Synthetic acid stress-tolerance modules improve growth robustness and lysine productivity of industrial Escherichia coli in fermentation at low pH.

BACKGROUND: During fermentation, industrial microorganisms encounter multiple stresses that inhibit cell growth and decrease fermentation yields, in particular acid stress, which is due to the accumulation of acidic metabolites in the fermentation medium. Although the addition of a base to the medium can counteract the effect of acid accumulation, the engineering of acid-tolerant strains is considered a more intelligent and cost-effective solution. While synthetic biology theoretically provides a novel approach for devising such tolerance modules, in practice it is difficult to assemble stress-tolerance modules from hundreds of stress-related genes. RESULTS: In this study, we designed a set of synthetic acid-tolerance modules for fine-tuning the expression of multi-component gene blocks comprising a member of the proton-consuming acid resistance system (gadE), a periplasmic chaperone (hdeB), and reactive oxygen species (ROS) scavengers (sodB and katE). Directed evolution was used to construct an acid-responsive asr promoter library, from which four variants were selected and used in the synthetic modules. The module variants were screened in a stepwise manner under mild acidic conditions (pH 5-6), first by cell growth using the laboratory Escherichia coli strain MG1655 cultured in microplates, and then by lysine production performance using the industrial lysine-producing E. coli strain MG1655 SCEcL3 cultured first in multiple 10-mL micro-bioreactors, and then in 1.3-L parallel bioreactors. The procedure resulted in the identification of a best strain with lysine titer and yield at pH 6.0 comparable to the parent strain at pH 6.8. CONCLUSION: Our results demonstrate a promising synthetic-biology strategy to enhance the growth robustness and productivity of E. coli upon the mildly acidic conditions, in both a general lab strain MG1655 and an industrial lysine-producing strain SCEcL3, by using the stress-responsive synthetic acid-tolerance modules comprising a limited number of genes. This study provides a reliable and efficient method for achieving synthetic modules of interest, particularly in improving the robustness and productivity of industrial strains.

Performance of Stepwise Screening Methods in Identifying Individuals at High Risk of Type 2 Diabetes in an Iranian Population.

BACKGROUND: Recent evidence recommended stepwise screening methods for identifying individuals at high risk of type 2 diabetes to be recruited in the lifestyle intervention programs for the prevention of the disease. This study aims to assess the performance of different stepwise screening methods that combine non-invasive measurements with lab-based measurements for identifying those with 5-years incident type 2 diabetes. METHODS: 3037 participants aged ≥30 years without diabetes at baseline in the Tehran Lipid and Glucose Study (TLGS) were followed. Thirty-two stepwise screening methods were developed by combining a non-invasive measurement (an anthropometric measurement (waist-to-height ratio, WtHR) or a score based on a non-invasive risk score [Australian Type 2 Diabetes Risk Assessment Tool, AUSDRISK]) with a lab-based measurement (different cut-offs of fasting plasma glucose [FPG] or predicted risk based on three lab-based prediction models [Saint Antonio, SA; Framingham Offspring Study, FOS; and the Atherosclerosis Risk in Communities, ARIC]). The validation, calibration, and usefulness of lab-based prediction models were assessed before developing the stepwise screening methods. Cut-offs were derived either based on previous studies or decision-curve analyses. RESULTS: 203 participants developed diabetes in 5 years. Lab-based risk prediction models had good discrimination power (area under the curves [AUCs]: 0.80-0.83), achieved acceptable calibration and net benefits after recalibration for population's characteristics and were useful in a wide range of risk thresholds (5%-21%). Different stepwise methods had sensitivity ranged 20%-68%, specificity 70%-98%, and positive predictive value (PPV) 14%-46%; they identified 3%-33% of the screened population eligible for preventive interventions. CONCLUSION: Stepwise methods have acceptable performance in identifying those at high risk of incident type 2 diabetes.

Development of phytoremediator screening strategy and exploration of Pennisetum aided chromium phytoremediation mechanisms in soil.

Screening of chromium (Cr) phytoremediators (i.e., hyperaccumulator plants and accumulation plants) is essential for the phytoremediation of Cr-contaminated soils but less tackled previously. In this study, we proposed a stepwise strategy for screening Cr phytoremediators and explored tolerance mechanism of the screened species. To achieve effective screening of Cr phytoremediators, seed germination, hydroponic, and pot experiment were performed sequentially, and an improved indicator system was established accordingly. Pennisetum was selected from nine plants, with its high growth rate and Cr remediation efficiency successfully demonstrated in the field. Antioxidant enzymes (i.e., superoxide dismutase (SOD) and catalase (CAT)) and photosynthesis under Cr stress were monitored for tracking the tolerance mechanism. Results showed that the enhanced SOD and CAT contributed to the strong tolerance of Pennisetum to Cr. The SOD and CAT were positively correlated with net photosynthetic rate (Pn), resulting in a phenomenon that Cr had no significant effect on Pn of Pennisetum even at 400 mg kg-1. The research findings helped obtain powerful Cr phytoremediators, deepen our understanding of the tolerance mechanisms associated with phytoremediation, and eventually facilitate effective Cr removal in soil.

Selection of plant growth promoting rhizobacteria sharing suitable features to be commercially developed as biostimulant products.

Plant biostimulants (PBs) are an eco-friendly alternative to chemical fertilisers because of their minimal or null impact on human health and environment, while ensuring optimal nutrient uptake and increase of crop yield, quality and tolerance to abiotic stress. Although there is an increasing interest on microbial biostimulants, the optimal procedure to select and develop them as commercial products is still not well defined. This work proposes and validates a procedure to select the best plant growth promoting rhizobacteria (PGPR) as potential active ingredients of commercial PBs. The stepwise screening strategy was designed based on literature analysis and consists of six steps: (i) determination of the target crop and commercial strategy, (ii) selection of growth media for the isolation of microbial candidates, (iii) screening for traits giving major agronomical advantages, (iv) screening for traits related to product development, (v) characterisation of the mode of action of PGPR and (vi) assessment of plant growth efficacy. The strategy was validated using a case study: PGPR combined with humic acids to be applied on tomato plants. Among 200 bacterial strains isolated from tomato rhizosphere, 39 % were able to grow in presence of humic acids and shared the ability to solubilise phosphate. After the screening for traits related to product development, only 6 % of initial bacterial strains were sharing traits suitable for the further development as potential PBs. In fact, the selected bacterial strains were able to produce high cell mass and tolerated drought, aspects important for the mass production and formulation. These bacterial strains were not able to produce antibiotics, establish pathogenic interaction with plants and did not belong to bacterial species associated to human, animal and plant diseases. Most importantly, five of the selected bacterial strains were able to promote tomato seedling vigour in experiments carried out in vitro. These bacterial strains were furtherly characterised for their ability to colonize effectively tomato plant roots, produce phytohormones and solubilise soil minerals. This characterisation led to the selection of two candidates that showed the ability to promote tomato plant growth in experiments carried out in greenhouse conditions. Overall, this work provides a flow diagram for the selection of PGPR candidates to be successfully developed and commercialized as PBs. The validation of the flow diagram led to the selection of two bacterial strains belonging to Pantoea and Pseudomonas genera, potential active ingredients of new commercial PBs.