Editing Streptomyces genome using target AID system fused with UGI-degradation tag.

The utilization of Streptomyces as a microbial chassis for developing innovative drugs and medicinal compounds showcases its capability to produce bioactive natural substances. Recent focus on the clustered regularly interspaced short palindromic repeat (CRISPR) technology highlights its potential in genome editing. However, applying CRISPR technology in certain microbial strains, particularly Streptomyces, encounters specific challenges. These challenges include achieving efficient gene expression and maintaining genetic stability, which are critical for successful genome editing. To overcome these obstacles, an innovative approach has been developed that combines several key elements: activation-induced cytidine deaminase (AID), nuclease-deficient cas9 variants (dCas9), and Petromyzon marinus cytidine deaminase 1 (PmCDA1). In this study, this novel strategy was employed to engineer a Streptomyces coelicolor strain. The target gene was actVA-ORF4 (SCO5079), which is involved in actinorhodin production. The engineering process involved introducing a specific construct [pGM1190-dcas9-pmCDA-UGI-AAV-actVA-ORF4 (SCO5079)] to create a CrA10 mutant strain. The resulting CrA10 mutant strain did not produce actinorhodin. This outcome highlights the potential of this combined approach in the genetic manipulation of Streptomyces. The failure of the CrA10 mutant to produce actinorhodin conclusively demonstrates the success of gene editing at the targeted site, affirming the effectiveness of this method for precise genetic modifications in Streptomyces.

3-Amino-4-hydroxybenzoic acid production from glucose and/or xylose via recombinant Streptomyces lividans.

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

Development of a strictly regulated xylose-induced expression system in Streptomyces.

BACKGROUND: Genetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces. Inducible promoters are useful for controlling gene expression, however only a limited number are applicable to Streptomyces. The aim of this study is to develop a controllable protein expression system based on an inducible promoter using sugar inducer, which has not yet been widely applied in Streptomyces. RESULTS: To determine a candidate promoter, inducible protein expression was first examined in Streptomyces avermitilis MA-4680 using various carbon sources. Xylose isomerase (xylA) promoter derived from xylose (xyl) operon was selected due to strong expression of xylose isomerase (XylA) in the presence of D-xylose. Next, a xylose-inducible protein expression system was constructed by investigating heterologous protein expression (chitobiase as a model protein) driven by the xylA promoter in Streptomyces lividans. Chitobiase activity was detected at high levels in S. lividans strain harboring an expression vector with xylA promoter (pXC), under both xylose-induced and non-induced conditions. Thus, S. avermitilis xylR gene, which encodes a putative repressor of xyl operon, was introduced into constructed vectors in order to control protein expression by D-xylose. Among strains constructed in the study, XCPR strain harboring pXCPR vector exhibited strict regulation of protein expression. Chitobiase activity in the XCPR strain was observed to be 24 times higher under xylose-induced conditions than that under non-induced conditions. CONCLUSION: In this study, a strictly regulated protein expression system was developed based on a xylose-induced system. As far as we could ascertain, this is the first report of engineered inducible protein expression in Streptomyces by means of a xylose-induced system. This system might be applicable for controllable expression of toxic products or in the field of synthetic biology using Streptomyces strains.

Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae.

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, ß-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.

Production of chemicals and proteins using biomass-derived substrates from a Streptomyces host.

Bioproduction using microbes from biomass feedstocks is of interest in regards to environmental problems and cost reduction. Streptomyces as an industrial microorganism plays an important role in the production of useful secondary metabolites for various applications. This strain also secretes a wide range of extracellular enzymes which degrade various biopolymers in nature, and it consumes these degrading substrates as nutrients. Hence, Streptomyces can be employed as a cell factory for the conversion of biomass-derived substrates into various products. This review focuses on the following two points: (1) Streptomyces as a producer of enzymes for degrading biomass-derived polysaccharides and polymers; and, (2) wild-type and engineered strains of Streptomyces as a host for chemical production from biomass-derived substrates.

Mannan endo-1,4-ß-mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for production of mannooligosaccharides from mannan polymers.

Mannan endo-1,4-ß-mannosidase (commonly known as ß-mannanase) catalyzes a random cleavage of the ß-D-1,4-mannopyranosyl linkage in mannan polymers. The enzyme has been utilized in biofuel production from lignocellulose biomass, as well as in production of mannooligosaccharides (MOS) for applications in feed and food industries. We aimed to obtain a ß-mannanase, for such mannan polymer utilization, from actinomycetes strains isolated in Indonesia. Strains exhibiting high mannanase activity were screened, and one strain belonging to the genus Kitasatospora was selected. We obtained a ß-mannanase from this strain, and an amino acid sequence of this Kitasatospora ß-mannanase showed a 58-71% similarity with the amino acid sequences of Streptomyces ß-mannanases. The Kitasatospora ß-mannanase showed a significant level of activity (944 U/mg) against locust bean gum (0.5% w/v) and a potential for oligosaccharide production from various mannan polymers. The ß-mannanase might be beneficial particularly in the enzymatic production of MOS for applications of mannan utilization.

Cloning and starch degradation profile of maltotriose-producing amylases from Streptomyces species.

The end products from starch hydrolysis by amylases have important applications in various industries. Here, two amylases derived from two Streptomyces species that hydrolyze soluble starch from potato produced maltotriose as the primary maltooligosaccharide product. The genes, annotated as putative glycoside hydrolases, were cloned and expressed in Streptomyces lividans. These amylases displayed hydrolysis activity from pH 3 to 9.5 and were not affected by Ca(2+.) Optimal production of maltotriose was between 20 and 30 °C at pH 6.5. At the optimal temperature, both amylases produced maltotriose-rich end products rather than either maltose or maltotetraose.

Turnover ability of a designed RNA acting as a template for chemical peptide ligation.

A designed self-folding RNA possessing two peptide-recognition motifs served as a template for the chemical ligation of two RNA-binding peptides under stoichiometric conditions. In this study, we investigated the turnover ability of this template RNA in facilitation of peptide ligation and found that the RNA exhibited modest turnover ability under conditions in which its 3D structure was marginally stable.

Designed RNAs with two peptide-binding units as artificial templates for native chemical ligation of RNA-binding peptides.

The template effect plays important roles not only in modern synthetic and enzymatic catalysis but also in the ancient "RNA-polypeptide (RNP) world," which has been postulated to be a crucial stage in the origin of life. To mimic primitive template catalysis of peptide ligations by RNAs, we previously reported the design and synthesis of a ternary RNP complex in which the ligation of two peptides was significantly facilitated by a template RNA with two peptide-binding units. However, RNA molecules also promoted the ligation reaction in a nonspecific manner through electrostatic interactions between RNA and basic peptides. In this study, we suppressed this effect by reducing the length of the original template derived from the Tetrahymena intron RNA. This modification, however, decreased the template ability for the specific reaction. As an alternative RNA that was as effective as the original template, we found that a self-dimerizing RNA was a promising template for peptide ligation without a nonspecific effect.

Toward a reciprocal evolution system between RNA and peptides as an artificial model for the early RNP world.

In the early stages of the evolution of life, RNA-polypeptide complexes (RNPs) have been suggested to play crucial roles. At a certain developmental stage of ancient RNPs, their RNA and polypeptide components could evolve in an interdependent manner to develop complex structures and functions. To mimic this possible process, we have designed an RNA molecule that can act as a template for chemical peptide ligation. This designed RNA possesses two peptide-binding sites that capture the two basic peptides. The designed RNA actually facilitated the peptide ligation. The resulting ligated peptide, which has two RNA binding sites, can in turn function as a trans-activator that enhances the intrinsic ribozymatic activity of the designed RNA.

Primitive templated catalysis of a peptide ligation by self-folding RNAs.

RNA-polypeptide complexes (RNPs), which play various roles in extant biological systems, have been suggested to have been important in the early stages of the molecular evolution of life. At a certain developmental stage of ancient RNPs, their RNA and polypeptide components have been proposed to evolve in a reciprocal manner to establish highly elaborate structures and functions. We have constructed a simple model system, from which a cooperative evolution system of RNA and polypeptide components could be developed. Based on the observation that several RNAs modestly accelerated the chemical ligation of the two basic peptides. We have designed an RNA molecule possessing two peptide binding sites that capture the two peptides. This designed RNA can also accelerate the peptide ligation. The resulting ligated peptide, which has two RNA-binding sites, can in turn function as a trans-acting factor that enhances the endonuclease activity catalyzed by the designed RNA.

Design and analysis of a structural RNA that acts as a template for peptide ligation.

An artificial RNA, which acts as a specific template for peptide ligation, was designed and constructed. Into the P4-P6 domain of the Tetrahymena intron RNA, two peptide binding motifs (boxB and RRE) were installed. Two peptides (N and Rev) were expected to bind to the RNA simultaneously, facilitating their ligation by an entropic effect. The peptide ligation actually proceeded effectively in the presence of the designer RNA. Analysis using mutant peptides or mutant RNAs demonstrated that the peptide ligation proceeded by forming a specific trimolecular RNP complex.

Synthesis and isomerization of imino-fused N-confused porphyrin.

[reaction: see text] A set of mutually interconvertable inner-bridged-type porphyrinoids, imino-fused N-confused porphyrins (IF-NCPs), which possess a [5.7.5] tricyclic ring in the core, were synthesized from a condensation reaction of 21-amino-substituted NCP and an arylaldehyde, and the structures were characterized by X-ray single-crystal analysis.