Design of a nanobiosystem with remote photothermal gene silencing in Chlamydomonas reinhardtii to increase lipid accumulation and production.

Research development in the precise control of gene expression in plant cells is an emerging necessity that would lead to the elucidation of gene function in these biological systems. Conventional gene-interfering techniques, such as micro-RNA and short interfering RNA, have limitations in their ability to downregulate gene expression in plants within short time periods. However, nanotechnology provides a promising new avenue with new tools to overcome these challenges. Here, we show that functionalized gold nanoparticles, decorated with sense and antisense oligonucleotides (FANSAO), can serve as a remote-control optical switch for gene interference in photosynthetic plant cells. We demonstrate the potential of employing LEDs as optimal light sources to photothermally dehybridize the oligonucleotides on the surface of metallic nanostructures, consequently inducing regulation of gene expression in plant cells. We show the efficiency of metallic nanoparticles in absorbing light from an LED source and converting it to thermal energy, resulting in a local temperature increase on the surface of the gold nanoparticles. The antisense oligonucleotides are then released due to the opto-thermal heating of the nanobiosystem composed of the metallic nanoparticles and the sense-antisense oligonucleotides. By applying this approach, we silenced the Carnitine Acyl Carnitine Translocase genes at 90.7%, resulting in the accumulation of lipid bodies in microalgae cells. These results exhibit the feasibility of using functionalized gold nanoparticles with sense and antisense oligonucleotides to enhance nucleic acid delivery efficiency and, most importantly, allow for temporal control of gene silencing in plant cells. These nanobiosystems have broad applications in the development and biosynthesis of biofuels, pharmaceuticals, and specialized chemicals.

LED control of gene expression in a nanobiosystem composed of metallic nanoparticles and a genetically modified E. coli strain.

BACKGROUND: Within the last decade, genetic engineering and synthetic biology have revolutionized society´s ability to mass-produce complex biological products within genetically-modified microorganisms containing elegantly designed genetic circuitry. However, many challenges still exist in developing bioproduction processes involving genetically modified microorganisms with complex or multiple gene circuits. These challenges include the development of external gene expression regulation methods with the following characteristics: spatial-temporal control and scalability, while inducing minimal permanent or irreversible system-wide conditions. Different stimuli have been used to control gene expression and mitigate these challenges, and they can be characterized by the effect they produce in the culture media conditions. Invasive stimuli that cause permanent, irreversible changes (pH and chemical inducers), non-invasive stimuli that cause partially reversible changes (temperature), and non-invasive stimuli that cause reversible changes in the media conditions (ultrasound, magnetic fields, and light). METHODS: Opto-control of gene expression is a non-invasive external trigger that complies with most of the desired characteristics of an external control system. However, the disadvantage relies on the design of the biological photoreceptors and the necessity to design them to respond to a different wavelength for every bioprocess needed to be controlled or regulated in the microorganism. Therefore, this work proposes using biocompatible metallic nanoparticles as external controllers of gene expression, based on their ability to convert light into heat and the capacity of nanotechnology to easily design a wide array of nanostructures capable of absorbing light at different wavelengths and inducing plasmonic photothermal heating. RESULTS: Here, we designed a nanobiosystem that can be opto-thermally triggered using LED light. The nanobiosystem is composed of biocompatible gold nanoparticles and a genetically modified E. coli with a plasmid that allows mCherry fluorescent protein production at 37 °C in response to an RNA thermometer. CONCLUSIONS: The LED-triggered photothermal protein production system here designed offers a new, cheaper, scalable switchable method, non-destructive for living organisms, and contribute toward the evolution of bioprocess production systems.

Biomass and lipid induction strategies in microalgae for biofuel production and other applications.

