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NOVEL ACTINOBACTERIAL DIVERSITY IN KAZAKHSTAN DESERTS SOILS AS A SOURCE OF NEW DRUG LEADS Arailym Ziyat*1, Professor Michael Goddfellow2, Ayaulym Nurgozhina1, Shynggys Sergazy1, Madiyar Nurgaziev1 Address(es): Arailym Ziyat, 1PI “National Laboratory Astana”, Centre for Life Sciences, Laboratory of Human Microbiome and Longevity, Kabanbay Batyr Ave. 53, 010000, Astana, Republic of Kazakhstan. 2Newcastle University, Faculty of Science, Agriculture & Engineering, School of Biology, NE1 7RU, Newcastle University, United Kingdom. *Corresponding author: [email protected] doi: 10.15414/jmbfs.2019.8.4.1057-1065 ARTICLE INFO ABSTRACT Received 24. 10. 2018 Discovering new metabolites, notably antibiotics, by isolation and screening novel actinomycetes from extreme habitats gave extraordinary results that can be adapted in the future for healthcare. However, it was little attention payed to desert soils in Central Revised 13. 11. 2018 Asia, such as from Kazakhstan. Accepted 13. 11. 2018 Taxonomic approach was to isolate selectively, dereplicate and classify actinomycetes from two Kazakhstan Deserts (Betpakdala and Published 1. 2. 2019 Usturt Plateu). The most representative isolates from colour-groups were describe via 16S rRNA gene sequence analysis. Relatively large number, of strains from environmental soil samples were classified into Streptomyces genera. Moreover, three strains Regular article from two different soil samples were identified as relatively close to Pseudonocarida genera. All representative isolates were screened for bioactive compound against wild type microorganisms, as a result, of it can be interpreted that approximately half of screened strains are likely to produce metabolites which inhibits cell growth. The results of this project demonstrate for the first time that arid regions of Kazakhstan soils are rich reservoirs of cultivable novel actinobacteria with the capacity to produce bioactive compounds that can be developed as drug leads for medicine. Keywords: Actinomycetes, Kazakhstan, 16sRNA gene sequence, Streptomyces genera INTRODUCTION have been found to be a rich source of new drug leads (Manivasagan et al. 2013). According to World Health Organization (WHO) antibiotic resistance has It is known that actinomycetes are common in extreme habitats, such as acidic become a huge problem that needs immediate attention (WHO 2014). New soils (Kim et al. 2003) or deep sediments (Maldonado et al. 2005). However, antibiotics are urgently needed to kill multi-drug resistant microbial pathogens little attention has been paid to actinomycetes diversity from desert soils, yet and to treat life threatening diseases, such as cancer (Genilloud 2014). For deserts cover almost one-quarter of the Earth’s land surface. example, Klebsiella pneumonia clones are carbapenemase-resistant, which The deserts are the arid and hyper-arid areas of lands; generally ecoregions appears as a major problem in the USA (Kumarasamy et al. 2010). An contain organisms that adapted to survive in arid environmental regions and estimated two million patients in the USA are infected every year by drug- geographically with large areas of soil or rock with low organic matter. resistant pathogens and around 23000 of them die (PharmTech 2013). Actinobacteria have been isolated from desert soils, including the soils from the Microbial natural products are still considered to be the most promising source of Amargosa desert in Nevada (Luedemann 1968), the Mojave desert in southwest new drugs (Cragg and Newman 2013). Shoichet (2013) states that natural United States (Garrity et al. 1996), the Mongolian desert (Kurapova et al. products contain a range of chemical structures optimised for biological 2012), the Namibian desert (Wink et al. 2003), the Sahara desert (Zitouni et al. interactions. However, compounds should follow “the rule of five” (Lipinski et 2005; Meklat et al. 2011), the Taklamakan desert in Xinjiang province, China al. 2012). Natural products are evolutionarily selected by their ability to enter and (Luo et al. 2012), the Thar desert in Rajastan, India (Harwani 2013) and the inhibit or kill bacterial cells and are therefore better than synthetically designed desert ecosystem in the northeast of the Qinghai-Tibet plateau (Ding et al. 2013). drugs which are basically not “tailored for microbial biology” (Wright 2014). However, Kazakhstan desert soil has not been explored for actinomycetes Amongst prokaryotes, members of the phylum Actinobacteria, notably diversity. Streptomyces strains, remain a unique source of novel bioactive compounds of The Kazakhstan Republic, located in Central Asia neighbouring the Russian therapeutic interest (Lucas et al. 2012). It is now known that the genomes of Federation and