法2所述的方法进行摇瓶发酵,并采用实验方法3中所述的HPLC方法测定各发酵液中的脱羧肌肽含量。
测定结果显示(图4),过表达pHW245的脱羧肌肽产量相对于pHW244增加了4倍,产量达到0.8g/L,进一步证明了基因HiEbony的催化效果,而且将组胺底物替换为组氨酸,可降低放大生产中底物投料成本。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (6)
1.一种非核糖体肽合成酶或其编码基因在生产脱羧肌肽中的应用,其特征在于,所述非核糖体肽合成酶为HiEbony,所述HiEbony的氨基酸序列为SEQ ID NO.1;
所述应用通过利用HiEbony上调的重组微生物发酵生产脱羧肌肽;或通过利用HiEbony上调的重组微生物的全细胞催化底物生产脱羧肌肽;
所述应用为在提高脱羧肌肽产量中的应用。
2.根据权利要求1所述的应用,其特征在于,所述编码基因的核苷酸序列为SEQ IDNO.2。
3.根据权利要求1所述的应用,其特征在于,所述全细胞包括培养液、细胞裂解液、细胞裂解液的上清部分或细胞裂解液的沉淀部分。
4.根据权利要求1所述的应用,其特征在于,所述底物包括β-丙氨酸、组胺或组氨酸中的任意一种或多种。
5.一种脱羧肌肽的生产方法,其特征在于,所述生产方法包括利用重组微生物发酵生产脱羧肌肽或重组微生物的全细胞催化底物生产脱羧肌肽;
所述的重组微生物为HiEbony上调的重组微生物;所述重组微生物为大肠杆菌,所述HiEbony的氨基酸序列为SEQ ID NO.1;
所述底物包括β-丙氨酸、组胺或组氨酸中的任意一种或多种。
6.根据权利要求5所述的生产方法,其特征在于,所述重组微生物中HiEbony的表达量或活性提高;或HiEbony的编码基因增加、拷贝数或活力提高。
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