Differential DNA methylation associated with multiple sclerosis and disease modifying treatments in an underrepresented minority population.

Black and Hispanic American patients frequently develop earlier onset of multiple sclerosis (MS) and a more severe disease course that can be resistant to disease modifying treatments. The objectives were to identify differential methylation of genomic DNA (gDNA) associated with disease susceptibility and treatment responses in a cohort of MS patients from underrepresented minority populations. Patients with MS and controls with non-inflammatory neurologic conditions were consented and enrolled under an IRB-approved protocol. Approximately 64% of donors identified as Black or African American and 30% as White, Hispanic-Latino. Infinium MethylationEPIC bead arrays were utilized to measure epigenome-wide gDNA methylation of whole blood. Data were analyzed in the presence and absence of adjustments for unknown covariates in the dataset, some of which corresponded to disease modifying treatments. Global patterns of differential methylation associated with MS were strongest for those probes that showed relative demethylation of loci with lower M values. Pathway analysis revealed unexpected associations with shigellosis and amoebiasis. Enrichment analysis revealed an over-representation of probes in enhancer regions and an under-representation in promoters. In the presence of adjustments for covariates that included disease modifying treatments, analysis revealed 10 differentially methylated regions (DMR's) with an FDR <1E-77. Five of these genes (ARID5B, BAZ2B, RABGAP1, SFRP2, WBP1L) are associated with cancer risk and cellular differentiation and have not been previously identified in MS studies. Hierarchical cluster and multi-dimensional scaling analysis of differential DNA methylation at 147 loci within those DMR's was sufficient to differentiate MS donors from controls. In the absence of corrections for disease modifying treatments, differential methylation in patients treated with dimethyl fumarate was associated with immune regulatory pathways that regulate cytokine and chemokine signaling, axon guidance, and adherens junctions. These results demonstrate possible associations of gastrointestinal pathogens and regulation of cellular differentiation with MS susceptibility in our patient cohort. This work further suggests that analyses can be performed in the presence and absence of corrections for immune therapies. Because of their high representation in our patient cohort, these results may be of specific relevance in the regulation of disease susceptibility and treatment responses in Black and Hispanic Americans.

Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase.

Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product inhibition via traditional methods remains challenging as many variants need to be tested against multiple substrate and product concentrations. Further, traditional screening methods are conducted in vitro, and resulting enzyme variants may perform differently in vivo in the context of whole-cell metabolism and regulation. In this study, we address these two problems by establishing a high-throughput screening method to alleviate product inhibition in an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, we engineered a highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) in an industrially relevant host, Pseudomonas putida KT2440. We subsequently applied the biosensor to detect the activity of a heterologously expressed UbiC that converts chorismate into 4HB and pyruvate. By using benzoate as a product surrogate that inhibits UbiC without activating the biosensor, we were able to efficiently create and screen a diversified library for UbiC variants with reduced product inhibition. Introduction of the improved UbiC enzyme variant into an experimental production strain for the industrial precursor cis,cis-muconic acid (muconate), enabled a >2-fold yield improvement for glucose to muconate conversion when the new UbiC variant was expressed from a plasmid and a 60% yield increase when the same UbiC variant was genomically integrated into the strain. Overall, this work demonstrates that by coupling a library of enzyme variants to whole-cell catalysis and biosensing, variants with reduced product inhibition can be identified, and that this improved enzyme can result in increased titers of a downstream molecule of interest.

A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution.

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators.