CN106947705A - 低产桔霉素高产红曲黄色素的基因重组紫色红曲霉M‑piy菌株及其制备方法和用途 - Google Patents
低产桔霉素高产红曲黄色素的基因重组紫色红曲霉M‑piy菌株及其制备方法和用途 Download PDFInfo
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Abstract
本发明属于微生物基因工程领域,涉及1株具有低产桔霉素高产红曲黄色素的基因重组紫色红曲霉M‑piy菌株及其制备方法和用途。该菌株分类名称为:紫色红曲霉Monascus purpureus,保藏编号:CCTCC NO:M 2016627,保藏日期:2016年11月9日。本发明利用基因工程手段将潮霉素抗性基因(hph)代替红曲霉色素相关基因(PigE),获得了1株低产桔霉素高产红曲黄色素的基因工程M‑piy菌株。本发明红曲黄色素产量较野生型菌株提高了约9.6倍,达到了9232.86U/g。另外,本发明M‑piy菌株较野生型菌株产桔霉素的发酵时间延迟,桔霉素含量显著降低,为工业上红曲霉菌种改良提供了新策略。
Description
技术领域
本发明属于微生物基因工程领域,涉及1株具有低产桔霉素高产红曲黄色素的基因重组紫色红曲霉M-piy菌株及其制备方法和用途。
背景技术
红曲霉是我国重要的食用和药用微生物,其发酵产生的主要次级代谢产物红曲色素是一种重要的天然色素。红曲色素安全性高、热稳定性强,对蛋白的着色性好不褪色,广泛的应用于食品加工、化妆品、保健品等行业。公认的红曲色素有6种,分为三种色调即橙色调、黄色调和红色调。红曲黄色素也是重要的食用色素,约占市场需求量的60%,市售食用黄色素产品以植物提取为主,相比之下,采用微生物发酵法进行天然食用黄色素的生产,不受季节限制、原料转化率高、成本低,是目前世界食用色素添加剂的发展趋势。目前工业上主要利用红曲红色素作为着色剂;而红曲黄色素仍然处于研究阶段,开发利用少、发酵产量低、发酵工艺复杂。因此,开发低产桔霉素高产红曲黄色素的基因重组菌株具有广阔的应用前景,可产生显著的经济效益。
自Blanc等(1995)研究发现某些红曲霉菌株能产生对人体有害的真菌毒素—桔霉素(citrinin)以来,红曲霉相关制品的安全性一直存在着争议,从而影响到我国红曲产品的出口。Hajjaj等研究结果表明,桔霉素与红曲色素同属于聚酮类化合物,桔霉素与红曲色素的合成开始于同一个途径和共同的合成前体,红曲霉色素相关基因的研究与基因工程菌株的开发有利于揭示桔霉素代谢途径,从根本上降低桔霉素含量。
王硕等(申请号:201510589619.5)利用基因工程手段将潮霉素抗性基因替代了桔霉素合成基因簇中加氧酶基因的开放阅读框,获得低产桔霉素高产色素的红曲霉基因工程菌株,该基因改造菌株与原始的野生株相比,桔霉素产量降低了96.48%,黄色素产量提高了89.9%(约6500U/g,吸收峰为410nm),橙色素提高172.1%,红色素提高140.6%。
常见的红曲霉菌株发酵产生的红曲色素基本上是红曲红色素、橙色素和黄色素的混合物,高效生产单一的红曲黄色素的菌株鲜见报道。目前常见的提高红曲黄色素产量的方法主要包括传统的菌种选育、优化发酵条件和改良提取工艺等。传统的育种方法主要是通过物理方法(紫外照射)和化学方法(化学诱变剂如:硫酸二乙酯、氯化锂、亚硝基胍)诱变提高黄色素产量,这种方法存在回复突变可能性,菌株易变异易退化,工业实际应用中面临很多挑战。另外,仅仅依靠优化发酵条件、改良提取工艺提高红曲黄色素产量能力有限,因此急需在了解代谢途径和关键编码基因的基础上,借助于基因工程技术对菌种进行定向改造。
发明内容
本发明的目的是提供1种低产桔霉素高产红曲黄色素的基因重组紫色红曲霉M-piy菌株、用途及其发酵生产方法。
本发明所述的1种低产桔霉素高产红曲黄色素的基因重组紫色红曲霉菌株命名为紫色红曲霉M-piy,即Monascus purpureus M-piy,保藏于武汉大学菌种保藏中心,保藏地址为:中国武汉武汉大学,保藏编号:CCTCC NO:M 2016627,保藏日期:2016年11月9日。其基因组上存在的色素合成相关基因PigE被敲除,由潮霉素基因hph基因序列所替代,因此该菌株含有外源潮霉素抗性。本发明所涉及的紫色红曲霉M-piy菌株在YES液体培养基中发酵19d只产生红曲黄色素,红曲黄色素产量较野生型菌株提高了9.6倍,达到了9232.86U/g。另外,本发明提供的基因工程M-piy菌株较野生型菌株产桔霉素的发酵时间延迟,桔霉素含量显著降低,发酵第7d才开始产生桔霉素,发酵第15d后桔霉素含量仅为0.31mg/L,是野生型菌株的0.22倍。本发明可为工业上红曲霉菌种改良提供了新策略。
