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Phenazine-1-carboxylic acid (PCA) and its derivative pyocyanin (PYO) are natural redox mediators in bioelectrochemical systems and have the potential to enable new bioelectrochemical production strategies. The native producer Pseudomonas aeruginosa harbors two identically structured operons in its genome, which encode the enzymes responsible for PCA synthesis [phzA1-G1 (operon 1), phzA2-G2 (operon 2)]. To optimize heterologous phenazines production in the biotech host Pseudomonas putida KT2440, we compared PCA production from both operons originating from P. aeruginosa strain PAO1 (O1.phz1 and O1.phz2) as well as from P. aeruginosa strain PA14 (14.phz1 and 14.phz2). Comparisons of phenazine synthesis and bioelectrochemical activity were performed between heterologous constructs with and without the combination with the genes phzM and phzS required to convert PCA to PYO. Despite a high amino acid homology of all enzymes of more than 97%, P. putida harboring 14.phz2 produced 4-times higher PCA concentrations (80 μg/mL), which resulted in 3-times higher current densities (12 μA/cm2) compared to P. putida 14.phz1. The respective PCA/PYO producer containing the 14.phz2 operon was the best strain with 80 μg/mL PCA, 11 μg/mL PYO, and 22 μA/cm2 current density. Tailoring phenazine production also resulted in improved oxygen-limited metabolic activity of the bacterium through enhanced anodic electron discharge. To elucidate the reason for this superior performance, a detailed structure comparison of the PCA-synthesizing proteins has been performed. The here presented characterization and optimization of these new strains will be useful to improve electroactivity in P. putida for oxygen-limited biocatalysis.

Phenazine-1-carboxylic acid (PCA) and its derivative pyocyanin (PYO) are natural redox mediators in bioelectrochemical systems and have the potential to enable new bioelectrochemical production strategies. The native producer Pseudomonas aeruginosa harbors two identically structured operons in its genome, which encode the enzymes responsible for PCA synthesis [ phzA1-G1 (operon 1), phzA2-G2 (operon 2)]. To optimize heterologous phenazines production in the biotech host Pseudomonas putida KT2440, we compared PCA production from both operons originating from P. aeruginosa strain PAO1 (O1.phz1 and O1.phz2) as well as from P. aeruginosa strain PA14 (14.phz1 and 14.phz2). Comparisons of phenazine synthesis and bioelectrochemical activity were performed between heterologous constructs with and without the combination with the genes phzM and phzS required to convert PCA to PYO. Despite a high amino acid homology of all enzymes of more than 97%, P. putida harboring 14.phz2 produced 4-times higher PCA concentrations (80 μg/mL), which resulted in 3-times higher current densities (12 μA/cm2) compared to P. putida 14.phz1. The respective PCA/PYO producer containing the 14.phz2 operon was the best strain with 80 μg/mL PCA, 11 μg/mL PYO, and 22 μA/cm2 current density. Tailoring phenazine production also resulted in improved oxygen-limited metabolic activity of the bacterium through enhanced anodic electron discharge. To elucidate the reason for this superior performance, a detailed structure comparison of the PCA-synthesizing proteins has been performed. The here presented characterization and optimization of these new strains will be useful to improve electroactivity in P. putida for oxygen-limited biocatalysis. [ABSTRACT FROM AUTHOR]

A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.

A novel cyclic peptide compound, KK-1, was originally isolated from the plantpathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species. [ABSTRACT FROM AUTHOR]

Keywords: microbial cell factories; metabolic engineering; synthetic biology; heterologous production; biologics and small molecule medicines I Escherichia coli i is reported as the most common cell factory for the production of both small molecules and biologics. Microbial cell factories, metabolic engineering, synthetic biology, heterologous production, biologics and small molecule medicines. [Extracted from the article]

Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20 °C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. qRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites.

Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis. [ABSTRACT FROM AUTHOR]

Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. qRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites.