Enhanced production of recombinant proteins in Corynebacterium glutamicum by construction a bicistronic gene expression system

Background: Corynebacterium glutamicum is a traditional food-grade industrial microorganism， in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum. Results: In this research, the Escherichia coli ( E. coli ) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-a (BoIFN-a), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type I N-terminal peptide (PINP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PINP was also performed in fed-batch cultivation, and the highest PINP production level was 1.2 g/L. Conclusion: In this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Enhanced production of recombinant proteins in Corynebacterium glutamicum by construction a bicistronic gene expression system

Background: Corynebacterium glutamicum is a traditional food-grade industrial microorganism， in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantages of low recombinant protein expression levels need to be solved. Here, according to the bacteria-specific polycistronic feature, trials have been made of inserting a leading peptide upstream of target genes as an expression enhancer, and we found it improving the expression level of proteins under the control of inducible tac promoter in C. glutamicum CGMCC1.15647 . Results: In this research, the Escherichia coli ( E. coli ) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic vectors were isolated and exhibited higher strength under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-a (BoIFN-a), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type I N-terminal peptide (PINP). All examined proteins were highly expressed compared with the original vector of tac promoter. Large-scale production of PINP was also performed in fed-batch cultivation, and the highest PINP production level was 1.2 g/L. Conclusions: In this study, we improved the strength of the inducible promoter tac promoter for C. glutamicum by screening and inserting fore-cistron in front of the target genes. Those vectors with bicistronic expression pattern have strong compatibility for expressing various heterogeneous proteins in high level. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Construction of Recombinant E.coli Expressing a Thermophilic Catalase and its Application in H2O2 Wastewater Treatment

The recombinant plasmid pETac-CATHis with tac promoter was constructed for the constitutive expression of a thermophilic catalase in E.coli. And the effects of cultivation conditions of two recombinant strains, BL21(DE3)/pETac-CATHis and BL21(DE3)pLysS/pETac-CATHis, such as initial pH, cultivation temperature, cultivation time, and loading volume on catalase activity were investigated. The optimal cultivation conditions were: the initial pH 7.0, cultivation temperature 37°C, cultivation time 12 hours, and loading volume 25mL in 100mL flask. Under the optimal cultivation condition, the highest catalase activities of the two recombinant strains were 291.7 U/mL and 349.5 U/mL, respectively. After adding catalase, H2O2in simulation H2O2wastewater containing phenol was decomposed completely very quickly. Declining of catalase activity was detected with enhancing the phenol concentration, probably because the phenol might be a kind of competitive inhibitor for catalase.

Characterization of the sodF gene region of Frankia sp. strain ACN14a and complementation of Escherichia coli sod mutant

The Frankia sp. strain ACN14a superoxide dismutase SodF was previously shown to be induced in response to Alnus glutinosa root exudates, and its gene was sequenced. We report here the sequence of the 9-kb genomic segment surrounding the sodF gene and further characterize this gene and its product. Nine ORFs coding for various proteins, such as regulators, acetyl-CoA transferases, and a bacterioferritin A next to the sodF gene, were found. Northern blot analysis showed that the sodF gene was expressed as a major 1-kb transcript, which indicates that it has its own promoter. The sodF gene strongly complemented an Escherichia coli triple mutant (sodA sodB recA), restoring aerobic growth when the gene was expressed from the synthetic tac promoter but when expressed from its own promoter showed only slight rescue, suggesting that it was poorly recognized by the E. coli RNA polymerase. It is noteworthy that this is the first time that a Frankia gene has been reported to complement an E. coli mutant. The superoxide dismutase activity of the protein was inactivated by hydrogen peroxide, indicating that the metal ligand is iron, which is supported by analysis of the protein sequence. Thus, the SodF protein induced in Frankia by root exudates is an iron-containing enzyme similar to the one present in the nodules.Key words: Frankia, iron superoxide dismutase, sodF, E. coli complementation.

Introduction of the phzH Gene of Pseudomonas chlororaphis PCL1391 Extends the Range of Biocontrol Ability of Phenazine-1-Carboxylic Acid-Producing Pseudomonas spp. Strains

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. s radicislycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2–79 and P. aureofaciens 30–84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2–79 and P. aureofaciens 30–84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.

Introduction of theSerratia marcescens chiAgene into an endophyticPseudomonas fluorescensfor the biocontrol of phytopathogenic fungi

An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 × 105CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.Key words: endophyte, biological control, chitinase.