Controllable detection threshold achieved through the toehold switch system in a mercury ion whole-cell biosensor.

Due to the toxicity of mercury and its harmful effects on human health, it is essential to establish a low-cost, highly sensitive and highly specific monitoring method with a wide detection range, ideally with a simple visual readout. In this study, a whole-cell biosensor with adjustable detection limits was developed for the detection of mercury ions in water samples, allowing controllable threshold detection with an expanded detection range. Gene circuits were constructed by combining the toehold switch system with lactose operon, mercury-ion-specific operon, and inducible red fluorescent protein gene. Using MATLAB for design and selection, a total of eleven dual-input single-output sensing logic circuits were obtained based on the basic logic of gene circuit construction. Then, biosensor DTS-3 was selected based on its fluorescence response at different isopropyl ß-D-Thiogalactoside (IPTG) concentrations, exhibiting the controllable detection threshold. At 5-20 µM IPTG, DTS-3 can achieve variable threshold detection in the range of 0.005-0.0075, 0.06-0.08, 1-2, and 4-6 µM mercury ion concentrations, respectively. Specificity experiments demonstrated that DTS-3 exhibits good specificity, not showing fluorescence response changes compared with other metal ions. Furthermore spiked sample experiments demonstrated its good resistance to interference, allowing it to distinguish mercury ion concentrations as low as 7.5 nM by the naked eye and 5 nM using a microplate reader. This study confirms the feasibility and performance of biosensor with controllable detection threshold, providing a new detection method and new ideas for expanding the detection range of biosensors while ensuring rapid and convenient measurements without compromising sensitivity.

Adsorption of Hg2+/Cr6+ by metal-binding proteins heterologously expressed in Escherichia coli.

BACKGROUND: Removal of heavy metals from water and soil is a pressing challenge in environmental engineering, and biosorption by microorganisms is considered as one of the most cost-effective methods. In this study, the metal-binding proteins MerR and ChrB derived from Cupriavidus metallidurans were separately expressed in Escherichia coli BL21 to construct adsorption strains. To improve the adsorption performance, surface display and codon optimization were carried out. RESULTS: In this study, we constructed 24 adsorption engineering strains for Hg2+ and Cr6+, utilizing different strategies. Among these engineering strains, the M'-002 and B-008 had the strongest heavy metal ion absorption ability. The M'-002 used the flexible linker and INPN to display the merRopt at the surface of the E. coli BL21, whose maximal adsorption capacity reached 658.40 µmol/g cell dry weight under concentrations of 300 µM Hg2+. And the B-008 overexpressed the chrB in the intracellular, its maximal capacity was 46.84 µmol/g cell dry weight under concentrations 500 µM Cr6+. While in the case of mixed ions solution (including Pb2+, Cd2+, Cr6+ and Hg2+), the total amount of ions adsorbed by M'-002 and B-008 showed an increase of up to 1.14- and 4.09-folds, compared to the capacities in the single ion solution. CONCLUSION: The construction and optimization of heavy metal adsorption strains were carried out in this work. A comparison of the adsorption behavior between single bacteria and mixed bacteria systems was investigated in both a single ion and a mixed ion environment. The Hg2+ absorption capacity is reached the highest reported to date with the engineered strain M'-002, which displayed the merRopt at the surface of chassis cell, indicating the strain's potential for its application in practical environments.

Microbial Consortium HJ-SH with Very High Degradation Efficiency of Phenanthrene.

