An integrated yeast-based process for cis,cis-muconic acid production.

Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.

Bioremediation of Petroleum Hydrocarbons in Seawater: Prospects of Using Lyophilized Native Hydrocarbon-Degrading Bacteria.

This work aimed to develop a bioremediation product of lyophilized native bacteria to respond to marine oil spills. Three oil-degrading bacterial strains (two strains of Rhodococcus erythropolis and one Pseudomonas sp.), isolated from the NW Portuguese coast, were selected for lyophilization after biomass growth optimization (tested with alternative carbon sources). Results indicated that the bacterial strains remained viable after the lyophilization process, without losing their biodegradation potential. The biomass/petroleum ratio was optimized, and the bioremediation efficiency of the lyophilized bacterial consortium was tested in microcosms with natural seawater and petroleum. An acceleration of the natural oil degradation process was observed, with an increased abundance of oil-degraders after 24 h, an emulsion of the oil/water layer after 7 days, and an increased removal of total petroleum hydrocarbons (47%) after 15 days. This study provides an insight into the formulation and optimization of lyophilized bacterial agents for application in autochthonous oil bioremediation.

Characteristics Associated with Acute-Phase Response following First Zoledronic Acid Infusion in Brazilian Population with Osteoporosis.

We aimed to evaluate characteristics associated with acute-phase response (APR) following first zoledronic acid infusion in a Brazilian cohort. This retrospective cohort study enrolled all adults with osteoporosis who underwent a first zoledronic acid infusion at our centre between June 2015 and June 2019. Clinical demographics (age, sex, diabetes, smoking, body mass index, and previous oral bisphosphonate use) and laboratory data (calcium, parathyroid hormone, renal function, and serum 25-hydroxyvitamin D and carboxy-terminal crosslinked telopeptide of type 1 collagen [CTX], both before and after infusion) were compared between patients with and without APR. We evaluated association magnitude between the presence of APR and clinical variables through logistic regression. This study enrolled 400 patients (women, 80%). APR was observed in 24.5% (n = 98) of patients. The mean symptom duration in days was 3.5 ± 2.8. Patients with APR were younger (67 ± 12 vs. 71 ± 11 years; p=0.001), used oral bisphosphonates less frequently (34% × 50%; p=0.005), and had greater baseline CTX (0.535 ng/mL [0.375, 0.697] × 0.430 [0.249, 0.681]; p=0.03) and ΔCTX (-69 [-76; -50] × -54 [-72; -23]; p=0.002) than those without APR. The other variables were similar between the groups. Only ΔCTX was associated (OR, 0.62; 95% CI 0.41-0.98) with APR after accounting for age and bisphosphonate use. APR occurred in 24.5% of the cohort. Younger age and absence of prior oral bisphosphonate use were associated with APR following first zoledronic acid infusion. APR was associated with ΔCTX (but no other variables) after adjusting for these factors.

Characteristics and outcomes of patients with cancer requiring admission to intensive care units: a prospective multicenter study.

OBJECTIVE: To evaluate the characteristics and outcomes of patients with cancer admitted to several intensive care units. Knowledge on patients with cancer requiring intensive care is mostly restricted to single-center studies. DESIGN: : Prospective, multicenter, cohort study. SETTING: Intensive care units from 28 hospitals in Brazil. PATIENTS: A total of 717 consecutive patients included over a 2-mo period. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: There were 667 (93%) patients with solid tumors and 50 (7%) patients had hematologic malignancies. The main reasons for intensive care unit admission were postoperative care (57%), sepsis (15%), and respiratory failure (10%). Overall hospital mortality rate was 30% and was higher in patients admitted because of medical complications (58%) than in emergency (37%) and scheduled (11%) surgical patients (p < .001). Adjusting for covariates other than the type of admission, the number of hospital days before intensive care unit admission (odds ratio [OR], 1.18; 95% confidence interval [CI], 1.01-1.37), higher Sequential Organ Failure Assessment scores (OR, 1.25; 95% CI, 1.17-1.34), poor performance status (OR, 3.40; 95% CI, 2.19 -5.26), the need for mechanical ventilation (OR, 2.42; 95% CI, 1.51-3.87), and active underlying malignancy in recurrence or progression (OR, 2.42; 95% CI, 1.51-3.87) were associated with increased hospital mortality in multivariate analysis. CONCLUSIONS: This large multicenter study reports encouraging survival rates for patients with cancer requiring intensive care. In these patients, mortality was mostly dependent on the severity of organ failures, performance status, and need for mechanical ventilation rather than cancer-related characteristics, such as the type of malignancy or the presence of neutropenia.

Improvement of cis,cis-Muconic Acid Production in Saccharomyces cerevisiae through Biosensor-Aided Genome Engineering.

