Engineering Papers⌕ Search

Engineering topics

Fenster, Jacob A.

Publications and source records attributed to Fenster, Jacob A..

African Swine Fever Virus Protein–Protein Interaction Prediction

The African swine fever virus (ASFV) is an often deadly disease in swine and poses a threat to swine livestock and swine producers. With its complex genome containing more than 150 coding regions, developing effective vaccines for this virus remains a challenge due to a lack of basic knowledge about viral protein function and protein–protein interactions between viral proteins and between viral and host proteins. In this work, we identified ASFV-ASFV protein–protein interactions (PPIs) using artificial intelligence-powered protein structure prediction tools. We benchmarked our PPI identification workflow on the Vaccinia virus, a widely studied nucleocytoplasmic large DNA virus, and found that it could identify gold-standard PPIs that have been validated in vitro in a genome-wide computational screening. We applied this workflow to more than 18,000 pairwise combinations of ASFV proteins and were able to identify seventeen novel PPIs, many of which have corroborating experimental or bioinformatic evidence for their protein–protein interactions, further validating their relevance. Two protein–protein interactions, I267L and I8L, I267L__I8L, and B175L and DP79L, B175L__DP79L, are novel PPIs involving viral proteins known to modulate host immune response.

59 BASIC BIOLOGICAL SCIENCES↗

Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p -coumarate

We report Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 hour quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

59 BASIC BIOLOGICAL SCIENCES↗

High-Throughput Functional Genomics for Energy Production

Functional genomics remains a foundational field for establishing genotype-phenotype relationships that enable strain engineering. High-throughput (HTP) methods accelerate the Design-Build-Test-Learn cycle that currently drives synthetic biology towards a forward engineering future. Trackable mutagenesis techniques including transposon insertion sequencing and CRISPR-Cas-mediated genome editing allow for rapid fitness profiling of a collection, or library, of mutants to discover beneficial mutations. Due to the relative speed of these experiments compared to adaptive evolution experiments, iterative rounds of mutagenesis can be implemented for next-generation metabolic engineering efforts to design complex production and tolerance phenotypes. Further, the expansion of these mutagenesis techniques to novel bacteria are opening up industrial microbes that show promise for establishing a bio-based economy.

59 BASIC BIOLOGICAL SCIENCES↗