Phenanthrene (PHE) is one of the model compounds of polycyclic aromatic hydrocarbons (PAHs). In this study, a natural PHE-degrading microbial consortium, named HJ-SH, with very high degradation efficiency was isolated from soil exposed to long-term PHE contamination. The results of GC analysis showed that the consortium HJ-SH degraded 98% of 100 mg/L PHE in 3 days and 93% of 1000 mg/L PHE in 5 days, an efficiency higher than that of any other natural consortia, and even most of the engineered strains and consortia reported so far. Seven dominating strains were isolated from the microbial consortium HJ-SH, named SH-1 to SH-7, which were identified according to morphological observation and 16S rDNA sequencing as Pseudomonas sp., Stenotrophomonas sp., Delftia sp., Pseudomonas sp., Brevundimonas sp., Curtobacterium sp., and Microbacterium sp., respectively. Among all the seven single strains, SH-4 showed the strongest PHE degradation ability, and had the biggest degradation contribution. However, it is very interesting that the microbial consortium can hold its high degradation ability only with the co-existence of all these seven single strains. Moreover, HJ-SH exhibited a very high tolerance for PHE, up to 4.5 g/L, and it can degrade some other typical organic pollutants such as biphenyl, anthracene, and n-hexadecane with the degradation ratios of 93%, 92% and 70%, respectively, under 100 mg/L initial concentration in 5 days. Then, we constructed an artificial consortium HJ-7 consisting of the seven single strains, SH-1 to SH-7. After comparing the degradation ratios, cell growth, and relative degradation rates, it was concluded that the artificial consortium HJ-7 with easier reproducibility, better application stability, and larger room for modification can largely replace the natural consortium HJ-SH. In conclusion, this research provided novel tools and new insights for the bioremediation of PHE and other typical organic pollutants using microbial consortia.

Engineering a Pseudomonas putida as living quorum quencher for biofilm formation inhibition, benzenes degradation, and environmental risk evaluation.

Bacterial communication interruption based on quorum quenching (QQ) has been proven its potential in biofilm formation inhibition and biofouling control. However, it would be more satisfying if QQ could be combined with the efficient degradation of contaminants in environmental engineering. In this study, we engineered a biofilm of Pseudomonas putida through introducing a QQ synthetic gene, which achieved both biofilm formation inhibition and efficient degradation of benzene series in wastewater. The aiiO gene introduced into the P. putida by heat shock method was highly expressed to produce QQ enzyme to degrade AHL-based signal molecules. The addition of this engineered P. putida reduced the AHLs concentration, quorum sensing gene expression, and connections of the microbial community network in activated sludge and therefore inhibited the biofilm formation. Meanwhile, the sodium benzoate degradation assay indicated an enhanced benzene series removal ability of the engineering bacteria on activated sludge. Besides, we also demonstrated a controllable environmental risk of this engineered bacteria through monitoring its abundance and horizontal gene transfer test. Overall, the results of this study suggest an alternative strategy to solve multiple environmental problems through genetic engineering means and provide support for the application of engineered bacteria in environmental biotechnology.

Association between constipation and major depression in adult Americans: evidence from NHANES 2005-2010.

Objective: Current studies on the association between constipation and depression is still insufficient. In this study, we investigated the detailed association between constipation and major depression among American adults. Methods: In this cross-sectional study, 12,352 adults aged 20 and older were selected from the National Health and Nutrition Examination Survey 2005-2010 for the sample. Constipation was defined as fewer than three defecation frequencies per week. For the assessment of major depression, the validated Patient Health Questionnaire-9 was used. Adjusted odds ratios (ORs) were calculated using multivariate logistic regression models. A subgroup analysis was carried out to ensure that the results were stable. Results: Of the 12,352 participants, 430 reported constipation, with a prevalence of 3.5%. Depression was reported in 1030 cases, indicating a prevalence rate of 8.3%. Patients with constipation were significantly more likely to have major depression (20.9%) than those without it (7.9%, p < 0.001). After adjusting for age, sex, race/ethnicity, marital status, education level, body mass index, vigorous physical activity, alcohol consumption, smoking status, poverty income ratio, diabetes, selective serotonin reuptake inhibitor use, liver disease, heart disease, pulmonary disease, hypertension, arthritis, cancer, dietary fiber intake, moisture intake, total fat intake, carbohydrates intake, and protein intake, constipation is significantly associated with major depression (OR: 2.20, 95%CI: 1.68-2.87, p < 0.001). Subgroup analyses by age, sex, dietary intake, risk behaviors, and common complications showed no statistically significant interactions (p > 0.05). Conclusion: In conclusion, this study showed that constipation were significantly associated with depression. When treating patients with constipation, it is necessary for clinicians to screen and evaluate depression, and provide timely and effective intervention for patients with depression to avoid further deterioration of the condition.