China is a landlocked country, with high mountain ranges along actinobacteria contain many biosynthetic gene clusters that code for known or the eastern and south-eastern borders. Deserts and semi-deserts make up around predicted specialist metabolites (Goodfellow and Fiedler 2010). Moreover, 58% of the country. natural habitats contain a wealth of undiscovered actinobacteria (Stach et al. Deserts are divided into two geomorphological regions: Turan lowland, Plato 2003). Usturt and Betpak-Dala (Turuzbekova 2014). Within these, many geological Despite these discoveries it has become increasingly difficult to find new landscapes exist: sand (Karakum), rocky (Betpak-Dala), gravelly (Ustyurt), clay bioactive compounds from actinobacteria isolated from well-studied habitats as (Hungry Steppe), saline (Kelkor) deserts (www.zoogeo365.ru 2011). screening such organisms leads to a costly rediscovery of known metabolites According to Gvozdetskii and Mihailov (1978) the average temperature in the (Busti et al. 2006). One way of addressing these problems is to selectively Betpakdala desert and in the Usturt Plateau is 26-28°C. In some years, the isolate, dereplicate (assign in to groups), and screen representatives of novel temperature reaches 40-60°C in the Usturt Plateau. Average annual rainfall is less actinomycetes from neglected and unexplored habitats. This taxonomic approach than 120 mm and 150 mm. Usturt Plateau is located between the Caspian and to bioprospecting has been used to isolate actinobacteria from extreme biomes on Aral seas. It is semi-enclosed by steep cliffs. The Usturt Plateau is 300 m plus the premise that, harsh environmental conditions will give rise to novel above sea level and generally flat with a number of shallow shafts (Muzbel, actinobacteria with the capacity to synthesise novel metabolites (Goodfellow and Karabaur) and hollows. Sarmatian limestone is the parent rock, which weathers Fiedler 2010; Goodfellow 2013). This hypothesis is tested by selecting, into gray-brown solonetzic soils, interbedded with gypsum. dereplicating and screening actinomycetes from marine habitats, notably deeper The Betpakdala desert is located between the lower reaches of the rivers Chu and sediments. To date, novel actinomycetes from desert and marine ecosystems Sarysu on the western shore of Lake Balkhash. The western part of Betpakdala – 1057 J Microbiol Biotech Food Sci / Ziyat et al. 2019 : 8 (4) 1057-1065 is an elevated plain of flat-lying folded Mesozoic and Paleogene loose rocks. The bulk pH values of the soil samples were determined following the procedure Above lie predominantly marine and continental Paleogene sediments (sand, described by Reed and Cummings (1945). Two grams of each soil sample were sandstone, clay, gravel). Flat sections alternate with undrained depressions in the transferred to 50 ml beakers and mixed thoroughly with demineralized water. The form of logs and closed cavities with salt marshes. soil samples were left for 2 hours, then the pH of each soil was determined, in Very little attention has been past to the microbial flora of Kazakhstan Deserts. triplicate, using a glass electrode pH meter (Model 320 Mettler-Toledo AG, Kutovaya et al. (2015) states that two dominant groups of bacteria are CH.8603, Schwerzenbach, Switzerland). represented in the Aridic Calcisols extreme hyper arid desert region: The soil samples were added to pre-weighed silica crucibles and the weight of the Proteobacteria (43.9%) and Actinobacteria (10%). Therefore, it is likely that the crucibles plus soil samples accurately taken. The samples were then dried to same amount of Actinobacteria can be isolated from other arid regions, such as constant weight in a hot oven of 105°C and re-weighed. This procedure was Ustyurt Plateau. carried out in triplicate and the moisture content of the samples recorded as the percentage loss of weight in the three measurements. The crucibles plus dry soil MATERIALS AND METHODS were then placed in a muffle furnace and the temperature slowly raised to 700°C and kept constant for 30 minutes in order to burn off the organic matter prior to Soil samples overnight cooling and reweighing. The average percentage loss as weighted for three measurements was record as the organic matter content. Two soil samples from the Kazakhstan Desert were studied (Table 2.1.). The soil samples from Kazakhstan was provided by the Director of Usturt National Selective isolation and enumeration of putative actinomycetes Preserve (Mangistauskaya region) and head of city administration Sholakkorgan, Sozak district, Southern Kazakhstan region (Betpakdala desert). The soil samples A gram of each soil sample was added to 4.5 ml of 1/4 strength Ringer’s solution -1/2 were stored in plastic bags at room temperature. (9 g NaCl, 0.42