Dynamic control systems that mimic natural regulation of catabolic pathways enable rapid production of lignocellulose-derived bioproducts

Expanding the catabolic repertoire of engineered microbial bioproduction hosts enables more efficient use of complex feedstocks such as lignocellulosic hydrolysates, but the deleterious effects of existing expression systems limit the maximum carry capacity for heterologous catabolic pathways. Here we demonstrate use of a conditionally beneficial oxidative xylose catabolic pathway to improve performance of a Pseudomonas putida strain that has been engineered for growth-coupled bioconversion of glucose into the valuable bioproduct cis,cis-muconic acid. In the presence of xylose, the pathway enhances growth rate, and therefor productivity, by >60%, but the metabolic burden of constitutive pathway expression reduces the its growth rate by >20% in the absence of xylose. To mitigate this growth defect, we develop a xylose biosensor based on the XylR transcription factor from Caulobacter crescentus NA1000 to autonomously regulate pathway expression. We generate a library of engineered xylose-sensitive promoters that cover a three order-of-magnitude range of expression levels to tune pathway expression. Using structural modeling to guide mutations, we engineer XylR with two and three orders-of-magnitude reduced sensitivity to xylose and L-arabinose, respectively. A previously developed heterologous xylose isomerase pathway is placed under control of the biosensor, which improves the growth rate with xylose as a carbon source by 10% over the original constitutively expressed pathway. Finally, the oxidative xylose catabolic pathway is placed under control of the biosensor, enabling the bioproduction strain to maintain the increased growth rate in the presence of xylose, without the growth defect incurred from constitutive pathway expression in the absence of xylose. Utilizing biosensors to autonomously regulate conditionally beneficial catabolic pathways is generalizable, and will be critical for engineering bioproduction hosts bacteria with the wide range of catabolic pathways required for bioconversion of complex feedstocks.

External Exposure to BTEX, Internal Biomarker Response, and Health Risk Assessment of Nonoccupational Populations near a Coking Plant in Southwest China

Benzene, toluene, ethylbenzene and xylene isomers (BTEX) have raised increasing concern due to their adverse effects on human health. In this study, a coking factory and four communities nearby were selected as the research area. Atmospheric BTEX samples were collected and determined by a preconcentrator GC–MS method. Four biomarkers in the morning urine samples of 174 participants from the communities were measured by LC–MS. The health risks of BTEX exposure via inhalation were estimated. This study aimed to investigate the influence of external BTEX exposure on the internal biomarker levels and quantitatively evaluate the health risk of populations near the coking industry. The results showed that the average total BTEX concentration in residential area was 7.17 ± 7.24 μg m−3. Trans,trans-muconic acid (T,T-MA) was the urinary biomarker with the greatest average level (127 ± 285 μg g−1 crt). Similar spatial trends can be observed between atmospheric benzene concentration and internal biomarker levels. The mean values of the LCR for male and female residents were 2.15 × 10−5 and 2.05 × 10−5, respectively. The results of the risk assessment indicated that special attention was required for the non-occupational residents around the area.

Catechol 1,2-Dioxygenase From Paracoccus sp. MKU1—A Greener and Cleaner Bio-Machinery for cis, cis-Muconic Acid Production by Recombinant E. coli

Cis, cis-muconic acid (ccMA) is known for its industrial importance as a precursor for the synthesis of several biopolymers. Catechol 1,2-dioxygenase (C12O) is involved in aromatic compounds catabolism and ccMA synthesis in a greener and cleaner way. This is the first study on C12O gene from a metabolically versatile Paracoccus sp. MKU1, which was cloned and expressed in E. coli to produce ccMA from catechol. From the E. coli transformant, recombinant C12O enzyme was purified and found to be a homotrimer with a subunit size of 38.6 kDa. The apparent Km and Vmax for C12O was 12.89 µM and 310.1 U.mg−1, respectively, evidencing high affinity to catechol than previously reported C12Os. The predicted 3D-structure of C12O from MKU1 consisted of five α-helices in N-terminus, one α-helix in C-terminus, and nine β-sheets in C-terminus. Moreover, a unique α-helix signature ‘EESIHAN’ was identified in C-terminus between 271 and 277 amino acids, however the molecular insight of conservative α-helix remains obscure. Further, fed-batch culture was employed using recombinant E. coli expressing C12O gene from Paracoccus sp. MKU1 to produce ccMA by whole-cells catalyzed bioconversion of catechol. With the successive supply of 120 mM catechol, the transformant produced 91.4 mM (12.99 g/L) of ccMA in 6 h with the purity of 95.7%. This single step conversion of catechol to ccMA using whole-cells reactions of recombinants did not generate any by-products in the reaction mixtures. Thus, the recombinant E. coli expressing high activity C12O from Paracoccus sp. MKU1 holds promise as a potential candidate for yielding high concentrations of ccMA at faster rates in low cost settings.

