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Geraniol is an important monoterpene alcohol with various industrial applications. The biological synthesis of geraniol requires the activity of geraniol synthase (GES). Despite several engineering efforts to improve catalytic rates of GES, overall efforts have been limited by the lack of a high-throughput screen. Here, we developed a coupled enzyme-based fluorogenic assay that can detect geraniol as well as other medium to long chain alcohols (C4-C9). Aided by this rapid screening capability, we performed saturation mutagenesis of GES of Catharanthus roseus and identified a mutation of F418 to Q that improved production of geraniol. This robust screening assay enables more high-throughput analysis and engineering of geraniol and other alcohols in S. cerevisiae and E. coli.

Within the Design-Build-Test Cycle for strain engineering, rapid product detection and selection strategies remain challenging and limit overall throughput. Here we summarize a wide variety of modalities that transduce chemical concentrations into easily measured absorbance, luminescence, and fluorescence signals. Specifically, we cover protein-based biosensors (including transcription factors), nucleic acid-based biosensors, coupled enzyme reactions, bioorthogonal chemistry, and fluorescent and chromogenic dyes and substrates as modalities for detection. We focus on the use of these methods for strain engineering and enzyme discovery and conclude with remarks on the current and future state of biosensor development for application in the metabolic engineering field.

Synthetic biology and metabolic engineering seek to re-engineer microbes into “living foundries” for the production of high value chemicals. Through a “design-build-test” cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals., Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.

Eukaryotic biochemistry is organized throughout the cell in and on membrane-bound organelles. When engineering metabolic pathways this organization is often lost, resulting in flux imbalance and a loss of kinetic advantages from enzyme colocalization and substrate channeling. Here, we develop a protein-based scaffold for colocalizing multienzyme pathways on the membranes of intracellular lipid droplets. Scaffolds based on the plant lipid droplet protein oleosin and cohesin-dockerin interaction pairs recruited upstream enzymes in yeast ester biosynthesis to the native localization of the terminal reaction step, alcohol-O-acetyltransferase (Atf1). The native localization is necessary for high activity and pathway assembly in close proximity to Atf1 increased pathway flux. Screening a library of scaffold variants further showed that pathway structure can alter catalysis and revealed an optimized scaffold and pathway expression levels that produced ethyl acetate at a rate nearly 2-fold greater than unstructured pathways. This strategy should prove useful in spatially organizing other metabolic pathways with key lipid droplet-localized and membrane-bound reaction steps.

Ethyl and acetate esters are naturally produced in various yeasts, plants, and bacteria. The biosynthetic pathways that produce these esters share a common reaction step, the condensation of acetyl/acyl-CoA with an alcohol by alcohol-O-acetyl/acyltransferase (AATase). Recent metabolic engineering efforts exploit AATase activity to produce fatty acid ethyl esters as potential diesel fuel replacements as well as short- and medium-chain volatile esters as fragrance and flavor compounds. These efforts have been limited by the lack of a rapid screen to quantify ester biosynthesis. Enzyme engineering efforts have also been limited by the lack of a high throughput screen for AATase activity. Here, we developed a high throughput assay for AATase activity and used this assay to discover a high activity AATase from tomato fruit, Solanum lycopersicum (Atf-S.l). Atf1-S.l exhibited broad specificity towards acyl-CoAs with chain length from C4 to C10 and was specific towards 1-pentanol. The AATase screen also revealed new acyl-CoA substrate specificities for Atf1, Atf2, Eht1, and Eeb1 from Saccharomyces cerevisiae, and Atf-C.m from melon fruit, Cucumis melo, thus increasing the pool of characterized AATases that can be used in ester biosynthesis of ester-based fragrance and flavor compounds as well as fatty acid ethyl ester biofuels.

Enzyme–DNA nanostructures were designed to introduce new substrate–enzyme interactions into their reactions, which altered enzyme kinetics in a predictable manner. The designed enzymes demonstrate a new strategy of enzyme engineering based on the rational design of intermolecular interactions outside of the active site that enhance and control enzyme kinetics. Binding interactions between tetramethylbenzidine and DNA attached to horseradish peroxidase (HRP) resulted in a reduced Michaelis constant (KM) for the substrate. The enhancement increased with stronger interactions in the micromolar range, resulting in a 2.6 fold increase in kcat/KM. The inhibition effect of 4-nitrobenzoic hydrazide on HRP was also significantly enhanced by tuning the binding to HRP–DNA. Lastly, binding of a nicotinamide adenine dinucleotide (NAD(H)) cofactor mimic, nicotinamide mononucleotide (NMN(H)), to an aldo-keto reductase (AdhD) was enhanced by introducing NMN(H)–DNA interactions.

