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

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

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

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

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

Novel Fusion \u3ci\u3eKTN1-PRKD1\u3c/i\u3e in Cribriform Adenocarcinoma of Salivary Glands Located in the Parotid Gland: Case Report Including Cytologic Findings

Background Cribriform adenocarcinoma of salivary glands (CASG) is a rare, predominantly minor salivary gland tumor first described in 1999. Because the tumor shares morphologic and molecular features with polymorphous adenocarcinoma (PAC), in 2017, the World Health Organization (WHO) included CASG within the spectrum of PAC. Almost 75% of CASG harbor molecular alterations in the PRKD (Protein kinase D) gene family, and some cases show ARID1A (AT-rich interaction domain 1A)-PRKD1 or DDX3X (DEAD-Box Helicase 3 X-Linked)-PRKD1 fusions. Case presentation A 39-year-old man presented with headache and painless right cheek mass of two years duration. Imaging showed a well-circumscribed, lobulated 1.7-centimeter mass located in the superficial lobe of the right parotid gland. Fine needle aspiration (FNA) of the mass revealed a “salivary gland neoplasm of uncertain malignant potential” (SUMP). Histopathology and immunohistochemical features of the resected tumor showed a primary salivary gland neoplasm with perineural invasion suggestive of cribriform adenocarcinoma of the salivary glands (CASG). Whole exome sequencing (WES) and transcriptome sequencing (RNAseq) of the tumor revealed a novel, intrachromosomal gene fusion: KTN1 (Kinectin1)-PRKD1. Sanger sequencing and Florescent insitu hybridization (FISH) break apart probe results subsequently confirmed the presence of the fusion. The patient recovered from surgery without complications. Conclusion We report a novel fusion KTN1-PRKD1 in Cribriform Adenocarcinoma of the Salivary Glands located in the parotid gland. Importantly, this KTN1 fusion partner may account for other reports of intrachromosomal fusions in CASG in which the PRKD1 gene partner was not identified

In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2–7 onto DNA. Helicase loading involves two MCM2–7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2–7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC–Cdc6 interaction and MCM2–7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2–7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2–7. To determine whether Cdc6 regulates MCM2–7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2–7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2–7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2–7 recruitment, show that ATPase activity is required for MCM2–7 hexamer dimerization and demonstrate that MCM2–7 hexamers are recruited to origins in a consecutive process

Engineering <i>Saccharomyces cerevisiae</i> for co-utilization of D-galacturonic acid and D-glucose from citrus peel waste

Pectin-rich agricultural byproducts are ideal feedstocks for biobased chemicals production. Here, the authors engineer the yeast, S. cerevisiae, in several steps to co-utilize d-galacturonic acid and d-glucose and demonstrate the potential of producing meso-galactaric acid from industrial orange peel

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action