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Abstract As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

Abstract Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the “moving-histidine” mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion.

Background: The purpose of this study is to evaluate patient reported outcomes after arthroscopic extensive débridement of the shoulder with subacromial decompression (SAD) for subacromial impingement using the Patient-Reported Outcomes Measurement Information System (PROMIS) system and evaluate if depression (Dep) (clinical or situational) impacts patients achieving a Minimal Clinically Important Difference (MCID). Methods: Preoperative PROMIS Physical function (PF), Mood, and Dep scores were obtained at the closest date prior to arthroscopic rotator cuff repair and postoperative scores were collected at every clinical visit thereafter. Final PROMIS score used for data analysis was determined by the patients final PROMIS value between 90 to 180 days. Clinical Dep was determined by patients having a formal diagnosis of “Depression or Major Depressive Disorder” at the time of their surgery. Situationally depressed patients, those without a formal diagnosis yet exhibited symptomatic depressive symptoms, were classified by having a PROMIS-Dep cutoff scores larger than 52.5. Results: A total of 136 patients were included for final statistical analysis. 13 patients had a clinical but not situational diagnosis of Dep, 86 patients were identified who had no instance of clinical or situational Dep (nondepressed). 35 patients were situationally depressed. All three cohorts demonstrated a significant improvement in postoperative PROMIS Dep, PI, and PF score relative to their preoperative value (P = .001). Situationally depressed patients achieved greater delta PROMIS-Dep compared to patients with major depressive disorder. Depressed patients had a higher chance of achieving MCID for PROMIS-Dep compared to nondepressed patients (P = .01). Logistic regression analysis demonstrated that underlying Dep did not alter the odds of obtaining MCID compared to nondepressed patients. Nonsmoking patients had significantly greater odds of achieving MCID for PF (P = .02). Discussion: Patients improved after undergoing SAD regardless of underlying Dep or depressive symptoms. Depressed patients exhibited greater change in PROMIS scores compared to nondepressed patients. Smoking remains a risk factor for postoperative outcomes in patients undergoing SAD for subacromial impingement. Identifying and counseling patients with underlying depressive symptoms without a formal major depressive disorder diagnosis may lead to improved outcomes. These findings may help guide clinicians in deciding who would benefit the most from this procedure.

Abstract Successes in biocatalytic polyester recycling have raised the possibility of deconstructing alternative polymers enzymatically, with polyamide (PA) being a logical target due to the array of amide-cleaving enzymes present in nature. Here, we screen 40 potential natural and engineered nylon-hydrolyzing enzymes (nylonases), using mass spectrometry to quantify eight compounds resulting from enzymatic nylon-6 (PA6) hydrolysis. Comparative time-course reactions incubated at 40-70 °C showcase enzyme-dependent variations in product distributions and extent of PA6 film depolymerization, with significant nylon deconstruction activity appearing rare. The most active nylonase, a NylCK variant we rationally thermostabilized (an N-terminal nucleophile (Ntn) hydrolase, NylCK-TS, T m = 87.4 °C, 16.4 °C higher than the wild-type), hydrolyzes 0.67 wt% of a PA6 film. Reactions fail to restart after fresh enzyme addition, indicating that substrate-based limitations, such as restricted enzyme access to hydrolysable bonds, prohibit more extensive deconstruction. Overall, this study expands our understanding of nylonase activity distribution, indicates that Ntn hydrolases may have the greatest potential for further development, and identifies key targets for progressing PA6 enzymatic depolymerization, including improving enzyme activity, product selectivity, and enhancing polymer accessibility.

Abstract Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl–ether bond cleavage. A large fraction of aromatic monomers in lignin, however, are linked by various carbon–carbon (C–C) bonds that are more challenging to cleave and limit the yields of aromatic monomers from lignin depolymerization. Here, we report a catalytic autoxidation method to cleave C–C bonds in lignin-derived dimers and oligomers from pine and poplar. The method uses manganese and zirconium salts as catalysts in acetic acid and produces aromatic carboxylic acids as primary products. The mixtures of the oxygenated monomers are efficiently converted to cis,cis-muconic acid in an engineered strain of Pseudomonas putida KT2440 that conducts aromatic O-demethylation reactions at the 4-position. This work demonstrates that autoxidation of lignin with Mn and Zr offers a catalytic strategy to increase the yield of valuable aromatic monomers from lignin.

ABSTRACTThere is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (“functional modules”), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets.IMPORTANCEThis study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

Purpose: To identify differences in performance on the Fundamentals of Arthroscopic Surgery Training (FAST) workstation between residents across different postgraduate years and training sites. Methods: During the 2018-2019 academic year, 102 orthopaedic surgery residents from 4 training sites completed 6 FAST modules. Failure was defined as either completion time exceeding benchmark time or commission of task-specific errors. With the exception of knot tying, each module was completed by participants twice—once with each hand serving as the camera hand. Time to completion (except for knot tying) and errors were recorded for each of the modules. Completion times and failure rates were compared between postgraduate years, seniority groups, and training sites. Results: In all modules for which time was recorded, except for the suture-passage module, there was no significant difference in time to completion based on seniority (P < .01 for suture passage and P > .05 for all others). Significant differences in completion time were observed between sites for all modules except for the suture-passage module (P = .957 for suture passage and P < .05 for all others). Site predicted failure by at least 1 measure (time or technical error) for all modules (P < .05) except for number probing and suture passage. Failure rate across training years varied for each module. Conclusions: Time to completion and rate of failure did not predictably decrease with level of training. Training site proved to be a significant predictor of performance. Factors such as hand dominance and familiarity with the equipment proved to be important considerations for some modules. Clinical Relevance: Objective assessment of arthroscopic skills among orthopaedic trainees is difficult. Using reproducible methodology to assess trainees on specific skills at all postgraduate years and at multiple training sites may provide important information about orthopaedic training.

Summary: Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin.

Enzymes have potential for recycling plastics such as PET, a polyester used in textiles and single-use packaging. Here, the authors identify and characterize additional PET-active biocatalysts and expand the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Muconic acid is a platform chemical with wide industrial applicability. Here, the authors report efficient muconate production from glucose and xylose by engineered Pseudomonas putida strain using adaptive laboratory evolution, metabolic modeling, and rational strain engineering strategies.

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

Lignin conversion to higher value products is essential to the economic viability of lignocellulosic biorefineries. Here, the authors demonstrate the bioconversion of alkali pretreated lignin to itaconic acid by dynamic two stage fermentation using a signal-amplified nitrogen-limitation biosensor.

Introduction: Optic capture of sutured scleral fixated posterior chamber intraocular lenses (PC IOLs) is an occasional complication resulting in blurred vision and discomfort. Methods: A retrospective study of the management of 18 eyes (3.6%) with optic capture out of 495 eyes with scleral fixated IOLs during the study period. 54 procedures were performed in the management of optic capture of sutured scleral fixated PC IOLs. An in-office technique was utilized to relieve the optic capture by repositioning the optic posterior to the iris. This technique was performed after topical anesthesia and topical 5% betadine with the patient stably positioned at the slit lamp. Using a 30-gauge needle, sometimes after a 15-degree paracentesis blade, the needle was advanced in a parallel plane above the iris until the tip reached the edge of the captured optic. The optic is engaged in the inferior periphery away from the central visual axis, and pushed gently posteriorly just enough to reposition the optic posterior to the iris. In some cases, pilocarpine 2% drops were utilized after the procedure to decrease the risk of recapture of the optic. Results: All 54 procedures were successfully performed in the office without significant pain or discomfort. Vision before optic capture, during optic capture, and at the first office visit after optic capture were comparable. There were not any cases of endophthalmitis, hyphema, iris trauma, iris prolapse or keratitis. While eight patients only had one episode of optic capture, 10 patients had multiple episodes of optic capture, all managed with this in office procedure. Recurrent optic capture occurred more frequently in eyes with fixation at less than 2 mm from the limbus than eyes with scleral fixation at 2 mm from the limbus. Conclusion: Reposition of the optic after pupillary capture of a scleral fixated PC IOL can be successfully performed in the office without discomfort or significant complications and is an alternative management option to a return to the operating room. This procedure may be especially important when there is poor access to the operating room or restricted access to the operating room as during the COVID19 pandemic.