New developments in nucleic acid nanotechnology and protein scaffold designs have enabled unparalleled control over the spatial organization of synthetic multienzyme cascade reactions. One of the goals of these new technologies is to create nanostructured enzyme cascade reactions that promote substrate channeling along the cascade and, in doing so, enhance cascade catalysis. The concept of substrate channeling has a long and rich history in biochemistry and has established methods of evaluation and quantification. In this Perspective, we review the most common of these methods and discuss them in the context of engineered multienzyme systems and natural bifunctional enzymes with known mechanisms of substrate channeling. In addition, we use experimental data and the results of simulations of coupled-enzyme reactions to develop a set of preliminary design rules for engineering multienzyme nanostructures. The design rules address the limitations on interenzyme distance and active site orientation in substrat...

Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.

Advances in DNA bionanotechnology have led to the ability to create structures with well-defined chemical and physical features at the nanoscale. Such nanostructures can be used to create spatially organized enzymatic cascades that promote substrate channeling and result in enhanced cascade kinetics. Here, we investigate the effects of substrate–scaffold interactions on the catalytic activity of an enzyme–DNA complex using horseradish peroxidase (HRP) and a nanoscale DNA scaffold with three addressable sites. Kinetic assays with a library of HRP substrates revealed that DNA scaffolding enhances HRP activity in a manner that is analogous to the Sabatier Principle. In this case, the binding of the substrate is to the scaffold and not to the catalyst, but the Sabatier trend holds: weak and strong binding substrates showed no enhancement in kinetics, whereas intermediately bound substrates result in >300% increase in enzyme activity.

BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism. Copyright © 2012 John Wiley & Sons, Ltd.

The C12O gene (catA gene) encodes for catechol 1,2-dioxygenase, which is a key enzyme involved in the first step catalysis of the aromatic ring in the ortho-cleavage pathway. This functional gene can be used as a marker to assess the catabolic potential of bacteria in bioremediation. C12OF and C12OR primers were designed based on the conserved regions of the CatA amino acid sequence of Actinobacteria for amplifying the catA gene from the genus Gordonia (16 Gordonia representing 11 species). The amplified catA genes (382 bp) were sequenced and analyzed. In the phylogenetic tree based on the translated catA amino acid sequences, all the Gordonia segregated clearly from other closely related genera. The sequence similarity of the catA gene in Gordonia ranged from 72.4% to 99.5%, indicating that the catA gene might have evolved faster than rrn operons or the gyrB gene at the inter-species level. A single nucleotide deletion of the catA gene was observed in Gordonia amicalis CC-MJ-2a, Gordonia rhizosphera and Gordonia sputi at nucleotide position 349. This deletion led to an encoding frame shift downstream of 11 amino acid residues, from WPSVAARAPAP to GHPWRPAHLHL, which was similar to most of the non-Gordonia Actinobacteria. Such variations might influence the catabolic activities or substrate utilization patterns of catechol 1,2-dioxygenase among Gordonia.

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.

BUD23 in Saccharomyces cerevisiae encodes for a class I methyltransferase, and deletion of the gene results in slow growth and random budding phenotypes. Herein, two BUD23 mutants defective in methyltransferase activity were generated to investigate whether the phenotypes of the null mutant might be correlated with a loss in enzymatic activity. Expression at the physiological level of both D77A and G57R mutants was able to rescue the phenotypes of the bud23-null mutant. The result implied that the methyltransferase activity of the protein was not necessary for supporting normal growth and bud site selection of the cells. High-level expression of Bud23 (G57R), but not Bud23 or Bud23 (D77A), in BUD23 deletion cells failed to complement these phenotypes. However, just like Bud23, Bud23 (G57R) was localized in a DAPI-poor region in the nucleus. Distinct behaviour in Bud23 (G57R) could not be originated from a mislocalization of the protein. Over-expression of Bud23 (G57R) in null cells also produced changes in actin organization and additional septin mutant-like phenotypes. Therefore, the absence of Bud23, Bud23 (G57R) at a high level might affect the cell division of yeast cells through an as yet unidentified mechanism.

Members of the Hsp70 protein family must work with other co-chaperones to exert their function. Herein, we identified a new Hsp70 co-chaperone, p49/STRAP, previously shown to interact with serum response factor. We demonstrated that a fraction of p49/STRAP was cytosolic, and that it interacted with the beta-sandwich domain of Hsp70. Although p49/STRAP had little effect on the intrinsic ATPase activity of Hsp70, it reduced the ATP-hydrolytic activity of Hsp70 stimulated by Hsp40, and inhibited the refolding activity of the Hsp70/Hsp40 system. Thus, p49/STRAP can be considered a bona fide co-chaperone of Hsp70.

