Determining the accuracy and suitability of common analytical techniques for sophorolipid biosurfactants.

To determine the performance of a sophorolipid biosurfactant production process, it is important to have accurate and specific analytical techniques in place. Among the most popular are the anthrone assay, gravimetric quantification (hexane:ethyl acetate extraction), and high-performance liquid chromatography (HPLC). The choice of analytical tool varies depending on cost, availability, and ease of use; however, these techniques have never been compared directly against one another. In this work, 75 fermentation broths with varying product/substrate concentrations were comprehensively tested with the 3 techniques and compared. HPLC-ultraviolet detection (198 nm) was capable of quantifying C18:1 subterminal hydroxyl diacetylated lactonic sophorolipid down to a lower limit of 0.3 g/L with low variability (<3.21%). Gravimetric quantification of the broths following liquid:liquid extraction with hexane and ethyl acetate showed some linearity (R2 = .658) when compared to HPLC but could not quantify lower than 11.06 g/L, even when no sophorolipids were detected in the sample, highlighting the non-specificity of the method to co-extract non-sophorolipid components in the final gravimetric measure. The anthrone assay showed no linearity (R2 = .129) and was found to cross-react with media components (rapeseed oil, corn steep liquor, glucose), leading to consistent overestimation of sophorolipid concentration. The appearance of poor biomass separation during sample preparation with centrifugation was noted and resolved with a novel sample preparation method with pure ethanol. Extensive analysis and comparisons of the most common sophorolipid quantification techniques are explored and the limitations/advantages are highlighted. The findings provide a guide for scientists to make an informed decision on the suitable quantification tool that meets their needs, exploring all aspects of the analysis process from harvest, sample preparation, and analysis.

Mechanistic Study of Glucose Photoreforming over TiO2-Based Catalysts for H2 Production.

Glucose is a key intermediate in cellulose photoreforming for H2 production. This work presents a mechanistic investigation of glucose photoreforming over TiO2 and Pt/m-TiO2 catalysts. Analysis of the intermediates formed in the process confirmed the α-scission mechanism of glucose oxidation forming arabinose (Cn-1 sugar) and formic acid in the initial oxidation step. The selectivity to sugar products and formic acid differed over Pt/TiO2 and TiO2, with Pt/TiO2 showing the lower selectivity to formic acid due to enhanced adsorption/conversion of formic acid over Pt/TiO2. In situ ATR-IR spectroscopy of glucose photoreforming showed the presence of molecular formic acid and formate on the surface of both catalysts at low glucose conversions, suggesting that formic acid oxidation could dominate surface reactions in glucose photoreforming. Further in situ ATR-IR of formic acid photoreforming showed Pt-TiO2 interfacial sites to be key for formic acid oxidation as TiO2 was unable to convert adsorbed formic acid/formate. Isotopic studies of the photoreforming of formic acid in D2O (with different concentrations) showed that the source of the protons (to form H2 at Pt sites) was determined by the relative surface coverage of adsorbed water and formic acid.

The effect of different storage media on the monomer elution and hardness of CAD/CAM composite blocks.

