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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (12): 21-31    DOI: 10.13523/j.cb.20181204
研究报告     
双启动子促进亮氨酸脱氢酶在Bacillus subtilis中表达及发酵研究 *
张玲,王男,金吕华,林荣,杨海麟()
江南大学工业生物技术教育部重点实验室 无锡 214122
To Promote the Expression of Leucine Dehydrogenase in Bacillus subtilis via Dual-Promoter and Fermentation Research
ZHANG Ling,WANG Nan,JIN Lv-hua,LIN Rong,YANG Hai-lin()
Key laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China
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摘要：

为了促进亮氨酸脱氢酶在Bacillus subtilis中高效表达,采取在质粒pMA5自带的启动子PHpaII之后分别添加诱导型和组成型启动子,考察双启动子对酶表达的影响。选取2种诱导型启动子(Pgrac、Pgl-M1)和4种组成型启动子(P43、Plaps、PHpaII、PamyQ)进行构建表达,其中组成型启动子PamyQ与PHpaII构成的双启动子效果最好,有效地将胞外活性提高到31.24U/ml,是单启动子PHpaII的3.4倍。在以上最优双启动子的基础上分别融合Sec途径和Tat途径的4种信号肽,但信号肽与双启动子共同作用并没有获得更高的酶活性。选用酶活最高的双启动子突变菌株Bacillus subtilis 168/pW6(PHpaII-PamyQ)进行7.5L发酵罐补料发酵产酶研究,LeuDH酶活达到217.96U/ml,是摇瓶水平的6.97倍,对工业上产亮氨酸脱氢酶有一定参考价值。
关键词： 亮氨酸脱氢酶;  枯草芽孢杆菌;  启动子;  信号肽;  发酵产酶    
Abstract:

In order to promote the efficient expression of leucine dehydrogenase in Bacillus subtilis, the inducible and constitutive promoters were inserted downstream of PHpaII, which is the native promoter of plasmid pMA5, to investigate the effect on LeuDH by dual-promoter. Two inducible promoters (Pgrac, Pglv-M1) and four constitutive promoters (P43, Plaps, PHpaII, PamyQ) were selected for expression of LeuDH. The dual promoter was consisted in the constitutive promoter PamyQ and PHpaII had the best performance, reaching 31.24U/ml, which was 3.4 times higher than that of the single promoter PHpaII. Four signal peptides of Sec pathway and Tat pathway were fused to behind the best dual-promoter PHpaII-PamyQ, respectively. However, this combination did not give higher enzyme activity. The strain B. subtlis 168/pW6 with the best activity of producing leucine dehydrogenase were analyzed in 7.5L fermenter. The enzyme activity reached 217.96U/ml, which was 6.97 times higher than the shake flask level. The results for industrial production of leucine dehydrogenase have a certain reference value.
Key words: Leucine dehydrogenase    Bacillus subtilis    Dual-promoter    Signal peptide    Fermentation enzyme production
收稿日期: 2018-06-11 出版日期: 2019-01-10
ZTFLH:  Q78  
基金资助: * 江苏省产学研(BY2016022-40);*国家轻工技术与工程一流学科自主课题(2018-23)
通讯作者: 杨海麟     E-mail: yanghailin@jiangnan.edu.cn
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引用本文:

张玲,王男,金吕华,林荣,杨海麟. 双启动子促进亮氨酸脱氢酶在Bacillus subtilis中表达及发酵研究 *[J]. 中国生物工程杂志, 2018, 38(12): 21-31.

ZHANG Ling,WANG Nan,JIN Lv-hua,LIN Rong,YANG Hai-lin. To Promote the Expression of Leucine Dehydrogenase in Bacillus subtilis via Dual-Promoter and Fermentation Research. China Biotechnology, 2018, 38(12): 21-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181204    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I12/21

引物 序列
Pgrac-F GGGAATTCCATATGAGCTATTGTAACATAATCGG
Pgrac-R CGCGGATCCTGATCCTTCCTCCTTTAATT
P43-F GGGAATTCCATATGTGATAGGTGGTATGTTTTCGC
P43-R CGCGGATCCGTGTACATTCCTCTCTTACCT
PHpaII-F GGGAATTCCATATGGGTGGAGATTTTTTGAGTGAT
PHpaII-R CGCGGATCCTAAATCGCTCCTTTTTAGGTG
PamyQ-F GGGAATTCCATATGGGCGGCGTTCTGTTTCTG
PamyQ-R CGCGGATCCATAAGGCAGTAAAGAGGTTTTG
SamyQ-F1 CGCGGATCCATGATTCAAAAACGAAAGCGG
SamyQ-R1 ATTCGAAGATCTCCAGGGTCATGTCGACTACGGCTGATGTTTTTGTAATCGGC
SamyQ-F2 GCCGATTACAAAAACATCAGCCGTAGTCGACATGACCCTGGAGATCTCCGAAT
SamyQ-R2 CGACGCGTTTAACGACGGCTAATGATATCGTGACCG
SsacB-F1 CGCGGATCCATGAACATCAAAAGTTTGC
SsacB-R1 ACGCGTCGACCGCAAACGCTTGAGTTGCGCCT
SphoD-F1 CGCGGATCCATGGCATACGACAGTCGTTTTG
SphoD-R1 ACGCGTCGACGGCCCCAACCGACTGGGCAATC
SywbN-F1 CGCGGATCCATGAGCGATGAACAAAAAAAGCC
SywbN-R1 ACGCGTCGACCGCAACGGCTGCCCCCGCCAT
表1  引物序列表
图1  诱导型启动子重组质粒构建示意图
图2  启动子Pgrac的PCR扩增(a)及质粒pW1 (PHpaII-Pgrac)的双酶切验证(b)
图3  含诱导型启动子重组菌生长曲线(a)及酶活曲线(b)
重组菌 培养时间(h) 胞外活性(U/ml) 胞内活性(U/ml) 总酶活(U/ml)
B. subtilis 168/pW1 (PHpaII-Pgrac) 24 1.87 18.61 20.58
B. subtilis 168/pW2 (PHpaII-Pglv-M1) 32 0.82 0.54 1.36
表2  含诱导型启动子Pgrac和Pglv-M1重组菌产酶比较
图4  含诱导型启动子重组菌产LeuDH发酵上清的SDS-PAGE
图5  组成型启动子PCR(a)及酶切验证(b)
图6  含组成型启动子重组菌生长曲线(a)和相应酶活测定(b)
重组菌 培养时间(h) 胞外活性(U/ml) 胞内活性 (U/ml) 总酶活 (U/ml)
B. subtilis 168/pW3(PHpaII-P43) 48 0.99 2.52 3.51
B. subtilis 168/pW4(PHpaII-Plaps) 48 0.32 3.69 4.01
B. subtilis 168/pW5(PHpaII-PHpaII) 48 0.80 1.11 1.91
B. subtilis 168/pW6(PHpaII-PamyQ) 48 31.24 36.89 68.31
表3  添加不同组成型型启动子重组菌产酶的比较
图7  含组成型启动子重组菌产LeuDH发酵上清的SDS-PAGE
图8  重组质粒pW6I (PHpaII-PamyQ,AmyQ)构建示意图
图9  基因融合PCR (a)和质粒pW6I (PHpaII-PamyQ, AmyQ)双酶切验证(b)
图10  双启动子融合信号肽重组质粒双酶切验证
图11  含双启动和信号肽重组菌生长曲线(a)和相应酶活测定(b)
重组菌 胞外酶活(U/ml) 胞内酶活(U/ml) 总酶活(U/ml)
B. subtilis168/pW6I (PHpaII-PamyQ, AmyQ) 0.44 0.65 1.09
B. subtilis168/pW6II (PHpaII-PamyQ, SacB) 1.69 3.47 5.16
B. subtilis168/pW6III (PHpaII-PamyQ, PhoD) 6.49 18.09 24.58
B. subtilis168/pW6IV (PHpaII-PamyQ, YwbN) 21.76 31.60 53.36
表4  各重组菌表达LeuDH酶活数据
图12  未补料(a)和补料(b)对重组菌发酵产酶影响
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