spp. are widely used model organisms in different areas of research. Despite the relevance of in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in spp. using a nuclease-null Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in and 300-fold repression in pyoverdine production in This inducible system enables the study of essential genes, as shown by depletions in ,, and that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first characterization of protospacer adjacent motif (PAM) site preferences of dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of dCas9, especially in GC-rich organisms. spp. are prevalent in a variety of environments, such as the soil, on the surface of plants, and in the human body. Although spp. are widely used as model organisms in different areas of research, existing tools to deplete a protein of interest in these organisms remain limited. We have developed a robust and inducible gene repression tool in, , and using the dCas9. This method of protein depletion is superior to existing methods, such as promoter replacements and addition of degradation tags, because it does not involve genomic modifications of the target protein, is titratable, and is capable of repressing multiple genes simultaneously. This gene repression system now enables easy depletion of specific proteins in, accelerating the study and engineering of this widely used model organism.
Microbial cell factories are a renewable source for the production of biofuels and valuable chemicals. Dynamic pathway regulation has proved successful in improving production of molecules by balancing flux between growth of cells and production of metabolites. Systems for autonomous induction of pathway regulation are increasingly being developed, which include metabolite responsive promoters, biosensors, and quorum sensing systems. Since engineering such systems are dependent on the available methods for controlling protein abundance in the desired host, we review recent tools used for gene repression at the transcriptional, post-transcriptional and post-translational levels in Escherichia coli and Saccharomyces cerevisiae. These approaches may facilitate pathway engineering for biofuel and biochemical production.
Engineering control of metabolic pathways is important to improving product titers and yields. Traditional methods such as overexpressing pathway enzymes and deleting competing ones are restricted by the interdependence of metabolic reactions and the finite nature of cellular resources. Here, we developed a metabolite valve that controls glycolytic flux through central carbon metabolism in Saccharomyces cerevisiae. In a Hexokinase 2 and Glucokinase 1 deleted strain (hxk2Δglk1Δ), glucose flux was diverted away from glycolysis and into a model pathway, gluconate, by controlling the transcription of Hexokinase 1 with the tetracycline transactivator protein (tTA). A maximum 10-fold decrease in hexokinase activity resulted in a 50-fold increase in gluconate yields, from 0.7% to 36% mol/mol of glucose. The reduction in glucose flux resulted in a significant decrease in ethanol byproduction that extended to semianaerobic conditions, as shown in the production of isobutanol. This proof-of-concept is one of the first demonstrations in S. cerevisiae of dynamic redirection of glucose from glycolysis and into a heterologous pathway.
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