The use of fossil fuels has been strongly related to critical problems currently affecting society, such as: global warming, global greenhouse effects and pollution. These problems have affected the homeostasis of living organisms worldwide at an alarming rate. Due to this, it is imperative to look for alternatives to the use of fossil fuels and one of the relevant substitutes are biofuels. There are different types of biofuels (categories and generations) that have been previously explored, but recently, the use of microalgae has been strongly considered for the production of biofuels since they present a series of advantages over other biofuel production sources: (a) they don't need arable land to grow and therefore do not compete with food crops (like biofuels produced from corn, sugar cane and other plants) and; (b) they exhibit rapid biomass production containing high oil contents, at least 15 to 20 times higher than land based oleaginous crops. Hence, these unicellular photosynthetic microorganisms have received great attention from researches to use them in the large-scale production of biofuels. However, one disadvantage of using microalgae is the high economic cost due to the low-yields of lipid content in the microalgae biomass. Thus, development of different methods to enhance microalgae biomass, as well as lipid content in the microalgae cells, would lead to the development of a sustainable low-cost process to produce biofuels. Within the last 10 years, many studies have reported different methods and strategies to induce lipid production to obtain higher lipid accumulation in the biomass of microalgae cells; however, there is not a comprehensive review in the literature that highlights, compares and discusses these strategies. Here, we review these strategies which include modulating light intensity in cultures, controlling and varying CO2 levels and temperature, inducing nutrient starvation in the culture, the implementation of stress by incorporating heavy metal or inducing a high salinity condition, and the use of metabolic and genetic engineering techniques coupled with nanotechnology.

The plant early recombinosome: a high security complex to break DNA during meiosis.

KEY MESSAGE: The formacion of numerous unpredictable DNA Double Strand Breaks (DSBs) on chromosomes iniciates meiotic recombination. In this perspective, we propose a 'multi-key lock' model to secure the risky but necesary breaks as well as a 'one per pair of cromatids' model for the topoisomerase-like early recombinosome. During meiosis, homologous chromosomes recombine at few sites of crossing-overs (COs) to ensure correct segregation. The initiation of meiotic recombination involves the formation of DNA double strand breaks (DSBs) during prophase I. Too many DSBs are dangerous for genome integrity: if these DSBs are not properly repaired, it could potentially lead to chromosomal fragmentation. Too few DSBs are also problematic: if the obligate CO cannot form between bivalents, catastrophic unequal segregation of univalents lead to the formation of sterile aneuploid spores. Research on the regulation of the formation of these necessary but risky DSBs has recently advanced in yeast, mammals and plants. DNA DSBs are created by the enzymatic activity of the early recombinosome, a topoisomerase-like complex containing SPO11. This opinion paper reviews recent insights on the regulation of the SPO11 cofactors necessary for the introduction of temporally and spatially controlled DSBs. We propose that a 'multi-key-lock' model for each subunit of the early recombinosome complex is required to secure the formation of DSBs. We also discuss the hypothetical implications that the established topoisomerase-like nature of the SPO11 core-complex can have in creating DSB in only one of the two replicated chromatids of early prophase I meiotic chromosomes. This hypothetical 'one per pair of chromatids' DSB formation model could optimize the faithful repair of the self-inflicted DSBs. Each DSB could use three potential intact homologous DNA sequences as repair template: one from the sister chromatid and the two others from the homologous chromosomes.

Nano-Regulation of Gene Expression in Chlamydomonas reinhardtii: Harnessing AuNPs for Remotely Switchable Lipid Biosynthesis via Antisense Oligonucleotides.

Antisense oligonucleotide (ASO)-mediated gene silencing has broad applications, spanning from biomedicine to agriculture, involving molecular biology, synthetic biology, and genetic manipulation. This research harnessed nanotechnology to augment ASO-mediated gene silencing, introducing a remotely switchable gene expression system for precise temporal control. We targeted lipid biosynthesis and accumulation enhancement in the photosynthetic eukaryote Chlamydomonas reinhardtii. Gold nanoparticles (AuNPs) transported double-stranded DNA (dsDNA), forming dsDNA-AuNP complexes. These complexes comprised 3'-thiolated sense strands attached to AuNPs and fluorescent antisense oligonucleotides. To avoid harmful laser effects on cells, we adopted a light-emitting diode (LED). Confocal microscopy confirmed dsDNA-AuNP internalization in C. reinhardtii. LED-triggered antisense release led to an 83% decrease in Citrate Synthase 2 (CIS 2) expression. Thiolated sense strand attachment postillumination inhibited antisense reannealing, enhancing gene silencing. This led to significant lipid body accumulation in cells, verified through fluorometric and fluorescence microscopy. This union of nanotechnology and ASO-mediated silencing provides gene regulation opportunities across sectors like biomedicine and agriculture. The system's remote switching capability underscores its potential in synthetic biology and genetic engineering. Our findings substantiate the utility of this approach for enhancing lipid biosynthesis in C. reinhardtii but also underscores its broader applicability to other organisms, fostering the development of novel solutions for pressing global challenges in energy, agriculture, and healthcare.

Crossover interference mechanism: New lessons from plants.

