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Verification of the integration of pOSV802 in S. coelicolor M145, S. lividans TK23, and S. albus J1074 chromosomes. (A) Principle of the PCR verification of the integration of the pOSV801 to pOSV812 vectors in the Streptomyces chromosomes (PCR 1 and PCR 2) (PCR 3, PCR verification before excision of modules 1 to 3). (B) PCR fragments obtained by PCR 1 (attL region; expected sizes, 913 bp for M145 and TK23, 888 bp for J1074) and by PCR 2 (attR region; expected sizes, 911 bp for M145 and TK23, 907 bp for J1074) on the three Streptomyces strains bearing pOSV802. No PCR amplification is expected when the genomic DNA of the wild-type Streptomyces strains is used as the matrix. MW corresponds to the molecular weight ladder (Thermo Scientific GeneRuler DNA ladder mix).
Source publication
One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example, antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites or to force the expression of otherwise silent natural biosynthetic gene clusters...
Citations
... For the detection and quantification of 1,3-diols or 3-hydroxy acids, 5 μl samples were injected onto a Phenomenex Kinetex XB-C18 LC column (2.6 μm, 100 mm × 3 mm, 100 Å) and analysed using the following HPLC protocol: buffer A: water with 0.1% (v/v) formic acid; buffer B: MeOH with 0.1% (v/v) formic acid; flow rate: 0. 42 For the detection and quantification of amino alcohols, 5 μl samples were injected onto an Agilent ZORBAX Eclipse Plus C18 LC column (3.5 μm, 4.6 mm × 150 mm) and analysed using the following HPLC protocol: buffer A: water with 0.1% (v/v) formic acid; buffer B: acetonitrile with 0.1% (v/v) formic acid; flow rate: 0.40 ml min −1 , 2% buffer B for 0.5 min, 2-13% buffer B gradient for 4.5 min, 13-80% buffer B gradient for 0.1 min; flow rate changed to 1.0 ml min −1 , 80% buffer B for 2 min, 80-2% buffer B gradient for 3.1 min, 2% buffer B for 1.1 min; flow rate changed to 0.4 ml min −1 , 2% buffer B for 1.1 min; mass detection range: m/z = 70-300. ...
Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here we present an approach that uses a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and nicotinamide adenine dinucleotide phosphate-dependent terminal thioreductases. Reduction of the terminal aldehyde with alcohol dehydrogenases enables the production of diols, oxidation enables the production of hydroxy acids and specific transaminases allow the production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A-specific acyltransferase in the extension module with methyl- or ethylmalonyl-coenzyme A-specific acyltransferase enables the production of branched-chain diols, amino alcohols and carboxylic acids in high titres. Use of our PKS platform in Streptomyces albus demonstrated the high tunability and efficiency of the platform.
... Such mobile genetic elements can either autonomously replicate in the cell cytoplasm or integrate into defined sites of the host chromosome. Although many naturally occurring mobile genetic elements, like bacteriophages, plasmids and transposons, have been found in Actinobacteria, only a few of them were applied to the genetic manipulation of actinomycetes [56,82]. ...
... Resistance genes originating from transposons of Gram-negative bacteria can also be applied for actinobacterial engineering, such as neomycin/kanamycin (aphII) and phleomycin (ble) from Tn5, gentamicin (aacC1) and apramycin (aac(3)IV) [20,91]. These resistance genes are expressed in Streptomyces from their own transposon promoters and seem to be functional in a wide range of actinobacterial strains [56,82,92]. ...
... The most used strong promoter is ermE*, which is a derivative of the ermE promoter and contains a trinucleotide deletion in the ermEp1 region of the erythromycin resistance gene from S. erythraeus [95]. Other used promoters include KasO from the SARP family regulator in S. coelicolor A3 and SF14 from the S. ghanaensis phage I19 genome, as well as promoters gapdh and rpsL from S. griseus, which have higher activity than the ermE* promoter (Table 1) [81,82,106,112,113]. ...
Organisms from the genus Streptomyces feature actinobacteria with complex developmental cycles and a great ability to produce a variety of natural products. These soil bacteria produce more than 2/3 of antibiotics used in medicine, and a large variety of bioactive compounds for industrial, medical and agricultural use. Although Streptomyces spp. have been studied for decades, the engineering of these bacteria remains challenging, and the available genetic tools are rather limited. Furthermore, most biosynthetic gene clusters in these bacteria are silent and require strategies to activate them and exploit their production potential. In order to explore, understand and manipulate the capabilities of Streptomyces spp. as a key bacterial for biotechnology, synthetic biology strategies emerged as a valuable component of Streptomyces research. Recent advancements in strategies for genetic manipulation of Streptomyces involving proposals of a large variety of synthetic components for the genetic toolbox, as well as new approaches for genome mining, assembly of genetic constructs and their delivery into the cell, allowed facilitation of the turnaround time of strain engineering and efficient production of new natural products at an industrial scale, but still have strain- and design-dependent limitations. A new perspective offered recently by technical advances in DNA sequencing, analysis and editing proposed strategies to overcome strain- and construct-specific difficulties in the engineering of Streptomyces. In this review, challenges and recent developments of approaches for Streptomyces engineering are discussed, an overview of novel synthetic biology strategies is provided and examples of successful application of new technologies in molecular genetic engineering of Streptomyces are highlighted.