Association between fecal incontinence and suicidal ideation in adult Americans: Evidence from NHANES 2005-2010.

OBJECTIVES: Currently, research on the relationship between fecal incontinence (FI) and suicidal ideation is very limited. This study aims to evaluate whether FI is associated with suicidal ideation among US adults. METHODS: In this cross-sectional study, 13,480 adults aged 20 years and older were selected from the National Health and Nutrition Examination Survey 2005-2010. Monthly loss of solid, liquid, or mucous stool was defined as FI. As part of the Patient Health Questionnaire-9, item 9 assessed suicidal ideation. Models of multivariate logistic regression were used to calculate adjusted odds ratios. A subgroup analysis was carried out to ensure that the results were stable. RESULTS: It was found that FI was associated with an increased risk of suicidal ideation after controlling for baseline characteristics, risk behaviors, and comorbid conditions such as depression (OR: 1.60, 95%CI: 1.24-2.08, P < 0.001). In subgroup analyses, FI remained significantly associated with suicidal ideation among participants aged 45 years and older, with odds ratios and 95% confidence intervals of 1.62 (1.11-2.38) and 2.49 (1.51-4.13), respectively. For age category <45 years, the association between FI and suicidal ideation weakened (OR: 1.02, 95%CI: 0.60-1.75, P = 0.932). CONCLUSIONS: In conclusion, this study showed that FI was significantly associated with suicidal ideation. Patients aged middle-aged and older are at high risk and should be the focus of screening and timely intervention for suicidal ideation.

Improvement of a highly sensitive and specific whole-cell biosensor by adding a positive feedback amplifier.

In this study, we designed a Cd2+ whole-cell biosensor with both positive and negative feedback cascade amplifiers in Pseudomonas putida KT2440 (LTCM) based on our previous design with only a negative feedback amplifier (TCM). The results showed that the newly developed biosensor LTCM was greatly improved compared to TCM. Firstly, the linear response range of LTCM was expanded while the maximum linear response range was raised from 0.05 to 0.1 µM. Meanwhile, adding a positive feedback amplifier further increased the fluorescence output signal of LTCM 1.11-2.64 times under the same culture conditions. Moreover, the response time of LTCM for detection of practical samples was reduced from 6 to 4 h. At the same time, LTCM still retained very high sensitivity and specificity, while its lowest detection limit was 0.1 nM Cd2+ and the specificity was 23.29 (compared to 0.1 nM and 17.55 in TCM, respectively). In summary, the positive and negative feedback cascade amplifiers effectively improved the performance of the biosensor LTCM, resulting in a greater linear response range, higher output signal intensity, and shorter response time than TCM while retaining comparable sensitivity and specificity, indicating better potential for practical applications.

Reconstruction and optimization of a Pseudomonas putida-Escherichia coli microbial consortium for mcl-PHA production from lignocellulosic biomass.

