A technology platform for in vitro transcription and translation of enzymes in micro compartments
September 15-19, 2019
Enzymes are crucial elements of all living cells. As biological catalysts they accelerate chemical reactions without being consumed in the process. The modern bioeconomy strives to identify new enzymes for improved usage in biotechnological applications such as the production of fine chemicals or pharmaceuticals. A promising source for the discovery of new enzymes are metagenomes. As there is no intermediary cultivation step for the extraction of the genetic material necessary, the genetic pool of a metagenome comprises genes of cultivable and non-cultivable microorganisms, which is beneficial for the detection of new enzymes. Here we introduce a new technology platform towards high efficient screening of whole metagenome libraries by combining in vitro compartmentation with a cell-free protein synthesis approach (Figure 1). A key element of this platform is a centrifugal microfluidic cartridge which encapsulates the metagenome library in up to 100’000 monodisperse droplets with a volume of 520 pl. The micro droplets are generated by centrifugal step emulsification (1) and are further transported into a standard reaction tube, decoupling the emulsification from the downstream processing. Based on substrate specificity, droplets with active enzymes are selected and a subsequent sequencing analysis allows the identification of the DNA sequence of these enzymes. The high number of generated micro droplets enables a high-throughput of large libraries and the high coverage increases the chance of finding new or rare enzymes. Compared to traditional approaches, the introduced all-in-one metagenome screening platform decreases screening time to a large extend by replacing heterologous expression with in vitro protein synthesis and massive screening. Further, the selection process minimizes the sequencing and annotation effort.
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Silvia Calabrese, Martin Schulz, Felix von Stetten, Nadine Borst, Sophia Probst, Gerassimos Kolaitis, and Joseph Sperl, "A technology platform for in vitro transcription and translation of enzymes in micro compartments" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/27