OBJECTIVE: This study aimed to assess the effect of different storage media on the hardness and monomer elution of CAD/CAM composite blocks. METHODS: Five resin-composite blocks (RCB), one polymer-infiltrated ceramic network (PICN) block (Enamic (EN)), one ceramic-filled poly ether ether ketone (PEEK) block (Dentokeep (DK)), and one feldspathic ceramic block. Microhardness was measured using a Vickers indenter tester (FM-700, Future Tech Corp., Japan). In addition 4 conventional resin-composites were investigated for monomer elution using high performance liquid chromatography (HPLC) after storage in different media for 3 months. The data were analysed by three-way ANOVA, two-way ANOVA, one-way ANOVA, Tukey's post hoc test and the independent t-test (α=0.05 for all tests). RESULTS: The specimens stored in the water had a hardness reduction ranging from 0.9% to 24.4%. In artificial saliva, the specimens had a hardness reduction ranging from 2.8% to 23.2%. The hardness reduction percentage in 75% Ethanol/Water (E/W) ranged between 3.8% and 35.3%. All materials, except GR (resin-composite block) and DK (Polyetheretherketone (PEEK)), showed a variable extent of monomer elution into 75% E/W with significantly higher amounts eluted from conventional composites. GRA and GND (conventional resin-composites) eluted TEGDMA in artificial saliva and GRA eluted TEGDMA in water. SIGNIFICANCE: The hardness of CAD/CAM composite blocks was affected by different storage media, and they were not as stable as ceramic, with PICN exhibited superior hardness stability to all of the resin-composite blocks in all the storage media and was comparable to ceramic block. The hardness reduction percentage of the CAD/CAM composite blocks was influenced by the filler loading and resin-matrix composition.Minimal or no monomer elution from CAD/CAM blocks was detected.

Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways.

S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy-SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site-directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co-factor, carboxy-S-adenosyl-l-ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1-carboxyethyl group to the substrate.

SelProm: A Queryable and Predictive Expression Vector Selection Tool for Escherichia coli.

The rapid prototyping and optimization of plasmid-based recombinant gene expression is one of the key steps in the development of bioengineered bacterial systems. Often, multiple genes or gene modules need to be coexpressed, and for this purpose compatible, inducible plasmid systems have been developed. However, inducible expression systems are not favored in industrial processes, due to their prohibitive cost, and consequently the conversion to constitutive expression systems is often desired. Here we present a set of constitutive-expression plasmids for this purpose, which were benchmarked using fluorescent reporter genes. To further facilitate the conversion between inducible and constitutive expression systems, we developed SelProm, a design tool that serves as a parts repository of plasmid expression strength and predicts portability rules between constitutive and inducible plasmids through model comparison and machine learning. The SelProm tool is freely available at http://selprom.synbiochem.co.uk .

Machine Learning of Designed Translational Control Allows Predictive Pathway Optimization in Escherichia coli.

The field of synthetic biology aims to make the design of biological systems predictable, shrinking the huge design space to practical numbers for testing. When designing microbial cell factories, most optimization efforts have focused on enzyme and strain selection/engineering, pathway regulation, and process development. In silico tools for the predictive design of bacterial ribosome binding sites (RBSs) and RBS libraries now allow translational tuning of biochemical pathways; however, methods for predicting optimal RBS combinations in multigene pathways are desirable. Here we present the implementation of machine learning algorithms to model the RBS sequence-phenotype relationship from representative subsets of large combinatorial RBS libraries allowing the accurate prediction of optimal high-producers. Applied to a recombinant monoterpenoid production pathway in Escherichia coli, our approach was able to boost production titers by over 60% when screening under 3% of a library. To facilitate library screening, a multiwell plate fermentation procedure was developed, allowing increased screening throughput with sufficient resolution to discriminate between high and low producers. High producers from one library did not translate during scale-up, but the reduced screening requirements allowed rapid rescreening at the larger scale. This methodology is potentially compatible with any biochemical pathway and provides a powerful tool toward predictive design of bacterial production chassis.

An automated Design-Build-Test-Learn pipeline for enhanced microbial production of fine chemicals.

The microbial production of fine chemicals provides a promising biosustainable manufacturing solution that has led to the successful production of a growing catalog of natural products and high-value chemicals. However, development at industrial levels has been hindered by the large resource investments required. Here we present an integrated Design-Build-Test-Learn (DBTL) pipeline for the discovery and optimization of biosynthetic pathways, which is designed to be compound agnostic and automated throughout. We initially applied the pipeline for the production of the flavonoid (2S)-pinocembrin in Escherichia coli, to demonstrate rapid iterative DBTL cycling with automation at every stage. In this case, application of two DBTL cycles successfully established a production pathway improved by 500-fold, with competitive titers up to 88 mg L-1. The further application of the pipeline to optimize an alkaloids pathway demonstrates how it could facilitate the rapid optimization of microbial strains for production of any chemical compound of interest.