The demand for non-petroleum-based, especially biodegradable plastics has been on the rise in the last decades. Medium-chain-length polyhydroxyalkanoate (mcl-PHA) is a biopolymer composed of 6-14 carbon atoms produced from renewable feedstocks and has become the focus of research. In recent years, researchers aimed to overcome the disadvantages of single strains, and artificial microbial consortia have been developed into efficient platforms. In this work, we reconstructed the previously developed microbial consortium composed of engineered Pseudomonas putida KT∆ABZF (p2-a-J) and Escherichia coli ∆4D (ACP-SCLAC). The maximum titer of mcl-PHA reached 3.98 g/L using 10 g/L glucose, 5 g/L octanoic acid as substrates by the engineered P. putida KT∆ABZF (p2-a-J). On the other hand, the maximum synthesis capacity of the engineered E. coli ∆4D (ACP-SCLAC) was enhanced to 3.38 g/L acetic acid and 0.67 g/L free fatty acids (FFAs) using 10 g/L xylose as substrate. Based on the concept of "nutrient supply-detoxification," the engineered E. coli ∆4D (ACP-SCLAC) provided nutrient for the engineered P. putida KT∆ABZF (p2-a-J) and it acted to detoxify the substrates. Through this functional division and rational design of the metabolic pathways, the engineered P. putida-E. coli microbial consortium could produce 1.30 g/L of mcl-PHA from 10 g/L glucose and xylose. Finally, the consortium produced 1.02 g/L of mcl-PHA using lignocellulosic hydrolysate containing 10.50 g/L glucose and 10.21 g/L xylose as the substrate. The consortium developed in this study has good potential for mcl-PHA production and provides a valuable reference for the production of high-value biological products using inexpensive carbon sources.

Engineering Pseudomonas putida To Produce Rhamnolipid Biosurfactants for Promoting Phenanthrene Biodegradation by a Two-Species Microbial Consortium.

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic contaminants that pose a significant environmental hazard. Phenanthrene is one of the model compounds for the study of biodegradation of PAHs. However, the biodegradation of phenanthrene is often limited by its low water solubility and dissolution rate. To overcome this limitation, we engineered a strain of Pseudomonas putida to produce rhamnolipid biosurfactants and thereby promote phenanthrene biodegradation by an engineered strain of Escherichia coli constructed previously in our lab. The E. coli-P. putida two-species consortium exhibited a synergistic effect of these two distinct organisms in degrading phenanthrene, resulting in an increase from 61.15 to 73.86% of the degradation ratio of 100 mg/L phenanthrene within 7 days. After additional optimization of the degradation conditions, the phenanthrene degradation ratio was improved to 85.73%. IMPORTANCE Polycyclic aromatic hydrocarbons (PAHs), which are recalcitrant, carcinogenic, and tend to bioaccumulate, are widespread and persistent environmental pollutants. Based on these characteristics, the U.S. Environmental Protection Agency has listed PAHs as priority contaminants. Although there are many methods to treat PAH pollution, these methods are mostly limited by the poor water solubility of PAHs, which is especially true for the biodegradation process. Recent evidence of PAH-contaminated sites suffering from increasingly severe impact has emerged. As a result, the need to degrade PAHs is becoming urgent. The significance of our study lies in the development of nonpathogenic strains of biosurfactant-producing Pseudomonas aeruginosa for promoting the degradation of phenanthrene by engineered Escherichia coli.

Prognostic Value of the Albumin-to-Globulin Ratio in Patients with Colorectal Cancer: A Meta-Analysis.

Recently, the albumin-to-globulin ratio (AGR) has been investigated as a prognostic parameter in patients with colorectal cancer (CRC); however, the results remain inconsistent. We aimed to quantitatively identify the prognostic role of the AGR in CRC through meta-analysis. Combined hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to assess the prognostic value of the AGR for overall survival (OS), disease-free survival (DFS)/progression-free survival (PFS), and cancer-specific survival (CSS). The association between the AGR and clinicopathological factors was investigated using pooled odds ratios (ORs) and 95% CIs. Eleven studies, comprising 8,397 patients, were included in this meta-analysis. Our results demonstrated that a low AGR was significantly associated with poor OS (HR, 2.58; 95% CI, 1.90-3.51; p < 0.001) and poor DFS/PFS (HR, 2.11; 95% CI, 1.46-3.05; p < 0.001) in CRC. However, the AGR was not a significant prognostic factor for CSS (HR, 1.008; 95% CI, 0.372-2.730; p = 0.988). In addition, a low AGR was associated with patients aged ≥60 years (OR, 1.71; 95% CI, 1.54-1.89; p < 0.001). A low AGR was significantly associated with worse OS and inferior DFS/PFS in patients with CRC. Thus, AGR can be used as a cost-effective and reliable prognostic marker for CRC in clinical practice.

Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralization.

Mineralization catalyzed by carbonic anhydrase (CA) is one of the most promising technologies for capturing CO2. In this work, Escherichia coli BL21(DE3) was used as the host, and the N-terminus of ice nucleation protein (INPN) was used as the carrier protein. Different fusion patterns and vectors were used to construct CA surface display systems for α-carbonic anhydrase (HPCA) from Helicobacter pylori 26695 and α-carbonic anhydrase (SazCA) from Sulfurihydrogenibium azorense. The surface display system in which HPCA was fused with INPN via a flexible linker and intermediate repeat sequences showed higher whole-cell enzyme activity, while the enzyme activity of the SazCA expression system was significantly higher than that of the HPCA expression system. The pET22b vector with the signal peptide PelB was more suitable for the cell surface display of SazCA. Cell fractionation and western-blot analysis indicated that SazCA and INPN were successfully anchored on the cell's outer membrane as a fusion protein. The enzyme activity of the surface display strain E-22b-IRLS (11.43 U·mL-1OD600 -1) was significantly higher than that of the intracellular expression strain E-22b-S (8.355 U·mL-1OD600 -1) under optimized induction conditions. Compared with free SazCA, E-22b-IRLS had higher thermal and pH stability. The long-term stability of SazCA was also significantly improved by surface display. When the engineered strain and free enzyme were used for CO2 mineralization, the amount of CaCO3 deposition catalyzed by the strain E-22b-IRLS on the surface (241 mg) was similar to that of the free SazCA and was significantly higher than the intracellular expression strain E-22b-S (173 mg). These results demonstrate that the SazCA surface display strain can serve as a whole-cell biocatalyst for CO2 capture and mineralization.

Artificial Consortium of Three E. coli BL21 Strains with Synergistic Functional Modules for Complete Phenanthrene Degradation.

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic and persistent organic pollutions that can accumulate in the environment. In this study, an aromatic ring cleavage module, a salicylic acid synthesis module, and a catechol metabolism module were respectively constructed in three Escherichia coli BL21 strains. Subsequently, the engineered strains were cocultured as an artificial consortium for the biodegradation of phenanthrene, a typical PHA. Single factor experiments and response surface methodology were used to identify the optimal degradation conditions, including an inoculation interval of 6 h, inoculation ratio of 1:1:1, and IPTG concentration of 2 mM. Under these conditions, the 7-day degradation ratio of 100 mg/L phenanthrene reached 72.67%. Moreover, the engineered Escherichia coli BL21 strains showed good phenanthrene degradation ability at substrate concentrations 10 mg/L up to 500 mg/L. Enzyme activity assays combined with gas chromatography-mass spectrometry measurements confirmed that the three engineered strains behaved as a synergistic consortium in the phenanthrene degradation process. Based on the analysis of the key metabolites, the engineered bacteria were supplemented at 7-day intervals in batches so that each engineered strain maintained its optimal degradation ability. The 21-day degradation ratio finally reached 90.66%, which was much higher than what was observed with simultaneous inoculation. These findings suggest that the three engineered strains with separate modules constructed in this study offer an attractive solution for removing PAHs from the environment.

Highly Sensitive Whole-Cell Biosensor for Cadmium Detection Based on a Negative Feedback Circuit.