... Such mobile genetic elements can either autonomously replicate in the cell cytoplasm or integrate into defined sites of the host chromosome. Although many naturally occurring mobile genetic elements like bacteriophages, plasmids and transposons have been found in Actinobacteria, only a few of them were applied for the genetic manipulation of actinomycetes [54,60]. ...
... Resistance genes originating from transposons of Gram-negative bacteria can also be applied for actinobacterial engineering, such as neomycin/kanamycin (aphII) and phleomycin (ble) from Tn5, gentamicin (aacC1 79) and apramycin (aac(3)IV). These resistance genes are expressed in Streptomyces from their own transposon promoters and seem to be functional in a wide range of actinobacterial strains [54,60,61]. ...
... The most used such strong promoter is ermE*, which is a derivative of the ermE promoter and contains a trinucleotide deletion in the ermEp1 region of the erythromycin resistance gene from S. erythraeus. Other used promoters include KasO from the SARP family regulator in S. coelicolor A3 and SF14 from the S. ghanaensis phage I19 genome, as well as promoters gapdh and rpsL from S. griseus, which have higher activity than the ermE* promoter [59,60,71]. ...
Organisms from the genus Streptomyces feature actinobacteria with complex developmental cycle and great ability to produce a variety of natural products, which is possible due to complicated crosstalk between primary and secondary metabolism. These soil bacteria produce more than 2/3 of antibiotics used in medicine, and a large variety of bioactive compounds for industry and agricultural use. Although Streptomyces spp. have been studied for decades, the engineering of these bacteria remains challenging, and available genetic tools rather limited. Recent advancements in genetic manipulation of Streptomyces involving proposal of CRISPR/Cas9-based workflows as well as synthetic components (e.g. promoters, ribosome-binding sites, terminators, reporter genes) allowed to facilitate the turnaround time of strain engineering, but still has strain-specific limitations. However, a new perspective offered by synthetic biology to exploit the potential of existing and novel pathways in primary and secondary metabolism allows combining of different biosynthetic steps originating from diverse bacteria using a limited toolbox. Manipulation of interplay between primary and secondary metabolism proposes strategies to overcome strain-specific difficulties in engineering, leveraging insights in Streptomyces-specific physiological features. In this review, developments of these approaches for Streptomyces engineering are discussed and an overview of the synthetic biology developments is provided.
... Similarly, a set of 12 standardized modular plasmids has been designed to facilitate the assembly of natural product BGCs, but only allow the iterative assembly of genes (or gene cassettes) using the BioBrick assembly method. In addition, they are all based on the E. coli p15A replicon, which limits the capacity of the cloned natural product BGCs [19]. ...
Streptomyces has enormous potential to produce novel natural products (NPs) as it harbors a huge reservoir of uncharacterized and silent natural product biosynthetic gene clusters (BGCs). However, the lack of efficient gene cluster engineering strategies has hampered the pace of new drug discovery. Here, we developed an easy-to-use, highly flexible DNA assembly toolkit for gene cluster engineering. The DNA assembly toolkit is compatible with various DNA assembling approaches including Biobrick, Golden Gate, CATCH, yeast homologous recombination-based DNA assembly and homing endonuclease-mediated assembly. This compatibility offers great flexibility in handling multiple genetic parts or refactoring large gene clusters. To demonstrate the utility of this toolkit, we quantified a library of modular regulatory parts, and engineered a gene cluster (act) using characterized promoters that led to increased production. Overall, this work provides a powerful part assembly toolkit that can be used for natural product discovery and optimization in Streptomyces.
... The schematic diagram of the 3 A assembly was shown in Fig. 2. Finally, positive clones can be easily obtained through antibiotic resistance-based positive and negative selection. Compared with previous RE-based cloning, the 3 A assembly eliminates the time-and labor-intensive steps such as column cleanup and agarose gel purification during plasmid construction, increasing the throughput of molecular cloning [27][28][29]. This system also supports the iterative assembly of genetic components, making it an ideal tool for high-throughput construction of expression element combinations for recombinant protein production [26,27,30]. ...
