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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (12): 21-31    DOI: 10.13523/j.cb.20181204
Orginal Article     
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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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     
Received: 11 June 2018      Published: 10 January 2019
ZTFLH:  Q78  
Corresponding Authors: Hai-lin YANG     E-mail: yanghailin@jiangnan.edu.cn
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Ling ZHANG
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Lv-hua JIN
Rong LIN
Hai-lin YANG
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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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181204     OR     https://manu60.magtech.com.cn/biotech/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
Table 1 List of primers
Fig.1 Construction procedure of recombinant plasmid with inducible promoters
Fig.2 PCR of promoter Pgrac (a) and restriction digestion (b) (a) M: DNA marker; 1: PCR verification of Pgrac (b) M1、M2: DNA marker; 1: pMA5-leudh digested with BamH I; 2: pW1 (PHpaII-Pgrac) double digested with BamH I and Nde I
Fig.3 Cell growth curves (a) and extracellular enzyme activity curves of different recombinants with inducible promoters (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
Table 2 Comparison of LeuDH activity by recombinants contained inducible promoter Pgrac and Pglv-M1
Fig.4 SDS-PAGE analysis of LeuDH extracellular activity contained inducible promoters M: Protein marker (Low); 1: B. subtilis 168/pMA5-leudh (PHpaII) (control); 2: Culture supernatants of B. subtilis 168/pW2 (PHpaII-Pglv-M1); 3: Culture supernatants of B. subtilis 168/pW1 (PHpaII-Pgrac)
Fig.5 PCR of consistant promoters (a) and restriction digestion (b) (a) M: marker; 1: PCR verification of PamyQ; 2: PCR verification of P43; 3: PCR verification of PHpaII (b) M: marker; 1: pMA5-leudh digested with BamH I (control); 2: pW3 (PHpaII-P43) double digested with BamH I and Nde I; 3: pW5 (PHpaII-PHpaII) double digested with BamH I and Nde I; 4: pW6 (PHpaII-PamyQ) double digested with BamH I and Nde I
Fig.6 Cell growth curves (a) and extracellular enzyme activity curves of different recombinants with constitutive promoters (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
Table 3 Comparison of LeuDH activity by recombinants contained different constitutive promoters
Fig.7 SDS-PAGE analysis of LeuDH extracellular activity contained consistive promoters M: Protein marker (Low); 1: Culture supernatants of B. subtilis 168/pMA5-leudh (PHpaII) (control); 2: Culture supernatants of B. subtilis 168/pW3 (PHpaII-P43); 3: Culture supernatants of B. subtilis 168/pW4 (PHpaII-Plaps); 4: Culture supernatants of B. subtilis 168/pW5 (PHpaII-PHpaII); 5: Culture supernatants of B. subtilis 168/pW6 (PHpaII-PamyQ)
Fig.8 Construction procedure of recombinant plasmid pW6I (PHpaII-PamyQ, AmyQ)
Fig. 9 Gene overlap PCR (a) and restriction digestion of plasmids pW6I (PHpaII-PamyQ, AmyQ) (b) (a) M1, M2: Marker; 1: PCR verification of AmyQ; 2: PCR verification of leudh; 3: PCR verification of AmyQ-leudh (b) M: Marker; 1: pW6I (PHpaII-PamyQ, AmyQ) double digested with BamH I and Sal I; 2: pW6 (PHpaII-PamyQ) double digested with BamH I and Sal I (control)
Fig.10 Restriction digestion of recombinant plasmids with double promoters and signal peptides M: Marker; 1: pMA5-leudh double digested with BamH I and Sal I (control); 2: pM6III (PHpaII-PamyQ, PhoD) double digested with BamH I and Sal I; 3: pW6II (PHpaII-PamyQ, SacB) double digested with BamH I and Sal I; 4: pW6IV (PHpaII-PamyQ, YwbN) double digested with BamH I and Sal I
Fig.11 Cell growth curves (a) and extracellular enzyme activity curves of different recombinants with dual promoter and signal peptides (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
Table 4 LeuDH activity of B. subtilis168/pW6 with different signal peptides
Fig. 12 Effect of no feeding (a) and feeding (b) on enzyme production by recombinant
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