Characterization of a Bi-directional Promoter OtrRp Involved in Oxytetracycline Biosynthesis.

Previous studies identified a MarR (multiple antibiotic resistance regulator) family transcription factor OtrR in the oxytetracycline biosynthetic gene cluster, which regulated the expression of an efflux pump OtrB. The genes otrB and otrR were divergent arranged and the inter-ORF (open reading frame) region between the two genes contained the promoter otrBp. In this study, we demonstrated that the reverse complementary sequence of otrBp contained the promoter of otrR, and its activity was also repressed by OtrR by sharing the same operator otrO within otrBp, and allosteric regulated by oxytetracycline.

Improvement of stress tolerance and riboflavin production of Bacillus subtilis by introduction of heat shock proteins from thermophilic bacillus strains.

In this study, stress tolerance devices consisting of heat shock protein (HSP) genes from thermophiles Geobacillus and Parageobacillus were introduced into riboflavin-producing strain Bacillus subtilis 446 to improve its stress tolerance and riboflavin production. The 12 HSP homologs were selected from 28 Geobacillus and Parageobacillus genomes according to their sequence clustering and phylogenetically analysis which represents the diversity of HSPs from thermophilic bacillus. The 12 HSP genes and 2 combinations of them (PtdnaK-PtdnaJ-PtgrpE and PtgroeL-PtgroeS) were heterologously expressed in B.

A novel signal transduction system for development of uric acid biosensors.

Uric acid (UA) is an important biomarker for clinical diagnosis. Here, we present a novel signal transduction system for the development of UA biosensors with the characteristics of stability and ease-of-use. In this system, bacterial allosteric transcription factor HucR was used as the bio-recognition element, and the competition between HucR and the restriction endonuclease HindIII-HF to bind to the designed DNA template was employed to enable signal transduction of UA recognized by HucR.

Development of small molecule biosensors by coupling the recognition of the bacterial allosteric transcription factor with isothermal strand displacement amplification.

Here, we demonstrate an easy-to-implement and general biosensing strategy by coupling the small-molecule recognition of the bacterial allosteric transcription factor (aTF) with isothermal strand displacement amplification (SDA) in vitro. Based on this strategy, we developed two biosensors for the detection of an antiseptic, p-hydroxybenzoic acid, and a disease marker, uric acid, using bacterial aTF HosA and HucR, respectively, highlighting the great potential of this strategy for the development of small-molecule biosensors.

New Intracellular Shikimic Acid Biosensor for Monitoring Shikimate Synthesis in Corynebacterium glutamicum.

The quantitative monitoring of intracellular metabolites with in vivo biosensors provides an efficient means of identifying high-yield strains and observing product accumulation in real time. In this study, a shikimic acid (SA) biosensor was constructed from a LysR-type transcriptional regulator (ShiR) of Corynebacterium glutamicum. The SA biosensor specifically responded to the increase of intracellular SA concentration over a linear range of 19.

An Autoregulated Fine-Tuning Strategy for Titer Improvement of Secondary Metabolites Using Native Promoters in Streptomyces.

Streptomycetes are well-known producers of biologically active secondary metabolites. Various efforts have been made to increase productions of these metabolites, while few approaches could well coordinate the biosynthesis of secondary metabolites and other physiological events of their hosts. Here we develop a universal autoregulated strategy for fine-tuning the expression of secondary metabolites biosynthetic gene clusters (BGCs) in Streptomyces species.

Improvement of oxytetracycline production mediated via cooperation of resistance genes in Streptomyces rimosus.

Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics. To increase the oxytetracycline (OTC) production in Streptomyces rimosus, we investigated the cooperative effect of three co-overexpressing OTC resistance genes: one gene encodes a ribosomal protection protein (otrA) and the other two express efflux proteins (otrB and otrC). Results indicated that combinational overexpression of otrA, otrB, and otrC (MKABC) exerted a synergetic effect.

Evidence for the formation of ScbR/ScbR2 heterodimers and identification of one of the regulatory targets in Streptomyces coelicolor.

The homologous transcriptional regulators ScbR and ScbR2 have previously been identified as γ-butyrolactone (GBL) and antibiotic receptors, respectively. They regulate diverse physiological processes in Streptomyces coelicolor in response to GBL and antibiotic signals. In this study, ScbR and ScbR2 proteins were shown to interact using a bacterial two-hybrid system where adenylate cyclase activity was reconstituted in Escherichia coli BTH101.

Engineered jadomycin analogues with altered sugar moieties revealing JadS as a substrate flexible O-glycosyltransferase.