Muconic acid is a potential platform chemical for the production of nylon, polyurethanes, and terephthalic acid. It is also an attractive functional copolymer in plastics due to its two double bonds. At this time, no economically viable process for the production of muconic acid exists. To harness novel genetic targets for improved production of cis,cis-muconic acid (CCM) in the yeast Saccharomyces cerevisiae, we employed a CCM-biosensor coupled to GFP expression with a broad dynamic response to screen UV-mutagenesis libraries of CCM-producing yeast. Via fluorescence activated cell sorting we identified a clone Mut131 with a 49.7% higher CCM titer and 164% higher titer of biosynthetic intermediate-protocatechuic acid (PCA). Genome resequencing of the Mut131 and reverse engineering identified seven causal missense mutations of the native genes (PWP2, EST2, ATG1, DIT1, CDC15, CTS2, and MNE1) and a duplication of two CCM biosynthetic genes, encoding dehydroshikimate dehydratase and catechol 1,2-dioxygenase, which were not recognized as flux controlling before. The Mut131 strain was further rationally engineered by overexpression of the genes encoding for PCA decarboxylase and AROM protein without shikimate dehydrogenase domain (Aro1pΔE), and by restoring URA3 prototrophy. The resulting engineered strain produced 20.8 g/L CCM in controlled fed-batch fermentation, with a yield of 66.2 mg/g glucose and a productivity of 139 mg/L/h, representing the highest reported performance metrics in a yeast for de novo CCM production to date and the highest production of an aromatic compound in yeast. The study illustrates the benefit of biosensor-based selection and brings closer the prospect of biobased muconic acid.

Hematopoietic stem cells: from the bone to the bioreactor.

The ex vivo expansion of human hematopoietic stem cells is a rapidly developing area with a broad range of biomedical applications. The mechanisms of renewal, differentiation and plasticity of stem cells are currently under intense investigation. However, the complexity of hematopoiesis, the heterogeneity of the culture population and the complex interplay between the culture parameters that significantly influence the proliferation and differentiation of hematopoietic cells have impaired the translation of small scale results to the highly demanded large-scale applications. The better understanding of these mechanisms is providing the basis for more rational approaches to the ex vivo expansion of hematopoietic stem cells. Efforts are now being made to establish a rational design of bioreactor systems, allowing the modeling and control of large-scale production of stem cells and the study of their proliferation and differentiation, under conditions as similar as possible to those in vivo.

Mycobacterium sp., Rhodococcus erythropolis, and Pseudomonas putida behavior in the presence of organic solvents.

This work aimed at studying the behavior and tolerance of Mycobacterium sp. NRRL B-3805, Rhodococcus erythropolis DCL14 and Pseudomonas putida S12 cells in the presence of various concentrations of water miscible (ethanol, butanol, and dimethylformamide, up to 50% v/v) and water immiscible solvents (dodecane, bis(2-ethylhexyl) phthalate and toluene, up to 5% v/v). When incubated in the presence of these solvents, the cells were found to have lower tolerance to butanol and toluene than to the remaining solvents. Nevertheless, the concentrations of solvents endured by the tested strains show that they are quite solvent-tolerant, confirming their potential as biocatalysts in nonconventional systems. Microscopic observation of samples showed that the hydrophobic Mycobacterium sp. and R. erythropolis cells were able to aggregate to protect the population under stress conditions. Comparison of the results obtained at the single cell level by fluorescence microscopy and colony development on agar plates indicated that the primary effects of most solvents tested were on the cell membrane and replicating capability of the cells.

Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain.

By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.

Recombinant Saccharomyces cerevisiae strain triggers acetate production to fuel biosynthetic pathways.

Although the metabolism and physiology of the growth of yeast strains has been extensively studied, many questions remain unanswered when the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. It was observed that whenever the strain needed to activate biosynthetic pathways, either for cutinase synthesis, or for the synthesis of the enzymes required for galactose intake, acetate production occurred. The on-line detection of acetate in the medium might prove useful for the control and the supervision of recombinant protein production processes using yeast. The volumes of acid and base added to control the pH throughout the time course of the cultivations were used to calculate an on-line estimator for acetate concentration.

Enhanced bioproduction of poly-3-hydroxybutyrate from wheat straw lignocellulosic hydrolysates.

Polyhydroxyalkanoates (PHAs) are bioplastics that can replace conventional petroleum-derived products in various applications. One of the major barriers for their widespread introduction in the market is the higher production costs compared with their petrochemical counterparts. In this work, a process was successfully implemented with high productivity based on wheat straw, a cheap and readily available agricultural residue, as raw material. The strain Burkholderia sacchari DSM 17165 which is able to metabolise glucose, xylose and arabinose, the main sugars present in wheat straw hydrolysates (WSHs), was used. Results in shake flask showed that B. sacchari cells accumulated about 70%gpoly(3-hydroxybutyrate)(P(3HB))/g cell dry weight (CDW) with a yield of polymer on sugars (YP/S) of 0.18g/g when grown on a mixture of commercial C6 and C5 sugars (control), while these values reached about 60%gP(3HB)/g CDW and 0.19g/g, respectively, when WSHs were used as carbon source. In fed-batch cultures carried out in 2L stirred-tank reactors (STRs) on WSH, a maximum polymer concentration of 105 g/L was reached after 61 hours of cultivation corresponding to an accumulation of 72% of CDW. Polymer yield and productivity were 0.22 gP(3HB)/g total sugar consumed and 1.6g/L hour, respectively. The selected feeding strategy successfully overcame the carbon catabolite repression (CCR) phenomenon observed with sugar mixtures containing hexoses and pentoses. This is the first work describing fed-batch cultivations aiming at PHA production using real lignocellulosic hydrolysates. Additionally, the P(3HB) volumetric productivities attained are by far the highest ever achieved on agricultural waste hydrolysates.

