Construction of a tunable promoter library to optimize gene expression in Methylomonas sp. DH-1, a methanotroph, and its application to cadaverine production

Background As methane is 84 times more potent than carbon dioxide in exacerbating the greenhouse effect, there is an increasing interest in the utilization of methanotrophic bacteria that can convert harmful methane into various value-added compounds. A recently isolated methanotroph, Methylomonas sp. DH-1, is a promising biofactory platform because of its relatively fast growth. However, the lack of genetic engineering tools hampers its wide use in the bioindustry. Results Through three different approaches, we constructed a tunable promoter library comprising 33 promoters that can be used for the metabolic engineering of Methylomonas sp. DH-1. The library had an expression level of 0.24–410% when compared with the strength of the lac promoter. For practical application of the promoter library, we fine-tuned the expressions of cadA and cadB genes, required for cadaverine synthesis and export, respectively. The strain with PrpmB-cadA and PDnaA-cadB produced the highest cadaverine titre (18.12 ± 1.06 mg/L) in Methylomonas sp. DH-1, which was up to 2.8-fold higher than that obtained from a non-optimized strain. In addition, cell growth and lysine (a precursor of cadaverine) production assays suggested that gene expression optimization through transcription tuning can afford a balance between the growth and precursor supply. Conclusions The tunable promoter library provides standard and tunable components for gene expression, thereby facilitating the use of methanotrophs, specifically Methylomonas sp. DH-1, as a sustainable cell factory. Graphical Abstract

Development of a Multi-Epitope Recombinant Protein for the Diagnosis of Human Visceral Leishmaniasis

Background: Iran is one of the endemic areas of Mediterranean Visceral Leishmaniasis, a disease caused by Leishmania infantum. In this work, we examined whether Proteína quimérica 10 (PQ10) recombinant protein is suitable for immunological diagnosis of human visceral leishmaniasis. Methods: The study was carried out in Tarbiat Modares University during 2016- 2018. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. Sequencing with proper primers was done, the expression, optimization of expression and protein purification were performed, and the purified recombinant protein was confirmed by western blot. The efficacy of PQ10 for serodiagnosis was evaluated with 50 positive and 50 negative serum samples, which confirmed by the direct agglutination test and collected from individuals living in the visceral leishmaniasis endemic areas of Iran. ELISA was performed with the PQ10 recombinant protein. Results: The 95% CI sensitivity of ELISA that was evaluated with sera from naturally infected individuals was 84%. The 95% CI specificity value of the ELISA determined with sera from healthy individuals (50 serum samples) and from individuals with other infectious diseases was 82%. The 95% CI positive predictive value (PPV) and negative predictive value (NPV) were exterminated 82.35% and 83.67%, respectively. Conclusion: We have used a recombinant synthetic protein to improve serodiagnosis of human visceral leishmaniasis. PQ10 could be useful for diagnosis of asymptomatic cases, as well as in the early phase of infections.

Publisher Correction: Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-21369-z

Production and Expression Optimization of Heterologous Serratiopeptidase

Background: Serratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the cloning and the expression optimization of serratiopeptidase. Methods: The heat-stable serratiopeptidase (5d7w) was selected as the template. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The recombinant protein was expressed in Esherichia coli BL21 and confirmed by SDS-PAGE and Western blot analysis. Different parameters such as expression vector, culture media, post-induction incubation temperature, inducer concentration, and post-induction incubation time were altered to obtain the highest amount of the recombinant protein. Results: Serratiopeptidase was successfully cloned and expressed under optimized conditions in E. coli which confirmed by western blot analysis. The optimal conditions of expression were determined using pQE30 as vector, cultivating the host bacteria in Terrific Broth (TB) medium, at 37º C, induction by IPTG concentration equal to 0.5 mM, and cells were harvested 4 h after induction. Conclusion: As serratiopeptidase is a multi-potent enzyme, the expressed recombinant protein can be considered as a valuable agent for pharmaceutical applications in further studies.