In the post-genomic era, the demand for faster and more efficient protein production has increased, both in public laboratories and industry. In addition, with the expansion of protein sequences in databases, the range of possible enzymes of interest for a given application is also increasing. Faced with peer competition, budgetary, and time constraints, companies and laboratories must find ways to develop a robust manufacturing process for recombinant protein production. In this review, we explore high-throughput technologies for recombinant protein expression and present a holistic high-throughput process development strategy that spans from genes to proteins. We discuss the challenges that come with this task, the limitations of previous studies, and future research directions.
... To further enhance the production of staurosporine, we amplified the complete BGC of staurosporine in the strain S-STA by site-specific recombination (SSR). Studies have shown that there were several additional attB sites (ΦBT1, R4, vwb, TG1) located in the chromosome of S. albus J1074 (Aubry et al. 2019;Baltz 2012) for stable genetic engineering. Considering the following two cases, only two of them could be selected for amplifying Fig. 2 Schematic illustration of selecting heterologous host for the production of staurosporine. ...
Staurosporine is the most well-known member of the indolocarbazole alkaloid family; it can induce apoptosis of many types of cells as a strong protein kinase inhibitor, and is used as an important lead compound for the synthesis of the antitumor drugs. However, the low fermentation level of the native producer remains the bottleneck of staurosporine production. Herein, integration of multi-copy biosynthetic gene cluster (BGC) in well characterized heterologous host and optimization of the fermentation process were performed to enable high-level production of staurosporine. First, the 22.5 kb staurosporine BGC was captured by CRISPR/Cas9-mediated TAR (transformation-associated recombination) from the native producer (145 mg/L), and then introduced into three heterologous hosts Streptomyces avermitilis (ATCC 31267), Streptomyces lividans TK24 and Streptomyces albus J1074 to evaluate the staurosporine production capacity. The highest yield was achieved in S. albus J1074 (750 mg/L), which was used for further production improvement. Next, we integrated two additional staurosporine BGCs into the chromosome of strain S-STA via two different attB sites (vwb and TG1), leading to a double increase in the production of staurosporine. And finally, optimization of fermentation process by controlling the pH and glucose feeding could improve the yield of staurosporine to 4568 mg/L, which was approximately 30-fold higher than that of the native producer. This is the highest yield ever reported, paving the way for the industrial production of staurosporine.
Keypoints
• Streptomyces albus J1074 was the most suitable heterologous host to express the biosynthetic gene cluster of staurosporine.
• Amplification of the biosynthetic gene cluster had obvious effect on improving the production of staurosporine.
• The highest yield of staurosporine was achieved to 4568 mg/L by stepwise increase strategy.
... When required, antibiotics were added to E. coli cultures in liquid (or on solid) medium at the following concentrations: ampicillin, 50 µg/mL (or 100 µg/mL in solid); apramycin, 25 µg/mL (or 50 µg/mL); hygromycin, 50 µg/mL (or 150 µg/mL); kanamycin, 25 µg/mL in liquid or solid medium. A. mediterranei strains were grown at 30 • C on GYM agar medium (32) for sporulation before the preparation of spore stocks, in TSB (Tryptic Soy Broth, Becton Dickinson) for DNA extraction and in MP5 (33) for rifamycin production. Conjugations between E. coli ET12567 harboring pUZ8002 (or pUZ8003) and A. mediterranei were carried out according to Kieser et al. (34) using MS medium complemented with 10 mM CaCl2 (35), instead of MgCl2. ...
Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis.
... In addition, several other integrases were identified ( Table 2). Recently, a modular and integrative vector that is easily compatible with vectors for cloning and assembly methods has been developed (Aubry et al., 2019). The antibiotic resistance cassette module and the integration system cassette module can be easily replaced with other cassettes by unique restriction sites, so multiple vector types can be generated from one backbone. ...
... The antibiotic resistance cassette module and the integration system cassette module can be easily replaced with other cassettes by unique restriction sites, so multiple vector types can be generated from one backbone. Because it has the advantage of being able to add a module, this method can contribute to resolving the difference in efficiency depending on the type of host or cloning strategy (Aubry et al., 2019). ...
Heterologous production of recombinant proteins is gaining increasing interest in biotechnology with respect to productivity, scalability, and wide applicability. The members of genus Streptomyces have been proposed as remarkable hosts for heterologous production due to their versatile nature of expressing various secondary metabolite biosynthetic gene clusters and secretory enzymes. However, there are several issues that limit their use, including low yield, difficulty in genetic manipulation, and their complex cellular features. In this review, we summarize rational engineering approaches to optimizing the heterologous production of secondary metabolites and recombinant proteins in Streptomyces species in terms of genetic tool development and chassis construction. Further perspectives on the development of optimal Streptomyces chassis by the design-build-test-learn cycle in systems are suggested, which may increase the availability of secondary metabolites and recombinant proteins.