Summary Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic‐catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole‐cell biocatalyst for funnelling lignin‐derived compounds to value‐added products, including its native carbon storage product, medium‐chain‐length polyhydroxyalkanoates (mcl‐PHA). In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl‐PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl‐PHA titre (g l−1) and a 20% and 100% increase in yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.

Diluted bitumen (dilbit) is an unconventional crude petroleum increasingly being extracted and transported to market by pipeline and tanker. Despite the transport of dilbit through terrestrial, aquatic, and coastal habitat important to diverse bird fauna, toxicity data are currently only available for fish and invertebrates. We used the zebra finch (Taeniopygia guttata) as a tractable, avian model system to investigate exposure effects of lightly weathered Cold Lake blend dilbit on survival, tissue residue, and a range of physiological and behavioural endpoints. Birds were exposed via oral gavage over 14-days with dosages of 0, 2, 4, 6, 8, 10, or 12 mL dilbit/kg bw/day. We identified an LD50 of 9.4 mL/kg/d dilbit, with complete mortality at 12 mL/kg/d. Mortality was associated with mass loss, external oiling, decreased pectoral and heart mass, and increased liver mass. Hepatic ethoxyresorufin-O-deethylase activity (EROD) was elevated in all dilbit-dosed birds compared with controls but there was limited evidence of sublethal effects of dilbit on physiological endpoints at doses

Seabirds are widely regarded as an invaluable bioindicator of environmental health. Matrices including eggs and feathers have been used as non-lethal means to assess contaminant burdens. We have developed a new approach for extraction of polycyclic aromatic compounds (PACs) from seabird plasma and serum based on automated microbead-beating homogenization and extraction. Commercially available bovine serum and plasma were purposely fortified with a suite of PACs separately at three dosing levels, placed inside a custom-made stainless-steel tube containing ceramic microbeads, and subjected to an extraction process using a Precellys tissue homogenizer. Tubes were shaken forcefully in three-dimensions, facilitating high mass-transfer of PACs from the matrix into the hexane extraction solvent. The accuracy of the method ranged from 55 to 120% and limits of detection and quantitation ranged from 0.1 to 8 and 0.2 to 27 pg/μL, respectively. The method exhibited good repeatability with both inter- and intra-day repeatability < 30%. The developed method represents an effective and efficient approach to extraction of PACs from important biological matrices.

Hydrogel buckle intrusion due to progressive swelling is a known complication, which usually requires surgical intervention due to vitreous hemorrhage, retinal detachment, or progressive encroachment into the optic nerve or macula. Hydrogel buckle surgery with successful retinal detachment repair was performed in this one-eyed physician in 1990, and there was slow progressive intrusion towards the macula and optic nerve without surgical intervention for 30 years and with visual acuity maintained at 20/40.

The ratio of syringyl (S) and guaiacyl (G) units in lignin has been regarded as a major factor in determining the maximum monomer yield. Here, the authors challenge this common conception using reductive catalytic fractionation in flow-through reactors as an analytical tool to depolymerize lignin in poplar with naturally varying S/G ratios.

