Lignin conversion to ß-ketoadipic acid by Pseudomonas putida via metabolic engineering and bioprocess development.

Bioconversion of a heterogeneous mixture of lignin-related aromatic compounds (LRCs) to a single product via microbial biocatalysts is a promising approach to valorize lignin. Here, Pseudomonas putida KT2440 was engineered to convert mixed p-coumaroyl- and coniferyl-type LRCs to ß-ketoadipic acid, a precursor for performance-advantaged polymers. Expression of enzymes mediating aromatic O-demethylation, hydroxylation, and ring-opening steps was tuned, and a global regulator was deleted. ß-ketoadipate titers of 44.5 and 25 grams per liter and productivities of 1.15 and 0.66 grams per liter per hour were achieved from model LRCs and corn stover-derived LRCs, respectively, the latter representing an overall yield of 0.10 grams per gram corn stover-derived lignin. Technoeconomic analysis of the bioprocess and downstream processing predicted a ß-ketoadipate minimum selling price of $2.01 per kilogram, which is cost competitive with fossil carbon-derived adipic acid ($1.10 to 1.80 per kilogram). Overall, this work achieved bioproduction metrics with economic relevance for conversion of lignin-derived streams into a performance-advantaged bioproduct.

Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p-coumarate.

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.

Production of itaconic acid from alkali pretreated lignin by dynamic two stage bioconversion.

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.

Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance.

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 â€‹+ â€‹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 â€‹h decrease in lag phase in 20 â€‹g/L pCA and a 2.4-fold increase in growth rate in 30 â€‹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 â€‹+ â€‹FA TALE-derived strains but did not affect growth in pCA â€‹+ â€‹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.

Glucose Responsiveness of ß-Cells Depends on Fatty Acids.

Glucose-stimulated insulin secretion (GSIS) is the gold standard for ß-cell function. Both experimental and clinical diabetology, i. e., preceding transplantation of isolated human islets, depend on functional testing. However, multiple factors influence GSIS rendering the comparison of different in vitro tests of glucose responsiveness difficult. This study examined the influence of bovine serum albumin (BSA)-coupled fatty acids on GSIS. Isolated islet preparations of human donors and of 12-months old mice displayed impaired GSIS in the presence of 0.5% FFA-free BSA compared to 0.5% BSA (fraction V, not deprived from fatty acids). In aged INS-1E cells, i. e. at a high passage number, GSIS became highly sensitive to FFA-free BSA. Readdition of 30 µM palmitate or 30 µM oleate to FFA-free BSA did not rescue GSIS, while the addition of 100 µM palmitate and the raise of extracellular Ca2+from 1.3 to 2.6 mM improved glucose responsiveness. A high concentration of palmitate (600 µM), which fully activates FFA1, largely restored insulin secretion. The FFA1-agonist TUG-469 also increased insulin secretion but to a lesser extent than palmitate. Glucose- and TUG-induced Ca2+oscillations were impaired in glucose-unresponsive, i. e., aged INS-1E cells. These results suggest that fatty acid deprivation (FFA-free BSA) impairs GSIS mainly through an effect on Ca2+sensitivity.

A Novel Hexavalent Capsular Polysaccharide Conjugate Vaccine (GBS6) for the Prevention of Neonatal Group B Streptococcal Infections by Maternal Immunization.

