Biology subjects

Garber, M. E.

Publications and source records attributed to Garber, M. E..

4 recordsLinked to original sources

bioRxiv2025

Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains challenging. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida, a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putidas native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a [~]16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing [~]900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid accumulation as key drivers of enhanced production. Techno-economic analysis elucidated the path to economic viability and confirmed the benefits of adding amino acids outweigh the additional costs. This study establishes a robust biosensor driven approach for optimizing other heterologous pathways, accelerating microbial cell factory development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/643695v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@533605org.highwire.dtl.DTLVardef@ff98fforg.highwire.dtl.DTLVardef@9592fdorg.highwire.dtl.DTLVardef@1e52993_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Pseudomonas response regulators produced in an E. coli heterologous expression host exhibit host-derived post-translational phosphorylation

In this report, we systematically characterize 32 response regulators (RRs) from a metal tolerant groundwater isolate, Pseudomonas stutzeri RCH2 to assess the impact of host-derived post-translational phosphorylation. As observed by distinct shifted bands in a phos-tag gel, 12 of the 24 detected RRs show homogenous mixtures of phosphorylated proteins or heterogenous mixtures of unphosphorylated and phosphorylated proteins. By evaluating the phosphorylation state of CzcR and CopR II under varying assay parameters, we found that changes to pH and exogenous addition of phospho-donors (e.g. acetyl phosphate) have little to no effect on phosphorylation state. By applying protein production conditions that decrease the pool of intracellular acetyl-phosphate in E. coli, we found a reduction in the phosphorylated population of CopR II when magnesium was added to the media, but observed no change in phosphorylated population when CopR II is expressed in E. coli BL21 (DE3){Delta} pta, a mutant with a metabolic disruption to the acetyl-phosphate pathway. Therefore, the specific mechanism of post-translational phosphorylation of RRs in E. coli remains obscure. These findings show the importance of characterizing the phosphorylations state of proteins when heterologously expressed, since their biochemical and physiological properties are dependent on post-translational modification.

biochemistry↗

bioRxiv2020

Evolutionarily Driven Domain Swap Alters Sigma Factor Dependence in Bacterial Signaling System

This report presents multi-genomes and experimental evidence that REC protein family expansion occurs when the emergence of new pathways give rise to functional discordance. Specificity between REC-domain containing response regulators with paired histidine kinases are under negative purifying selection, constrained by the presence of other bacterial two-component systems signaling cascades that share sequence and structural identity. Presuming that the two-component systems can evolve by neutral drift when these constraints are relaxed, how might the REC protein family expand when constraints remain intact? Using an unsupervised machine learning approach to observe the sequence landscape of REC domains across long phylogenetic distances, we find that within-gene-recombination, a subcategory of gene conversion, switched the effector domain, and consequently the regulatory context of a duplicated response regulator from transcriptional regulation by {sigma}54 to {sigma}70. We determined that the recombined response regulator diverged from its parent by positive episodic diversifying selection, giving rise to two new residues. Functional experiments of the parent of recombined response regulators in our model system, Pseudomonas putida KT2440, revealed that the parent and recombined response regulators sense and respond to carboxylic acids and that the two new residues in the recombined regulator form a new interaction interface and prevent crosstalk. Overall, our study finds genetic perturbations can create conditions of functional discordance, whereby the REC protein family can evolve by positive diversifying selection.

evolutionary biology↗

bioRxiv2019

Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida

Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000x more sensitive to exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 g/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ~6 g/L. The high sensitivity and range of OplR is a powerful tool towards the development of novel routes to the biological synthesis of caprolactam. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/700484v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@ff5b8corg.highwire.dtl.DTLVardef@d365aaorg.highwire.dtl.DTLVardef@b79b18org.highwire.dtl.DTLVardef@27732f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