Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring.

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. RESULTS: We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO2 and NaHCO3, and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO2 were 5-7 times higher than the ones grown with 20 mM NaHCO3, at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO3- to CO2. The highest RuBisCO activity of 2.13 nmol of NAD+/ µg of Chl-a/ min was observed with 50 µg of protein and 5% CO2. Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited  ~ 3.4-fold slower reaction rate (0.37 min-1) than the biochemical assay when using 5% CO2. We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO3 as compared to the cells that were grown without NaHCO3 supplementation. CONCLUSIONS: Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

Comprehensive review of water management and wastewater treatment in food processing industries in the framework of water-food-environment nexus.

Food processing is among the greatest water-consuming industries with a significant role in the implementation of sustainable development goals. Water-consuming industries such as food processing have become a threat to limited freshwater resources, and numerous attempts are being carried out in order to develop and apply novel approaches for water management in these industries. Studies have shown the positive impact of the new methods of process integration (e.g., water pinch, mathematical optimization, etc.) in maximizing water reuse and recycle. Applying these methods in food processing industries not only significantly supported water consumption minimization but also contributed to environmental protection by reducing wastewater generation. The methods can also increase the productivity of these industries and direct them to sustainable production. This interconnection led to a new subcategory in nexus studies known as water-food-environment nexus. The nexus assures sustainable food production with minimum freshwater consumption and minimizes the environmental destructions caused by untreated wastewater discharge. The aim of this study was to provide a thorough review of water-food-environment nexus application in food processing industries and explore the nexus from different aspects. The current study explored the process of food industries in different sectors regarding water consumption and wastewater generation, both qualitatively and quantitatively. The most recent wastewater treatment methods carried out in different food processing sectors were also reviewed. This review provided a comprehensive literature for choosing the optimum scenario of water and wastewater management in food processing industries.

Regulatory Role of Circadian Clocks on ABA Production and Signaling, Stomatal Responses, and Water-Use Efficiency under Water-Deficit Conditions.

Plants deploy molecular, physiological, and anatomical adaptations to cope with long-term water-deficit exposure, and some of these processes are controlled by circadian clocks. Circadian clocks are endogenous timekeepers that autonomously modulate biological systems over the course of the day-night cycle. Plants' responses to water deficiency vary with the time of the day. Opening and closing of stomata, which control water loss from plants, have diurnal responses based on the humidity level in the rhizosphere and the air surrounding the leaves. Abscisic acid (ABA), the main phytohormone modulating the stomatal response to water availability, is regulated by circadian clocks. The molecular mechanism of the plant's circadian clock for regulating stress responses is composed not only of transcriptional but also posttranscriptional regulatory networks. Despite the importance of regulatory impact of circadian clock systems on ABA production and signaling, which is reflected in stomatal responses and as a consequence influences the drought tolerance response of the plants, the interrelationship between circadian clock, ABA homeostasis, and signaling and water-deficit responses has to date not been clearly described. In this review, we hypothesized that the circadian clock through ABA directs plants to modulate their responses and feedback mechanisms to ensure survival and to enhance their fitness under drought conditions. Different regulatory pathways and challenges in circadian-based rhythms and the possible adaptive advantage through them are also discussed.

One cell-two wells bio-refinery: Demonstrating cyanobacterial chassis for co-production of heterologous and natural hydrocarbons.

In order to improve the potential of cyanobacterial cell factories, Synechococcus sp. PCC7002 was engineered as 'one cell-two wells bio-refinery', for ethylene ('heterologous' hydrocarbon) and carotenoids ('natural' metabolites) production, and demonstrating its outdoor performance. Although the cultures showed better production outdoor, they experienced multiple collapses during scale-up. Hence, flux balance analysis was performed which predicted higher ethylene production with increase in carbon input under outdoor light conditions. Furthermore, FBA predicted that ethylene production will not increase beyond a threshold carbon input flux, owing to limitations on ribulose-1,5-bisphosphate regeneration. Hence, a bicarbonate-supplementation strategy was devised. Cultures grown outdoor at optimal bicarbonate concentration (20 g/L) resulted in improved growth (0.141/h) and ethylene productivity (1.88 mL/L.h) for > 10 days, with enhanced carotenoid titres (40.4 mg/L). In a 100 L air-lift photo-bioreactor; cultures exhibited efficient ethylene (2.464 mL/L.h) and biomass (0.3 g/L.d) productivities, and carotenoids titres (64.4 mg/L), establishing a significant step towards commercialization.

Interactions of microalgae-bacteria consortia for nutrient removal from wastewater: A review.

Nitrogen and phosphorus pollution can cause eutrophication, resulting in ecosystem disruption. Wastewater treatment systems employing microalgae-bacteria consortia have the potential to enhance the nutrient removal efficiency from wastewater through mutual interaction and synergetic effects. The knowledge and control of the mechanisms involved in microalgae-bacteria interaction could improve the system's ability to transform and recover nutrients. In this review, a critical evaluation of recent literature was carried out to synthesize knowledge related to mechanisms of interaction between microalgae and bacteria consortia for nutrient removal from wastewater. It is now established that microalgae can produce oxygen through photosynthesis for bacteria and, in turn, bacteria supply the required metabolites and inorganic carbon source for algae growth. Here we highlight how the interaction between microalgae and bacteria is highly dependent on the nitrogen species in the wastewater. When the nitrogen source is ammonium, the generated oxygen by microalgae has a positive influence on nitrifying bacteria. When the nitrogen source is nitrate, the oxygen can have an inhibitory effect on denitrifying bacteria. However, some strains of microalgae have the capability to supply hydrogen gas for hydrogenotrophic denitrifiers as an energy source. Recent literature on biogranulation of microalgae and bacteria and its application for nutrient removal and biomass recovery is also discussed as a promising approach. Significant research challenges remain for the integration of microalgae-bacteria consortia into wastewater treatment processes including microbial community control and process stability over long time horizons.

Cultivation of Mixed Microalgae Using Municipal Wastewater: Biomass Productivity, Nutrient Removal, and Biochemical Content.

BACKGROUND: Microalgal biotechnology has gained much attention previously. Monoculture algae cultivation has been carried out extensively in the last decades. However, although the mixed microalgae cultivation has some advantageous over pure cultures, there is still a lack of knowledge about the performance of mixed cultures. OBJECTIVE: In this study, it has been tried to investigate all growth aspects of marine and freshwater microalgal species in a mixed culture and their biological effects on biomass growth and composition based on wastewater nutrient consumption. MATERIAL AND METHODS: Three algal species of Chlorella vulgaris, Scenedesmus obliquus, and Nannochloropsis sp. were cultivated in saline wastewater individually, then the effects of mixing the three strains on biomass productivity, nutrient removal efficiency, chlorophyll, carotenoid, and lipid content were investigated. RESULTS: The obtained results revealed that the mixed culture of three strains showed the highest biomass productivity of 191 mg. L-1.d-1. Also, while there were no significant differences between the performance of mono and mixed culture of algal species in the removal efficiency of wastewater nutrients, the three-strain microalgal mixed culture showed the highest values of 3.5 mg.L-1.d-1 and 5.75 mg.L-1.d-1 in the removal rate of phosphate and nitrate, respectively. In terms of total chlorophyll and carotenoid per produced biomass, however, the mixed culture of three species showed the lowest values of 4.08 and 0.6 mg. g biomass-1, respectively. CONCLUSIONS: The finding proves the potential of attractive and economically feasible mixed microalgae cultivation for high percentage nutrient removal and microalgal biomass production.