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Polyhydroxyalkanoates (PHAs) are promising alternatives to petroleum-based plastics, owing to their biodegradability and superior material properties. Here, the controllable biosynthesis of scl- co -mcl PHA containing 3-hydroxybutyrate (3HB) and mcl 3-hydroxyalkanoates was achieved in Pseudomonas chlororaphis HT66. First, key genes involved in fatty acid β-oxidation, the de novo fatty acid biosynthesis pathway, and the phaC1 - phaZ - phaC2 operon were deleted to develop a chassis strain. Subsequently, an acetoacetyl-CoA reductase gene phaB and a PHA synthase gene phaC with broad substrate specificity were heterologously expressed for producing and polymerizing the 3HB monomer with mcl 3-hydroxyalkanoates under the assistance of native β-ketothiolase gene phaA . Furthermore, the monomer composition of scl- co -mcl PHA was regulated by adjusting the amount of glucose and dodecanoic acid supplemented. Notably, the cell dry weight and scl- co -mcl PHA content reached 14.2 g/L and 60.1 wt %, respectively, when the engineered strain HT11Δ:: phaCB was cultured in King's B medium containing 5 g/L glucose and 5 g/L dodecanoic acid. These results demonstrated that P. chlororaphis can be a platform for producing scl- co -mcl PHA and has the potential for industrial application.

The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel organisms in industrial biotechnology is becoming increasingly evident. The nonmodel organism Pseudomonas chlororaphis is a safe plant growth promoting bacterium for the production of phenazine compounds; however, its application is seriously hindered due to the lack of an effective gene expression precise regulation toolkit. In this study, we constructed a library of 108 promoter-5'-UTR (PUTR) and characterized them through fluorescent protein detection. Then, 6 PUTRs with stable low, intermediate, and high intensities were further characterized by report genes lacZ encoding β-galactosidase from Escherichia coli K12 and phzO encoding PCA monooxygenase from P. chlororaphis GP72 and thus developed as a static gene expression regulation system. Furthermore, the stable and high-intensity expressed P MOK_RS0128085 UTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity P MOK_RS0116635 UTR regulated by cell density, resulting in a dynamic gene expression regulation system. In summary, this study established two sets of static and dynamic regulatory systems for P. chlororaphis , providing an effective toolkit for fine-tuning gene expression and reprograming the metabolism flux.

Aims: Phenazines, such as phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA), 2-hydroxyphenazine (2-OH-PHZ), are a class of secondary metabolites secreted by plant-beneficial Pseudomonas. Ps. chlororaphis GP72 utilizes glycerol to synthesize PCA, 2-OH-PCA and 2-OH-PHZ, exhibiting broad-spectrum antifungal activity. Previous studies showed that the addition of dithiothreitol (DTT) could increase the phenazines production in Ps. chlororaphis GP72AN. However, the mechanism of high yield of phenazine by adding DTT is still unclear., Methods and Results: In this study, untargeted and targeted metabolomic analysis were adopted to determine the content of metabolites. The results showed that the addition of DTT to GP72AN affected the content of metabolites of central carbon metabolism, shikimate pathway and phenazine competitive pathway. Transcriptome analysis was conducted to investigate the changed cellular process, and the result indicated that the addition of DTT affected the expression of genes involved in phenazine biosynthetic cluster and genes involved in phenazine competitive pathway, driving more carbon flux into phenazine biosynthetic pathway. Furthermore, genes involved in antioxidative stress, phosphate transport system and mexGHI-opmD efflux pump were also affected by adding DTT., Conclusion: This study demonstrated that the addition of DTT altered the expression of genes related to phenazine biosynthesis, resulting in the change of metabolites involved in central carbon metabolism, shikimate pathway and phenazine competitive pathway., Significance and Impact of the Study: This work expands the understanding of high yield of phenazine by the addition of DTT and provides several targets for increasing phenazine production., (© 2022 Society for Applied Microbiology.)

