Photosynthetic chemical production in cyanobacteria is a promising technology for renewable energy, CO₂ mitigation, and fossil fuel replacement. Metabolic engineering has enabled a direct ...biosynthetic process from CO₂ fixation to chemical feedstocks and biofuels, without requiring costly production, storage, and breakdown of cellulose or sugars. However, direct production technology is challenged by a need to achieve high-carbon partitioning to products in order to be competitive. This review discusses principles for the design of biosynthetic pathways in cyanobacteria and describes recent advances in relevant genetic tools. This field is at a critical juncture in assessing the strength and feasibility of carbon partitioning. To address this, we have included the stoichiometry of reducing equivalents and carbon conservation for heterologous pathways, and a method for calculating product yields against a theoretical maximum.
Conversion of CO₂ for the synthesis of chemicals by photosynthetic organisms is an attractive target for establishing independence from fossil reserves. However, synthetic pathway construction in ...cyanobacteria is still in its infancy compared with model fermentative organisms. Here we systematically developed the 2,3-butanediol (23BD) biosynthetic pathway in Synechococcus elongatus PCC7942 as a model system to establish design methods for efficient exogenous chemical production in cyanobacteria. We identified 23BD as a target chemical with low host toxicity, and designed an oxygen-insensitive, cofactor-matched biosynthetic pathway coupled with irreversible enzymatic steps to create a driving force toward the target. Production of 23BD from CO₂ reached 2.38 g/L, which is a significant increase for chemical production from exogenous pathways in cyanobacteria. This work demonstrates that developing strong design methods can continue to increase chemical production in cyanobacteria.
The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One useful but challenging situation is a switch between life ...and death depending on environment. Here are presented “essentializer” and “cryodeath” circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 105 after 10 days of growth in the mammalian gut.
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•Construction of kill switches that respond to environmental cues•Reasonable evolutionary stability achieved through toxin/antitoxin approach•Temperature was used to contain bacterial growth to the mammalian gut
Biocontainment of microorganisms is becoming an increasingly relevant issue as functional uses for transgenic organisms outside of a controlled laboratory become readily available. By limiting bacterial growth with evolutionarily stable kill switches, it is possible to impart some level of control over which environments a transgenic organism is viable in.
Although carbonyl oxides, "Criegee intermediates," have long been implicated in tropospheric oxidation, there have been few direct measurements of their kinetics, and only for the simplest compound ...in the class, CH₂OO. Here, we report production and reaction kinetics of the next larger Criegee intermediate, CH₃CHOO. Moreover, we independently probed the two distinct CH₃CHOO conformers, syn- and anti-, both of which react readily with SO₂ and with NO₂. We demonstrate that anti-CH₃CHOO is substantially more reactive toward water and SO₂ than is syn-CH₃CHOO. Reaction with water may dominate tropospheric removal of Criegee intermediates and determine their atmospheric concentration. An upper limit is obtained for the reaction of syn-CH₃CHOO with water, and the rate constant for reaction of anti-CH₃CHOO with water is measured as 1.0 × 10⁻¹⁴ ± 0.4 × 10⁻¹⁴ centimeter³ second⁻¹.
Summary Background Vulval and vaginal cancers among younger women are often related to infection with human papillomavirus (HPV). These cancers are preceded by high-grade vulval intraepithelial ...neoplasia (VIN2–3) and vaginal intraepithelial neoplasia (VaIN2–3). Our aim was to do a combined analysis of three randomised clinical trials to assess the effect of a prophylactic quadrivalent HPV vaccine on the incidence of these diseases. Methods 18 174 women (16–26 years) were enrolled and randomised to receive either quadrivalent HPV6/11/16/18 L1 virus-like-particle vaccine or placebo at day 1, and months 2 and 6. Individuals underwent detailed anogenital examination at day 1, 1 month after dose three, and at 6–12-month intervals for up to 48 months. Suspect genital lesions were biopsied and read by a panel of pathologists and vaccine HPV type-specific DNA testing was done. The primary endpoint was the combined incidence of VIN2–3 or VaIN2–3 associated with HPV16 or HPV18. Primary efficacy analyses were done in a per-protocol population. Findings The mean follow-up time was 3 years. Among women naive to HPV16 or HPV18 through 1 month after dose three (per-protocol population; vaccine n=7811; placebo n=7785), the vaccine was 100% effective (95% CI 72–100) against VIN2–3 or VaIN2–3 associated with HPV16 or HPV18. In the intention-to-treat population (which included 18 174 women who, at day 1, could have been infected with HPV16 or HPV18), vaccine efficacy against VIN2–3 or VaIN2–3 associated with HPV16 or HPV18 was 71% (37–88). The vaccine was 49% (18–69) effective against all VIN2–3 or VaIN2–3, irrespective of whether or not HPV DNA was detected in the lesion. The most common treatment-related adverse event was injection-site pain. Interpretation Prophylactic administration of quadrivalent HPV vaccine was effective in preventing high-grade vulval and vaginal lesions associated with HPV16 or HPV18 infection in women who were naive to these types before vaccination. With time, such vaccination could result in reduced rates of HPV-related vulval and vaginal cancers.