Analysis of long-term monomer elution from bulk-fill and conventional resin-composites using high performance liquid chromatography.

OBJECTIVE: The aim of this study was to assess monomer elution from bulk-fill and conventional resin-composites stored in different media using high performance liquid chromatography (HPLC) for up to 3 months. METHODS: Six bulk-fill (SureFil SDR, Venus Bulk Fill, X-tra base, Filtek Bulk Fill flowable, Sonic Fill, and Tetric EvoCeram Bulk Fill) and eight conventional resin-composites (Grandioso Flow, Venus Diamond Flow, X-Flow, Filtek Supreme XTE, Grandioso, Venus Diamond, TPH Spectrum, and Filtek Z250) were tested. Cylindrical samples (n=5) were immersed in water, 70% ethanol/water solution (70% E/W), and artificial saliva and stored at 37°C for 24h, 1 month, and 3 months. The storage solutions were analysed with HPLC. Data were analysed with repeated measures ANOVA, one-way ANOVA, and Tukey post hoc test at α=0.05. RESULTS: Monomers detected in water and artificial saliva were TEGDMA, DEGDMA, UDMA, and TCD-DI-HEA. No eluted monomers were detected from X-tra base and Sonic fill in these media. All monomers showed a variable extent of elution into 70% E/W with significantly higher amounts than those detected in water and artificial saliva. Significantly higher elution was detected from UDMA-BisEMA based composites compared to BisGMA and BisGMA-BisEMA based systems in 70% E/W. The rate of elution into different media varied between different monomers and was highly dependent on the molecular weight of the eluted compounds. SIGNIFICANCE: Elution from bulk-fill resin-composites is comparable to that of conventional materials despite their increased increment thickness. Monomer elution is highly dependent on the hydrophobicity of the base monomers and the final network characteristics of the resin-matrix.

Qualitative and quantitative characterization of monomers of uncured bulk-fill and conventional resin-composites using liquid chromatography/mass spectrometry.

OBJECTIVES: The aim of this study was to assess the resin matrix monomer composition of selected bulk-fill and conventional resin-composite materials using reverse phase liquid chromatography coupled with electron spray ionization mass spectrometry. MATERIAL AND METHODS: Six bulk-fill (SureFil SDR, Venus Bulk Fill, X-tra base, Filtek Bulk Fill flowable, Sonic Fill, and Tetric EvoCeram Bulk Fill) and eight conventional resin-composites (Grandioso Flow, Venus Diamond Flow, X-Flow, Filtek Supreme XTE, Grandioso, Venus Diamond, TPH Spectrum, and Filtek Z250) were tested. For assessment of resin composition and relative monomer amounts, uncured resin-composites were analysed with reverse phase liquid chromatography/electron spray ionization mass spectrometry. logP values (a measure of hydrophobicity) of detected compounds were calculated and their correlation to reverse phase liquid chromatography retention time was explored. Data were analysed with one-way ANOVA, Tukey post hoc test, Pearson correlation and regression analyses at α=0.05. RESULTS: The main monomers detected were BisGMA, UDMA, TEGDMA, and BisEMA. Monomers were detected at variable combinations in different materials with significantly different relative amounts. Other monomers were detected including HDDMA, DEGDMA, TCD-DI-HEA, and SDR-UDMA in Grandioso flow, X-flow, Venus Diamond, and SureFil SDR respectively. A positive correlation between logP and reverse phase liquid chromatography retention time was detected (r(2)=0.62, p=0.004). CONCLUSIONS: Resin composition of bulk-fill resin-composites is comparable to that of conventional materials with the exception of SureFil SDR. The relative hydrophobicity of dental monomers can be determined by their reverse phase liquid chromatography retention time.