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Establishment of toolkit and T7RNA polymerase/promoter system in Shewanella oneidensis MR-1
Journal of the Taiwan Institute of Chemical Engineers ( IF 5.7 ) Pub Date : 2020-02-25 , DOI: 10.1016/j.jtice.2020.02.003
Ying-Chen Yi , I-Son Ng

Shewanella oneidensis MR-1 is a well-known electrogenic bacterium for its respiratory and extracellular electron transfer (EET) capability. However, the genetic toolkits, including promoters, replication origins and biological parts are still rarely used. In this study, constitutive promoters of pLacI, pJ23100, pJ23105, pJ23109, and pTet expressing super-folder green fluorescent protein (sfGFP) were verified in combination with replication origins of pBR322 and p15A. The optimal genetic module was obtained from the pLacI promoter and pBR322 origin, which the specific fluorescence intensity in the MR-1 reached 5518 a.u./g-DCW. T7RNA polymerase (T7RNAP) was also integrated into MR-1 chromosome (i.e., MR1::T7R) by homologous recombination to establish the T7 system. Thus, an expression vector was constructed under T7 promoter and a mobilization gene cluster, which was overexpressed on red fluorescence protein (RFP), carbonic anhydrase (CA), and heme-related proteins. The optimal condition for induction of isopropyl β-d-1-thiogalactopyranoside (IPTG) was determined by the RFP fluorescence intensity, which was 0.5 mM IPTG after 2 h incubation. Moreover, the carbon dioxide capture was enhanced with CAs which activity reached to 12,106 WAU/mg, while the productivity of valuable 5-aminolevuinic acid, a pro-drug for cancer therapy, increased by 3.96-folds with overexpression of HemD in MR1::T7R. The results proved the feasibility of the functional T7 system in Shewanella species.



中文翻译:

沙瓦氏假单胞菌MR-1的工具包和T7RNA聚合酶/启动子系统的建立

沙瓦氏菌(Shewanella oneidensis) MR-1因其呼吸和细胞外电子转移(EET)功能而闻名。但是,遗传工具包,包括启动子,复制起点和生物学部分,仍然很少使用。在这项研究中,结合pBR322和p15A的复制起点,验证了表达超文件夹绿色荧光蛋白(sfGFP)的pLacI,pJ23100,pJ23105,pJ23109和pTet的组成型启动子。从pLacI启动子和pBR322来源获得了最佳的遗传模块,其中MR-1中的比荧光强度达到5518 au / g-DCW。T7RNA聚合酶(T7RNAP)也被整合到MR-1染色体((,MR1 :: T7R)通过同源重组建立T7系统。因此,在T7启动子和动员基因簇下构建了表达载体,该动员基因簇在红色荧光蛋白(RFP),碳酸酐酶(CA)和血红素相关蛋白上过表达。诱导异丙基β- d -1-硫代半乳糖吡喃糖苷(IPTG)的最佳条件由RFP荧光强度确定,孵育2 h后为0.5 mM IPTG。此外,CAs的活性达到12,106 WAU / mg,可提高二氧化碳的捕获量,而MR1中HemD的过表达则有价值的5-氨基乙酰丙酸(一种用于癌症治疗的前药)的生产率提高了3.96倍: T7R。结果证明了功能性T7系统在希瓦氏菌属中的可行性。

更新日期:2020-02-25
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