(228ep) Mass Spectrometry-Based Screening for Protein Engineering

Mass spectrometry imaging (MSI) has been instrumental to characterize spatial chemical heterogeneity of biological systems due to genetic diversity, cell differentiation, and intra-species interactions. Here we applied MSI in screening microbial strain libraries for directed protein evolution. First, we sought to produce designer rhamnolipids with defined molecular compositions by modifying the substrate specificity of the biosynthetic enzymes. In particular, rhamnolipids are a class of glycolipids with varying lipid chain lengths. We targeted the RhlA protein, which is responsible for the lipid moiety synthesis, and performed error-prone PCR for variant creation. The abundance ratios of different rhamnolipids were directly measured using MSI from individual colonies of the strain libraries. As a result, RhlA mutants were isolated with modified preferences towards the beta-hydroxylacyl ACP substrates, enabling production of rhamnolipids with specific lipid chain lengths. Second, we tried to activate cryptic pathways for natural product (NP) production in high throughput. We isolated strains with spontaneous mutations in ribosomal proteins, as ribosomal mutations may activate secondary metabolism (ribosome engineering). Using MSI, the isolated strains were rapidly screened to discover new peptidic NPs, and the results were used to guide the fermentation and purification of new compounds for structural elucidation. Overall, we established a metabolomics-based screening platform that may greatly expand the range of phenotypes that can be improved using protein engineering.