Post-translation digital data encoding into the genomes of mammalian cell populations.

High resolution cellular signal encoding is critical for better understanding of complex biological phenomena. DNA-based biosignal encoders alter genomic or plasmid DNA in a signal dependent manner. Current approaches involve the signal of interest affecting a DNA edit by interacting with a signal specific promoter which then results in expression of the effector molecule (DNA altering enzyme). Here, we present the proof of concept of a biosignal encoding system where the enzyme terminal deoxynucleotidyl transferase (TdT) acts as the effector molecule upon directly interacting with the signal of interest. A template independent DNA polymerase (DNAp), TdT incorporates nucleotides at the 3' OH ends of DNA substrate in a signal dependent manner. By employing CRISPR-Cas9 to create double stranded breaks in genomic DNA, we make 3'OH ends available to act as substrate for TdT. We show that this system can successfully resolve and encode different concentrations of various biosignals into the genomic DNA of HEK-293T cells. Finally, we develop a simple encoding scheme associated with the tested biosignals and encode the message "HELLO WORLD" into the genomic DNA of HEK-293T cells at a population level with 91% accuracy. This work demonstrates a simple and engineerable system that can reliably store local biosignal information into the genomes of mammalian cell populations.

Engineering Ca2+-Dependent DNA Polymerase Activity.

Advancements in synthetic biology have provided new opportunities in biosensing, with applications ranging from genetic programming to diagnostics. Next generation biosensors aim to expand the number of accessible environments for measurements, increase the number of measurable phenomena, and improve the quality of the measurement. To this end, an emerging area in the field has been the integration of DNA as an information storage medium within biosensor outputs, leveraging nucleic acids to record the biosensor state over time. However, slow signal transduction steps, due to the time scales of transcription and translation, bottleneck many sensing-DNA recording approaches. DNA polymerases (DNAPs) have been proposed as a solution to the signal transduction problem by operating as both the sensor and responder, but there is presently a lack of DNAPs with functional sensitivity to many desirable target ligands. Here, we engineer components of the Pol δ replicative polymerase complex of Saccharomyces cerevisiae to sense and respond to Ca2+, a metal cofactor relevant to numerous biological phenomena. Through domain insertion and binding site grafting to Pol δ subunits, we demonstrate functional allosteric sensitivity to Ca2+. Together, this work provides an important foundation for future efforts in the development of DNAP-based biosensors.

Recording Temporal Signals with Minutes Resolution Using Enzymatic DNA Synthesis.

Employing DNA as a high-density data storage medium has paved the way for next-generation digital storage and biosensing technologies. However, the multipart architecture of current DNA-based recording techniques renders them inherently slow and incapable of recording fluctuating signals with subhour frequencies. To address this limitation, we developed a simplified system employing a single enzyme, terminal deoxynucleotidyl transferase (TdT), to transduce environmental signals into DNA. TdT adds nucleotides to the 3'-ends of single-stranded DNA (ssDNA) in a template-independent manner, selecting bases according to inherent preferences and environmental conditions. By characterizing TdT nucleotide selectivity under different conditions, we show that TdT can encode various physiologically relevant signals such as Co2+, Ca2+, and Zn2+ concentrations and temperature changes in vitro. Further, by considering the average rate of nucleotide incorporation, we show that the resulting ssDNA functions as a molecular ticker tape. With this method we accurately encode a temporal record of fluctuations in Co2+ concentration to within 1 min over a 60 min period. Finally, we engineer TdT to allosterically turn off in the presence of a physiologically relevant concentration of calcium. We use this engineered TdT in concert with a reference TdT to develop a two-polymerase system capable of recording a single-step change in the Ca2+ signal to within 1 min over a 60 min period. This work expands the repertoire of DNA-based recording techniques by developing a novel DNA synthesis-based system that can record temporal environmental signals into DNA with a resolution of minutes.

Characterizing and predicting carboxylic acid reductase activity for diversifying bioaldehyde production.

Chemicals with aldehyde moieties are useful in the synthesis of polymerization reagents, pharmaceuticals, pesticides, flavors, and fragrances because of their high reactivity. However, chemical synthesis of aldehydes from carboxylic acids has unfavorable thermodynamics and limited specificity. Enzymatically catalyzed reductive bioaldehyde synthesis is an attractive route that overcomes unfavorable thermodynamics by ATP hydrolysis in ambient, aqueous conditions. Carboxylic acid reductases (Cars) are particularly attractive, as only one enzyme is required. We sought to increase the knowledge base of permitted substrates for four Cars. Additionally, the Lys2 enzyme family was found to be mechanistically the same as Cars and two isozymes were also tested. Our results show that Cars prefer molecules where the carboxylic acid is the only polar/charged group. Using this data and other published data, we develop a support vector classifier (SVC) for predicting Car reactivity and make predictions on all carboxylic acid metabolites in iAF1260 and Model SEED.

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin.

Anthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (+)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

Expanding the chemical space of polyketides through structure-guided mutagenesis of Vitis vinifera stilbene synthase.

Several natural polyketides (PKs) have been associated with important pharmaceutical properties. Type III polyketide synthases (PKS) that generate aromatic PK polyketides have been studied extensively for their substrate promiscuity and product diversity. Stilbene synthase-like (STS) enzymes are unique in the type III PKS class as they possess a hydrogen bonding network, furnishing them with thioesterase-like properties, resulting in aldol condensation of the polyketide intermediates formed. Chalcone synthases (CHS) in contrast, lack this hydrogen-bonding network, resulting primarily in the Claisen condensation of the polyketide intermediates formed. We have attempted to expand the chemical space of this interesting class of compounds generated by creating structure-guided mutants of Vitis vinifera STS. Further, we have utilized a previously established workflow to quickly compare the wild-type reaction products to those generated by the mutants and identify novel PKs formed by using XCMS analysis of LC-MS and LC-MS/MS data. Based on this approach, we were able to generate 15 previously unreported PK molecules by exploring the substrate promiscuity of the wild-type enzyme and all mutants using unnatural substrates. These structures were specific to STSs and cannot be formed by their closely related CHS-like counterparts.

Antimicrobial mechanism of resveratrol-trans-dihydrodimer produced from peroxidase-catalyzed oxidation of resveratrol.

Plant polyphenols are known to have varying antimicrobial potencies, including direct antibacterial activity, synergism with antibiotics and suppression of bacterial virulence. We performed the in vitro oligomerization of resveratrol catalyzed by soybean peroxidase, and the two isomers (resveratrol-trans-dihydrodimer and pallidol) produced were tested for antimicrobial activity. The resveratrol-trans-dihydrodimer displayed antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus (minimum inhibitory concentration (MIC) = 15.0, 125, and 62.0 µM, respectively) and against Gram-negative Escherichia coli (MIC = 123 µM, upon addition of the efflux pump inhibitor Phe-Arg-ß-naphthylamide). In contrast, pallidol had no observable antimicrobial activity against all tested strains. Transcriptomic analysis implied downregulation of ABC transporters, genes involved in cell division and DNA binding proteins. Flow cytometric analysis of treated cells revealed a rapid collapse in membrane potential and a substantial decrease in total DNA content. The active dimer showed >90% inhibition of DNA gyrase activity, in vitro, by blocking the ATP binding site of the enzyme. We thus propose that the resveratrol-trans-dihydrodimer acts to: (1) disrupt membrane potential; and (2) inhibit DNA synthesis. In summary, we introduce the mechanisms of action and the initial evaluation of an active bactericide, and a platform for the development of polyphenolic antimicrobials.

Enzymatic formation of a resorcylic acid by creating a structure-guided single-point mutation in stilbene synthase.

A novel C17 resorcylic acid was synthesized by a structure-guided Vitis vinifera stilbene synthase (STS) mutant, in which threonine 197 was replaced with glycine (T197G). Altering the architecture of the coumaroyl binding and cyclization pocket of the enzyme led to the attachment of an extra acetyl unit, derived from malonyl-CoA, to p-coumaroyl-CoA. The resulting novel pentaketide can be produced strictly by STS-like enzymes and not by Chalcone synthase-like type III polyketide synthases; due to the unique thioesterase like activity of STS-like enzymes. We utilized a liquid chromatography mass spectrometry-based data analysis approach to directly compare the reaction products of the mutant and wild type STS. The findings suggest an easy to employ platform for precursor-directed biosynthesis and identification of unnatural polyketides by structure-guided mutation of STS-like enzymes.

Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering.