Although many whole-cell biosensors (WCBs) for the detection of Cd2+ have been developed over the years, most lack sensitivity and specificity. In this paper, we developed a Cd2+ WCB with a negative feedback amplifier in P. putida KT2440. Based on the slope of the linear detection curve as a measure of sensitivity, WCB with negative feedback amplifier greatly increased the output signal of the reporter mCherry, resulting in 33% greater sensitivity than in an equivalent WCB without the negative feedback circuit. Moreover, WCB with negative feedback amplifier exhibited increased Cd2+ tolerance and a lower detection limit of 0.1 nM, a remarkable 400-fold improvement compared to the WCB without the negative feedback circuit, which is significantly below the World Health Organization standard of 27 nM (0.003 mg/L) for cadmium in drinking water. Due to the superior amplification of the output signal, WCB with negative feedback amplifier can provide a detectable signal in a much shorter time, and a fast response is highly preferable for real field applications. In addition, the WCB with negative feedback amplifier showed an unusually high specificity for Cd2+ compared to other metal ions, giving signals with other metals that were between 17.6 and 41.4 times weaker than with Cd2+. In summary, the negative feedback amplifier WCB designed in this work meets the requirements of Cd2+ detection with very high sensitivity and specificity, which also demonstrates that genetic negative feedback amplifiers are excellent tools for improving the performance of WCBs.

Construction of cadmium whole-cell biosensors and circuit amplification.

Owing to the prevalence of cadmium contamination and its serious hazards, it is important to establish an efficient and low-cost monitoring technique for the detection of the heavy metal cadmium. In this study, we first designed 30 cadmium whole-cell biosensors (WCBs) using different combinations of detection elements, reporting elements, and the host. The best performing WCB KT-5-R with Pseudomonas putida KT2440 as the host and composed of CadR and mCherry was selected for further analysis and engineering. In order to enhance its sensitivity, a positive feedback amplifier was added or the gene dosage of the reporter gene was increased. The WCB with the T7RNAP amplification module, p2T7RNAPmut-68, had the best performance and improved tolerance to cadmium with a detection limit of 0.01 µM, which is the WHO standard. It also showed excellent specificity toward cadmium when assayed with mixed metal ions. This study demonstrated the power of circuit engineering in WCB design and provided valuable insights for the development of other WCBs. KEY POINTS: • KT-5-R was selected after prescreening and engineered for better performance. • Using multi-copy reporters and the T7RNAP amplifier greatly improved the performance. • p2T7RNAPmut-68 had a detection limit of 0.01 µM and improved tolerance to cadmium.

Effect of High Suspension and Low Incision Surgery Based on Traditional Ligation of Chinese Medicine in Treatment of Mixed Haemorrhoids: A Multi-centre, Randomized, Single-Blind, Non-inferiority Clinical Trial.

OBJECTIVE: To observe the clinical effect of high suspension and low incision (HSLI) surgery on mixed haemorrhoids, compared with Milligan-Morgan haemorrhoidectomy. METHODS: A multi-centre, randomized, single-blind, non-inferiority clinical trial was performed. Participants with mixed haemorrhoids from Xiyuan Hospital of China Academy of Chinese Medical Sciences, Beijing Rectum Hospital, Air Force Medical Center of People's Liberation Army of China, and Puyang Hospital of Traditional Chinese Medicine were enrolled from September 2016 to March 2018. By using a blocked randomization scheme, participants were assigned to two groups. The experimental group was treated with HSLI, while the control group was treated with Milligan-Morgan haemorrhoidectomy. The primary outcome was the clinical effect evaluated at 12 weeks after operation. The secondary outcomes included the number of haemorrhoids treated during the operation, pain scores, use of analgesics, postoperative oedema, wound healing, incidence of anal stenosis, anorectal manometry after operation, as well as surgical duration, length of stay and total hospitalization expenses. A safety evaluation was also conducted. RESULTS: In total, 246 eligible participants were enrolled, with 123 cases in each group. There was no significant difference in the clinical effect between the two groups (100.00% vs. 99.19%, P>0.05). Compared with the control group, the number of external haemorrhoids treated during the operation and the pain scores after operation were significantly reduced in the experimental group (P<0.05 or P<0.01); the patient number with wound healing at 2 weeks after operation and the functional length of anal canal at 12 weeks after operation were significantly increased in the experimental group (P<0.05). There was no significant difference in the incidence of anal stenosis, the numbers of patients using analgesics and patients with postoperative oedema between the two groups after operation (P>0.05). The surgical duration and length of stay in the experimental group were significantly longer than those in the control group, and the total hospitalization expense was significantly higher than that in the control group (all P<0.05). No adverse events were reported in either group during the whole trial or follow-up period. CONCLUSION: HSLI had the advantages of preserving the skin of anal canal completely, alleviating postsurgical pain and promoting rapid recovery after operation. (Registration No. ChiCTR1900022883).

