Sorting for secreted molecule production using a biosensor-in-microdroplet approach.

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.

Bidirectional titration of yeast gene expression using a pooled CRISPR guide RNA approach.

Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae's metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches.

Enhanced scale and scope of genome engineering and regulation using CRISPR/Cas in Saccharomyces cerevisiae.

Although only 6 years old, the CRISPR system has blossomed into a tool for rapid, on-demand genome engineering and gene regulation in Saccharomyces cerevisiae. In this minireview, we discuss fundamental CRISPR technologies, tools to improve the efficiency and capabilities of gene targeting, and cutting-edge techniques to explore gene editing and transcriptional regulation at genome scale using pooled approaches. The focus is on applications to metabolic engineering with topics including development of techniques to edit the genome in multiplex, tools to enable large numbers of genetic modifications using pooled single-guide RNA libraries and efforts to enable programmable transcriptional regulation using endonuclease-null Cas enzymes.

CRISPR-PIN: Modifying gene position in the nucleus via dCas9-mediated tethering.

Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair, genetic recombination, and gene expression. Here, we present CRISPR-PIN, a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae. This approach utilizes a cohesin-dockerin interaction between dCas9 and a perinuclear protein. In doing so, we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery. We demonstrate the utility of this approach for two applications: the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci. In both cases, we obtain results on par with prior reports using traditional, more cumbersome genetic systems. Thus, CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.

Recent advancements in fungal-derived fuel and chemical production and commercialization.

Due to unsustainable petroleum supply and poor yields from plant and animal sources, there is an increased effort to engineer microbial hosts for renewable chemical production. When compared to microbes such as Escherichia coli, fungal hosts show advantages due to their natural robust tolerance for industrial fermentation. Synthetic biology has focused on implementing heterologous pathways and manipulating native flux towards downstream products to achieve industrial productivity, titers, and yields. This review highlights recent advances in the engineering of yeasts for fuels and other molecules. As the field progresses, strains with improved productivities will begin to compete with the traditional chemical-based industrial approaches.

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae.

Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts. Next, we devise and validate a scheme to allow modular construction of tRNA-sgRNA (TST) operons using an iterative Type IIs digestion/ligation extension approach, termed CRISPR-Ligation Extension of sgRNA Operons (LEGO). This approach enables facile construction of large TST operons. We demonstrate this utility by constructing a metabolic rewiring prototype for 2,3-butanediol production in 2 distinct yeast strain backgrounds. These results demonstrate that our approach can act as a surrogate for traditional genetic modification on a much shorter design-cycle timescale.

Promoter and Terminator Discovery and Engineering.

Control of gene expression is crucial to optimize metabolic pathways and synthetic gene networks. Promoters and terminators are stretches of DNA upstream and downstream (respectively) of genes that control both the rate at which the gene is transcribed and the rate at which mRNA is degraded. As a result, both of these elements control net protein expression from a synthetic construct. Thus, it is highly important to discover and engineer promoters and terminators with desired characteristics. This chapter highlights various approaches taken to catalogue these important synthetic elements. Specifically, early strategies have focused largely on semi-rational techniques such as saturation mutagenesis to diversify native promoters and terminators. Next, in an effort to reduce the length of the synthetic biology design cycle, efforts in the field have turned towards the rational design of synthetic promoters and terminators. In this vein, we cover recently developed methods such as hybrid engineering, high throughput characterization, and thermodynamic modeling which allow finer control in the rational design of novel promoters and terminators. Emphasis is placed on the methodologies used and this chapter showcases the utility of these methods across multiple host organisms.

Enabling Graded and Large-Scale Multiplex of Desired Genes Using a Dual-Mode dCas9 Activator in Saccharomyces cerevisiae.

Standard approaches for dCas9-based modification of gene expression are limited in the ability to multiplex targets, establish streamlined cassettes, and utilize commonly studied Pol II promoters. In this work, we repurpose the dCas9-VPR activator to act as a dual-mode activator/repressor that can be programmed solely on the basis of target position at gene loci. Furthermore, we implement this approach using a streamlined Pol II-ribozyme system that allows expression of many sgRNAs from a single transcript. By "stepping" dCas9-VPR within the promoter region and ORF we create graded activation and repression (respectively) of target genes, allowing precise control over multiplexed gene modulation. Expression from the Pol II system increased the net amount of sgRNA production in cells by 3.88-fold relative to the Pol III SNR52 promoter, leading to a significant improvement in dCas9-VPR repression strength. Finally, we utilize our Pol II system to create galactose-inducible switching of gene expression states and multiplex constructs capable of modulating up to 4 native genes from a single vector. Our approach represents a significant step toward minimizing DNA required to assemble CRISPR systems in eukaryotes while enhancing the efficacy (greater repression strength), scale (more sgRNAs), and scope (inducibility) of dCas9-mediated gene regulation.

Systematic testing of enzyme perturbation sensitivities via graded dCas9 modulation in Saccharomyces cerevisiae.

Dissecting genotype-phenotype relationships in a high-throughput and scalable manner is still an unresolved problem facing metabolic engineers. While the RNA-guided nuclease Cas9 has been repurposed as a programmable transcription regulator, its application has typically been limited to binary on/off regulation and thus misses informative and potentially optimal intermediate levels of gene expression. In this work, we establish a rapid method for fine-tuned, graded expression of pathway enzymes via dCas9 regulation by varying sgRNA target location as the dominant parameter. Next, we utilize this technique to produce graded gene expression and Systematically Test Enzyme Perturbation Sensitivities (STEPS) to identify rate limiting steps in metabolic pathways. Specifically, we utilize this approach in an iterative fashion for the glycerol biosynthesis pathway and ultimately achieve a 5.7-fold increase in titer. We then demonstrate the portability of this approach by applying it to the pentose phosphate pathway in two distinct strain backgrounds. In doing so, we identify and alleviate pathway bottlenecks resulting in a 7.8-fold increase in 3-dehydroshikimate titer and the identification of 3 unique targets for xylose catabolism. This technique easily scales with DNA synthesis, a rapidly decreasing cost, and thus we envision that this technique can be used to complement genome-scale metabolic models by experimentally mapping the flux sensitivity of the entire genome to desired phenotypes.

Highly selective condensation of biomass-derived methyl ketones as a source of aviation fuel.

Aviation fuel (i.e., jet fuel) requires a mixture of C9 -C16 hydrocarbons having both a high energy density and a low freezing point. While jet fuel is currently produced from petroleum, increasing concern with the release of CO2 into the atmosphere from the combustion of petroleum-based fuels has led to policy changes mandating the inclusion of biomass-based fuels into the fuel pool. Here we report a novel way to produce a mixture of branched cyclohexane derivatives in very high yield (>94 %) that match or exceed many required properties of jet fuel. As starting materials, we use a mixture of n-alkyl methyl ketones and their derivatives obtained from biomass. These synthons are condensed into trimers via base-catalyzed aldol condensation and Michael addition. Hydrodeoxygenation of these products yields mixtures of C12 -C21 branched, cyclic alkanes. Using models for predicting the carbon number distribution obtained from a mixture of n-alkyl methyl ketones and for predicting the boiling point distribution of the final mixture of cyclic alkanes, we show that it is possible to define the mixture of synthons that will closely reproduce the distillation curve of traditional jet fuel.