Pd–Au Bimetallic Catalysts for the Hydrogenation of Muconic Acid to Bio-Adipic Acid

The hydrogenation reaction of muconic acid, produced from biomass using fermentative processes, to bio-adipic acid is one of the most appealing green emerging chemical process. This reaction can be promoted by catalysts based on a metal belonging to the platinum group, and the use of a second metal can preserve and increase their activity. Pd–Au bimetallic nanoparticle samples supported on high-temperature, heat-treated carbon nanofibers were prepared using the sol immobilization method, changing the Pd–Au molar ratio. These catalysts were characterized by TEM, STEM, and XPS analysis and tested in a batch reactor pressurized with hydrogen, where muconic acid dissolved in water was converted to adipic acid. The synthesized Pd–Au bimetallic catalysts showed higher activity than monometallic Au and Pd material and better stability during the recycling tests. Moreover, the selectivity toward the mono-unsaturated changed by decreasing the Pd/Au molar ratio: the higher the amount of gold, the higher the selectivity toward the intermediates.

Factors Affecting Adverse Health Effects of Gasoline Station Workers

This cross-sectional study examined the risk factors affecting adverse health effects from benzene exposure among gasoline station workers in Khon Kean province, Thailand. An interview questionnaire of adverse symptoms relating to benzene toxicity was administered to 151 workers. Area samplings for benzene concentration and spot urine for tt-muconic acid (tt-MA), a biomarker of benzene exposure, were collected. The factors associated with adverse symptoms were analysed by using multiple logistic regression. It was found that these symptoms mostly affected fuelling workers (77.5%), and the detected air benzene reached an action level or higher than 50% of NIOSH REL (>50 ppb). The top five adverse symptoms, i.e., fatigue, headache, dizziness, nasal congestion, and runny nose, were reported among workers exposed to benzene. More specific symptoms of benzene toxicity were chest pain, bleeding/epistaxis, and anaemia. The detected tt-MA of workers was 506.7 ug/g Cr (IQR), which was a value above the BEI and higher than that of asymptomatic workers. Risk factors significantly associated with adverse symptoms, included having no safety training experience (ORadj = 5.22; 95% CI: 2.16–12.58) and eating during work hours (ORadj = 16.08; 95% CI: 1.96–131.74). This study urges the tightening of health and safety standards at gasoline stations to include training and eating restrictions while working in hazardous areas.

Synthesis and Characterization of Metal-Organic Polymer based on Fe(III)

In this work, a coordination polymer based on iron trinuclear acetate was synthesized. The coordination polymer was obtained in two stages. At the first stage [Fe3O(CH3CO2)6(H2O)3]·2H2O – tricyclic iron acetate was synthesized. As the dicarboxylate ligand, we chose muconic acid, which is not toxic to the body. Double bonds in its structure create additional nodes for interaction with drugs. The optimal conditions for the synthesis of a coordination polymer based on tricyclic iron acetate and muconic acid were selected: solvothermal synthesis at 78 °C, autogenous pressure and using ethanol as a solvent. The resulting coordination polymer is a nanosized mesoporous framework with a narrow pore distribution, an average radius of~1.18 nm, and a developed specific surface area of 512.1 m2/g. The composition and crystal structure of tricyclic iron acetate and a coordination polymer based on it have been confirmed by the methods of elemental and X-ray phase analysis.

Investigation of Adsorption Capacity of Metal-Organic Polymers

In this work, we studied a biocompatible hybrid material based on iron (III) and muconic acid oxoclusters. It has been shown that coordination polymers are a promising class as functional materials for various purposes (as sorbents, catalysts, conductors, storage materials, etc.). The adsorption capacity of the obtained adsorbent for removing dyes from the prepared solution was in the following order: CR> MB> MV. From the results of the study, we can conclude that the dye Congo red is best suited for adsorption by the coordination polymer. The maximum absorption of the dye on organometallic coordination polymers occurs in the pH range 5 - 7 with adsorption of ~ 90%, which is important for the potential practical application of such coordination polymers as carriers for drug delivery.