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Burkholderia sacchari using wheat straw hydrolysates and gamma-butyrolactone.

Burkholderia sacchari DSM 17165 is able to grow and produce poly(3-hydroxybutyrate) both on hexoses and pentoses. In a previous study, wheat straw lignocellulosic hydrolysates (WSH) containing high C6 and C5 sugar concentrations were shown to be excellent carbon sources for P(3HB) production. Using a similar feeding strategy developed for P(3HB) production based on WSH, fed-batch cultures were developed aiming at the production of the copolymer P(3HB-co-4HB) (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) by B. sacchari. The ability of this strain to synthesize P(3HB-co-4HB) was first shown in shake flasks using gamma-butyrolactone (GBL) as precursor of the 4HB units. Fed-batch cultures using glucose as carbon source (control) and GBL were developed to achieve high copolymer productivities and 4HB incorporations. The attained P(3HB-co-4HB) productivity and 4HB molar% were 0.7g/(Lh) and 4.7molar%, respectively. The 4HB incorporation was improved to 6.3 and 11.8molar% by addition of 2g/L propionic and acetic acid, respectively. When WSH were used as carbon source under the same feeding conditions, the values achieved were 0.5g/(Lh) and 5.0molar%, respectively. Burkholderia sacchari, a strain able to produce biopolymers based on xylose-rich lignocellulosic hydrolysates, is for the first time reported to produce P(3HB-co-4HB) using gamma butyrolactone as precursor.

Outcomes for patients with cancer admitted to the ICU requiring ventilatory support: results from a prospective multicenter study.

BACKGROUND: This study was undertaken to evaluate the clinical characteristics and outcomes of patients with cancer requiring nonpalliative ventilatory support. METHODS: This was a secondary analysis of a prospective cohort study conducted in 28 Brazilian ICUs evaluating adult patients with cancer requiring invasive mechanical ventilation (MV) or noninvasive ventilation (NIV) during the first 48 h of their ICU stay. We used logistic regression to identify the variables associated with hospital mortality. RESULTS: Of 717 patients, 263 (37%) (solid tumors = 227; hematologic malignancies = 36) received ventilatory support. NIV was initially used in 85 patients (32%), and 178 (68%) received MV. Additionally, NIV followed by MV occurred in 45 patients (53%). Hospital mortality rates were 67% in all patients, 40% in patients receiving NIV only, 69% when NIV was followed by MV, and 73% in patients receiving MV only (P < .001). Adjusting for the type of admission, newly diagnosed malignancy (OR, 3.59; 95% CI, 1.28-10.10), recurrent or progressive malignancy (OR, 3.67; 95% CI, 1.25-10.81), tumoral airway involvement (OR, 4.04; 95% CI, 1.30-12.56), performance status (PS) 2 to 4 (OR, 2.39; 95% CI, 1.24-4.59), NIV followed by MV (OR, 3.00; 95% CI, 1.09-8.18), MV as initial ventilatory strategy (OR, 3.53; 95% CI, 1.45-8.60), and Sequential Organ Failure Assessment score (each point except the respiratory domain) (OR, 1.15; 95% CI, 1.03-1.29) were associated with hospital mortality. Hospital survival in patients with good PS and nonprogressive malignancy and without tumoral airway involvement was 53%. Conversely, patients with poor functional capacity and cancer progression had unfavorable outcomes. CONCLUSIONS: Patients with cancer with good PS and nonprogressive disease requiring ventilatory support should receive full intensive care, because one-half of these patients survive. On the other hand, provision of palliative care should be considered the main goal for patients with poor PS and progressive underlying malignancy.

Kinetic analysis of the ex vivo expansion of human hematopoietic stem/progenitor cells.

The total cell expansion of human umbilical cord blood (CB) and adult bone marrow (BM) CD34+-enriched cells cultured in supplemented serum-free media, either over irradiated human feeder layers or in stroma-free systems, were characterized by a simple kinetic model using only two parameters: the specific cell expansion rate, mu, and the death rate constant, k(k). Both CB and BM cells can expand at approximately the same rate (0.21 day(-1)) in this culture system however, cell death depends on the presence of stroma and the environment in which the cells are cultured.