Reconstruction of highly efficient biosynthesis pathways is essential for the production of valuable plant secondary metabolites in recombinant microorganisms. In order to improve the titer of green tea catechins in Escherichia coli, combinatorial strategies were employed using the ePathBrick vectors to express the committed catechin pathway: flavanone 3ß-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR). Three F3H, three DFR, and two LAR genes originating from different plant species were selected and synthesized, to create 18 pathway variants to be screened in E. coli. Constructs containing F3H(syn) originally from Camellia sinensis, DFR(syn) from Anthurium andraeanum, C. sinensis, or Fragaria ananass, and LAR(syn) from Desmodium uncinatum (p148, p158 and p168) demonstrated high conversion efficiency with either eriodictyol or naringenin as substrate. A highly efficient construct was created by assembling additional copies of DFR(syn) and LAR(syn) enabling a titer of 374.6 ± 43.6 mg/L of (+)-catechin. Improving the NADPH availability via the ΔpgiΔppc mutation, BLΔpgiΔppc-p148 produced the highest titer of catechin at 760.9 ± 84.3 mg/L. After utilizing a library of scaffolding proteins, the strain BLΔpgiΔppc-p168-759 reached the highest titer of (+)-catechin of 910.9 ± 61.3 mg/L from 1.0 g/L of eriodictyol in batch culture with M9 minimal media. The impact of oxygen availability on the biosynthesis of catechin was also investigated.

Design and kinetic analysis of a hybrid promoter-regulator system for malonyl-CoA sensing in Escherichia coli.

Malonyl-CoA is the rate-limiting precursor involved in the chain elongation reaction of a range of value-added pharmaceuticals and biofuels. Development of malonyl-CoA responsive sensors holds great promise in overcoming critical pathway limitations and optimizing production titers and yields. By incorporating the Bacillus subtilis trans-regulatory protein FapR and the cis-regulatory element fapO, we constructed a hybrid promoter-regulatory system that responds to a broad range of intracellular malonyl-CoA concentrations (from 0.1 to 1.1 nmol/mgDW) in Escherichia coli. Elimination of regulatory protein and nonspecific DNA cross-communication leads to a sensor construct that exhibits malonyl-CoA-dependent linear phase kinetics with increased dynamic response range. The sensors reported in this study could potentially control and optimize carbon flux leading to robust biosynthetic pathways for the production of malonyl-CoA-derived compounds.

Pathway and protein engineering approaches to produce novel and commodity small molecules.

Nature has provided us the basis of designing the most integrated and efficacious production platforms. Cell factories via their millions of years of evolution have nearly perfected each of their production systems. We have been trying to imitate, utilize and tweak this system to our advantage by using slightly overlapping and greatly interdependent approaches such as metabolic engineering and systems biology to make nature work for us in an efficient and robust way, without producing toxic waste and/or unnecessary side products. Systems biology, metabolic engineering and 'omics' technologies have paved the way for protein and pathway engineering. To this end we will talk about the recent advances in production of novel pharmaceutical and commodity small molecules by designing novel proteins and pathways.

Engineering plant metabolism into microbes: from systems biology to synthetic biology.

Plant metabolism represents an enormous repository of compounds that are of pharmaceutical and biotechnological importance. Engineering plant metabolism into microbes will provide sustainable solutions to produce pharmaceutical and fuel molecules that could one day replace substantial portions of the current fossil-fuel based economy. Metabolic engineering entails targeted manipulation of biosynthetic pathways to maximize yields of desired products. Recent advances in Systems Biology and the emergence of Synthetic Biology have accelerated our ability to design, construct and optimize cell factories for metabolic engineering applications. Progress in predicting and modeling genome-scale metabolic networks, versatile gene assembly platforms and delicate synthetic pathway optimization strategies has provided us exciting opportunities to exploit the full potential of cell metabolism. In this review, we will discuss how systems and synthetic biology tools can be integrated to create tailor-made cell factories for efficient production of natural products and fuel molecules in microorganisms.

ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli.

Harnessing cell factories for producing biofuel and pharmaceutical molecules has stimulated efforts to develop novel synthetic biology tools customized for modular pathway engineering and optimization. Here we report the development of a set of vectors compatible with BioBrick standards and its application in metabolic engineering. The engineered ePathBrick vectors comprise four compatible restriction enzyme sites allocated on strategic positions so that different regulatory control signals can be reused and manipulation of expression cassette can be streamlined. Specifically, these vectors allow for fine-tuning gene expression by integrating multiple transcriptional activation or repression signals into the operator region. At the same time, ePathBrick vectors support the modular assembly of pathway components and combinatorial generation of pathway diversities with three distinct configurations. We also demonstrated the functionality of a seven-gene pathway (~9 Kb) assembled on one single ePathBrick vector. The ePathBrick vectors presented here provide a versatile platform for rapid design and optimization of metabolic pathways in E. coli.