Tryptophan (TRP) is an essential dietary amino acid that, unless otherwise committed to protein synthesis, undergoes metabolism via the Tryptophan-Kynurenine (TRP-KYN) pathway in vertebrate organisms. TRP and its metabolites have key roles in diverse physiological processes including cell growth and maintenance, immunity, disease states and the coordination of adaptive responses to environmental and dietary cues. Changes in TRP metabolism can alter the availability of TRP for protein and serotonin biosynthesis as well as alter levels of the immune-active KYN pathway metabolites. There is now considerable evidence which has shown that the TRP-KYN pathway can be influenced by various stressors including glucocorticoids (marker of chronic stress), infection, inflammation and oxidative stress, and environmental toxicants. While there is little known regarding the role of TRP metabolism following exposure to environmental contaminants, there is evidence of linkages between chemically induced metabolic perturbations and altered TRP enzymes and KYN metabolites. Moreover, the TRP-KYN pathway is conserved across vertebrate species and can be influenced by exposure to xenobiotics, therefore, understanding how this pathway is regulated may have broader implications for environmental and wildlife toxicology. The goal of this narrative review is to (1) identify key pathways affecting Trp-Kyn metabolism in vertebrates and (2) highlight consequences of altered tryptophan metabolism in mammals, birds, amphibians, and fish. We discuss current literature available across species, highlight gaps in the current state of knowledge, and further postulate that the kynurenine to tryptophan ratio can be used as a novel biomarker for assessing organismal and, more broadly, ecosystem health.

Targeted proteomics is a mass spectrometry-based protein quantification technique with high sensitivity, accuracy, and reproducibility. As a key component in the multi-omics toolbox of systems biology, targeted liquid chromatography-selected reaction monitoring (LC-SRM) measurements are critical for enzyme and pathway identification and design in metabolic engineering. To fulfill the increasing need for analyzing large sample sets with faster turnaround time in systems biology, high-throughput LC-SRM is greatly needed. Even though nanoflow LC-SRM has better sensitivity, it lacks the speed offered by microflow LC-SRM. Recent advancements in mass spectrometry instrumentation significantly enhance the scan speed and sensitivity of LC-SRM, thereby creating opportunities for applying the high speed of microflow LC-SRM without losing peptide multiplexing power or sacrificing sensitivity. Here, we studied the performance of microflow LC-SRM relative to nanoflow LC-SRM by monitoring 339 peptides representing 132 enzymes in Pseudomonas putida KT2440 grown on various carbon sources. The results from the two LC-SRM platforms are highly correlated. In addition, the response curve study of 248 peptides demonstrates that microflow LC-SRM has comparable sensitivity for the majority of detected peptides and better mass spectrometry signal and chromatography stability than nanoflow LC-SRM.

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) individually and in combination (pCA ​+ ​FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 ​h decrease in lag phase in 20 ​g/L pCA and a 2.4-fold increase in growth rate in 30 ​g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of pCA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in pCA ​+ ​FA TALE-derived strains but did not affect growth in pCA ​+ ​FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

Organic amendments can be effective at controlling some soil-borne pathogens. This work entailed two parts; the first involved two field experiments that investigated the effects of four different composts derived from agricultural wastes on soil load of Verticillium dahliae in organic and conventional bell pepper (Capsicum annuum L.) systems. The composts were prepared using dairy and horse manure (DC), grape pomace (GC), olive pomace-dairy manure mix (ODC) or mixed crop residue (CRC). For each system, one 25 t ha−1 compost application was made in May 2018 before bell pepper planting. Soil samples were taken before application and four times after application, approximately every six weeks. Plant growth and yield were also monitored. The second part involved lab experiments that tested the effects of extracts of the above composts on the growth of V. dahliae on selective media in Petri dishes. All four compost treatments significantly suppressed soil pathogen populations in June, two weeks after the application of treatments, with plant-based amendments presenting greater effects than DC. These suppressive effects disappeared within eight weeks (for DC) or 14 weeks (for GC, ODC and CRC) after application. The effects of composts on soil chemical properties, V. dahliae abundance and bell pepper growth and yield parameters were generally similar in both organic and conventional fields. In the Petri dish assays, plant composts reduced V. dahliae growth by 40–60 %, especially during the first week after application.

