Establishment of toolkit and T7RNA polymerase/promoter system in Shewanella oneidensis MR-1

Abstract

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.