Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2_IIA3_IIB_IIA1_II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET.     

Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Polyhydroxyalkanoates (PHA) have found widespread medical applications due to their biocompatibility and biodegradability, while further chemical modification requires functional groups on PHA. Halomonas bluephagenesis, a non-model halophilic bacterium serving as a chassis for the Next Generation Industrial Biotechnology (NGIB), was successfully engineered to express heterologous PHA synthase (PhaC) and enoyl coenzyme-A hydratase (PhaJ) from Aeromonas hydrophila 4AK4, along with a deletion of its native phaC gene to synthesize the short chain-co-medium chain-length PHA copolymers, namely poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhex-5-enoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyhex-5-enoate). After optimizations of the expression cassette and ribosomal binding site combined with introduction of endogenous acyl-CoA synthetase (fadD), the resulting recombinant strain H. bluephagenesis TDR4 achieved a remarkably high 3-hydroxyhexenoate (3HHxE) molar ratio of 35% when grown on glucose and 5-hexenoic acid as co-substrates. The total ratio of side chain consisting of 3HHx and 3HHxE monomers in the terpolymer can approach 44 mol%. H. bluephagenesis TDR4 was grown to a cell dry mass (CDM) of 30 g/L containing approximately 20% poly(3-hydroxybutyrate-co-22.75 mol% 3-hydroxy-5-hexenoate) in a 48-h of open and unsterile fermentation with a 5-hexenoic acid conversion efficiency of 91%. The resulted functional PHA containing 12.5 mol% 3-hydroxy-5-hexenoate exhibits more than 1000% elongation at break. The engineered H. bluephagenesis TDR4 can be used as an experimental platform to produce functional PHA.     

Validation of low-dose lung cancer PET-CT protocol and PET image improvement using machine learning

PurposeTo conduct a simplified lesion-detection task of a low-dose (LD) PET-CT protocol for frequent lung screening using 30% of the effective PETCT dose and to investigate the feasibility of increasing clinical value of low-statistics scans using machine learning.MethodsWe acquired 33 SD PET images, of which 13 had actual LD (ALD) PET, and simulated LD (SLD) PET images at seven different count levels from the SD PET scans. We employed image quality transfer (IQT), a machine learning algorithm that performs patch-regression to map parameters from low-quality to high-quality images. At each count level, patches extracted from 23 pairs of SD/SLD PET images were used to train three IQT models – global linear, single tree, and random forest regressions with cubic patch sizes of 3 and 5 voxels. The models were then used to estimate SD images from LD images at each count level for 10 unseen subjects. Lesion-detection task was carried out on matched lesion-present and lesion-absent images.ResultsLD PET-CT protocol yielded lesion detectability with sensitivity of 0.98 and specificity of 1. Random forest algorithm with cubic patch size of 5 allowed further 11.7% reduction in the effective PETCT dose without compromising lesion detectability, but underestimated SUV by 30%.ConclusionLD PET-CT protocol was validated for lesion detection using ALD PET scans. Substantial image quality improvement or additional dose reduction while preserving clinical values can be achieved using machine learning methods though SUV quantification may be biased and adjustment of our research protocol is required for clinical use.     

In-planta production of the biodegradable polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC): Stacking reduced biomass recalcitrance with value-added co-product

2-Pyrone-4,6-dicarboxylic acid (PDC), a chemically stable intermediate that naturally occurs during microbial degradation of lignin by bacteria, represents a promising building block for diverse biomaterials and polyesters such as biodegradable plastics. The lack of a chemical synthesis method has hindered large-scale utilization of PDC and metabolic engineering approaches for its biosynthesis have recently emerged. In this study, we demonstrate a strategy for the production of PDC via manipulation of the shikimate pathway using plants as green factories. In tobacco leaves, we first showed that transient expression of bacterial feedback-resistant 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG) and 3-dehydroshikimate dehydratase (QsuB) produced high titers of protocatechuate (PCA), which was in turn efficiently converted into PDC upon co-expression of PCA 4,5-dioxygenase (PmdAB) and 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (PmdC) derived from Comamonas testosteroni. We validated that stable expression of AroG in Arabidopsis in a genetic background containing the QsuB gene enhanced PCA content in plant biomass, presumably via an increase of the carbon flux through the shikimate pathway. Further, introducing AroG and the PDC biosynthetic genes (PmdA, PmdB, and PmdC) into the Arabidopsis QsuB background, or introducing the five genes (AroG, QsuB, PmdA, PmdB, and PmdC) stacked on a single construct into wild-type plants, resulted in PDC titers of ~1% and ~3% dry weight in plant biomass, respectively. Consistent with previous studies of plants expressing QsuB, all PDC producing lines showed strong reduction in lignin content in stems. This low lignin trait was accompanied with improvements of biomass saccharification efficiency due to reduced cell wall recalcitrance to enzymatic degradation. Importantly, most transgenic lines showed no reduction in biomass yields. Therefore, we conclude that engineering plants with the proposed de-novo PDC pathway provides an avenue to enrich biomass with a value-added co-product while simultaneously improving biomass quality for the supply of fermentable sugars. Implementing this strategy into bioenergy crops has the potential to support existing microbial fermentation approaches that exploit lignocellulosic biomass feedstocks for PDC production.     

Design,synthesis and identification of N,N-dibenzylcinnamamide (DBC) derivatives as novel ligands for α-synuclein fibrils by SPR evaluation system

PET imaging of α-synuclein (α-syn) deposition in the brain will be an effective tool for earlier diagnosis of Parkinson's disease (PD) due to α-syn aggregation is the widely accepted biomarker for PD. However, the necessary PET radiotracer for imaging is clinically unavailable until now. The lead compound discovery is the first key step for the study. Herein, we initially established an efficient biologically evaluation system well in high throughput based on SPR technology, and identified a novel class of N, N-dibenzylcinnamamide (DBC) compounds as α-syn ligands through the assay. These compounds were proved to have high affinities against α-syn aggregates (K_D < 10 nM), which well met the requirement of binding activity for the PET probe. These DBC compounds were firstly reported as α-syn ligands herein and the preliminary obtained structure has been further modified into F-labeled ones. Among them, a high-affinity tracer (5–41) with 1.03 nM (K_D) has been acquired, indicating its potential as a new lead compound for developing PET radiotracer.     

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.     

Proteomic Analysis of Trichomonas vaginalis Phagolysosome,Lysosomal Targeting,and Unconventional Secretion of Cysteine Peptidases

The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.     

Pretreatment of outer membrane vesicle and subsequent infection with influenza virus induces a long-lasting adaptive immune response against broad subtypes of influenza virus

Influenza is an acute respiratory disease and a global health problem. Although influenza vaccines are commercially available, frequent antigenic changes in hemagglutinin might render them less effective or unavailable. We previously reported that modified outer membrane vesicle (fmOMV) provided immediate and robust protective immunity against various subtypes of influenza virus. However, the effect was transient because it was innate immunity-dependent. In this study, we investigated the effects of consecutive administration of fmOMV and influenza virus on the adaptive immune response and long-term protective immunity against influenza virus. When the mice were pretreated with fmOMV and subsequently infected with influenza virus, strong influenza-specific antibody and T cell responses were induced in both systemic and lung mucosal compartments without pathogenic symptoms. Upon the secondary viral challenge at week 4, the mice given fmOMV and influenza virus exhibited almost complete protection against homologous and heterologous viral challenge. More importantly, this strong protective immunity lasted up to 18 weeks after the first infection. These results show that pretreatment with fmOMV and subsequent infection with influenza virus efficiently induces broad and long-lasting protective immunity against various virus subtypes, suggesting a novel antiviral strategy against newly-emerging viral diseases without suitable vaccines or therapeutics.     

A Novel Monoclonal Antibody Degrades the Thyrotropin Receptor Autoantibodies in Graves' Disease

ObjectiveAutoantibodies against the thyrotropin receptor (TSH-R-Ab) are key mediators for the pathogenesis of Graves' disease (GD). TSH-R-Ab degradation was evaluated using several immunoassays within an exploratory, controlled trial in patients with GD receiving a monoclonal antibody (mAb) targeting the neonatal crystallizable fragment receptor (FcRn).MethodsSerial measurements of TSH-R-Ab serum levels were performed using 3 different binding and cell-based assays in patients with GD either on medication or on placebo.ResultsIn contrast to the placebo group, in which no changes were observed, a 12-week mAb therapy led to an early and significant decrease (>60%) in the serum TSH-R-Ab levels in patients with thyroidal and extrathyroidal GD, as unanimously shown in all 3 assays. These marked changes were noted already at week 7 post baseline (P <.0001 for the binding immunoassay and for the luciferase (readout) bioassay). The 3 TSH-R-Ab binding and bioassays were highly correlated in the samples of both study groups (binding immunoassay vs luciferase bioassay, r =.91, P <.001, binding vs cyclic adenosine monophosphate (cAMP) bioassay, r = 0.86, P <.001, and luciferase vs cAMP bioassay, r = 0.71, P =.006). The serological results correlated with the course of the extrathyroidal clinical parameters of GD, that is, clinical activity score and proptosis.ConclusionTargeting the FcRn markedly reduces the disease-specific TSH-R-Ab in patients with GD. The novel and rapid TSH-R-Ab bioassay improves diagnosis and management of patients with GD.     

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO₂ using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO₂ to carboxylic acids.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

The identification and conservation of climate refugia for two Colombian endemic titi (Plecturocebus) monkeys

Natural resource managers face the challenge of developing conservation plans for key species and given that anthropogenic climate change (CC) effects on biodiversity are becoming increasingly evident, the new challenge is to properly incorporate CC adaptation strategies into such plans. Thus, the objective of this study is to evaluate the potential CC effects on the climatically suitable areas for two Colombian endemic titi monkeys Plecturocebus ornatus and P. caquetensis and to identify the prospective climate refugia as macro-ecological adaptation strategies for each species. A detailed ecological niche modeling (ENM) approach was applied with the maximum entropy algorithm, using presence records and different sets of bioclimatic variables describing baseline (1960–1990) and future climates (∼2070). Models of future climatic suitability were generated using projections of variables under a stabilization (RCP4.5) and business as usual (RCP8.5) scenarios with data from two general circulation models (GCMs) describing storylines of increasing (CESM1_CAM5) and decreasing (CSIRO_ACCESS1_3) rainfall patterns. The results for both species indicate that in a warmer future, opposite rainfall patterns and choice of the bioclimatic variables may lead to divergent responses on the extent and geographic distribution of their climatic niche, which varied from regions gaining, losing, and retaining suitability in potential climate refugia. Moreover, CC represents a serious threat for P. caquetensis and P. ornatus since their ranges may be largely exposed to novel climates. Their baseline climatic suitability area is projected to shrink and shift to higher elevations in the Andes mountains, and the climate refugia identified for both species are poorly covered by protected areas. Therefore, the climate refugia identified in this work and the management recommendations offered should be considered by species conservation plans to contribute to the selection of priority regions for conservation actions. The modeling approach reveals the uncertainties arising from the selection of bioclimatic variables and GCMs in ENM, which can be replicated to identify climate refugia targeting different species of conservation concern.     

Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.     

Deconvoluting the biology and druggability of protein lipidation using chemical proteomics

Lipids are indispensable cellular building blocks, and their post-translational attachment to proteins makes them important regulators of many biological processes. Dysfunction of protein lipidation is also implicated in many pathological states, yet its systematic analysis presents significant challenges. Thanks to innovations in chemical proteomics, lipidation can now be readily studied by metabolic tagging using functionalized lipid analogs, enabling global profiling of lipidated substrates using mass spectrometry. This has spearheaded the first deconvolution of their full scope in a range of contexts, from cells to pathogens and multicellular organisms. Protein N-myristoylation, S-acylation, and S-prenylation are the most well-studied lipid post-translational modifications because of their extensive contribution to the regulation of diverse cellular processes. In this review, we focus on recent advances in the study of these post-translational modifications, with an emphasis on how novel mass spectrometry methods have elucidated their roles in fundamental biological processes.     

Clinical Applicability of Patient- and Clinician-Reported Outcome Tools in the Management of Patients With Acromegaly

ObjectiveWe aimed to assess treatment outcomes and disease control status in patients with acromegaly using patient- and clinician-reported outcome tools and to analyze correlations among different components of both tools.MethodsThis cross-sectional study included 72 patients from a national referral center with a median follow-up of 8 (5-12) years. The baseline SAGIT score at diagnosis was determined retrospectively, whereas the follow-up SAGIT and acromegaly quality of life questionnaire (AcroQoL) results were assessed at the most recent visit and by additional telephone interviews.ResultsAll SAGIT subscores decreased significantly from baseline to follow-up (global score from 14 to 4 [P < .001]). The SAGIT scores at baseline did not discriminate the current disease control status. However, a higher baseline SAGIT score and subscore T were associated with uncontrolled disease after the first-line treatment. Diagnostic delay was correlated with baseline S, A, G, and global SAGIT scores. At the follow-up, the global SAGIT score discriminated between cured/controlled and uncontrolled groups (4 vs 6 [P = .007]). The AcroQoL score was 69.3, with the personal relations subscale being the least affected and the physical scale being the most affected. There was no difference in the AcroQoL score between patients classified as uncontrolled or cured/controlled. At baseline and follow-up, there were significant negative correlations between S and A subscores and AcroQoL score. A higher body mass index, the presence of swelling, joint symptoms, headaches, sleep apnea, and hypertension significantly impaired quality of life.ConclusionOur results emphasize the complementary nature of the patient- and clinician-reported outcome tools in acromegaly management. We identified modifiable signs, symptoms, and comorbidities as treatment targets that might help clinicians improve quality of life in this population.     

Call for biotechnological approach to degrade plastic in the era of COVID-19 pandemic

Plastic pollution is a global issue and has become a major concern since Coronavirus disease (COVID)-19. In developing nations, landfilling and illegal waste disposal are typical ways to dispose of COVID-19-infected material. These technologies worsen plastic pollution and other human and animal health problems. Plastic degrades in light and heat, generating hazardous primary and secondary micro-plastic. Certain bacteria can degrade artificial polymers using genes, enzymes, and metabolic pathways. Microorganisms including bacteria degrade petrochemical plastics slowly. High molecular weight, strong chemical bonds, and excessive hydrophobicity reduce plastic biodegradation. There is not enough study on genes, enzymes, and bacteria-plastic interactions. Synthetic biology, metabolic engineering, and bioinformatics methods have been created to biodegrade synthetic polymers. This review will focus on how microorganisms' degrading capacity can be increased using recent biotechnological techniques.     

Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid

Valorization of lignin, an abundant component of plant cell walls, is critical to enabling the lignocellulosic bioeconomy. Biological funneling using microbial biocatalysts has emerged as an attractive approach to convert complex mixtures of lignin depolymerization products to value-added compounds. Ideally, biocatalysts would convert aromatic compounds derived from the three canonical types of lignin: syringyl (S), guaiacyl (G), and p-hydroxyphenyl (H). Pseudomonas putida KT2440 (hereafter KT2440) has been developed as a biocatalyst owing in part to its native catabolic capabilities but is not known to catabolize S-type lignin-derived compounds. Here, we demonstrate that syringate, a common S-type lignin-derived compound, is utilized by KT2440 only in the presence of another energy source or when vanAB was overexpressed, as syringate was found to be O-demethylated to gallate by VanAB, a two-component monooxygenase, and further catabolized via extradiol cleavage. Unexpectedly, the specificity (k_cat/K_M) of VanAB for syringate was within 25% that for vanillate and O-demethylation of both substrates was well-coupled to O₂ consumption. However, the native KT2440 gallate-cleaving dioxygenase, GalA, was potently inactivated by 3-O-methylgallate. To engineer a biocatalyst to simultaneously convert S-, G-, and H-type monomers, we therefore employed VanAB from Pseudomonas sp. HR199, which has lower activity for 3MGA, and LigAB, an extradiol dioxygenase able to cleave protocatechuate and 3-O-methylgallate. This strain converted 93% of a mixture of lignin monomers to 2-pyrone-4,6-dicarboxylate, a promising bio-based chemical. Overall, this study elucidates a native pathway in KT2440 for catabolizing S-type lignin-derived compounds and demonstrates the potential of this robust chassis for lignin valorization.     

Immunopeptidomic Analyses of Colorectal Cancers With and Without Microsatellite Instability

Colorectal cancer is the second leading cause of cancer death worldwide, and the incidence of this disease is expected to increase as global socioeconomic changes occur. Immune checkpoint inhibition therapy is effective in treating a minority of colorectal cancer tumors; however, microsatellite stable tumors do not respond well to this treatment. Emerging cancer immunotherapeutic strategies aim to activate a cytotoxic T cell response against tumor-specific antigens, presented exclusively at the cell surface of cancer cells. These antigens are rare and are most effectively identified with a mass spectrometry–based approach, which allows the direct sampling and sequencing of these peptides. Although the few tumor-specific antigens identified to date are derived from coding regions of the genome, recent findings indicate that a large proportion of tumor-specific antigens originate from allegedly noncoding regions. Here, we employed a novel proteogenomic approach to identify tumor antigens in a collection of colorectal cancer–derived cell lines and biopsy samples consisting of matched tumor and normal adjacent tissue. The generation of personalized cancer databases paired with mass spectrometry analyses permitted the identification of more than 30,000 unique MHC I–associated peptides. We identified 19 tumor-specific antigens in both microsatellite stable and unstable tumors, over two-thirds of which were derived from noncoding regions. Many of these peptides were derived from source genes known to be involved in colorectal cancer progression, suggesting that antigens from these genes could have therapeutic potential in a wide range of tumors. These findings could benefit the development of T cell–based vaccines, in which T cells are primed against these antigens to target and eradicate tumors. Such a vaccine could be used in tandem with existing immune checkpoint inhibition therapies, to bridge the gap in treatment efficacy across subtypes of colorectal cancer with varying prognoses. Data are available via ProteomeXchange with identifier PXD028309.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.