本发明还提供了一种具有低产桔霉素高产红曲黄色素的基因重组紫色红曲霉的制备方法。本发明所述的基因重组紫色红曲霉的制备方法包括以下步骤:
(1)从野生型菌株基因组DNA中扩增目的基因PigE上游臂和下游臂基因序列,从质粒pKD1中扩增潮霉素抗性基因hph序列。(2)利用双接头(Double-joint)PCR方法将PigE上下游臂基因和hph基因三个基因片段连接,构建敲除盒。(3)构建基因敲除载体:KpnⅠ、XbaⅠ两种限制性内切酶双酶切敲除盒和质粒pKO1B,再用T4DNA连接酶(ligase)连接。(4)将敲除载体转入空载的根癌农杆菌AGL-1菌株,后利用根癌农杆菌介导的转化法转化红曲霉。(5)潮霉素抗性平板上筛选转化子,转化子PCR验证。
附图说明
图1基于同源重组的PigE基因敲除原理图。
图2敲除盒构建相关PCR产物电泳图谱;其中:M,DL5000;1,入突变菌株;PigE基因上游同源臂;2,PigE基因下游同源臂;3,潮霉素抗性基因;4,敲除盒基因片段。
图3PigE基因敲除成功的缺失突变株PCR验证电泳图;其中:M,DL5000;1,野生型菌株;2,PigE基因缺失突变株;3,T-DNA插入突变菌株。
图4野生型菌株与M-piy菌株的菌落及显微结构特征图。
图5野生型菌株与M-piy菌株的菌丝色素提取液全波长扫描图谱。
图6野生型菌株与M-piy菌株菌丝黄色素色价图。
图7野生型菌株与M-piy菌株发酵液桔霉素含量图。
保藏声明
紫色红曲霉(Monascus purpureus)M-piy菌株,该保藏于武汉大学菌种保藏中心,保藏地址为:中国武汉武汉大学,保藏编号:CCTCC NO:M 2016627,保藏日期:2016年11月9日;其基因组上存在的色素合成相关基因PigE被敲除,由潮霉素基因hph基因序列所替代。
具体实施方式
下面对本发明具体实施方式做一个详细说明。
实施例1:PigE基因敲除载体的构建:基于同源重组的PigE基因敲除原理如图1所示。
(1)PigE基因上下游臂和hph基因的扩增:利用GeneBank中公布的红色红曲霉(Monascus ruber)中PigE基因序列(登录号:KF285431.1),设计两对引物P1、P2和P3、P4分别扩增靶基因PigE基因的翻译起始区和终止密码子区侧翼序列,引物序列如表1所示,实验结果如图2所示。PigE基因5’侧翼序列和3’侧翼序列PCR扩增体系如表2所示。PigE基因5’侧翼序列PCR反应条件如下:98℃预变性4min,98℃变性30s,64℃退火30s,72℃延伸1min,循环数为30个,72℃终延伸10min,12℃保存。PigE基因3’侧翼序列PCR反应条件如下:98℃预变性4min,98℃变性30s,68℃退火30s,72℃延伸1min,循环数为30个,72℃终延伸10min,12℃保存。
表1敲除载体构建中设计的引物
注:P1和P2用于扩增PigE基因上游臂,P3和P4用于扩增PigE基因下游臂,加下划线部分序列为hph基因部分序列。hph-F和hph-R用于从pCB1003载体中扩增hph基因。P1和P4用于进行三段连接反应,双下划线部分分别为KpnⅠ和XbaⅠ酶切位点。
表2 PigE基因5’侧翼序列和3’侧翼序列PCR扩增体系
(2)Double-joint PCR法连接PigE基因上下游臂、hph基因:PigE基因的5’侧翼序列、3’侧翼序列、hph基因序列加入PCR管中,进行两轮PCR反应。两轮PCR反应体系分别如表3、表4所示。第一轮PCR反应条件如下:98℃预变性4min,98℃变性30s,55℃退火10min,72℃延伸4min,循环数为15个,72℃终延伸10min,12℃保存。第二轮PCR反应条件如下:98℃预变性3min,98℃变性30s,68℃退火30s,72℃延伸3min,循环数为30个,72℃终延伸10min,12℃保存。
表3第一轮PCR反应体系
反应成分 | 体积(μL) |
