Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces.

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining.

Targeted activation of silent natural product biosynthesis pathways by reporter-guided mutant selection.

The continuously increasing genome sequencing data has revealed numerous cryptic pathways, which might encode novel secondary metabolites with interesting biological activities. However, utilization of this hidden potential has been hindered by the observation that many of these gene clusters remain silent (or poorly expressed) under laboratory conditions. Here we present reporter-guided mutant selection (RGMS) as an effective and widely applicable method for targeted activation of silent gene clusters in the native producers.

An engineered strong promoter for streptomycetes.

Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor, the native kasOp is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasOp was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasOp3.

A simplified diphenylamine colorimetric method for growth quantification.

Cell growth needs to be monitored in biological studies and bioprocess optimization. In special circumstances, such as microbial fermentations in media containing insoluble particles, accurate cell growth quantification is a challenge with current methods. Only the Burton method is applicable in such circumstances.

Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples.

We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ (max)=390 nm) is converted to a red product (λ (max)=486 nm).

Induction of holomycin production and complex metabolic changes by the argR mutation in Streptomyces clavuligerus NP1.

In bacteria, arginine biosynthesis is tightly regulated by a universally conserved regulator, ArgR, which regulates the expression of arginine biosynthetic genes, as well as other important genes. Disruption of argR in Streptomyces clavuligerus NP1 resulted in complex phenotypic changes in growth and antibiotic production levels. To understand the metabolic changes underlying the phenotypes, comparative proteomic studies were carried out between NP1 and its argR disruption mutant (designated CZR).

[Evaluation of the activities of two promoters in Streptomycetes by reporter gene method].

Objective: The mutated promoter of the erythromycin resistance gene (PermE *) is a strong promoter generally used in streptomycetes, and we evaluated the expression activities of a new promoter (Psf) and PermE * in Streptomycetes.

Methods: We used kanamycin resistance gene(neo) and catechol 2,3-dioxygenase gene (xylE) as reporters.

Results: Both promoters exhibited high level of promoter activities in Streptomyces clavuligerus NRRL3585, Streptomyces coelicolor M145, Streptomyces venezuelae ISP5230 and Streptomyces lividans TK54.

[Establishment of a cell-based high-throughput screening model for PPARdelta agonists].

To establish a new high-throughput screening model for the agonist of PPARdelta, PPARdelta gene was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR), and subcloned to pGEM-T Vector for sequencing, then the PPARdelta fragment was excised by restriction enzymes, and inserted into pTARGET Vector to construct expression vector pTARGET-ppARdelta. Insert three copies of PPRE into pGl3-promoter vector to construct expression vector pGl3-PPRE x 3-luc. The vector pTARGET-ppARdelta was transiently cotransfected with pGl3-PPRE x 3-luc into different cell lines to assay the expression levels of luciferase.