An Engineered Strong Promoter for Streptomycetes

Liu

Appl Biochem Biotechnol

et al. 2011

Escherichia coli is one of the most commonly used host strains for recombinant protein production. More and more research works on the production of recombinant protein indicate that extracellular production throughout a culture medium is more convenient and attractive compared to intracellular production. In present work, inducing temperature and isopropyl β-D: -1-thiogalactopyranoside (IPTG) concentration were investigated to decrease the formation of inclusion body and increase the amount of soluble recombinant cutinase initially. Enzyme activity in the culture medium reached to 118.9 U/ml at 64 h of culture, and no inclusion body was detected in cytoplasm under the inducement condition of 0.2 mM IPTG and 30°C. In addition, it was found that a large amount of cutinase had been accumulated in periplasm since 16-h cultivation under the same inducement condition. Therefore, glycine and surfactant sodium taurodeoxycholate (TDOC) were further used to promote the leakage of recombinant cutinase from periplasm. Supplied with 100 mM glycine and 1 mM TDOC, the amount of cutinase in periplasm decreased remarkably, and the activity in the culture medium reached to 146.2 and 149.2 U/ml after 54 h of culturing, respectively.

Section: Effect Of Surfactant On Translocation Of Recombinant Cutinasmentioning

confidence: 99%

Study on Improvement of Extracellular Production of Recombinant Thermobifida fusca Cutinase by Escherichia coli

Liu

Appl Biochem Biotechnol

et al. 2011

“…TX-TL can provide a tool to rapidly prototype the cellular machinery of synthetic biology hosts [17]. Herein, we provide evidence for the development of a high-activity S. venezuelae TX-TL system utilizing the kasOp* promoter as a standard for cell-extract optimization [18]. In summary, we demonstrate high-yield synthesis of up to 1.3 µM superfolder GFP (sfGFP), prototype a tetO-TetR gene expression tool [19] and synthesize a selection of enzymes from the S. rimosus (ATCC 10970) oxytetracycline pathway [20].…”

Section: Introductionmentioning

confidence: 98%

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

Moore

Lai

Needham

et al. 2017

Biotechnology Journal

Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.

Proc. Natl. Acad. Sci. U.S.A.

“…S4) reconfirmed that the engineered short and strong promoter was less likely to interfere with the global regulatory factors (36), in comparison with the previously reported regulatory elements, thereby providing a great opportunity to rationally design the pathway-specific regulatory system for a gene cluster. Therefore, the characterized synthetic promoter and RBS libraries are of high application value for activation and optimization of cryptic secondary metabolic pathways.…”

Section: Characterization Of Native or Synthetic Promoters And Rbss Inmentioning

confidence: 56%

“…As shown in Fig. 4A, the kasOp* promoter, originally obtained by Wang and coworkers (36), exhibited the strongest activity among all the promoters. Its activity is 20-fold higher than that of the widely used ermEp* promoter.…”

Section: Characterization Of Native or Synthetic Promoters And Rbss Inmentioning

confidence: 65%

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

Bai

Zhang

Zhao

et al. 2015

Self Cite

168

197

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. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.synthetic biology | natural product | flow cytometry | single-cell resolution | modular regulatory elements S treptomycetes are well known as the most abundant source of bioactive secondary metabolites (1), including medically important antimicrobial agents [e.g., chloramphenicol from Streptomyces venezuelae (2)], agricultural chemicals [e.g., avermectin from Streptomyces avermitilis (3)], and anticancer agents and immunosuppressants [e.g., rapamycin from Streptomyces hygroscopicus (4)]. However, the increasing difficulty of discovering novel drugs via traditional high-throughput screening and the "one strain many compounds" approach is frustrating pharmaceutical productivity (5, 6). Deciphering the genome sequences of Streptomyces surprisingly established the presence of a plethora of gene clusters encoding for yet-unobserved molecules, even in intensively investigated Streptomyces coelicolor A3 (2), revealing a much higher potential of novel bioactive agent production than originally anticipated (7,8). Therefore, the enormous number of natural products that have been obtained likely represent only a tiny portion of the repertoire of bioactive compounds that can possibly be produced. This has brought about extensive research into applied genomics aimed at investigating these new gene clusters, generally referred to as "cryptic," "silent," or "orphan" (9-11). With data on more than 12,000 in-house draft bacterial genomes, the potential for the discovery of a number of novel chemicals encrypted in silent biosynthetic gene clusters has been detected by genome mining.Many new strategies have been documented for "awakening" poorly expressed and/or silent gene clusters in Strept...