A powerful hybrid puc operon promoter tightly regulated by both IPTG and low oxygen level

Abstract

Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photosynthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI(q)-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl beta-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.

Increased Production of Coenzyme $Q_{10}$ from Genetic Engineered <i>Rhodobacter sphaeroides</i> Overexpressing UbiG

“…sphaeroides strains were grown at 30C in malate minimal medium (Remes et al, 2014). Growth under micro-aerobic conditions was performed as described in our previous study (Hu et al, 2010). E. coli strains were cultivated vigorously in flasks at 37C in Luria-Bertani medium.…”

“…LH1 is one of the most important photosynthetic apparatus encoded by puf operon (Hu et al, 2002). puf operon promoter is normally regulated by oxygen tension and light intensity (Hu, et al, 2010). A powerful promoter and optimal growth conditions are very important for largest production of CoQ10 in genetic engineered Rb.…”

Human neutrophil peptide 3 could be functionally expressed in Rhodobacter sphaeroides

“…After sequencing verification, the HNP3 gene fragment was cut by SacI and XbaI and then ligated into the previously constructed Rb. sphaeroides expression vector pRK-pucPlacI q puc1B His10 1AC (Hu et al, 2010), resulting in the HNP3 expression vector pRKpucPlacI q puc1B-HNP3 His10 -1AC, as shown in Fig. 2.…”

Spectral properties of LH2 exhibit very similar even when heterologously express LH2 with &lt;i&gt;β&lt;/i&gt;-subunit fusion protein in &lt;i&gt;Rhodobacter&lt;/i&gt; &lt;i&gt;sphaeroides&lt;/i&gt;

“…Rb. sphaeroides CQU68 cell cultures expressing LH2 or LH2 with the β-subunit-GFP fusion protein complexes were shifted from aerobic conditions to semi-aerobic conditions at an OD 600 of 0.5 -1.0, and continuously incubated for about 8 hours after adding the isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM [27]. LH2 and LH2 with the β-subunit-GFP fusion protein complexes were one-step Figure 1.…”

“…In our previous study, we constructed a novel Rb. sphaeroides expression system which could be employed to evaluate heterologous protein expression levels in real-time using LH2 characteristic absorption at near infra-red regions [27][28][29]. In the present study, we lie emphasis on the spectral properties of LH2 when heterologously express LH2 with -subunit fusion protein in Rb.…”