Showing 1 - 3 of 3 results
1.
Anti-Pdc1p Nanobody as a Genetically Encoded Inhibitor of Ethanol Production Enables Dual Transcriptional and Post-translational Controls of Yeast Fermentations.
Abstract:
Microbial fermentation provides a sustainable method of producing valuable chemicals. Adding dynamic control to fermentations can significantly improve titers, but most systems rely on transcriptional controls of metabolic enzymes, leaving existing intracellular enzymes unregulated. This limits the ability of transcriptional controls to switch off metabolic pathways, especially when metabolic enzymes have long half-lives. We developed a two-layer transcriptional/post-translational control system for yeast fermentations. Specifically, the system uses blue light to transcriptionally activate the major pyruvate decarboxylase PDC1, required for cell growth and concomitant ethanol production. Switching to darkness transcriptionally inactivates PDC1 and instead activates the anti-Pdc1p nanobody, NbJRI, to act as a genetically encoded inhibitor of Pdc1p accumulated during the growth phase. This dual transcriptional/post-translational control improves the production of 2,3-BDO and citramalate by up to 100 and 92% compared to using transcriptional controls alone in dynamic two-phase fermentations. This study establishes the NbJRI nanobody as an effective genetically encoded inhibitor of Pdc1p that can enhance the production of pyruvate-derived chemicals.
2.
Light-Induced Nanobody-Mediated Targeted Protein Degradation for Metabolic Flux Control.
Abstract:
In metabolic engineering, increasing chemical production usually involves manipulating the expression levels of key enzymes. However, limited synthetic tools exist for modulating enzyme activity beyond the transcription level. Inspired by natural post-translational mechanisms, we present targeted enzyme degradation mediated by optically controlled nanobodies. We applied this method to a branched biosynthetic pathway, deoxyviolacein, and observed enhanced product specificity and yield. We then extend the biosynthesis pathway to violacein and show how simultaneous degradation of two target enzymes can further shift production profiles. Through the redirection of metabolic flux, we demonstrate how targeted enzyme degradation can be used to minimize unwanted intermediates and boost the formation of desired products.
3.
Optogenetics Illuminates Applications in Microbial Engineering.
Abstract:
Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe-host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 13 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
