Improvement of chloramphenicol production in Streptomyces venezuelae ATCC 10712 by overexpression of the aroB and aroK genes catalysing steps in the shikimate pathway.

Streptomyces venezuelae ATCC 10712 produces chloramphenicol in small amounts. To enhance chloramphenicol production, two genes, aroB and aroK, encoding rate-limiting enzymes of the shikimate pathway were overexpressed using the expression vector pIJ86 under the control of the strong constitutive ermE* promoter. The recombinant strains, S. venezuelae/pIJ86-aroB and S. venezuelae/pIJ86-aroK, produced 2.5- and 4.3-fold greater amounts respectively of chloramphenicol than wild type at early stationary phase of growth. High transcriptional levels of aroB and aroK genes were detected at the early exponential growth of both recombinant strains and consistent with the enhanced expression of pabB gene encoding an early enzyme in chloramphenicol biosynthesis. The results suggested that the increment of carbon flux was directed towards intermediates in the shikimate pathway required for the production of chorismic acid, and consequently resulted in the enhancement of chloramphenicol production. This work is the first report of a convenient genetic approach to manipulate primary metabolite genes in S. venezuelae in order to increase chloramphenicol production.

Plant growth enhancing effects by a siderophore-producing endophytic streptomycete isolated from a Thai jasmine rice plant (Oryza sativa L. cv. KDML105).

An endophytic Streptomyces sp. GMKU 3100 isolated from roots of a Thai jasmine rice plant (Oryza sativa L. cv. KDML105) showed the highest siderophore production on CAS agar while phosphate solubilization and IAA production were not detected. A mutant of Streptomyces sp. GMKU 3100 deficient in just one of the plant growth promoting traits, siderophore production, was generated by inactivation of a desD-like gene encoding a key enzyme controlling the final step of siderophore biosynthesis. Pot culture experiments revealed that rice and mungbean plants inoculated with the wild type gave the best enhancement of plant growth and significantly increased root and shoot biomass and lengths compared with untreated controls and siderophore-deficient mutant treatments. Application of the wild type in the presence or absence of ferric citrate significantly promoted plant growth of both plants. The siderophore-deficient mutant clearly showed the effect of this important trait involved in plant-microbe interaction in enhancement of growth in rice and mungbean plants supplied with sequestered iron. Our results highlight the value of a substantial understanding of the relationship of the plant growth promoting properties of endophytic actinomycetes to the plants. Endophytic actinomycetes, therefore, can be applied as potentially safe and environmentally friendly biofertilizers in agriculture.

Modulation of salt tolerance in Thai jasmine rice (Oryza sativa L. cv. KDML105) by Streptomyces venezuelae ATCC 10712 expressing ACC deaminase.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a plant growth promoting (PGP) trait found in beneficial bacteria including streptomycetes and responsible for stress modulation. The ACC deaminase gene, acdS, of S. venezuelae ATCC 10712 was cloned into an expression plasmid, pIJ86, to generate S. venezuelae/pIJ86-acdS. Expression of acdS and production of ACC deaminase of S. venezuelae/pIJ86-acdS were significantly higher than the unmodified strain. The ACC deaminase-overexpressing mutant and the wild type control were inoculated into Thai jasmine rice (Oryza sativa L. cv. KDML105) under salt stress conditions. S. venezuelae on its own augmented rice growth and significantly increased more tolerance to salinity by reduction of ethylene, reactive oxygen species (ROS) and Na+ contents, while accumulating more proline, total chlorophyll, relative water content (RWC), malondialdehyde (MDA), and K+ than those of uninoculated controls. The overproducer did not alter chlorophyll, RWC, or MDA further-while it did boost more shoot weight and elongation, and significantly regulated salt tolerance of rice by increasing proline and reducing ethylene and Na+ contents further than that of the wild type. This work is the first illustration of the beneficial roles of S. venezuelae to enhance plant fitness endophytically by promotion of growth and salt tolerance of rice.

Molecular interaction of 1-aminocyclopropane-1-carboxylate deaminase (ACCD)-producing endophytic Streptomyces sp. GMKU 336 towards salt-stress resistance of Oryza sativa L. cv. KDML105.

1-aminocyclopropane-1-carboxylate deaminase (ACCD)-producing endophytic Streptomyces sp. GMKU 336 and its ACCD-deficient mutant were inoculated into Thai jasmine rice Khao Dok Mali 105 cultivar (Oryza sativa L. cv. KDML105) under salt stress (150 mM NaCl) conditions. The results clearly indicated that Streptomyces sp. GMKU 336 significantly increased plant growth, chlorophyll, proline, K+, Ca+, and water contents; but decreased ethylene, reactive oxygen species (ROS), Na+, and Na+/K+ ratio when compared to plants not inoculated and those inoculated with the ACCD-deficient mutant. Expression profiles of stress responsive genes in rice in association with strain GMKU 336 were correlated to plant physiological characteristics. Genes involved in the ethylene pathway, ACO1 and EREBP1, were significantly down-regulated; while acdS encoding ACCD in Streptomyces sp. GMKU 336 was up-regulated in vivo. Furthermore, genes involved in osmotic balance (BADH1), Na+ transporters (NHX1 and SOS1), calmodulin (Cam1-1), and antioxidant enzymes (CuZn-SOD1 and CATb) were up-regulated; whereas, a gene implicated in a signaling cascade, MAPK5, was down-regulated. This work demonstrates the first time that ACCD-producing Streptomyces sp. GMKU 336 enhances growth of rice and increases salt tolerance by reduction of ethylene via the action of ACCD and further assists plants to scavenge ROS, balance ion content and osmotic pressure.