Improving Microbial Tolerance to Biomass Hydrolysates Using Massively Parallel Genome Engineering | AIChE

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Improving Microbial Tolerance to Biomass Hydrolysates Using Massively Parallel Genome Engineering

Lignocellulosic biomass represents an attractive feedstock for microbial biotechnology. A current challenge for utilization of these promising carbon sources comes from fact that they contain a mixture of compounds that inhibit growth of microbial strains. Therefore, engineering industrial strains for tolerance and robust growth in the presence of inhibitory molecules is essential for performance.

Engineering complex phenotypes, such as tolerance, relies on exploratory approaches like directed evolution that require generation of significant genetic diversity. Traditional mutagenesis strategies carry the risk of introducing unwanted mutations that may have deleterious effects and are difficult to parse out. On the other hand, targeted and trackable genome engineering allows to control the edits and directly tie them to the phenotype.

This app note describes the application of the OnyxTM platform for massively parallel targeted strain engineering to generate a genetically diverse population for pooled cultivations under selective pressure. Inscripta’s E. coli strain library was engineered to create 3,676 genome-wide promoter insertions and 3,966 knockouts. The engineered cell libraries were then grown in the presence of four growth-inhibitory compounds found in biomass hydrolysates and sequenced using Inscripta’s rapid barcode sequencing assay to identify significantly enriched or depleted variants.

The results of the study cover:

  • Enrichment and depletion profiles of engineered strain variants in the presence of biomass growth inhibitors
  • Target Identification: thousands of novel genotype-phenotype links for biomass hydrolysate inhibitors
  • The advantage of massively parallel genome-edited libraries over traditional omics experiments

Read the app note to discover more insights.