Pseudomonas chlororaphis is a non-pathogenic, plant growth-promoting rhizobacterium that secretes phenazine compounds with broad-spectrum antibiotic activity. Currently available genome-editing methods for P. chlororaphis are based on homologous recombination (HR)-dependent allelic exchange, which requires both exogenous DNA repair proteins (e.g. λ-Red-like systems) and endogenous functions (e.g. RecA) for HR and/or providing donor DNA templates. In general, these procedures are time-consuming, laborious and inefficient. Here, we established a CRISPR-assisted base-editing (CBE) system based on the fusion of a rat cytidine deaminase (rAPOBEC1), enhanced-specificity Cas9 nickase (eSpCas9pp D10A ) and uracil DNA glycosylase inhibitor (UGI). This CBE system converts C:G into T:A without DNA strands breaks or any donor DNA template. By engineering a premature STOP codon in target spacers, the hmgA and phzO genes of P. chlororaphis were successfully interrupted at high efficiency. The phzO-inactivated strain obtained by base editing exhibited identical phenotypic features as compared with a mutant obtained by HR-based allelic exchange. The use of this CBE system was extended to other P. chlororaphis strains (subspecies LX24 and HT66) and also to P. fluorescens 10586, with an equally high editing efficiency. The wide applicability of this CBE method will accelerate bacterial physiology research and metabolic engineering of non-traditional bacterial hosts., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The take-all disease of wheat is one of the most serious diseases in the field of food security in the world. There is no effective biological pesticide to prevent the take-all disease of wheat. 2-Hydroxyphenazine (2-OH-PHZ) was reported to possess a better inhibitory effect on the take-all disease of wheat than phenazine-1-carboxylic acid, which was registered as "Shenqinmycin" in China in 2011. The aim of this study was to construct a 2-OH-PHZ high-producing strain by strain screening, genome sequencing, genetic engineering, and fermentation optimization. First, the metabolites of the previously screened new phenazine-producing Pseudomonas sp. strain were identified, and the taxonomic status of the new Pseudomonas sp. strain was confirmed through 16S rRNA and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Then, the new Pseudomonas sp. strain was named Pseudomonas chlororaphis subsp. aurantiaca LX24, which is a new subspecies of P. chlororaphis that can synthesize 2-OH-PHZ. Next, the draft genome of strain LX24 was determined, and clusters of orthologous group (COG) analysis, KEGG analysis, and gene ontology (GO) analysis of strain LX24 were performed. Furthermore, the production of 2-OH-PHZ increased to 351.7 from 158.6 mg/L by deletion of the phenazine synthesis negative regulatory genes rpeA and rsmE in strain LX24. Finally, the 2-OH-PHZ production of strain LX24 reached 677.1 mg/L after fermentation optimization, which is the highest production through microbial fermentation reported to date. This work provides a reference for the efficient production of other pesticides and antibiotics.

Polyhydroxyalkanoates (PHAs) have been reported with agricultural and medical applications in virtue of their biodegradable and biocompatible properties. Here, we systematically engineered three modules for the enhanced biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA) in Pseudomonas chlororaphis HT66. The phzE , fadA , and fadB genes were deleted to block the native phenazine pathway and weaken the fatty acid β-oxidation pathway. Additionally, a PHA depolymerase gene phaZ was knocked out to prevent the degradation of mcl-PHA. Three genes involved in the mcl-PHA biosynthesis pathway were co-overexpressed to increase carbon flux. The engineered strain HT4Δ:: C1C2J exhibited an 18.2 g/L cell dry weight with 84.9 wt % of mcl-PHA in a shake-flask culture, and the 3-hydroxydodecanoate (3HDD) monomer was increased to 71.6 mol %. Thermophysical and mechanical properties of mcl-PHA were improved with an enriched ratio of 3HDD. This study demonstrated a rational metabolic engineering approach to enhance the production of mcl-PHA with the enriched dominant monomer and improved material properties.

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.

2-Hydroxyphenazine (2-OH-PHZ) is an effective biocontrol antibiotic secreted by Pseudomonas chlororaphis GP72AN and is transformed from phenazine-1-carboxylic acid (PCA). PCA is the main component of the recently registered biopesticide "Shenqinmycin". Previous research showed that 2-OH-PHZ was better in controlling wheat take-all disease than PCA; however, 2-OH-PHZ production was low under natural conditions. Herein, we confirmed that PCA induced reactive oxygen species in its host P. chlororaphis GP72AN and that the addition of DTT improved PCA production by 1.8-fold, whereas the supplementation of K 3 [Fe(CN) 6 ] and H 2 O 2 increased the conversion rate of PCA to 2-OH-PHZ. Finally, a two-stage fermentation strategy combining the addition of DTT at 12 h and H 2 O 2 at 24 h enhanced 2-OH-PHZ production. Taken together, the two-stage fermentation strategy was designed to enhance 2-OH-PHZ production for the first time, and it provided a valuable reference for the fermentation of other antibiotics.

