Abstract: A system and method for controlling metabolic enzymes or pathways in cells to produce a chemical above the levels of a wild-type strain is disclosed. The system utilizes cells, including yeasts, bacteria, and molds, having at least two genes capable of being controlled bi-directionally with light, where one gene is turned from off to on when exposed to light and another gene is turned from on to off when exposed to light, the two genes reversing when the light is turned off. Cells may utilize any number of sequences that benefit chemical production, including sequences that: encode for constitutive transcription of light-activated transcription factor fusions; encode for a metabolic enzyme; encode for a repressor; induce expression of metabolic enzymes; and an endogenous or exogenous activator expressed by a constitutive promoter, inducible promoter, or gene circuit.

Abstract: A system and method for controlling metabolic enzymes or pathways in cells to produce a chemical above the levels of a wild-type strain is disclosed. The system utilizes cells, including yeasts, bacteria, and molds, having at least two genes capable of being controlled bi-directionally with light, where one gene is turned from off to on when exposed to light and another gene is turned from on to off when exposed to light, the two genes reversing when the light is turned off. Cells may utilize any number of sequences that benefit chemical production, including sequences that: encode for constitutive transcription of light-activated transcription factor fusions; encode for a metabolic enzyme; encode for a repressor; induce expression of metabolic enzymes; and an endogenous or exogenous activator expressed by a constitutive promoter, inducible promoter, or gene circuit.

Abstract: Provided herein are programmable condensate protein systems and nucleic acid constructs encoding the same. The protein system enables modular targeting of proteins of interest. Protein-peptide interaction domains (PPIDs) are incorporated to functionalize engineered condensates with the attributes of the recruited protein, resulting in a modular system that allows for diverse facile and reprogrammable applications, including in enzyme clustering of metabolic pathways. Colocalizing specific metabolic enzymes in these condensates results in functionalized organelles with which can be used to manipulate the output of engineered metabolic pathways for the production of a pharmaceutical precursor.

Abstract: Disclosed is a technique for constructing optogenetic amplifier and inverter circuits utilizing transcriptional activator/repressor pairs, in which expression of the transcriptional activator or repressor, respectively, is controlled by light-controlled transcription factors. This system is demonstrated utilizing the quinic acid regulon system from Neurospora crassa, or Q System, a transcriptional activator/repressor system. This is also demonstrated utilizing the galactose regulon from Saccharomyces cerevisiae, or GAL System. Such optogenetic amplifier circuits enable multi-phase microbial fermentations, in which different light schedules are applied in each phase to dynamically control different metabolic pathways for the production of proteins, fuels or chemicals.

Abstract: Microbial consortia exert great influence over the physiology of humans, animals, plants, and ecosystems. However, difficulty in controlling their composition and population dynamics have limited their application in medicine, agriculture, biotechnology, and the environment. The approach disclosed herein provides an effective method to dynamically control population compositions in microbial consortia, which we demonstrate in the context of co-culture fermentations for chemical production. Co-culture fermentations can improve chemical production from complex biosynthetic pathways over monocultures by distributing enzymes across multiple strains, thereby reducing metabolic burden, overcoming endogenous regulatory mechanisms, or exploiting natural traits of different microbial species. However, stabilizing and optimizing microbial sub-populations for maximal chemical production remains a major obstacle in the field.

Abstract: A method for producing metabolites that are heavy alcohols, and particularly branched-chain alcohols is provided, involving contacting a suitable substrate with recombinant microorganisms. The microorganisms contain at least one deletion, disruptions, or mutations from the GLN gene family, VPS gene family, GNP gene family, AVT gene family, GCN gene family, or YDR391C, and combinations thereof, and overproduce the heavy alcohol as compared to a wild-type yeast strain.

Abstract: Disclosed herein are optogenetic circuits for the bacterium Escherichia coli that induce gene expression in darkness and repress it under blue light. Applying them to metabolic engineering improves chemical production compared to chemically induced controls in light-controlled fermentations. More particularly, these circuits can be used to control protein production with light. The system and method use light as a suitable alternative to chemical induction for microbial production of chemicals and proteins.

Abstract: Provided herein is a system and method of optogenetically inducibly clustering metabolic enzymes for the production of chemicals using cell factories. More particularly, the described inducible protein clustering approach clusters metabolic enzymes by, e.g., a change in illumination conditions (either a switch from dark to light or from light to dark). Performing this clustering leads to an increase in the production of metabolites by the clustered enzymes. In some embodiments, a light-sensitive domain may be replaced with any inducible domain.

Abstract: A system and method for controlling metabolic enzymes or pathways in cells to produce a chemical above the levels of a wild-type strain is disclosed. The system utilizes cells, including yeasts, bacteria, and molds, having at least two genes capable of being controlled bi-directionally with light, where one gene is turned from off to on when exposed to light and another gene is turned from on to off when exposed to light, the two genes reversing when the light is turned off. Cells may utilize any number of sequences that benefit chemical production, including sequences that: encode for constitutive transcription of light-activated transcription factor fusions; encode for a metabolic enzyme; encode for a repressor; induce expression of metabolic enzymes; and an endogenous or exogenous activator expressed by a constitutive promoter, inducible promoter, or gene circuit.

Abstract: The instant invention describes methods of identifying compounds that modulate the activity of Sir2 enzymes. Sir2 enzymes form a unique class Of NAD+ dependent deacetylases required for diverse biological processes including transcriptional silencing, regulation of apoptosis, fat mobilization, and lifespan regulation. Sir2 activity is regulated by nicotinamide, a non-competitive inhibitor that promotes a base exchange reaction at the expense of deacetylation.