Limited information exists regarding contemporary and historical emissions for many anthropogenic chemicals, especially for contaminants of emerging concern (CECs). This study examined temporal trends of several perfluoroalkyl substances (PFASs), polybrominated diphenyl ethers (PBDEs), and hexabromocyclododecanes (HBCDs) in three beluga whale (Delphinapterus leucas) populations from the Canadian Arctic [Hendrickson Island (HI), NT; Pangnirtung (PG), NU; and Sanikiluaq (SQ), NU] collected from 1982 to 2013. The confounding factors of animal size, age, and sex were included in the analyses, but were only significant for some CECs. The strongest temporal resolution was obtained from HI, which showed increasing trends in PBDEs (4.8%/year) and HBCDs (2.9%/year), and decreases in perfluoroalkylcarboxylic acids (PFCAs, −6.0%/year). Concentrations of perfluorooctanesulfonate (PFOS) were relatively stable between 1984 and 2010, increasing substantially in 2011; trends in perfluorooctanesulfonamide (PFOSA) concentrations were not strictly linear over the study period. PBDE, HBCD, and PFCA trends in the HI population were opposite/different to that of many other arctic animals (e.g., polar bears) for the same time period. Trends were inconsistent among locations, suggesting that regional differences in dietary exposure and/or sources may also impact these trends. The effects of climate-change-driven processes on the exposure and distribution of CECs are currently not well understood, highlighting a need for ongoing contaminant monitoring.

Beluga whales (Delphinapterus leucas) are facing profound changes in their habitat, with impacts expected at the individual and population level. Detecting and monitoring exposure and response to environmental stressors is necessary for beluga conservation and management of human activities. Cortisol has proven as a useful tool to assess stress on wildlife. Cortisol was measured in three blubber layers and plasma in subsistence-hunted beluga whales from the summers of 2007 to 2010 using an HPLC/MS/MS. We assessed the effect of biological and biochemical factors. Cortisol ranged from undetectable to 17.8 ng/g in blubber and 2.5 to 61.2 ng/mL in plasma. Concentrations were highest in the inner blubber layer likely reflecting circulating levels. All tissues were significantly higher in 2008 for reasons that remain unclear. Cortisol levels were on par with resting levels in captive belugas. Best fit models for cortisol revealed age to be an important determinant along with length and blubber thickness. Lack of relationships with biochemical factors such as organic contaminants suggests current cortisol levels are not significantly influenced by present contaminant concentrations. Our findings support the use of middle and outer blubber tissues for an integrated measure of chronic stress that are less subject to the influence of acute stress.

Catabolizing lignin-derived aromatic compounds requires an aryl-O-demethylation step. Here the authors present the structures of GcoA and GcoB, a cytochrome P450-reductase pair that catalyzes aryl-O-demethylations and show that GcoA displays broad substrate specificity, which is of interest for biotechnology applications.

Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators. Keywords: Whole cell biosensor, Aromatic catabolism, Transcription factor, PcaU, Shikimate

Cellobiohydrolases (CBHs) are critical for natural and industrial biomass degradation but their structure–activity relationships are not fully understood. Here, the authors present the biochemical and structural characterization of two CBHs, identifying protein regions that confer enhanced CBH activity.

Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36 h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72 h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24 h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72 h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). These results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals.