Cinnabarinic acid is a valuable phenoxazinone that has broad applications in the pharmaceutical, chemical, and dyeing industries. However, few studies have investigated the production of cinnabarinic acid or its derivatives using genetically engineered microorganisms. Herein, an efficient synthetic pathway of cinnabarinic acid was designed and constructed in Pseudomonas chlororaphis GP72 for the first tim, which was more straightforward and robust than the known eukaryotic biosynthetic pathways. First, we screened and identified trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) dehydrogenases from Escherichia coli MG1655 (encoded by entA), Streptomyces sp. NRRL12068 (encoded by bomO) and Streptomyces chartreusis NRRL3882 (encoded by calB 3 ) based on the structural similarity of the substrate and product, and the DHHA dehydrogenase encoded by calB 3 was selected for the synthesis of cinnabarinic acid due to its high DHHA conversion rate. Subsequently, cinnabarinic acid was synthesized by the expression of the DHHA dehydrogenase CalB 3 and the phenoxazinone synthase CotA in the DHHA-producing strain P. chlororaphis GP72, resulting in a cinnabarinic acid titer of 20.3 mg/L at 48 hr. Further fermentation optimization by the addition of Cu 2+ , H 2 O 2 , and with adding glycerol increased cinnabarinic acid titer to 136.2 mg/L in shake flasks. The results indicate that P. chlororaphis GP72 may be engineered as a microbial cell factory to produce cinnabarinic acid or its derivatives from renewable bioresources., (© 2019 Wiley Periodicals, Inc.)

Phenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. Pseudomonas chlororaphis HT66 is a PCN-producing, non-pathogenic biocontrol strain, and we obtained the mutant P. chlororaphis P3, which produces 4.7 times more PCN than the wild-type HT66 strain. To reveal the cause of PCN production enhancement in P3 and find potential factors related to PCN biosynthesis, an iTRAQ-based quantitative proteomic analysis was used to study the expression changes between the two strains. Of the 452 differentially expressed proteins, most were functionally mapped into PCN biosynthesis pathway or other related metabolisms. The upregulation of proteins, including PhzA/B, PhzD, PhzF, PhzG, and PhzH, involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3. A number of proteins that function primarily in energy production, amino acid metabolism, and secondary metabolism played important roles in PCN biosynthesis. Notably, proteins involved in the uptake and conversion of phosphate, inorganic nitrogen sources, and iron improved the PCN production. Furthermore, the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P. chlororaphis. This study provides valuable clues to better understand the biosynthesis, excretion and regulation of PCN in Pseudomonas and also provides potential gene targets for further engineering high-yield strains.

Simple Summary: Phenazine compounds are widely used in agricultural control and the medicine industry. Synthesizing phenazine compounds through microbial fermentation often requires a complex and relatively expensive culture medium, which greatly limits the large-scale industrial production of phenazine compounds by fermentation. Therefore, a cost-effective minimal medium (GDM) for the efficient synthesis of phenazine compounds by Pseudomonas chlororaphis was developed in this work. Using the GDM, the production of phenazine compounds by P. chlororaphis reached 1073.5 mg/L, which was 1.3 times that achieved using a complex medium, while the cost of the GDM was only 10% that of a complex medium (e.g., the KB medium). To improve the utilization of glycerol in the GDM, the glycerol metabolic pathway was enhanced by the genetic engineering of the strain, and the titer of phenazine-1-carboxylic acid of the engineered strain was further improved, which is a promising process for industrial production. Phenazine compounds are widely used in agricultural control and the medicine industry due to their high inhibitory activity against pathogens and antitumor activity. The green and sustainable method of synthesizing phenazine compounds through microbial fermentation often requires a complex culture medium containing tryptone and yeast extract, and its cost is relatively high, which greatly limits the large-scale industrial production of phenazine compounds by fermentation. The aim of this study was to develop a cost-effective minimal medium for the efficient synthesis of phenazine compounds by Pseudomonas chlororaphis. Through testing the minimum medium commonly used by Pseudomonas, an ME medium for P. chlororaphis with a high production of phenazine compounds was obtained. Then, the components of the ME medium and the other medium were compared and replaced to verify the beneficial promoting effect of Fe2+ and NH4+ on phenazine compounds. A cost-effective general defined medium (GDM) using glycerol as the sole carbon source was obtained by optimizing the composition of the ME medium. Using the GDM, the production of phenazine compounds by P. chlororaphis reached 1073.5 mg/L, which was 1.3 times that achieved using a complex medium, while the cost of the GDM was only 10% that of a complex medium (e.g., the KB medium). Finally, by engineering the glycerol metabolic pathway, the titer of phenazine-1-carboxylic acid reached the highest level achieved using a minimum medium so far. This work demonstrates how we systematically analyzed and optimized the composition of the medium and integrated a metabolic engineering method to obtain the most cost-effective fermentation strategy. [ABSTRACT FROM AUTHOR]