Actinomycetes and fungi isolated from Maliau Basin, Sabah and the screening for novel secondary metabolites against eukaryotic signal transduction and Mycobacterium

Maliau Basin is one of the world s hot spot of biodiversity. Maliau Basin Sabah has such forest diversity such as the heath forest and the submontane hill forest that contain the family dipterocarpaceae. The unique geological landscape which gives rise to the multitude of water falls. The Basin supports a myriad of flora, fauna and microbes. These microbes serve the main purpose of biodegrading organic materials and enriching the soil s nutrients. Actinomycetes and microfungi have been known to be prolific producers of bioactive secondary metabolites, which affect signal transduction and cell cycle. The search for novel pharmaceutical products requires a very large collection of new actinomycetes and fungi to be isolated from new micro niches. Certain microorganism may have a tendency to dominate or co-exist with other microorganisms in a certain niche based on the organic materials being degraded or its ability to produce certain compounds. Based on this assumption, we have sampled the soils just below the leaf litter under identified trees. We have successfully isolated 98 strains of actinomycetes and 254 strains of microfungi from the soil samples of Maliau Basin. Acetone extracts from these microbes have been tested against various yeast based screening systems. We screened for inhibitors against mitogen-activated protein kinase (MAPK) pathway by targeting the activity of MKK1 and MSG5 proteins, which are involved in the yeast cell wall integrity system. Overexpression, under induction by galactose utilizing the GAL1 promoter of a hyperactive mutant MKK1P386 leads to cell lysis even in the presence of D- sorbitol (1M) as an osmoprotectant. No actinomycetes and filamentous fungi were found to inhibit against MAPK kinase (MKK1) in yeast and 3 actinomycetes strains H7897, H7944 and H7973 were shown to be toxic towards yeast in the MKK1P386 screening system. The introduction of a dual specificity (tyrosine and serine/threonine) phosphatase MSG5 acting on Mpkl (MAP kinase) into the MKKlp386 system led to cell growth and inhibitor of this phosphatase will lead to growth inhibition (cell lysis). No actinomycetes and filamentous fungi were found to inhibit against protein phosphatase (MSG5) in yeast. Five actinomycetes strain H7897, H7944, H7970, H7973 and H8528 showed toxicity to yeast in MSG5 screening system. In the Ras/Raf protein interaction, the yeast two-hybrid system with cloned mammalian Ras and Raf proteins was used. No yeast growth will be observed on medium without histidine if the inhibitor blocks the Ras/Raf protein interaction as the histidine biosynthetic gene (.HIS3) would not be transcribed. The screening done in the Ras/Raf protein interaction involves the inhibitory effect in which the protein-protein interaction was blocked. H7897, an actinomycete was found to be a potential inhibitor against Ras/Raf screening system and 4 actinomycetes strains (H7915, H7940, H7944 and H7976) were toxic against yeast in Ras/Raf screening system. Another strain, H7944, an actinomycete (a non-Streptomyces) from Maliau Basin soil was screened as toxic to yeast and found to have decrease phosphorylation of mammalian MEK1/2 and ERK1/2 kinases in the Western blotting analysis. Ras/Raf protein interaction may be affected as evidenced by lowered (3- galactosidase synthesis. For the serine/ threonine protein phosphatase type 1 (PP1) inhibitor screening, the yeast that possesses one catalytic subunit (Glc7p) was used as the target for the screening. The screening system uses two alleles of the Glc7p which are GLC7, the wild type allele (PAY704-1) and glc7-10, the temperature sensitive mutant (PAY700-4). The mutant allele causes cell wall integrity defects and cell cycle arrest at 37°C but rescued by high osmolarity (1M D-sorbitol). An inhibitor acting on Glc7p in the wild type would behaved same as the mutant phenotype as that caused by the mutant allele, with growth inhibition at 37°C but rescued by D- sorbitol (1M). Two Penicillium strains (H9307 and H9318) and 4 unknown fungi (H9114, H9213, H9316 and H9317) isolated from Maliau Basin soil showed up as positive in the screen.In in vitro system, three strains (H9307, H9317 and H9318) also decreased dephosphorylation by Glc7p of yeast as well as the mammalian PP1 gamma.MycobacteriumIn the screening for inhibitors against isocitrate lyase (ICL) in Mycobacterium, there are 3 extracts (H7893, H7910 and H7944) shown toxicity against both the wild type M. smegmatis (H8000) and transformed M. smegmatis (H8012). These extracts inhibited the growth of Mycobacterium on both glucose and acetate plates.