CULTURE-INDEPENDENT METHOD FOR SCREENING AND IDENTIFYING MICROBIAL ENZYME-ENCODING GENES USING MICRODROPLET-BASED SINGLE CELL GENOMICS K. Nakamura 1 , R. Iizuka 1* , T. Yoshida 2 , Y. Hatada 2 , Y. Takaki 2 , S. Nishi 2 , A. Iguchi 3 , D. H. Yoon 3 , T. Sekiguchi 3 , S. Shoji 3 , and T. Funatsu 1* 1 The University of Tokyo, Japan; 2 Japan Agency for Marine-Earth Science and Technology, Japan; and 3 Waseda University, Japan ABSTRACT Environmental microbes are a great source of industrially valuable enzymes with high and unique catalytic activities. However, a vast majority of microbes remain unculturable and thus are not accessible by culture-based methods. Here, we present a rapid and efficient method to screen and identify enzyme- encoding genes from environmental microbes in a culture-independent manner. This method combines activity-based single cell screening using microdroplets and single cell genomics. Using this method, we successfully identified 13 novel β-glucosidase genes from uncultured marine bacteria. This method will facilitate the identification of genes encoding industrially valuable enzymes. KEYWORDS: Microdroplet, Single cell genomics, Enzyme, Screening INTRODUCTION Enzymes have been increasingly used in a wide range of industrial applications because of their prominent properties such as substrate specificity and efficiency, generally at mild pH values, temperatures, and pressures. The most enzymes are identified from environmental microbes using culture-based methods, and industrially valuable enzymes are often obtained from newly identified microbes. However, a vast majority of microbes have not been cultivated in the laboratory and thus their resources cannot be accessed by using culture-based methods. Recently, culture-independent metagenomic approaches have been successfully applied to access the untapped genetic resources. However, the results of these approaches are not outstanding, despite requiring a huge amount of time, cost, and effort. Thus, we have developed a rapid and efficient method for the screening and identification of enzyme-encoding genes from environmental microbes in a culture-independent manner, with a combination of activity-based single cell screening using microdroplets and single cell genomics. METHODOLOGY A schematic representation of our workflow is shown in Figure 1a. Using a microfluidic device, environmental microbes are first encapsulated at a single-cell level in picoliter-sized water-in-oil (W/O) microdroplets with a fluorogenic substrate for the target enzyme (Figure 1a, step 1, and Figure 1b). Microfluidic systems enable the generation of uniform-sized microdroplets and the rapid isolation of single cells in individual compartments. Following incubation at an ambient temperature, the microdroplets are observed under a fluorescence microscope to screen and collect those containing a fluorescent microbe, which exhibits the desired enzymatic activity (Figure 1a, step 2, and Figure 1c). Each fluorescent microbe is recovered from the microdroplets and then subjected to whole genome amplification using multiple displacement amplification (MDA) with phi29 DNA polymerase (Figure 1a, step 3). The resulting MDA products are subjected to next generation sequencing (Figure 1a, step 4) and bioinformatics analysis to identify the genes encoding the target enzymes (Figure 1a, step 5). The entire process can be completed in 4–5 days and is cost-effective. 513 978-0-9798064-8-3/μTAS 2015/$20©15CBMS-0001 19 th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA