Engineering Pseudomonas putida KT2440 for Dipicolinate Production via the Entner-Doudoroff Pathway.

Dipicolinic acid (DPA), a cyclic diacid, has garnered significant interest due to its potential applications in antimicrobial agents, antioxidants, chelating reagents, and polymer precursors. However, its natural bioproduction is limited since DPA is only accumulated in Bacillus and Clostridium species during sporulation. Thus, heterologous production by engineered strains is of paramount importance for developing a sustainable biological route for DPA production. Pseudomonas putida KT2440 has emerged as a promising host for the production of various chemicals thanks to its robustness, metabolic versatility, and genetic tractability. The dominant Entner-Doudoroff (ED) pathway for glucose metabolism in this strain offers an ideal route for DPA production due to the advantage of NADPH generation and the naturally balanced flux between glyceraldehyde-3-phosphate and pyruvate, which are both precursors for DPA synthesis. In this study, DPA production via the ED pathway was in silico designed in P. putida KT2440. The systematically engineered strain produced dipicolinate with a titer of 11.72 g/L from glucose in a 5 L fermentor. This approach not only provides a sustainable green route for DPA production but also expands our understanding of the metabolic potential of the ED pathway in P. putida KT2440.

Adaptive laboratory evolution of Rhodococcus rhodochrous DSM6263 for chlorophenol degradation under hypersaline condition.

BACKGROUND: Normally, a salt amount greater than 3.5% (w/v) is defined as hypersaline. Large amounts of hypersaline wastewater containing organic pollutants need to be treated before it can be discharged into the environment. The most critical aspect of the biological treatment of saline wastewater is the inhibitory/toxic effect exerted on bacterial metabolism by high salt concentrations. Although efforts have been dedicated to improving the performance through the use of salt-tolerant or halophilic bacteria, the diversities of the strains and the range of substrate spectrum remain limited, especially in chlorophenol wastewater treatment. RESULTS: In this study, a salt-tolerant chlorophenol-degrading strain was generated from Rhodococcus rhodochrous DSM6263, an original aniline degrader, by adaptive laboratory evolution. The evolved strain R. rhodochrous CP-8 could tolerant 8% NaCl with 4-chlorophenol degradation capacity. The synonymous mutation in phosphodiesterase of strain CP-8 may retard the hydrolysis of cyclic adenosine monophosphate (cAMP), which is a key factor reported in the osmoregulation. The experimentally verified up-regulation of intracellular cAMP level in the evolved strain CP-8 contributes to the improvement of growth phenotype under high osmotic condition. Additionally, a point mutant of the catechol 1,2-dioxygenase, CatAN211S, was revealed to show the 1.9-fold increment on activity, which the mechanism was well explained by molecular docking analysis. CONCLUSIONS: This study developed one chlorophenol-degrading strain with extraordinary capacity of salt tolerance, which showed great application potential in hypersaline chlorophenol wastewater treatment. The synonymous mutation in phosphodiesterase resulted in the change of intracellular cAMP concentration and then increase the osmotic tolerance in the evolved strain. The catechol 1,2-dioxygenase mutant with improved activity also facilitated chlorophenol removal since it is the key enzyme in the degradation pathway.

Effects of Surgical Timing of Facial Nerve Decompression for Patients With Severe Bell's Palsy.

Context: Bell's palsy is a form of idiopathic, facial nerve palsy. Initial treatment includes the use of oral corticosteroids and/or antiviral agents, but facial paralysis may persist. Some surgeons suggest that surgical decompression of the facial nerve can be a beneficial, but the optimal surgical approach, extent of nerve decompression, and timing of surgery remain unclear. Objective: This study intended to evaluate the efficacy of delayed, facial nerve decompression for severe Bell's palsy (BP) and to explore the relationship of opportunity timing for operations, with postoperative recovery for facial nerve function. Design: The research team performed a retrospective study. Setting: The study took place at Beijing Tiantan Hospital of Capital Medical University in Beijing, China. Participants: Participants were 45 patients who had been diagnosed with BP between 2015 and 2021 and who had undergone facial nerve decompression using the transmastoid approach, between 30 and 180 days after the onset of BP. According to the operation's timing, the research team divided the participants into three groups, consisting of participants who underwent surgery: (1) at 30-60-days after BP onset-19 participants, (2) at 61-90 days after BP onset-18 participants, and (3) at more than 90 days after BP onset-8 participants. Outcome Measures: The research team: (1) analyzed participants' demographic and preoperative and postoperative clinical characteristics, (2) compared the surgical outcomes with participants' House-Brackmann (HB) scales, and (3) analyzed the factors affecting the recovery of facial nerve function using logistic regression. Results: Decompression surgery was effective for 29 participants (64.4%), with similar rates for participants who underwent surgery after 30-60 days (73.7%) and 61-90 days (77.8%), but the surgery' success was significantly higher for those groups than for participants who underwent surgery after >90 days (12.5%), with P = .008 and P = .003, respectively. Multivariate logistic regression analysis showed that disease duration was the only factor significantly associated with the effectiveness of surgery (odds ratio = 120.337; 95% confidence interval 2.997-4832.267, P = .011). Conclusions: For patients with severe Bell's palsy with ineffective conservative treatment, surgery performed 30 to 90 days after the onset of paralysis can have therapeutic benefits, whereas surgery performed after 3 months is relatively ineffective.

High-Yield Production of Propionate from 1,2-Propanediol by Engineered Pseudomonas putida KT2440, a Robust Strain with Highly Oxidative Capacity.

Bio-based propionate attracts increasing attention owing to its green nature and specific food additive market. To date, the time-consuming and costly fermentation process by strict anaerobes makes propionate production not ideal. In this study, we designed a new route for propionate production, in which 1,2-propanediol was first dehydrated to propionaldehyde and then to propionate by taking advantage of the robust oxidization capacity of the Pseudomonas putida KT2440 strain. The high atom economy (0.97 g/g) in this proposed pathway is more advantageous than the previous l-threonine-derived route (0.62 g/g). The molecular mechanism of the extraordinary oxidation capacity of P. putida KT2440 was first deciphered. The propionate production was realized in P. putida KT2440 by screening suitable glycerol dehydratases and optimizing the expression to eliminate the formation of 1-propanol and the accumulation of the intermediate propionaldehyde. The engineered strain produced propionate with a molar conversion rate of >99% from 1,2-propanediol. A high titer of 46.5 g/L pure propionic acid with a productivity of 1.55 g/L/h and a mass yield of 0.96 g/g was achieved in fed-batch biotransformation. Thus, this study provides another idea for the production of high-purity bio-based propionate from renewable materials with high atom economy.

Mercury bioremediation by engineered Pseudomonas putida KT2440 with adaptationally optimized biosecurity circuit.

Hazardous materials, such as heavy metals, are the major sources of health risk. Using genetically modified organisms (GMOs) to dispose heavy metals has the advantages of strong environmental compatibility and high efficiency. However, the biosecurity of GMOs used in the environment is a major concern. In this study, a self-controlled genetic circuit was designed and carefully fine-tuned for programmable expression in Pseudomonas putida KT2440, which is a widely used strain for environmental bioremediation. The cell behaviours were controlled by automatically sensing the variation of Hg2+ concentration without any inducer requirement or manual interventions. More than 98% Hg2+ was adsorbed by the engineered strain with a high cell recovery rate of 96% from waterbody. The remaining cells were killed by the suicide module after the mission was accomplished. The escape frequency of the engineered P. putida strain was lower than 10-9 , which meets the recommendation of US NIH guideline for GMOs release (<10-8 ). The same performance was achieved in a model experiment by using natural lake water with addition of Hg2+ . The microbial diversity analysis further confirmed that the remediation process made little impact on the indigenous ecosystem. Thus, this study provides a practical method for environmental remediation by using GMOs.

Enhanced removal of P(V), Mo(VI) and W(VI) generated oxyanions using Fe-MOF as adsorbent from hydrometallurgical waste liquid: Exploring the influence of ionic polymerization.

Environmentally hazardous P(V), Mo(VI) and W(VI) generated oxyanions exist widely in the waste liquid of nonferrous hydrometallurgy. In this work, Fe-MOF material was simply prepared via solvothermal synthesis and then used as an adsorbent to remove P(V), Mo(VI) and W(VI) oxyanions from hydrometallurgical waste liquid. Several important parameters, including solution pH, oxyanion concentration, contact time, adsorbent amount, temperature and coexistent heavy metal ions, were systematically investigated. The results demonstrate that adsorption process was almost pH-independent over a broad range of pH 3.0-10.0. The adsorption efficiency was strongly associated with the chemical species of oxyanions. The higher polymerisation degree of oxyanions was more favourable for removal efficiency. Additionally, the maximum removal efficiencies for P(V), Mo(VI) and W(VI) oxyanions under optimum conditions were approximately 100%. Furthermore, the adsorption kinetics and isotherms of oxyanions on the adsorbent separately belonged to the pseudo-second-order and Langmuir isotherm models. XPS analysis revealed that inner-sphere complexation played a dominant role in the adsorption removal process. Fe-MOFs with pH-independent properties, abundant binding sites and high stability are prospective adsorbents for treating waste liquids in the hydrometallurgical industry.

One major facilitator superfamily transporter is responsible for propionic acid tolerance in Pseudomonas putida KT2440.

Propionic acid (PA) has been widely used as a food preservative and chemical intermediate in the agricultural and pharmaceutical industries. Environmental and friendly biotechnological production of PA from biomass has been considered as an alternative to the traditional petrochemical route. However, because PA is a strong inhibitor of cell growth, the biotechnological host should be not only able to produce the compound but the host should be robust. In this study, we identified key PA tolerance factors in Pseudomonas putida KT2440 strain by comparative transcriptional analysis in the presence or absence of PA stress. The identified major facilitator superfamily (MFS) transporter gene cluster of PP_1271, PP_1272 and PP_1273 was experimentally verified to be involved in PA tolerance in P. putida strains. Overexpression of this cluster improved tolerance to PA in a PA producing strain, what is useful to further engineer this robust platform not only for PA synthesis but for the production of other weak acids.

An efficient separation for metal-ions from wastewater by ion precipitate flotation: Probing formation and growth evolution of metal-reagent flocs.

Hazardous metal pollution became a severe environmental issue in China. An efficient precipitation-flotation process was developed to achieve fast removal for metal-ions from wastewater. Structure and strength of precipitate particles/flocs significantly influence the flotation removal of metal-ions. Formation and growth-evolution of precipitate flocs in precipitate flotation were studied by stage analysis of precipitate particles-formation, flocs-regulation and flotation separation. The results demonstrate that early formed precipitates MHA(humics-metal complexing particles) have small particle size, high fractal dimension, low strength and recovery factor. The addition of Fe3+ and CTAB(cetyl trimethyl ammonium bromide) reagents make the precipitate particles aggregated to flocs(MHA-Fe, MHA-Fe-CTAB) much more large, loose, coarse, and small-density. The final generated MHA-Fe-CTAB flocs are hard to be broken up, easy to be recovered and efficient to be separated by flotation process. The flotation removal of MHA-Fe-CTAB flocs is clearly higher than that of MHA or MHA-Fe. The flotation results of MHA-Fe-CTAB are as follows: flotation removal of 98.7 ± 0.40%-99.9 ± 0.10%, residual TOC of 0.96 ± 0.38-1.35 ± 0.41 mg/L and turbidity of 0.44 ± 0.09-0.63 ± 0.16 NTU. Introducing Fe3+ and CTAB reagents into flotation solution contributes to the growth-evolution of precipitate flocs, which could intensify the metal-ions removal via precipitate flotation process and result in more ideal purification indexes for metal-containing wastewater.

[Blink restoration by the functional electrical stimulation in unilateral facial nerve palsy rabbits].

OBJECTIVE: Tocompare the effects of different waveforms and parameters of electrical stimulation to elicit a blink, and construct a functional electrical stimulation (FES) system to restore synchronous blink in unilateral facial nerve palsy (FNP). METHODS: Firstly, twenty-four rabbits were surgically induced unilateral FNP and were divided into three groups, who received square, sine and triangle pulse wareforms, respectirely. Both the healthy and the paralysis eyelids of the rabbits received pulse train stimulation to produce a blink in both eyes. For each rabbit, twenty-seven combinations of frequencies (25 Hz, 50 Hz and 100 Hz) and nine pulse widths (1-9 ms) were stimulated. The threshold amplitude and electric charge to elicit a blink was compared between different waveforms and different parameters. Secondly, a FES system was constructed to treat six surgically induced unilateral FNP rabbit chosen in the twenty-four rabbits, it consisted by an electromyogram (EMG) amplifier module which record the EMG of the healthy muscle, and a stimulator which received the EMG input and output a pulse train stimulation when triggered by the EMG. RESULTS: When the carrier frequency of the pulse train was 25 Hz, it was not able to induce a smooth blink. However, when the carrier frequencies were 50 Hz and 100 Hz, a smooth blink could be induced. The voltage required by 100 Hz was lower than 50 Hz, but it cost more electric charge. The amplitude that square waveforms required was far lower than sine and triangle, but the electric charge between the three waveforms was similar. Synchronous blink could be restored in the six unilateral FNP rabbits with the FES system. CONCLUSIONS: To elicit a blink, square pulse train delivered in 50 Hz is a preferable option. The motion of the healthy eyelids as a source of information for stimulation of the paralyzed sides can restore the synchronous blink in unilateral FNP rabbits.

[Survey of quality of life scale for patients with congenital ear malformation].

OBJECTIVE: To explore the congenital ear malformation (CEM)-specific quality of life (QOL) and examine the effects of total ear reconstruction surgery for QOL of CEM patients. METHODS: A self-composed QOL scale was used for 129 patients with congenital external and middle ear malformation. All patients were requested to fill in the QOL scale before and 1 month after ear reconstruction surgery. RESULTS: The level of QOL varied according to the degree of ear malformation. The total QOL score of patients with unilateral and bilateral CEM was 28.5 ± 18.4 and 51.6 ± 23.6, respectively. The total QOL score of patients pre- and post-operation was 21.0 ± 14.0 and 14.2 ± 9.7 respectively. Physiological functions, psychological status and social interactions of the patients were of statistical significance after ear reconstruction surgery compared to that of at pre-operation. CONCLUSION: Congenital ear malformation-specific QOL scale can show sensitively the changes of QOL of CEM patients. And ear reconstruction surgery is beneficial for the patients.