BACKGROUND: Group B streptococcus (GBS) causes serious diseases in newborn infants, often resulting in lifelong neurologic impairments or death. Prophylactic vaccination of pregnant women prior to delivery could provide comprehensive protection, as early onset and late-onset disease and maternal complications potentially could be addressed. METHODS: Capsular polysaccharide conjugate vaccine GBS6 was designed using surveillance data yielded by whole-genome sequencing of a global collection of recently recovered GBS isolates responsible for invasive neonatal GBS disease. Capsular polysaccharides were isolated, oxidized using sodium periodate, and conjugated to CRM197 by reductive amination in dimethyl sulfoxide. Immune responses in mice and rhesus macaques were measured in a multiplex Luminex immunoglobulin G (IgG) assay and opsonophagocytic activity assays. RESULTS: The optimized conjugates were immunogenic, alone and in combination, in mice and rhesus macaques, inducing IgG antibodies that mediated opsonophagocytic killing. Active immunization of murine dams with GBS6 prior to mating resulted in serotype-specific protection of pups from a lethal challenge with GBS. Protection following passive administration of serotype-specific IgG monoclonal antibodies to dams demonstrated conclusively that anticapsular polysaccharide IgG alone is sufficient for protection. CONCLUSIONS: The findings support the ongoing clinical evaluation of maternal GBS6 vaccination as a potential alternative method to prevent GBS disease in infants.

Engineering ß-oxidation in Yarrowia lipolytica for methyl ketone production.

Medium- and long-chain methyl ketones are fatty acid-derived compounds that can be used as biofuel blending agents, flavors and fragrances. However, their large-scale production from sustainable feedstocks is currently limited due to the lack of robust microbial biocatalysts. The oleaginous yeast Yarrowia lipolytica is a promising biorefinery platform strain for the production of methyl ketones from renewable lignocellulosic biomass due to its natively high flux towards fatty acid biosynthesis. In this study, we report the metabolic engineering of Y. lipolytica to produce long- and very long-chain methyl ketones. Truncation of peroxisomal ß-oxidation by chromosomal deletion of pot1 resulted in the biosynthesis of saturated, mono-, and diunsaturated methyl ketones in the C13-C23 range. Additional overexpression and peroxisomal targeting of a heterologous bacterial methyl ketone biosynthesis pathway yielded an initial titer of 151.5 mg/L of saturated methyl ketones. Dissolved oxygen concentrations in the cultures were found to substantially impact cell morphology and methyl ketone biosynthesis. Bioreactor cultivation under optimized conditions resulted in a titer of 314.8 mg/L of total methyl ketones, representing more than a 6000-fold increase over the parental strain. This work highlights the potential of Y. lipolytica to serve as chassis organism for the biosynthesis of acyl-thioester derived long- and very long-chain methyl ketones.

O-Acetylation is essential for functional antibody generation against Staphylococcus aureus capsular polysaccharide.

Staphylococcus aureus produces an antiphagocytic polysaccharide capsule to evade neutrophil-mediated killing. Many vaccines against encapsulated bacterial pathogens require generation of functional anti-capsular antibodies to mediate protection against infection and disease. Here it is shown that the generation of such antibody responses to S. aureus in vivo and in vitro requires the presence of O-acetyl modifications on the capsular polysaccharides. O-acetylation of S. aureus capsular polysaccharide therefore should be monitored carefully during vaccine development and production. This finding may provide additional insight into the previous failure of a S. aureus capsular polysaccharide conjugate vaccine.

Neutrophil killing of Staphylococcus aureus in diabetes, obesity and metabolic syndrome: a prospective cellular surveillance study.

BACKGROUND: Obesity, metabolic syndrome (MetS), and diabetes are frequent in surgical populations and can enhance susceptibility to postoperative surgical site infections. Reduced neutrophil function has been linked with diabetes and risk of Staphylococcus aureus infection. Therefore, neutrophil function in diabetic and obese subjects (± MetS) was assessed in this prospective serological and cellular surveillance study to determine whether vaccines administered to protect against infections after surgery could be effective in these populations. METHODS: Neutrophil function (chemotaxis, phagocytosis, and opsonophagocytic killing of S. aureus) was assessed in subjects classified according to diabetes status, body mass index, and presence/absence of MetS. Neutrophils were characterized within functional subsets by flow cytometry. A serologic assay was used to measure baseline antibody presence to each antigen in SA4Ag: capsular polysaccharide (CP) type 5, CP8, recombinant mutant Clumping factor A (rmClfA), and recombinant Manganese transport protein C (rMntC). RESULTS: Neutrophil function was similar for comorbid and healthy cohorts, with no significant between-group differences in cell counts, migration, phagocytosis ability, neutrophil subset proportions, and S. aureus killing ability when neutrophils were isolated 3-6 months apart (Visit 1 [n = 90] and Visit 2 [n = 70]) and assessed. Median pre-existing antibody titers to CP5, CP8, and rmClfA were comparable for all cohorts (insufficient subjects with rMntC titers for determination). CONCLUSIONS: MetS, diabetes, and obesity do not impact in vitro neutrophil function with regard to S. aureus killing, suggesting that if an effective S. aureus vaccine is developed it may be effective in individuals with these comorbidities.

