Genes to jeans: A green solution to blue denim

Dyeing jeans to produce the classic blue tones we love is an extremely dirty process. In addition to the hazardous chemicals involved in indigo synthesis, excess reducing agent is required to solubilize the crystals for dyeing the cotton fibers. Despite many efforts to circumvent the need for this reducing agent to limit cost and environmental damage, it remains instrumental to the current dye mill process. We are implementing a fermentation strategy where the air-reactive indigo precursor indoxyl is biosynthesized from tryptophan. Indoxyl is stabilized in a soluble form by glucosylation. Upon treatment of the glucoside to cotton, a betaglucosidase can be added to reform the air-reactive indoxyl that oxidizes to indigo crystals in the fibers. We have replaced the glucose biochemical protecting group with other biochemical protecting groups for altering the chemical behavior of the product in the production host and the ease of deprotection on cotton. We have also explored this strategy of adding biochemical protecting groups for controlled reactivity for other molecules of interes

CRISPR-guided DNA polymerase enabling diversification of all nucleotides in a tunable window

The capacity to diversify genetic codes advances our understanding and engineering of biological systems. A method to continuously diversify user-defined regions of a genome without requiring the integration of nucleic acid libraries would enable forward genetic approaches in systems not amenable to high efficiency homologydirected integration, rapid evolution of biotechnologically useful activity through accelerated and parallelized rounds of mutagenesis and selection, and cell lineage tracking. Here we developed EvolvR, the first system that can continuously diversify all nucleotides within a tunable window length at user-defined loci. Our results demonstrate that EvolvR enables multiplexed and continuous diversification of user-defined genomic loci that will be useful for a broad range of basic and biotechnological applications

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli

The field of metabolic engineering has the potential to produce a wide variety of chemicals in both an inexpensive and ecologically-friendly manner. Heterologous expression of novel combinations of enzymes promises to provide new or improved synthetic routes towards a substantially increased diversity of small molecules. Recently, we constructed a synthetic pathway to produce d-glucaric acid, a molecule that has been deemed a “top-value added chemical” from biomass, starting from glucose. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To synthetically increase the effective concentration of myo-inositol, polypeptide scaffolds were built from protein–protein interaction domains to co-localize all three pathway enzymes in a designable complex as previously described (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of increased numbers of MIOX-targeted domains was much less significant. We determined that the scaffolds directly increased the specific MIOX activity and that glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed an approximately 5-fold improvement in product titers over the non-scaffolded control, and a 50% improvement over the previously reported highest titers. These results further validate the utility of these synthetic scaffolds as a tool for metabolic engineering.United States. Office of Naval Research (Young Investigator Program, Grant No. N000140510656)Synthetic Biology Engineering Research CenterNational Science Foundation (U.S.) (Grant No. EEC-0540879)National Science Foundation (U.S.) (Grant No. CBET-0756801

Identification and characterization of a galacturonic acid transporter from Neurospora crassa and its application for Saccharomyces cerevisiae fermentation processes

BACKGROUND: Pectin-rich agricultural wastes potentially represent favorable feedstocks for the sustainable production of alternative energy and bio-products. Their efficient utilization requires the conversion of all major constituent sugars. The current inability of the popular fermentation host Saccharomyces cerevisiae to metabolize the major pectic monosaccharide D-galacturonic acid (D-GalA) significantly hampers these efforts. While it has been reasoned that the optimization of cellular D-GalA uptake will be critical for the engineering of D-GalA utilization in yeast, no dedicated eukaryotic transport protein has been biochemically described. Here we report for the first time such a eukaryotic D-GalA transporter and characterize its functionality in S. cerevisiae. RESULTS: We identified and characterized the D-GalA transporter GAT-1 out of a group of candidate genes obtained from co-expression analysis in N. crassa. The N. crassa Δgat-1 deletion strain is substantially affected in growth on pectic substrates, unable to take up D-GalA, and impaired in D-GalA-mediated signaling events. Moreover, expression of a gat-1 construct in yeast conferred the ability for strong high-affinity D-GalA accumulation rates, providing evidence for GAT-1 being a bona fide D-GalA transport protein. By recombinantly co-expressing D-galacturonate reductase or uronate dehydrogenase in yeast we furthermore demonstrated a transporter-dependent conversion of D-GalA towards more reduced (L-galactonate) or oxidized (meso-galactaric acid) downstream products, respectively, over a broad concentration range. CONCLUSIONS: By utilizing the novel D-GalA transporter GAT-1 in S. cerevisiae we successfully generated a transporter-dependent uptake and catalysis system for D-GalA into two products with high potential for utilization as platform chemicals. Our data thereby provide a considerable first step towards a more complete utilization of biomass for biofuel and value-added chemicals production

Design and implementation of a biomolecular concentration tracker

As a field, synthetic biology strives to engineer increasingly complex artificial systems in living cells. Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise. In this work, we use synthetic protein scaffolds as a modular and tunable mechanism for concentration tracking through negative feedback. Input to the circuit initiates scaffold production, leading to colocalization of a two-component system and resulting in the production of an inhibitory antiscaffold protein. Using a combination of modeling and experimental work, we show that the biomolecular concentration tracker circuit achieves dynamic protein concentration tracking in Escherichia coli and that steady state outputs can be tuned

Selection of chromosomal DNA libraries using a multiplex CRISPR system.

The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function

Genomewide and Enzymatic Analysis Reveals Efficient d-Galacturonic Acid Metabolism in the Basidiomycete Yeast Rhodosporidium toruloides.

Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid- and terpene-derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6 sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed the potential of R. toruloides to assimilate d-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. d-Galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with those of d-glucose and d-xylose in R. toruloides A genomewide analysis by combined transcriptome sequencing (RNA-seq) and RB-TDNA-seq revealed those genes with high relevance for fitness on d-galacturonic acid. While R. toruloides was found to utilize the nonphosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for d-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies with basidiomycetes.IMPORTANCE The switch from the traditional fossil-based industry to a green and sustainable bioeconomy demands the complete utilization of renewable feedstocks. Many currently used bioconversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. d-Galacturonic acid is a plant component of bioconversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeast Rhodosporidium toruloides has been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods and physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of d-galacturonic acid. This study identified an efficient pathway for uronic acid conversion to guide future engineering efforts and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus

BglBricks: A flexible standard for biological part assembly

<p>Abstract</p> <p>Background</p> <p>Standard biological parts, such as BioBricks™ parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks™ assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon.</p> <p>Results</p> <p>Here, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the <it>E. coli </it>genome.</p> <p>Conclusions</p> <p>The BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.</p

De novo design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering

Design and implementation of a synthetic biomolecular concentration tracker

As a field, synthetic biology strives to engineer increasingly complex artificial systems in living cells. Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise. In this work, we use synthetic protein scaffolds as a modular and tunable mechanism for concentration tracking through negative feedback. Input to the circuit initiates scaffold production, leading to colocalization of a two-component system and resulting in the production of an inhibitory antiscaffold protein. Using a combination of modeling and experimental work, we show that the biomolecular concentration tracker circuit achieves dynamic protein concentration tracking in Escherichia coli and that steady state outputs can be tuned