Abstract Background The production of value-added chemicals alongside biofuels from lignocellulosic hydrolysates is critical for developing economically viable biorefineries. Here, the production of propionic acid (PA), a potential building block for C3-based chemicals, from corn stover hydrolysate is investigated using the native PA-producing bacterium Propionibacterium acidipropionici. Results A wide range of culture conditions and process parameters were examined and experimentally optimized to maximize titer, rate, and yield of PA. The effect of gas sparging during fermentation was first examined, and N2 was found to exhibit improved performance over CO2. Subsequently, the effects of different hydrolysate concentrations, nitrogen sources, and neutralization agents were investigated. One of the best combinations found during batch experiments used yeast extract (YE) as the primary nitrogen source and NH4OH for pH control. This combination enabled PA titers of 30.8 g/L with a productivity of 0.40 g/L h from 76.8 g/L biomass sugars, while successfully minimizing lactic acid production. Due to the economic significance of downstream separations, increasing titers using fed-batch fermentation was examined by changing both feeding media and strategy. Continuous feeding of hydrolysate was found to be superior to pulsed feeding and combined with high YE concentrations increased PA titers to 62.7 g/L and improved the simultaneous utilization of different biomass sugars. Additionally, applying high YE supplementation maintains the lactic acid concentration below 4 g/L for the duration of the fermentation. Finally, with the aim of increasing productivity, high cell density fed-batch fermentations were conducted. PA titers increased to 64.7 g/L with a productivity of 2.35 g/L h for the batch stage and 0.77 g/L h for the overall process. Conclusion These results highlight the importance of media and fermentation strategy to improve PA production. Overall, this work demonstrates the feasibility of producing PA from corn stover hydrolysate.

The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.

Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Moreover, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates. Keywords: Pseudomonas putida KT2440, Levoglucosan kinase, B-glucosidase, Cellobiosan, Pyrolysis, Biofuels

The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCA decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92±0.48 g/L muconate. This study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions. Keywords: Protocatechuate decarboxylase, Pseudomonas Putida KT2440, cis,cis-Muconate, Muconic acid, Lignin valorization

Self-driving labs (SDLs) combine fully automated experiments with artificial intelligence (AI) that decides the next set of experiments. Taken to their ultimate expression, SDLs could usher a new paradigm of scientific research, where the world is probed, interpreted, and explained by machines for human benefit. While there are functioning SDLs in the fields of chemistry and materials science, we contend that synthetic biology provides a unique opportunity since the genome provides a single target for affecting the incredibly wide repertoire of biological cell behavior. However, the level of investment required for the creation of biological SDLs is only warranted if directed toward solving difficult and enabling biological questions. Here, we discuss challenges and opportunities in creating SDLs for synthetic biology.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions.

Generating routes for entities in virtual environments, such as simulated vehicles or synthetic human characters, is a long-standing problem, and route planning algorithms have been developed and studied for some time. Existing route planning algorithms, including the widely used A* algorithm, are generally intended to achieve optimality in some metric, such as minimum length or minimum time. Comparatively little attention has been given to route realism, defined as the similarity of the algorithm-generated route to the route followed by real humans in the same terrain with the same constraints and goals. Commercial game engines have seen increasing use as a context for research. To study route realism in a game engine, two developments were needed: a quantitative metric for measuring route realism and a game engine able to capture route data needed to compute the realism metric. Enhancements for recording route data for both synthetic characters and human players were implemented within the Unreal Tournament 2004 game engine. A methodology for assessing the realism of routes and other behaviors using a quantitative metric was developed. The enhanced Unreal Tournament 2004 game engine and the realism assessment methodology were tested by capturing data required to calculate a metric of route realism.

Seminal fluid is an important part of the ejaculate of internally fertilizing animals. This fluid contains substances that nourish and activate sperm for successful fertilization. Additionally, it contains components that influence female physiology to further enhance fertilization success of the sperm donor, possibly beyond the recipient's optimum. Although evidence for such substances abounds, few studies have unraveled their identities, and focus has been exclusively on separate-sex species. We present the first detailed study into the seminal fluid composition of a hermaphrodite (Lymnaea stagnalis). Eight novel peptides and proteins were identified from the seminal-fluid-producing prostate gland and tested for effects on oviposition, hatching and consumption. The gene for the protein found to suppress egg mass production, Ovipostatin, was sequenced, thereby providing the first fully-characterized seminal fluid substance in a simultaneous hermaphrodite. Thus, seminal fluid peptides and proteins have evolved and can play a crucial role in sexual selection even when the sexes are combined.