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| Construction of Bidirectional Promoter Probe Vector and Its Application in Corynebacterium glutamicum |
| CAO Yu, YU Xin-Yu, LIU Xiu-Xia, BAI Zhong-Hu* |
| College of Biotechnology/Key Laboratory of Industrial Biotechnology/National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, Wuxi 214122, China |
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Abstract As one of the common expression elements in nature, the bidirectional promoters (BDPs) can provide new tools for genetic circuit design and metabolic pathway assembly and optimization. However, there is a lack of high-throughput screening and characterization methods for BDPs. In order to obtain the efficient BDPs for multi-gene regulation of metabolic pathways based on genome-level high-throughput screening, a BDPs probe vector p19BDP based on fluorescence generated by the interaction of the green fluorescent sensor for transiently expressed proteins 1 (gSTEP1) and the sensor for transiently expressed proteins tag (STEPtag) was constructed. The gSTEP1 and STEPtag gene were amplified by PCR, and ligated to the p19-0 vector in a "head-to-head" manner. After inserting the BDPs sequence between the two genes, the BDPs activity was assessed by detecting the fluorescence intensity. The p19BDP was used to characterize the activity of 8 BDPs screened by transcriptome data analysis in Corynebacterium glutamicum. The results showed that the fluorescence intensity of the BDPcat and BDPicl were 15.9 and 6.2 times higher than those before induction, respectively, the fluorescence intensity was positively correlated with the concentration of inducer in a certain range. For the other 6 endogenous BDPs (BDP1~BDP6), the fluorescence intensity of BDP6 was 6.8 times higher than that of the control promoter BDPcat (uninduced). The probe vecotr did not affect the growth of recombinant strain, and its fluorescence value remained stable after the formation of gSTEP1/STEPtag complex. The above results showed that the probe vector p19BDP could characterize sensitively and effectively the strength of BDPs in C. glutamicum. This study provides a powerful tool for high-throughput mining of natural BDPs in C. glutamicum and a new idea for characterizing BDPs in prokaryote.
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Received: 21 April 2022
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Corresponding Authors:
*baizhonghu@jiangnan.edu.cn
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