dNTP | 2 |
purified PigE 5,-flanking amplicon | 1 |
purified PigE 3,-flanking amplicon | 1 |
purified hph amplicon | 3 |
5×prime STAR buffer | 5 |
Prime STAR HS DNA Polymersae | 0.25 |
ddH2O | Up to 25 |
表4第二轮PCR反应体系
反应成分 | 体积(μL) |
dNTP | 4 |
LA PCR buffer | 2 |
LF | 1 |
LR | 1 |
Template | 0.5 |
LA Taq DNA Polymersae | 0.2 |
ddH2O | Up to 25 |
PigE基因敲除盒和pKO1B质粒用XbaⅠ和KpnⅠ双酶切,酶切后敲除盒用DNA清洁试剂盒纯化,pKO1B质粒酶切后跑电泳割胶回收,后用T4DNA Ligase连接,连接产物转化根癌农杆菌AGL-1菌株,菌液PCR验证,验证敲除载体。
实施例2:根癌农杆菌介导的基因转化及敲除子的筛选
1培养基
PDA培养基:马铃薯20%、葡萄糖2%、琼脂粉2.0%、pH 5.0~5.5。
LB液体培养基:胰蛋白胨1%、酵母提取物0.5%、NaCl 1%、pH 7.0。
IM液体培养基:pH 4.9,0.01mol/L的磷酸氢钾缓冲液0.8mL,硫酸镁-氯化钠溶液20mL,1%CaCl2·H2O溶液1mL,50%甘油溶液10mL,20%NH4NO3溶液2.5mL,20%葡萄糖溶液10mL,补充蒸馏水至1000mL。
使用时,每1mL培养基中加入0.01%FeSO4 10μL,100g/L 2-(N-吗啡啉)乙磺酸(MES)10μL和0.1mol/L乙酰丁香酮(AS)4μL,混匀后马上使用,用于农杆菌的诱导培养。
Co-IM培养基:同IM,但是20%葡萄糖溶液只加5mL。使用时,先将培养基熔化,待温度稍降低后,每1mL培养基中加入0.01%FeSO4 10μL,100g/L MES 10μL和0.1mol/L AS8μL,混匀后马上使用,用于培养红曲霉孢子和农杆菌。
2实验方法
2.1红曲霉分生孢子悬浮液的制备
将野生型红曲霉菌株接种于PDA培养基,30℃培养21d,后用5mL无菌水清洗培养平板,洗脱液经4层擦镜纸过滤,滤液即为红曲霉孢子悬浮液。将红曲霉孢子悬浮液离心后用无菌水重悬,血球计数板计数,配成浓度为105孢子/mL的红曲霉分生孢子悬浮液。
2.2根癌农杆菌的活化与诱导培养
将含有敲除载体的农杆菌AGL-1菌株在含有50μg/mL卡那霉素(Kan)、50μg/mL利福平(Rif)抗性的LB培养平板上划线培养2d。后挑取农杆菌单菌落接种于2mL含有50μg/mL卡那霉素抗性的LB液体培养基中,28℃、180r/min摇床培养12-16h。培养后的农杆菌菌液离心弃上清,菌体沉淀用诱导培养基IM(加入1mL 100mg/L MES,1mL 0.01%FeSO4,400μL0.1mol/L AS)稀释,后28℃180r/min培养5-6h,至农杆菌菌液OD600=0.5。
2.3农杆菌与红曲霉孢子洗脱液共培养
将制备的红曲霉孢子悬浮液5000r/min离心10min,红曲霉孢子用经诱导活化培养获得的农杆菌菌液稀释,血球计数板计数稀释至红曲霉孢子悬浮液浓度为1×105个/mL。取上述红曲霉孢子与农杆菌混合液200μL涂布于硝酸纤维素膜上,后将硝酸纤维素膜贴于Co-IM培养基(加入1mL 100mg/L MES,1mL 0.01%FeSO4,800μL 0.1mol/L AS)表面,28℃培养3d。
2.4hph抗性平板筛选转化子
将硝酸纤维素膜转移到PDA选择培养基上(50μg/mL潮霉素、500μg/mL头孢霉素),30℃培养7d,将长出来的单菌落转接至含有50μg/mL潮霉素抗性的PDA平板上培养,抗性平板上再次长出的转化子接种于无抗的PDA平板上30℃培养7d,保藏于25%的甘油中储存于-80℃冰箱备用。
2.5PCR验证基因重组PigE缺失突变株
提取步骤2.4中保藏的红曲霉转化子菌株基因组DNA,分别用引物hph-F/hph-R、PigE-F/Pig-R、P1/P4进行扩增,验证基因重组PigE缺失突变株,实验结果如图3所示。
