Employing a biochemical protecting group for a sustainable indigo dyeing strategy.

Indigo is an ancient dye uniquely capable of producing the signature tones in blue denim; however, the dyeing process requires chemical steps that are environmentally damaging. We describe a sustainable dyeing strategy that not only circumvents the use of toxic reagents for indigo chemical synthesis but also removes the need for a reducing agent for dye solubilization. This strategy utilizes a glucose moiety as a biochemical protecting group to stabilize the reactive indigo precursor indoxyl to form indican, preventing spontaneous oxidation to crystalline indigo during microbial fermentation. Application of a ß-glucosidase removes the protecting group from indican, resulting in indigo crystal formation in the cotton fibers. We identified the gene coding for the glucosyltransferase PtUGT1 from the indigo plant Polygonum tinctorium and solved the structure of PtUGT1. Heterologous expression of PtUGT1 in Escherichia coli supported high indican conversion, and biosynthesized indican was used to dye cotton swatches and a garment.

Bioproduction of a betalain color palette in Saccharomyces cerevisiae.

Betalains are a family of natural pigments found exclusively in the plant order Caryophyllales. All members of this chemical family are biosynthesized through the common intermediate betalamic acid, which is capable of spontaneously condensing with various primary and secondary amines to produce betalains. Of particular interest is the red-violet betanin, most commonly obtained from Beta vulgaris (beet) as a natural food dye. We demonstrate the first complete microbial production of betanin in Saccharomyces cerevisiae from glucose, an early step towards a fermentation process enabling rapid, on-demand production of this natural dye. A titer of 17mg/L was achieved, corresponding to a color intensity obtained from 10g/L of beetroot extract. Further, we expanded the spectrum of betalain colors by condensing betalamic acid with various amines fed to an engineered strain of S. cerevisiae. Our work establishes a platform for microbial production of betalains of various colors as a potential alternative to land- and resource-intensive agricultural production.

An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose.

Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.

Application of a Palladium-Catalyzed C-H Functionalization/Indolization Method to Syntheses of cis-Trikentrin†A and Herbindole†B.

We describe herein formal syntheses of the indole alkaloids cis-trikentrin A and herbindole B from a common meso-hydroquinone intermediate prepared by a ruthenium-catalyzed [2+2+1+1] cycloaddition that has not been used previously in natural product synthesis. Key steps include a sterically demanding Buchwald-Hartwig amination as well as a unique C(sp(3) )-H amination/indole formation. Studies toward a selective desymmetrization of the meso-hydroquinone are also reported.

Exploration of Acetylation as a Base-Labile Protecting Group in Escherichia coli for an Indigo Precursor.

Biochemical protecting groups are observed in natural metabolic pathways to control reactivity and properties of chemical intermediates; similarly, they hold promise as a tool for metabolic engineers to achieve the same goals. Protecting groups come with costs: lower yields from carbon, metabolic load to the production host, deprotection catalyst costs and kinetics limitations, and wastewater treatment of the group. Compared to glycosyl biochemical protection, such as glucosyl groups, acetylation can mitigate each of these costs. As an example application where these benefits could be valuable, we explored acetylation protection of indoxyl, the reactive precursor to the clothing dye, indigo. First, we demonstrated denim dyeing with chemically sourced indoxyl acetate by deprotection with base, showing results comparable to industry-standard denim dyeing. Second, we modified an Escherichia coli production host for improved indoxyl acetate stability by the knockout of 14 endogenous hydrolases. Cumulatively, these knockouts yielded a 67% reduction in the indoxyl acetate hydrolysis rate from 0.22 mmol/g DCW/h to 0.07 mmol/g DCW/h. To biosynthesize indoxyl acetate, we identified three promiscuous acetyltransferases which acetylate indoxyl in vivo. Indoxyl acetate titer, while low, was improved 50%, from 43 µM to 67 µM, in the hydrolase knockout strain compared to wild-type E. coli. Unfortunately, low millimolar concentrations of indoxyl acetate proved to be toxic to the E. coli production host; however, the principle of acetylation as a readily cleavable and low impact biochemical protecting group and the engineered hydrolase knockout production host should prove useful for other metabolic products.

Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways.

Compartmentalization of enzymes into organelles is a promising strategy for limiting metabolic crosstalk and improving pathway efficiency, but improved tools and design rules are needed to make this strategy available to more engineered pathways. Here we focus on the Saccharomyces cerevisiae peroxisome and develop a sensitive high-throughput assay for peroxisomal cargo import. We identify an enhanced peroxisomal targeting signal type 1 (PTS1) for rapidly sequestering non-native cargo proteins. Additionally, we perform the first systematic in vivo measurements of nonspecific metabolite permeability across the peroxisomal membrane using a polymer exclusion assay. Finally, we apply these new insights to compartmentalize a two-enzyme pathway in the peroxisome and characterize the expression regimes where compartmentalization leads to improved product titre. This work builds a foundation for using the peroxisome as a synthetic organelle, highlighting both promise and future challenges on the way to realizing this goal.

The khmer software package: enabling efficient nucleotide sequence analysis.

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at  https://github.com/dib-lab/khmer/.

Cell-free protein synthesis: search for the happy middle.

Cell-free protein synthesis (CFPS) is a versatile technique gaining popularity because it allows researchers access to on-demand production of proteins. This Commentary by Zachary Russ and John Dueber discusses the latest research article by Rui Gan and Michael Jewett, which reports a convenient and cost-effective methodology for the preparation of Saccharomyces cerevisiae-based CFPS reactions.

Mapping the moral boundaries of biological engineering.

The following essay was written by a sophomore undergraduate student majoring in Bioengineering at the University of Maryland, Mr. Zachary Russ. Mr. Russ was one of 174 students who submitted a 1000-1200 word essay to the 4th Annual Bioethics Contest sponsored by the Institute of Biological Engineering (IBE). A group of professionals in Biological Engineering assessed and ranked the essays in a blinded process. Five semi-finalists were invited to present their essays at a session at the annual meeting of IBE in Santa Clara, CA on March 21, 2009. Five judges scored all the presentation at the annual meeting and selected Mr. Russ's contribution as the overall winner (1st Place).

Synthetic biology: enormous possibility, exaggerated perils.

The following essay was written by a freshman undergraduate student majoring in Bioengineering at the University of Maryland, Mr. Zachary Russ. Mr. Russ was one of 94 students who submitted a 1000 to 1200 word essay to the 3rd Annual Bioethics Essay Contest sponsored by the Institute of Biological Engineering (IBE). A group of professionals in Biological Engineering assessed and ranked the essays in a blinded process. Five semi-finalists were invited to present their essays at a session at the annual meeting of IBE in Chapel Hill, NC on March 8, 2008. Five judges scored the presentations at the annual meeting and selected Mr. Russ's contribution as the overall winner (1st Place). Below is his essay.