The burning of fossil reserves, and subsequent release of carbon into the atmosphere is depleting the supply of carbon-based molecules used for synthetic materials including plastics, oils, ...medicines, and glues. To provide for future society, innovations are needed for the conversion of waste carbon (CO2) into organic carbon useful for materials. Chemical production directly from photosynthesis is a nascent technology, with great promise for capture of CO2 using sunlight. To improve low yields, it has been proposed that photosynthetic capacity can be increased by a relaxation of bottlenecks inherent to growth. The limits of carbon partitioning away from growth within the cell and the effect of partitioning on carbon fixation are not well known. Here we show that expressing genes in a pathway between carbon fixation and pyruvate increases partitioning to 2,3-butanediol (23BD) and leads to a 1.8-fold increase in total carbon yield in the cyanobacterium Synechococcus elongatus PCC 7942. Specific 2,3-butanediol production increases 2.4-fold. As partitioning increases beyond 30%, it leads to a steep decline in total carbon yield. The data suggests a local maximum for carbon partitioning from the Calvin Benson cycle that is scalable with light intensity.
•A carbon sink pathway increased total carbon productivity in cyanobacteria.•This method led to 1.8-fold increase in total carbon yield.•Specific 2,3-butanediol production increased 2.4-fold.•Partitioning to the product beyond 30% led to a steep decline in total carbon yield.
A vital goal of renewable technology is the capture and re-energizing of exhausted CO2 into usable carbon products. Cyanobacteria fix CO2 more efficiently than plants, and can be engineered to ...produce carbon feedstocks useful for making plastics, solvents, and medicines. However, fitness of this technology in the economy is threatened by low yields in engineered strains. Robust engineering of photosynthetic microorganisms is lagging behind model microorganisms that rely on energetic carbon, such as Escherichia coli, due in part to slower growth rates and increased metabolic complexity. In this work we show that protein expression from characterized parts is unpredictable in Synechococcus elongatus sp. strain PCC 7942, and may contribute to slow development. To overcome this, we apply a combinatorial approach and show that modulation of the 5'-untranslated region (UTR) can produce a range of protein expression sufficient to optimize chemical feedstock production from CO2.
•Enzyme activity used to monitor protein expression in a production pathway.•Effect of RBS in S. elongatus PCC7942 observed to differ from standard models.•Balanced expression from a single operon decreased toxicity and increased titer.•Modulation from RBS characterized in vivo varied when expressed in an operon.•Pathway expression is significantly less predictable than that of common reporters.
In nature, microbes interact antagonistically, neutrally, or beneficially. To shed light on the effects of positive interactions in microbial consortia, we introduced metabolic dependencies and ...metabolite overproduction into four bacterial species. While antagonistic interactions govern the wild-type consortium behavior, the genetic modifications alleviated antagonistic interactions and resulted in beneficial interactions. Engineered cross-feeding increased population evenness, a component of ecological diversity, in different environments, including in a more complex gnotobiotic mouse gut environment. Our findings suggest that metabolite cross-feeding could be used as a tool for intentionally shaping microbial consortia in complex environments.
Microbial communities are ubiquitous in nature. Bacterial consortia live in and on our body and in our environment, and more recently, biotechnology is applying microbial consortia for bioproduction. As part of our body, bacterial consortia influence us in health and disease. Microbial consortium function is determined by its composition, which in turn is driven by the interactions between species. Further understanding of microbial interactions will help us in deciphering how consortia function in complex environments and may enable us to modify microbial consortia for health and environmental benefits.
Asthma is a common chronic disease amongst children. Epidemiological studies showed that the mortality rate of asthma in children is still high worldwide. Asthma control is therefore essential to ...minimize asthma exacerbations, which can be fatal if the condition is poorly controlled. Frequent monitoring could help to detect asthma progression and ensure treatment effectiveness. Although subjective asthma monitoring tools are available, the results vary as they rely on patients' self-perception. Emerging evidence suggests several objective tools could have the potential for monitoring purposes. However, there is no consensus to standardise the use of objective monitoring tools. In this review, we start with the prevalence and severity of childhood asthma worldwide. Then, we detail the latest available objective monitoring tools, focusing on their effectiveness in paediatric asthma management. Publications of spirometry, fractional exhaled nitric oxide (FeNO), hyperresponsiveness tests and electronic monitoring devices (EMDs) between 2016 and 2023 were included. The potential advantages and limitations of each tool were also discussed. Overall, this review provides a summary for researchers dedicated to further improving objective paediatric asthma monitoring and provides insights for clinicians to incorporate different objective monitoring tools in clinical practices.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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