2.6基因重组PigE缺失突变株菌落及显微结构特征
经筛选获得了1株菌落色泽发生显著变化的基因工程M-piy菌株。野生型菌株与M-piy菌株的菌落及显微结构特征如图4所示。M-piy菌株除红曲色素种类和产量发生变化外,M-piy菌株的生长速度、菌落、菌丝、分生孢子和闭囊壳等与野生型菌株没有明显差异。
实施例3:PigE基因缺失M-piy菌株黄色素色价及桔霉素含量的检测
1培养基
PDA培养基:马铃薯20%、葡萄糖2%、琼脂粉2.0%、pH 5.0~5.5。用于菌株活化培养。
PD液体培养基:马铃薯20%、葡萄糖2%、pH 5.0~5.5。用于菌株种子培养。
YES液体培养基:酵母提取物4%、蔗糖16%、pH 5.5~6.0。用于菌株发酵培养。
2实验方法
2.1 M-piy菌株菌丝黄色素色价的检测
将保藏的红曲霉野生型菌株和PigE基因缺失M-piy菌株分别接种到PDA培养基上,28℃活化培养7d。用打孔器取3个边缘菌饼(直径8mm)转接到装有100mL PD培养液的250mL三角瓶中,28℃、180r/min摇床上培养3d用作种子液。再将种子液按10%转接到YES液体培养基中(250mL三角瓶装100mL)、28℃、180r/min摇床上分别培养5d、7d、9d、11d、13d、15d、17d、19d,取样检测不同培养天数下红曲霉野生型菌株与M-piy菌株菌丝黄色素的色价。
称取0.05g红曲霉干菌丝,加入4mL 70%乙醇,漩涡震荡,60℃温育1h,12000rpm离心10min,取上清液测其在300nm-600nm处全波长扫描图谱,用分光光度计检测OD值(410nm、370nm)并计算黄色素色价。黄色素色价计算公式:黄色素色价=吸收峰处OD值×稀释倍数×20U/g
2.2红曲霉发酵液桔霉素含量的检测
培养方法同2.1。分别取不同培养时间发酵液(包括菌丝)1mL放入5mL的离心管中,加入等体积的甲苯-乙酸乙酯-甲酸(7:3:1),剧烈震荡混匀,12000r/min离心10min,取上清液。采用高效液相色谱(HPLC)法检测桔霉素含量,色谱条件是:Agilent C18柱(4.6mm×250mm,5μm);流动相是乙腈(A):酸水(B,磷酸调节pH 2.5)=75:25;λ=331nm、流速1mL/min、柱温30℃、进样体积20μL。
3实验结果
3.1 M-piy菌株色素提取液扫描图谱
野生型菌株和M-piy菌株菌丝色素提取液300nm-600nm波长扫描图谱见图5,M-piy菌株菌丝色素提取液只有一个吸收峰,吸收峰为370nm左右;而野生型菌株菌丝色素提取液有2个吸收峰,吸收峰分别为410nm和520nm左右。
3.2 M-piy菌株菌丝红曲黄色素的色价
野生型菌株和M-piy菌株菌丝色素提取液黄色素色价见图6。M-piy菌株只产生红曲黄色素,红曲黄色素产量较野生型菌株提高了约9.6倍,达到了9232.86U/g,而野生型菌株红曲黄色素产量仅为958.23U/g。
3.3 M-piy菌株发酵液桔霉素的含量
野生型菌株和M-piy菌株发酵液桔霉素含量检测结果见图7。M-piy菌株产桔霉素的发酵时间较原始野生型菌株延长,桔霉素含量显著降低,发酵7d后才开始产生桔霉素,发酵15d后桔霉素含量仅为0.31mg/L,是野生型菌株的0.22倍。
<110>浙江师范大学
<120>低产桔霉素高产红曲黄色素的基因重组紫色红曲霉M-piy菌株及其制备方法和用途
<160>8
<210>1
<211>34
<212>DNA
<213>人工序列
<400>1
GGGGTACCCC CGACAGCATC TCCCGTGTTG AAGT 34
<210>2
<211>46
<212>DNA
<213>人工序列
<400>2
GCTCCTTCAA TATCATCTTC TCTCGCTTTC TTTGGTCGGA GTTATC 46
<210>3
<211>45
<212>DNA
<213>人工序列
<400>3
TAGAGTAGAT GCCGACCGAA CAAGAGGAAT CCAGTTTCAT TAGAG 45
<210>4
<211>35
<212>DNA
<213>人工序列
<400>4
GCTCTAGAGC TCTGGCAGTA TTTTCGCTTT TCCGC 35
<210>5
<211>25
<212>DNA