... However, within the field of specialized NP synthetic biology, even though there are multifarious vectors for large DNA fragment cloning, few such standard vectors have been constructed. It is well known that the size (from a few kb to more than 100 kb) of NP BGCs, the genomic GC content, and the repeat or similar sequence in the PKS or NRPS (nonribosomal peptide synthase) genes can affect the choice of vectors for BGC cloning (Aubry et al., 2019). Thus, vectors that are flexible and adapted to various assembly methods are preferred. ...
... However, these vectors were mainly used for monocistronic gene expression (Phelan et al., 2017). A set of 12 standardized and modular (three different resistance markers and four orthogonal integration systems) vectors based on model SEVA plasmids were designed to allow for the assembly of NP BGCs through various cloning methods in Streptomyces species (Aubry et al., 2019). In these vectors, the FLP (flippase) recombination system was also incorporated for the recycling of antibiotic markers and for reducing unwanted homologous recombination when several vectors are used simultaneously (Aubry et al., 2019). ...
... A set of 12 standardized and modular (three different resistance markers and four orthogonal integration systems) vectors based on model SEVA plasmids were designed to allow for the assembly of NP BGCs through various cloning methods in Streptomyces species (Aubry et al., 2019). In these vectors, the FLP (flippase) recombination system was also incorporated for the recycling of antibiotic markers and for reducing unwanted homologous recombination when several vectors are used simultaneously (Aubry et al., 2019). It can be expected that through the modularization and orthogonalization of key vector elements, including orthogonal integration systems, origins of replication, antibiotic selection markers, and a variety of cargoes with specific applications, a suitable vector can be quickly designed to efficiently assemble or clone large DNA fragments. ...
Microbial natural products (NPs) are a major source of pharmacological agents. Most NPs are synthesized from specific biosynthetic gene clusters (BGCs). With the rapid increase of sequenced microbial genomes, large numbers of NP BGCs have been discovered, regarded as a treasure trove of novel bioactive compounds. However, many NP BGCs are silent in native hosts under laboratory conditions. In order to explore their therapeutic potential, a main route is to activate these silent NP BGCs in heterologous hosts. To this end, the first step is to accurately and efficiently capture these BGCs. In the past decades, a large number of effective technologies for cloning NP BGCs have been established, which has greatly promoted drug discovery research. Herein, we describe recent advances in strategies for BGC cloning, with a focus on the preparation of high-molecular-weight DNA fragment, selection and optimization of vectors used for carrying large-size DNA, and methods for assembling targeted DNA fragment and appropriate vector. The future direction into novel, universal, and high-efficiency methods for cloning NP BGCs is also prospected.
... Advances in sequencing technics [75] and bioinformatics tools for genome mining [63] facilitate cost-effective sequencing and a fast identification of BGCs and other potential targets for genetic engineering. Molecular biology enzymes [76][77][78], fast cloning strategies and synthetic biology which involves vectors and genetic parts (e.g., attachment sites, replicons, selection markers, promoters, terminators) [79][80][81][82][83][84] were optimized for introduction and maintenance of additional or new genetic material in actinomycetes strains. Methods for transfer of the generated construct into the cell (e.g., protoplast transformation, conjugation) were established [74,85]. ...
... In the past, usually single genetic parts were introduced or exchanged in plasmids or other vehicles to build new constructions. Using synthetic biology this concept was recently advanced in order to combine several genetic parts (also called modules) at once ("plug-and-play") (Section 2.2.2) [80]. ...
... Another example of modularly assembled, standardized vectors which were developed for refactoring of BGCs in actinomycetes, such as Streptomyces species, are the systems reported by Aubry et al. [80]. A set of twelve constructs (pOSV801-pOSV812) provides combinations of different resistance cassettes and four orthogonal integration sites. ...
The first antibiotic-producing actinomycete (Streptomyces antibioticus) was described by Waksman and Woodruff in 1940. This discovery initiated the “actinomycetes era”, in which several species were identified and demonstrated to be a great source of bioactive compounds. However, the remarkable group of microorganisms and their potential for the production of bioactive agents were only partially exploited. This is caused by the fact that the growth of many actinomycetes cannot be reproduced on artificial media at laboratory conditions. In addition, sequencing, genome mining and bioactivity screening disclosed that numerous biosynthetic gene clusters (BGCs), encoded in actinomycetes genomes are not expressed and thus, the respective potential products remain uncharacterized. Therefore, a lot of effort was put into the development of technologies that facilitate the access to actinomycetes genomes and activation of their biosynthetic pathways. In this review, we mainly focus on molecular tools and methods for genetic engineering of actinomycetes that have emerged in the field in the past five years (2015–2020). In addition, we highlight examples of successful application of the recently developed technologies in genetic engineering of actinomycetes for activation and/or improvement of the biosynthesis of secondary metabolites.
