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

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

BBF RFC 21: BglBricks Assembly Standard

The BglBricks standard has been developed as an alternative to the original XbaI/SpeI standard primarily to provide a solution to the issue of generating translational-fusion parts. When joined together in a standard assembly reaction, BglBricks parts contain a scar sequence of GGATCT between the two parts. This sequence conveniently translates as Gly-Ser in the zero frame, a commonly-used linker in protein engineering. BglBricks has previously been called the “BglBrick” standard, and is also referred to as Assembly standard 21. A consolidated description of the standard is available at: http://openwetware.org/wiki/Template:AndersonLab:BglBrick_Standar

Production of β‑ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae

Background Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plantslike raspberries and rosesby the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid -ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP. Results Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of -ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased -ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of -ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase. Conclusions An efficient -ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 genethe highest -ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.This work was funded by grants COPEC-UC 6C-063 and FONDECYT No 1130822, and the Novo Nordisk Foundation

Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.ope