<213>人工序列
<400>5
CGAGAGAAGA TGATATTGAA GGAGC 25
<210>6
<211>25
<212>DNA
<213>人工序列
<400>6
TCTTGTTCGG TCGGCATCTA CTCTA 25
<210>7
<211>22
<212>DNA
<213>人工序列
<400>7
AAAGCACATC TAGGATTTAT AG 22
<210>8
<211>23
<212>DNA
<213>人工序列
<400>8
ATTAATCTTC TGGTCAATGCG AAT 23
Claims (7)
1.紫色红曲霉(Monascus purpureus)M-piy菌株,该保藏于武汉大学菌种保藏中心,保藏地址为:中国武汉武汉大学,保藏编号:CCTCC NO:M 2016627,保藏日期:2016年11月9日;该菌株基因组上存在的色素合成相关基因PigE被敲除,由潮霉素基因hph基因序列所替代。
2.权利要求1所述的紫色红曲霉M-piy菌株的制备方法,其特征在于该方法包括以下的步骤:
1)PigE基因上下游臂和hph基因的扩增:
利用GeneBank中公布的红色红曲霉(Monascus ruber)中PigE基因序列,设计两对引物P1、P2和P3、P4分别扩增靶基因PigE基因的翻译起始区和终止密码子区侧翼序列,引物序列如下所述:
P1和P2用于扩增PigE基因上游臂,P3和P4用于扩增PigE基因下游臂,加下划线部分序列为hph基因部分序列;hph-F和hph-R用于从pCB1003载体中扩增hph基因;P1和P4用于进行三段连接反应,双下划线部分分别为Kpn Ⅰ和Xba Ⅰ酶切位点;
PigE基因5’侧翼序列和3’侧翼序列PCR扩增体系如下:
PigE基因5’侧翼序列PCR反应条件如下:98℃预变性4min,98℃变性30s,64℃退火30s,72℃延伸1min,循环数为30个,72℃终延伸10min,12℃保存;PigE基因3’侧翼序列PCR反应条件如下:98℃预变性4min,98℃变性30s,68℃退火30s,72℃延伸1min,循环数为30个,72℃终延伸10min,12℃保存;
2)Double-joint PCR法连接PigE基因上下游臂、hph基因:
PigE基因的5’侧翼序列、3’侧翼序列、hph基因序列加入PCR管中,进行两轮PCR反应;第一轮PCR反应体系如下:
第一轮PCR反应条件如下:98℃预变性4min,98℃变性30s,55℃退火10min,72℃延伸4min,循环数为15个,72℃终延伸10min,12℃保存;
第二轮PCR反应体系如下:
第二轮PCR反应条件如下:98℃预变性3min,98℃变性30s,68℃退火30s,72℃延伸3min,循环数为30个,72℃终延伸10min,12℃保存;
PigE基因敲除盒和pKO1B质粒用XbaⅠ和KpnⅠ双酶切,酶切后敲除盒用DNA清洁试剂盒纯化,pKO1B质粒酶切后跑电泳割胶回收,后用T4 DNA Ligase连接,连接产物转化根癌农杆菌AGL-1菌株,菌液PCR验证,验证敲除载体;
3)红曲霉分生孢子悬浮液的制备
将野生型红曲霉菌株接种于PDA培养基,30℃培养21d,后用5mL无菌水清洗培养平板,洗脱液经4层擦镜纸过滤,滤液即为红曲霉孢子悬浮液;将红曲霉孢子悬浮液离心后用无菌水重悬,血球计数板计数,配成浓度为105孢子/mL的红曲霉分生孢子悬浮液;
4)根癌农杆菌的活化与诱导培养
将含有敲除载体的农杆菌AGL-1菌株在含有50μg/mL卡那霉素(Kan)、50μg/mL利福平(Rif)抗性的LB培养平板上划线培养2d;后挑取农杆菌单菌落接种于2mL含有50μg/mL卡那霉素抗性的LB液体培养基中,28℃、180r/min摇床培养12-16h;培养后的农杆菌菌液离心弃上清,菌体沉淀用诱导培养基IM稀释,诱导培养基IM加入1mL 100mg/L MES,1mL0.01%FeSO4,400μL 0.1mol/L AS,后28℃180r/min培养5-6h,至农杆菌菌液OD600=0.5;
5)农杆菌与红曲霉孢子洗脱液共培养