Advanced glycation endproducts (AGEs) in saliva of patients with multiple myeloma - a pilot study.

Multiple myeloma (MM) is a hematological cancer with underlying causes associated with increased oxidative stress. Through signaling of their receptor RAGE, advanced glycation endproducts (AGEs) are known to increase oxidative stress associated with malignant transformation. In the present study, we have demonstrated that the levels of these compounds are increased in the saliva of myeloma patients with bone lesions. This data may provide a potential marker for bone lesions in MM and a potential target for the treatment of myeloma by blocking the AGEs or their receptor.

Colon capsule endoscopy: detection of colonic polyps compared with conventional colonoscopy and visualization of extracolonic pathologies.

BACKGROUND: Conventional colonoscopy (CC) is the gold standard for diagnostic examination of the colon. However, the overall acceptance of this procedure is low due to patient fears of complications or embarrassment. Colon capsule endoscopy (CCE) represents a minimally invasive, patient-friendly procedure that offers complete visualization of the entire intestine. OBJECTIVE: To assess the PillCam Colon 2 (Given Imaging Ltd, Israel) capsule with regard to feasibility, sensitivity and specificity for the detection of colonic pathologies and additional recorded extracolonic findings. METHODS: CCE was performed before CC in patients indicated for CC for known or suspected colonic disease. The results of both techniques were compared with regard to polyp detection. Additionally, bowel preparation and extracolonic pathologies were analyzed. RESULTS: Twenty-four patients (mean age 51.1 years) were included in the analysis. Visualization of the colon was complete in 23 CCs and 17 CCEs. No adverse events or major technical failures occurred. CC detected 47 polyps and CCE detected 43 polyps of any size (per-finding sensitivity 90.9%, specificity 67.6%). The accuracy of CCE in detecting polyp carriers was 81.5% (per-patient analysis). On average, the colon was adequately cleansed in 90.1% of patients. CCE identified esophageal, gastric and small bowel pathologies in seven (24%), nine (38%) and 14 (58%) patients, respectively. CONCLUSIONS: CCE proved to be technically feasible and safe. Acceptable sensitivity and moderate specificity levels in polyp detection were recorded. Bowel preparation was adequate in most patients. Because extracolonic pathologies were effectively visualized, new indications for the PillCam Colon 2 may be defined.

Staphylococcus aureus manganese transport protein C is a highly conserved cell surface protein that elicits protective immunity against S. aureus and Staphylococcus epidermidis.

Staphylococcus aureus and other staphylococci cause severe human disease, and there are currently no vaccines available. We evaluated whether manganese transport protein C (MntC), which is conserved across the staphylococcal species group, could confer protection against S. aureus and Staphylococcus epidermidis. In vivo analysis of S. aureus MntC expression revealed that expression occurs very early during the infectious cycle. Active immunization with MntC was effective at reducing the bacterial load associated with S. aureus and S. epidermidis infection in an acute murine bacteremia model. Anti-MntC monoclonal antibodies have been identified that can bind S. aureus and S. epidermidis cells and are protective in an infant rat passive protection model and induce neutrophil respiratory burst activity. This is the first description of a protein that has the potential to provide protection across the staphylococcal species group.