Browsing by Author "Lazar, John Tyler"

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    Engineering Optogenetic Control of Bacterial Metabolism in Stationary Phase of Growth
    (2023-09-06) Lazar, John Tyler; Tabor, Jeffrey J; Thyer, Ross
    Genetically-encoded sensors are used to induce metabolite production in bacterial fermentations. However, these sensors are typically optimized for exponential growth phase rather than stationary phase where the majority of metabolite production occurs. In the first portion of this work, we find that our exponential phase-optimized green light-activated E. coli two-component gene regulatory system CcaSR is effectively non-functional in stationary phase. We show that the major causes of failure are stationary-phase specific mutation of the plasmid-borne biosynthetic pathway used to produce the required chromophore phycocyanobilin (PCB) and accumulation of the response regulator CcaR leading to very high leaky target gene expression. To address these problems, we move the PCB biosynthetic pathway into the chromosome and re-optimize expression of the component enzymes, and re-balance CcaR expression for stationary phase . The resulting CcaSRstat system exhibits low levels of leakiness and an 80-fold activation of target gene expression in stationary phase. Notably, our stationary phase-optimized CcaSRstat system is not functional in exponential phase, a feature that may have benefits for metabolic engineering and other applications. In the second portion of this work, we combine CcaSRstat with static and periodic illumination patterns to achieve high levels of production of several industrially-relevant phenylpropanoid metabolites such as p-coumaric acid and bisdemethoxycurcumin. We then proceed to demonstrate that our optimal light signals at the 0.5 mL light plate apparatus (LPA) volume scale to 25 mL optogenetic bioreactors. CcaSRstat is a useful tool for optimizing bacterial metabolite production and could be used to control bacterial behaviors in other non-growth environments such as the gastrointestinal tract or soil. Our work lays the foundation for increasing the exploration space of dynamic control of metabolic pathways while also providing valuable insight into design considerations of biosensors in the stationary phase of growth.
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    Optogenetic control of gut bacterial metabolism to promote longevity
    (eLife Sciences Publications, Ltd, 2020) Hartsough, Lucas A.; Park, Mooncheol; Kotlajich, Matthew V.; Lazar, John Tyler; Han, Bing; Lin, Chih-Chun J.; Musteata, Elena; Gambill, Lauren; Wang, Meng C.; Tabor, Jeffrey J.; Bioengineering
    Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.