Recent advances in constructing artificial microbial consortia for the production of medium-chain-length polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are a class of high-molecular-weight polyesters made from hydroxy fatty acid monomers. PHAs produced by microorganisms have diverse structures, variable physical properties, and good biodegradability. They exhibit similar physical properties to petroleum-based plastics but are much more environmentally friendly. Medium-chain-length polyhydroxyalkanoates (mcl-PHAs), in particular, have attracted much interest because of their low crystallinity, low glass transition temperature, low tensile strength, high elongation at break, and customizable structure. Nevertheless, high production costs have hindered their practical application. The use of genetically modified organisms can reduce production costs by expanding the scope of substrate utilization, improving the conversion efficiency of substrate to product, and increasing the yield of mcl-PHAs. The yield of mcl-PHAs produced by a pure culture of an engineered microorganism was not high enough because of the limitations of the metabolic capacity of a single microorganism. The construction of artificial microbial consortia and the optimization of microbial co-cultivation have been studied. This type of approach avoids the addition of precursor substances and helps synthesize mcl-PHAs more efficiently. In this paper, we reviewed the design and construction principles and optimized control strategies for artificial microbial consortia that produce mcl-PHAs. We described the metabolic advantages of co-cultivating artificial microbial consortia using low-value substrates and discussed future perspectives on the production of mcl-PHAs using artificial microbial consortia.

Optimization of a Two-Species Microbial Consortium for Improved Mcl-PHA Production From Glucose-Xylose Mixtures.

Polyhydroxyalkanoates (PHAs) have attracted much attention as a good substitute for petroleum-based plastics, especially mcl-PHA due to their superior physical and mechanical properties with broader applications. Artificial microbial consortia can solve the problems of low metabolic capacity of single engineered strains and low conversion efficiency of natural consortia while expanding the scope of substrate utilization. Therefore, the use of artificial microbial consortia is considered a promising method for the production of mcl-PHA. In this work, we designed and constructed a microbial consortium composed of engineered Escherichia coli MG1655 and Pseudomonas putida KT2440 based on the "nutrition supply-detoxification" concept, which improved mcl-PHA production from glucose-xylose mixtures. An engineered E. coli that preferentially uses xylose was engineered with an enhanced ability to secrete acetic acid and free fatty acids (FFAs), producing 6.44 g/L acetic acid and 2.51 g/L FFAs with 20 g/L xylose as substrate. The mcl-PHA producing strain of P. putida in the microbial consortium has been engineered to enhance its ability to convert acetic acid and FFAs into mcl-PHA, producing 0.75 g/L mcl-PHA with mixed substrates consisting of glucose, acetic acid, and octanoate, while also reducing the growth inhibition of E. coli by acetic acid. The further developed artificial microbial consortium finally produced 1.32 g/L of mcl-PHA from 20 g/L of a glucose-xylose mixture (1:1) after substrate competition control and process optimization. The substrate utilization and product synthesis functions were successfully divided into the two strains in the constructed artificial microbial consortium, and a mutually beneficial symbiosis of "nutrition supply-detoxification" with a relatively high mcl-PHA titer was achieved, enabling the efficient accumulation of mcl-PHA. The consortium developed in this study is a potential platform for mcl-PHA production from lignocellulosic biomass.

The prognostic role of age in primary cutaneous B-cell lymphoma: a proposal derived from a population-based registry.