Members of the metabolically diverse genus Gordonia were isolated from various biotopes including pristine and polluted sites around Taiwan. Identification, comparison and diversity assessment based on the gyrB gene were carried out using a newly developed primer pair for gyrB .T he 16S rRNA gene was also sequenced for comparison. A 1.2-kb fragment of the gyrB gene of 17 Gordonia strains including type strains was determined by direct sequencing of PCR amplified fragments. A total of 25 strains (8 of which were retrieved from a public database) of the genus Gordonia form a distinct phyletic line in the GyrB-based tree and are separated from other closely related species of genera of the suborder Corynebacterineae. Sequence similarity of the gyrB sequence from twelve Gordonia type strains ranged from 79.3 to 97.2%, corresponding to between 270 and 41 nucleotide differences, while there was only a 0.3–3.8% difference in 16S rRNA gene sequence similarity at the interspecies level. Phylogenetic analysis based on the GyrB sequence deduced from the gyrB gene is consistent with that of DNA–DNA hybridization results and provides a better discrimination within the species of Gordonia compared to the 16S rRNA gene. The present study demonstrates that gyrB gene analysis will aid in describing novel species belonging to the genus Gordonia.  2005 Elsevier SAS. All rights reserved.

In nature we find many examples of enzymes and multienzyme stuctures where catalysis is enhanced by well-designed molecular interactions between the enzymes and their substrates. Two compelling examples are the enzyme superoxide dismutase (SOD) and the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR). SOD, one of the fastest known enzymes (kcat » 1.5 ´ 109 M-1s-1), uses charge complementarity to produce substrate-enzyme interactions that enhance enzyme kinetics by directing the substrate to the enzyme’s active site. A positively charge patch on the surface TS-DHFR restricts diffusion of a negatively charged reaction intermediate to a pre-defined channel between two active sites, sequestering the intermediate along the enzyme’s surface and preventing diffusion to the bulk. This bounded diffusion promotes substrate channeling, enhancing pathway catalysis by protecting the intermediate from undesired side reactions. These examples are informative: They suggest that rational design of substrate-enzyme interactions can be used to enhance enzyme catalysis. In this work we engineer new enzyme structures with quantifiable binding interactions between the enzyme and its substrate. We hypothesize that the engineered molecular interactions will lead to increases in local substrate concentrations thereby enhancing enzyme catalysis. We confirm this hypothesis by demonstrating control over the apparent Michaelis constant (KM) of horseradish peroxidase (HRP) modified with a double stranded DNA structure that exhibits sequence dependent binding of phenolic HRP substrates. We extend this work to a second experimental system and demonstrate enhanced catalysis with an alcohol dehydrogenase (AdhD) through rationally designed molecular interactions between the NAD+ co-factor mimic nicotinamide mononucleotide (NMN+) and a double stranded DNA structure conjugated near the enzyme’s active site. Demonstration of rationally designed kinetic enhancements with enzyme co-factors such as NMN+ is an important demonstration with respect to bioelectrocatalysis as many such systems rely on this type of co-factor. We aim to extend this work towards other co-factor and substrates relevant to electrochemical biosensor and enzymatic biofuel cells.

In nature we find many examples of bifunctional enzymes that effectively process cascade reactions. For example, the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) couples two active sites on the same polypeptide structure via an electrostatic patch. Interactions between the negatively charged cascade intermediate, dihydrofolate, and the positively charged enzyme surface create an environment for bounded diffusion between active sites, thus promoting substrate channeling. This is a useful example from which we can draw design inspiration for synthetic enzyme cascades for multi-step reactions in biofuel cells. To this end, our research group is designing new nanostructured multi-enzyme complexes to study the effects of cascade structure on reaction kinetics. Our overall goal is to turn our understanding of these relationships into a generalized set of design rules that can be used to engineer optimized cascade catalysis. The first step is to investigate interactions between multi-enzyme scaffolds and cascade substrates. The TS-DHFR example suggests that substrate-scaffold interactions are important and can be beneficial to cascade catalysis. Here, we demonstrate that DNA scaffolds can enhance the kinetics of assembled enzymes and that these enhancements are related to the binding energy of the substrate and DNA scaffold. A model system of horseradish peroxidase (HRP) with phenolic substrates and a triangular DNA scaffold (sides ~ 25 nm in length) showed increased enzyme activity with one, two, and three HRPs assembled on the scaffold over freely diffusing HRP modified with short single stranded DNA. Interestingly, the enhancements in activity mimicked the Sabatier principle and a plot of the kinetic enhancement as a function of substrate-DNA binding energy followed the trends of a volcano plot commonly described in heterogeneous catalysis. No enhancement in enzyme activity was observed when the binding of the substrate to the scaffold was weak (kd = 10 mM). No enhancement occurred when binding was strong (kd = 4 mM). With intermediate binding (kd = 100 – 1000 mM) the enhancement in enzyme activity of HRP assembled on a DNA scaffold over freely diffusing enzymes was significant. We hypothesize that the enhancement was due to an increase in local concentration of the substrate resulting from substrate-DNA interactions. We confirm this hypothesis by demonstrating control over the apparent Michaelis constant of HRP-DNA nanostructures by tuning the interactions between substrates and DNA scaffold. These findings represent an important first step in designing multi-enzyme complexes and demonstrate that interactions between substrates and the scaffolds must be considered when engineering such structures. Our current work is focused on extending these findings to controlling the local concentration of enzyme co-factors used in biofuel cell anodes for multi-step oxidation cascades.