Deep mutational scanning of an alanine racemase from Streptococcus thermophilus

It is of fundamental interest in evolutionary biology to link the effect of mutations in a protein to the fitness of an organism. One of the best methods to investigate this effect is to perform a deep mutational scan (DMS). This involves creating a library of protein mutants which are subject to selection in diverse conditions and consequently high throughput sequencing is employed to monitor the fate of mutants. In this study, in the first section we introduce a novel method of creating large chromosomally encoded libraries (upto 10^9 variants) by a two-step scarless gene replacement using a cat-oroP cassette in Streptococcus thermophilus. We use this method to create a library of 10^8 variants of alanine racemase (Alr) cloned at its native locus in the chromosome. Then in the second section, the Alr library was grown in two conditions, in presence and absence of selection pressure for function. Growth under functional selection, created a map of residues essential to alanine racemase activity expressed under native conditions. We observe that 44% of substitutions were deleterious for activity. In absence of selection pressure for function, we observe that the majority of substitutions (67%) had neutral effects on fitness. In both conditions, certain substitutions were selected which may be involved in restricting entry to the active site. In the third section, we engineered a S. thermophilus strain by inserting a SARS-CoV-2 protease recognition sequence in the Alr protein. This mutant displayed conditional auxotrophy on expressing the protease. This strain was used to screen libraries of peptidic inhibitors to identify SARS-CoV-2 protease inhibitors.(SC - Sciences) -- UCL, 202