PURPOSE: A few prognostic predicting systems existed for primary cutaneous B-cell lymphoma (PCBCL). However, none of them took age into consideration. We sought to declare the prognostic role of age in PCBCL. MATERIALS AND METHODS: A total of 4859 patients were identified in the surveillance, epidemiology and end results data, spanning 1975-2016. The association between age and survival was determined using unadjusted and adjusted proportional hazard analysis. RESULTS: There was a uni-modal distribution of age, with most in the seventh decade (22%), followed by the sixth and eighth decades (19%). As a continuous variable, age was demonstrated to have an adverse effect on overall survival (OS, HR, 1.077, 95% CI 1.073-1.082, p = 0.000) and cancer-specific survival (CSS, HR, 1.099, 95% CI 1.092-1.106, p = 0.000) after adjusted proportional hazard analysis. Patients aged ≤ 60 years had significantly higher survival rates than those aged > 60 (5-year OS was 93% vs 64%, 10-year OS was 90% vs 45%, p = 0.000; 5-year CSS was 98% vs 80%, 10-year CSS 96% was 62%, p = 0.000). Similar survival trends were also observed in different sub-group analyses, including disease with different stages (p = 0.000), different histology subgroups (p = 0.000), and different sites (p = 0.000). CONCLUSION: Age has an important effect on overall survival and cancer-specific survival. The addition of age to the primary site, histology, and stage improves predicting long-term outcomes in cutaneous B-cell lymphoma.

A four-microorganism three-step fermentation process for producing medium-chain-length polyhydroxyalkanoate from starch.

In this study, a four-microorganism three-step fermentation process was established for producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) from starch, which was used as the sole carbon source. The four microorganisms used for this process were Aspergillus niger, Saccharomyces cerevisiae L2612, Acetobacter orientalis, and Pseudomonas putida KT2440-acs. The initial carbon source starch concentration was set to 30 g/L, the maximum glucose concentration reached 17.66 g/L at 48 h after starch hydrolysis, and then, 2.36 g/L of acetic acid was obtained at 96 h. The final output of mcl-PHA was 0.5 g/L at 144 h, overall productivity for mcl-PHA was 3.47 mg/(L·h) and the total starch to mcl-PHA yield for the process was 16.67 mg/g. Although the overall yield and conversion rate of this process were not high, this is the first attempt to produce mcl-PHA using starch as a substrate, and it provides a feasible strategy for producing PHA from kitchen waste. The production process of mcl-PHA with a clear flora structure and short fermentation cycle was realized.

Display of lead-binding proteins on Escherichia coli surface for lead bioremediation.

Cell surface display of heavy metal-binding proteins has been used to enhance the adsorption capacity of heavy metals and the engineered microbial cells can be potentially used for the bioremediation of heavy metals. In this study, the proteins PbrR, PbrR691, and PbrD from the Cupriavidus metallidurans strain CH34 were displayed on the extracellular membrane of Escherichia coli BL21 cells, with the N-domain of ice-nucleation protein as the anchor protein to achieve specific adsorption of lead ions (Pb2+ ) and bioremediation of lead in the soil. The localization of fusion proteins was confirmed by western blot analysis. We investigated the effects of fusion pattern, expression level, heavy metal concentration, and the presence of other heavy metal ions on the adsorption of Pb2+ by these engineered bacteria, and the optimal linker peptide (flexible linker) and inducer concentration (0.5 mM) were obtained. The engineered bacteria showed specific selectivity and strong adsorption capacity for Pb2+ . The maximum Pb2+ adsorption capacity of strains displaying the three proteins (PbrR, PbrR691, and PbrD) were 942.1-, 754.3-, and 864.8-µmol/g cell dry weight, respectively, which was the highest reported to date. The engineered E. coli bacteria were also applied to Pb2+ -contaminated soil and the detoxification effects were observed via the seed germination test and the growth of Nicotiana benthamiana in comparison with the control BL21, which provides the proof-of-concept for in situ remediations of Pb2+ -contaminated water or soil.