Glycosyltransferases (GTs)-mediated glycodiversification studies have drawn significant attention recently, with the goal of generating bioactive compounds with improved pharmacological properties by diversifying the appended sugars. The key to achieving glycodiversification is to identify natural and/or engineered flexible GTs capable of acting upon a broad range of substrates. Here, we report the use of a combinatorial biosynthetic approach to probe the substrate flexibility of JadS, the GT in jadomycin biosynthesis, towards different non-native NDP-sugar substrates, enabling us to identify six jadomycin B analogues with different sugar moieties.

A platform for the development of novel biosensors by configuring allosteric transcription factor recognition with amplified luminescent proximity homogeneous assays.

A wide range of chemicals can be sensed by allosteric transcription factors (aTFs) in bacteria. Herein, we report a biosensing platform by using isolated aTFs as recognition elements in vitro. Moreover, a general strategy to increase the sensitivity of the aTF-based biosensors is provided.

Engineering deacetoxycephalosporin C synthase as a catalyst for the bioconversion of penicillins.

7-aminodeacetoxycephalosporanic acid (7-ADCA) is a key intermediate of many clinically useful semisynthetic cephalosporins that were traditionally prepared by processes involving chemical ring expansion of penicillin G. Bioconversion of penicillins to cephalosporins using deacetoxycephalosporin C synthase (DAOCS) is an alternative and environmentally friendly process for 7-ADCA production. Arnold Demain and co-workers pioneered such a process.

Heterologous expression of oxytetracycline biosynthetic gene cluster in Streptomyces venezuelae WVR2006 to improve production level and to alter fermentation process.

Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts.

Bioluminescent Probe for Detecting Mercury(II) in Living Mice.

A novel bioluminescence probe for mercury(II) was obtained on the basis of the distinct deprotection reaction of dithioacetal to decanal, so as to display suitable sensitivity and selectivity toward mercury(II) over other ions with bacterial bioluminescence signal. These experimental results indicated such a probe was a novel promising method for mercury(II) bioluminescence imaging in environmental and life sciences ex vivo and in vivo.

[Efficient production of polyketide products in Streptomyces hosts - A review].

Polyketides represent an important class of structurally and functionally diverse secondary metabolites with high economic value. Among bacteria, Streptomycetes are the main producers of polyketides. To enhance polyketide production in Streptomyces hosts, rational metabolic engineering approaches have been applied, such as overexpressing rate-limiting enzymes, or transcriptional activator, increasing the supply of precursor, removing feedback inhibition by end products and heterologous expression of polyketide biosynthetic gene clusters.

[The modified bacterial two-hybrid system].

Bacterial two-hybrid system is a newly developed method for studying protein-protein interactions. However, in our studies of the interaction of regulatory proteins in Streptomyces, it was found that the bacterial two-hybrid system is not sensitive enough by the blue-and-white selection on X-gal plate. To overcome this drawback, the reason of false positive clone was firstly determined, which was the disturbance of other direct or indirect regulation on lacZ promoter.

Overproduction and identification of butyrolactones SCB1-8 in the antibiotic production superhost Streptomyces M1152.

Gamma-butyrolactones (GBLs) are signalling molecules that control antibiotic production in Streptomyces bacteria. The genetically engineered strain S. coelicolor M1152 was found to overproduce GBLs SCB1-3 as well as five novel GBLs named SCB4-8.

Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts.

Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.

[Evaluation of penicillin expandase mutants and complex substrate inhibition characteristics at high concentrations of penicillin G].

Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models.

Discovery of New Substrates for LuxAB Bacterial Bioluminescence.

In this article, four novel substrates with long halftime have been designed and synthesized successfully for luxAB bacterial bioluminescence. After in vitro and in vivo biological evaluation, these molecules can emit obvious bioluminescence emission with known bacterial luciferase, thus indicating a new promising approach to developing the bacterial bioluminescent system.

Cloning, expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from Thermoanaerobacter tengcongensis.

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa.

[Progress in transcriptional studies].

Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system.

Genome-wide identification and characterization of reference genes with different transcript abundances for Streptomyces coelicolor.

The lack of reliable reference genes (RGs) in the genus Streptomyces hampers effort to obtain the precise data of transcript levels. To address this issue, we aimed to identify reliable RGs in the model organism Streptomyces coelicolor. A pool of potential RGs containing 1,471 genes was first identified by determining the intersection of genes with stable transcript levels from four time-series transcriptome microarray datasets of S.

Genome-wide identification and evaluation of constitutive promoters in streptomycetes.

Background: Streptomycetes attract a lot of attention in metabolic engineering and synthetic biology because of their well-known ability to produce secondary metabolites. However, the available constitutive promoters are rather limited in this genus.

Results: In this work, constitutive promoters were selected from a pool of promoters whose downstream genes maintained constant expression profiles in various conditions.