Liang Tianxin, Sun Jun, Ju Shuyun, Su Shenyi, Yang Lirong, Wu Jianping
Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China.
Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, China.
Front Chem. 2021 Jul 16;9:664967. doi: 10.3389/fchem.2021.664967. eCollection 2021.
KT2440 has become an attractive chassis for heterologous expression with the development of effective genetic manipulation tools. Improving the level of transcriptional regulation is particularly important for extending the potential of KT2440 in heterologous expression. Although many strategies have been applied to enhance the heterologous expression level in KT2440, it was still at a relatively low level. Herein we constructed a T7-like expression system in KT2440, mimicking the pET expression system in , which consisted of T7-like RNA polymerase (MmP1) integrated strain and the corresponding expression vector for the heterologous expression enhancement. With the optimization of the insertion site and the copy number of RNA polymerase (RNAP), the relative fluorescence intensity (RFI) of the super-folder green fluorescent protein (sfGFP) was improved by 1.4-fold in MmP1 RNAP integrated strain. The induction point and IPTG concentration were also optimized. This strategy was extended to the gene-reduced strain EM42 and the expression of sfGFP was improved by 2.1-fold. The optimal RNAP integration site was also used for introducing T7 RNAP in KT2440 and the expression level was enhanced, indicating the generality of the integration site for the T7 expression system. Compared to other inducible expression systems in KT2440, the heterologous expression level of the Mmp1 system and T7 system were more than 2.5 times higher. Furthermore, the 3.6-fold enhanced expression level of a difficult-to-express nicotinate dehydrogenase from JA1 verified the efficiency of the T7-like expression system in KT2440. Taken together, we constructed and optimized the T7-like and T7 expression system in , thus providing a set of applicable chassis and corresponding plasmids to improve recombinant expression level, expecting to be used for difficult-to-express proteins.
随着有效基因操作工具的发展，KT2440已成为用于异源表达的有吸引力的底盘。提高转录调控水平对于扩展KT2440在异源表达中的潜力尤为重要。尽管已应用许多策略来提高KT2440中的异源表达水平，但仍处于相对较低的水平。在此，我们在KT2440中构建了一个类似T7的表达系统，模仿大肠杆菌中的pET表达系统，该系统由整合了类似T7 RNA聚合酶（MmP1）的菌株和用于增强异源表达的相应表达载体组成。通过优化RNA聚合酶（RNAP）的插入位点和拷贝数，在整合了MmP1 RNAP的菌株中，超折叠绿色荧光蛋白（sfGFP）的相对荧光强度（RFI）提高了1.4倍。还对诱导点和异丙基-β-D-硫代半乳糖苷（IPTG）浓度进行了优化。该策略扩展到基因减少菌株EM42，sfGFP的表达提高了2.1倍。最佳的RNAP整合位点也用于在KT2440中引入T7 RNAP，表达水平得到提高，表明该整合位点对T7表达系统具有通用性。与KT2440中的其他诱导表达系统相比，Mmp1系统和T7系统的异源表达水平高出2.5倍以上。此外，来自JA1的一种难以表达的烟酸脱氢酶的表达水平提高了3.6倍，验证了类似T7的表达系统在KT2440中的效率。综上所述，我们在KT2440中构建并优化了类似T7和T7的表达系统，从而提供了一组适用的底盘和相应的质粒以提高重组表达水平，有望用于表达困难的蛋白质。