将制备的红曲霉孢子悬浮液5000r/min离心10min,红曲霉孢子用经诱导活化培养获得的农杆菌菌液稀释,血球计数板计数稀释至红曲霉孢子悬浮液浓度为1×105个/mL;取上述红曲霉孢子与农杆菌混合液200μL涂布于硝酸纤维素膜上,后将硝酸纤维素膜贴于Co-IM培养基表面,Co-IM培养基加入1mL 100mg/L MES,1mL 0.01%FeSO4,800μL 0.1mol/L AS,28℃培养3d;
6)筛选获得紫色红曲霉M-piy菌株。
3.权利要求1所述的紫色红曲霉M-piy菌株用于生产红曲黄色素。
4.一种红曲黄色素的生产方法,其特征在于该方法包括以下的步骤:
1)将保藏的紫色红曲霉M-piy菌株接种到PDA培养基上,28℃活化培养7d;
2)用打孔器取3个边缘菌饼转接到装有100mL PD培养液的250mL三角瓶中,28℃、180r/min摇床上培养3d用作种子液;
3)再将种子液按10%质量百分比转接到YES液体培养基中、28℃、180r/min摇床上分别培养11~19d。
5.根据权利要求4所述的一种红曲黄色素的生产方法,其特征在于,PDA培养基按质量百分比计包括以下组分:马铃薯20%、葡萄糖2%、琼脂粉2.0%、pH 5.0~5.5。
6.根据权利要求4所述的一种红曲黄色素的生产方法,其特征在于,PD液体培养基按质量百分比计包括以下组分:马铃薯20%、葡萄糖2%、pH 5.0~5.5。
7.根据权利要求4所述的一种红曲黄色素的生产方法,其特征在于,YES液体培养基按质量百分比计包括以下组分:酵母提取物4%、蔗糖16%、pH 5.5~6.0。
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CN112608853A (zh) * | 2020-12-24 | 2021-04-06 | 浙江工业大学 | 一株不产桔霉素的重组红曲菌及其构建方法 |
CN114736814A (zh) * | 2022-04-25 | 2022-07-12 | 浙江工业大学 | 一株高产monacolin k的重组红曲菌及其构建方法与应用 |
CN115287280A (zh) * | 2022-09-05 | 2022-11-04 | 安徽农业大学 | 一种增强红曲霉紫外诱变效果的方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105385604A (zh) * | 2015-09-17 | 2016-03-09 | 天津科技大学 | 低产桔霉素高产色素的Winter4红曲霉菌株 |
-
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105385604A (zh) * | 2015-09-17 | 2016-03-09 | 天津科技大学 | 低产桔霉素高产色素的Winter4红曲霉菌株 |
Non-Patent Citations (1)
Title |
---|
QINGPEI LIU等: "MpigE , a gene involved in pigment biosynthesis in Monascus ruber M7", 《APPL MICROBIOL BIOTECHNOL》 * |
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CN110331101A (zh) * | 2019-04-11 | 2019-10-15 | 北京工商大学 | 紫色红曲菌mokH基因缺失菌株的构建方法 |
CN112608853A (zh) * | 2020-12-24 | 2021-04-06 | 浙江工业大学 | 一株不产桔霉素的重组红曲菌及其构建方法 |
CN114736814A (zh) * | 2022-04-25 | 2022-07-12 | 浙江工业大学 | 一株高产monacolin k的重组红曲菌及其构建方法与应用 |
CN114736814B (zh) * | 2022-04-25 | 2024-02-09 | 浙江工业大学 | 一株高产monacolin k的重组红曲菌及其构建方法与应用 |
CN115287280A (zh) * | 2022-09-05 | 2022-11-04 | 安徽农业大学 | 一种增强红曲霉紫外诱变效果的方法 |
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