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An auto-inducible phosphate-controlled expression system of Bacillus licheniformis.

ABSTRACT: BACKGROUND:A promoter that drives high-level, long-term expression of the target gene under substrate limited growth conditions in the absence of an artificial inducer would facilitate the efficient production of heterologous proteins at low cost. A novel phosphate-regulated expression system was constructed using the promoter of the phytase encoding gene phyL from Bacillus licheniformis for the overexpression of proteins in this industrially relevant host. RESULTS:It is shown that the phyL promoter enables a strong overexpression of the heterologous genes amyE and xynA in B. licheniformis when cells were subjected to phosphate limitation. Whether B. licheniformis can use phytate as an alternative phosphate source and how this substrate influences the PphyL controlled gene expression under growth conditions with limited inorganic phosphate concentrations were also investigated. It is shown that B. licheniformis cells are able to use sodium phytate as alternative phosphate source. The addition of small amounts of sodium phytate (? 5?mM) to the growth medium resulted in a strong induction and overexpression of both model genes in B. licheniformis cells under phosphate limited growth conditions. CONCLUSIONS:The PphyL controlled expression of the investigated heterologous genes in B. licheniformis is strongly auto-induced under phosphate limited conditions. The proposed PphyL expression system enables an overexpression of target genes in B. licheniformis under growth conditions, which can be easily performed in a fed-batch fermentation process.

SUBMITTER: Trung NT

PROVIDER: S-EPMC6327384 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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An auto-inducible phosphate-controlled expression system of Bacillus licheniformis.

Trung Nguyen Thanh NT Hung Nguyen Minh NM Thuan Nguyen Huy NH Canh Nguyen Xuan NX Schweder Thomas T Jürgen Britta B

BMC biotechnology 20190109 1

<h4>Background</h4>A promoter that drives high-level, long-term expression of the target gene under substrate limited growth conditions in the absence of an artificial inducer would facilitate the efficient production of heterologous proteins at low cost. A novel phosphate-regulated expression system was constructed using the promoter of the phytase encoding gene phyL from Bacillus licheniformis for the overexpression of proteins in this industrially relevant host.<h4>Results</h4>It is shown tha ...[more]

PMID: 30626366

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Project description:BACKGROUND: After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction) prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli. RESULT: The newly designed pLAI expression system places the expression of recombinant proteins in Escherichia coli under control of the regulatory genes of the lux regulon of Vibrio fischeri's Quorum Sensing (QS) system.The pLAI system allows a tight regulation of the recombinant gene allowing a negligible basal expression and expression only at high cell density. Sequence optimization of regulative genes of QS of V. fischeri for expression in E. coli upgraded the system to high level expression. Moreover, partition of regulative genes between the plasmid and the host genome and introduction of a molecular safety lock permitted tighter control of gene expression. CONCLUSION: Coupling gene expression to cell density using cell-to-cell communication provides a promising approach for recombinant protein production. The system allows the control of expression of the target recombinant gene independently from external inducers or drastic changes in metabolic conditions and enabling tight regulation of expression.

Project description:BackgroundTo study the biological function of miRNAs, and to achieve sustained or conditional gene silencing with siRNAs, systems that allow controlled expression of these small RNAs are desirable. Methods for cell delivery of siRNAs include transient transfection of synthetic siRNAs and expression of siRNAs in the form of short hairpins using constitutive RNA polymerase III promoters. Systems employing constitutive RNA polymerase II promoters have been used to express miRNAs. However, for many experimental systems these methods do not offer sufficient control over expression.ResultsWe present an inducible mammalian expression system that allows for the conditional expression of short hairpin RNAs that are processed in vivo to generate miRNAs or siRNAs. Using modified nuclear receptors in a two hybrid format and a synthetic ligand, the Rheoswitch system allows rapid and reversible induction of mRNA expression. We evaluated the system's properties using miR-122 as a model miRNA. A short hairpin encoding miR-122 cloned into the expression vector was correctly processed to yield mature miRNA upon induction with ligand and the amount of miRNA produced was commensurate with the concentration of ligand. miR-122 produced in this way was capable of silencing both endogenous target genes and appropriately designed reporter genes. Stable cell lines were obtained, resulting in heritable, consistent and reversible expression of miR-122, a significant advantage over transient transfection. Based on these results, obtained with a microRNA we adapted the method to produce a desired siRNA by designing short hairpins that can be accurately and efficiently processed.ConclusionWe established an Inducible expression system with a miR-122 backbone that can be used for functional studies of miRNAs and their targets, in heterologous cells that do not normally express the miRNA. Additionally we demonstrate the feasibility of using the miR-122 backbone to express shRNA with a desired siRNA guide strand for inducible RNAi silencing.

Dataset Information

An auto-inducible phosphate-controlled expression system of Bacillus licheniformis.

Publications

An auto-inducible phosphate-controlled expression system of Bacillus licheniformis.

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