BackgroundThe biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA). PCA has been a commercialized antifungal pesticide registered as “Shenqinmycin” in China since 2011. It is found that 2-OH-PHZ shows stronger fungistatic and bacteriostatic activity to some pathogens than PCA. 2-OH-PHZ could be developed as a potential antifungal pesticide. But the yield of 2-OH-PHZ generally is quite low, such as P. chlororaphis GP72, the production of 2-OH-PHZ by the wide-type strain is only 4.5 mg/L, it is necessary to enhance the yield of 2-OH-PHZ for its application in agriculture.ResultsDifferent strategies were used to improve the yield of 2-OH-PHZ: knocking out the negative regulatory genes, enhancing the shikimate pathway, deleting the competing pathways of 2-OH-PHZ synthesis based on chorismate, and improving the activity of PhzO which catalyzes the conversion of PCA to 2-OH-PHZ, although the last two strategies did not give us satisfactory results. In this study, four negative regulatory genes (pykF, rpeA, rsmE and lon) were firstly knocked out of the strain GP72 genome stepwise. The yield of 2-OH-PHZ improved more than 60 folds and increased from 4.5 to about 300 mg/L. Then six key genes (ppsA, tktA, phzC, aroB, aroD and aroE) selected from the gluconeogenesis, pentose phosphate and shikimate pathways which used to enhance the shikimate pathway were overexpressed to improve the production of 2-OH-PHZ. At last a genetically engineered strain that increased the 2-OH-PHZ production by 99-fold to 450.4 mg/L was obtained.ConclusionsThe 2-OH-PHZ production of P. chlororaphis GP72 was greatly improved through disruption of four negative regulatory genes and overexpression of six key genes, and it is shown that P. chlororaphis GP72 could be modified as a potential cell factory to produce 2-OH-PHZ and other phenazine biopesticides by genetic and metabolic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0529-0) contains supplementary material, which is available to authorized users.

No abstract

A conductive hydrogel is a kind of polymer material having substantial potential applications with various properties, including high toughness, self-recoverability, electrical conductivity, transparency, freezing resistance, stimuli responsiveness, stretchability, self-healing, and strain sensitivity. Herein, according to the current research status of conductive hydrogels, properties of conductive hydrogels, preparation methods of different conductive hydrogels, and their application in different fields, such as sensor and actuator fabrication, biomedicine, and soft electronics, are introduced. Furthermore, the development direction and application prospects of conductive hydrogels are proposed.

Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic β-amino acid that can be used for the synthesis of chiral materials and nonnatural peptides. The aim of this study was to accumulate DHHA by engineering Pseudomonas chlororaphis GP72, a nonpathogenic strain that produces phenazine-1-carboxylic acid and 2-hydroxyphenazine. First, the phzF deletion mutant DA1 was constructed, which produced 1.91 g/L DHHA. Moreover, rpeA and pykF were disrupted and then ppsA and tktA were co-expressed in strain DA1. The resulting strain DA4 increased DHHA concentration to 4.98 g/L, which is 2.6-fold than that of DA1. The effects of the addition of glucose, glycerol, L-tryptophan, and Feon DHHA production were also investigated. Strain DA4 produced 7.48 g/L of DHHA in the culture medium in the presence of 12 g/L glucose and 3 mM Fe, which was 1.5-fold higher than the strain in the original fermentation conditions. These results indicate the potential of P. chlororaphis GP72 as a DHHA producer.

Bacillus subtilis is a widely distributed aerobic Gram-positive species of bacteria. As a tool in the lab, it has the advantages of nonpathogenicity and limited likelihood of becoming drug resistant. It is a probiotic strain that can be directly used in humans and animals. It can be induced to produce spores under nutrient deficiency or other adverse conditions. B. subtilis spores have unique physical, chemical, and biochemical characteristics. Expression of heterologous antigens or proteins on the surface of B. subtilis spores has been successfully performed for over a decade. As an update and supplement to previously published research, this paper reviews the latest research on spore surface display technology using B. subtilis. We have mainly focused on the regulation of spore coat protein expression, display and application of exogenous proteins, and identification of developing research areas of spore surface display technology.

Phenazine-1-carboxylic acid (PCA), the primary active ingredient of Shenqinmycin, was awarded the China Pesticide Certificate in 2011 due to its excellent antibacterial action. Phenazine-1-carboxamide (PCN) is a derivative of PCA, which is modified by the phzH gene, and its anti-bacterial effect is better than that of PCA. At present, PCN can be produced via Pseudomonas fermentation using an opportunistic pathogen, Pseudomonas aeruginosa. Qlu-1 is an environmentally friendly strain of Pseudomonas chlororaphis that can produce phenazine derivatives. We replaced the phzO gene with the phzH gene from P. aeruginosa to achieve PCN accumulation. Different strategies were used to enhance PCN production: knocking out of negative regulatory factors, enhancing the shikimate pathway by gene overexpression and gene knocking, and using fed-batch fermentation. Finally, an engineered strain of P. chlororaphis was produced, which produced 11.45 g/L PCN. This achievement indicates that Qlu-1 could be modified as a potential microbial cell factory for PCN production by metabolic engineering.

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.

Trehalose, a stable nonreducing disaccharide, protects biomolecules against environmental stress. However, trehalose production using secretory trehalose synthase (TreS) by Bacillus subtilis has not been well studied. In this study, a mutant TreS was successfully secreted and expressed in B. subtilis WB800N. The extracellular enzyme activity of TreS regulated by the P43 promoter and SPPhoD signal peptide in recombinant B. subtilis WB800N reached 23080.6 ± 1119.4 U/L in a 5-L fermenter after optimizing the culture medium, while xpF, skfA, lytC, and sdpC were knocked out. To reduce maltose consumption, malP and amyE corresponding to maltose transporters were further deleted. To simplify the trehalose production process, we invented a fermentation-coupling biocatalysis process involving recombinant bacteria fermentation to secrete TreS and simultaneous conversion of maltose to trehalose by TreS and found that the conversion rate of maltose to trehalose reached 75.5%, suggesting that this is an efficient strategy for large-scale trehalose production using recombinant B. subtilis.