Plants are the source of our understanding of several fundamental biological principles. It is well known that Gregor Mendel discovered the laws of Genetics in peas and that maize was used for the discovery of transposons by Barbara McClintock. Plant models are still useful for the understanding of general key biological concepts. In this article, we will focus on discussing the recent plant studies that have shed new light on the mysterious mechanisms of meiotic crossover (CO) interference, heterochiasmy, obligatory CO, and CO homeostasis. Obligatory CO is necessary for the equilibrated segregation of homologous chromosomes during meiosis. The tight control of the different male and female CO rates (heterochiasmy) enables both the maximization and minimization of genome shuffling. An integrative model can now predict these observed aspects of CO patterning in plants. The mechanism proposed considers the Synaptonemal Complex as a canalizing structure that allows the diffusion of a class I CO limiting factor linearly on synapsed bivalents. The coarsening of this limiting factor along the SC explains the interfering spacing between COs. The model explains the observed coordinated processes between synapsis, CO interference, CO insurance, and CO homeostasis. It also easily explains heterochiasmy just considering the different male and female SC lengths. This mechanism is expected to be conserved in other species.

Microalgal co-cultivation -recent methods, trends in omic-studies, applications, and future challenges.

The burgeoning human population has resulted in an augmented demand for raw materials and energy sources, which in turn has led to a deleterious environmental impact marked by elevated greenhouse gas (GHG) emissions, acidification of water bodies, and escalating global temperatures. Therefore, it is imperative that modern society develop sustainable technologies to avert future environmental degradation and generate alternative bioproduct-producing technologies. A promising approach to tackling this challenge involves utilizing natural microbial consortia or designing synthetic communities of microorganisms as a foundation to develop diverse and sustainable applications for bioproduct production, wastewater treatment, GHG emission reduction, energy crisis alleviation, and soil fertility enhancement. Microalgae, which are photosynthetic microorganisms that inhabit aquatic environments and exhibit a high capacity for CO2 fixation, are particularly appealing in this context. They can convert light energy and atmospheric CO2 or industrial flue gases into valuable biomass and organic chemicals, thereby contributing to GHG emission reduction. To date, most microalgae cultivation studies have focused on monoculture systems. However, maintaining a microalgae monoculture system can be challenging due to contamination by other microorganisms (e.g., yeasts, fungi, bacteria, and other microalgae species), which can lead to low productivity, culture collapse, and low-quality biomass. Co-culture systems, which produce robust microorganism consortia or communities, present a compelling strategy for addressing contamination problems. In recent years, research and development of innovative co-cultivation techniques have substantially increased. Nevertheless, many microalgae co-culturing technologies remain in the developmental phase and have yet to be scaled and commercialized. Accordingly, this review presents a thorough literature review of research conducted in the last few decades, exploring the advantages and disadvantages of microalgae co-cultivation systems that involve microalgae-bacteria, microalgae-fungi, and microalgae-microalgae/algae systems. The manuscript also addresses diverse uses of co-culture systems, and growing methods, and includes one of the most exciting research areas in co-culturing systems, which are omic studies that elucidate different interaction mechanisms among microbial communities. Finally, the manuscript discusses the economic viability, future challenges, and prospects of microalgal co-cultivation methods.

Development of a Theoretical Model That Predicts Optothermal Energy Conversion of Gold Metallic Nanoparticles.

Gold nanoparticles (AuNPs) can be found in different shapes and sizes, which determine their chemical and physical characteristics. Physical and chemical properties of metallic NPs can be tuned by changing their shape, size, and surface chemistry; therefore, this has led to their use in a wide variety of applications in many industrial and academic sectors. One of the features of metallic NPs is their ability to act as optothermal energy converters, where they absorb light at a specific wavelength and heat up their local nanosurfaces. This feature has been used in many applications where metallic NPs get coupled with thermally responsive systems to trigger an optical response. In this study, we synthesized AuNPs that are spherical in shape with an average diameter of 20.07 nm. This work assessed simultaneously theoretical and experimental techniques to evaluate the different factors that affect heat generation at the surface of AuNPs when exposed to a specific light wavelength. The results indicated that laser power, concentration of AuNPs, time × laser power interaction, and time illumination, were the most important factors that contributed to the temperature change exhibited in the AuNPs solution. We report a regression model that allows predicting heat generation and temperature changes with residual standard errors of less than 4%. These results are highly relevant in the future design and development of applications where metallic NPs are incorporated into systems to induce a temperature change triggered by light exposure.