A variety of chemicals have been produced through metabolic engineering approaches, and enhancing biosynthesis performance can be achieved by using enzymes with high catalytic efficiency. Accordingly, a number of efforts have been made to discover enzymes in nature for various applications. In addition, enzyme engineering approaches have been attempted to suit specific industrial purposes. However, a significant challenge in enzyme discovery and engineering is the efficient screening of enzymes with the desired phenotype from extensive enzyme libraries. To overcome this bottleneck, genetically encoded biosensors have been developed to specifically detect target molecules produced by enzyme activity at the intracellular level. Especially, the biosensors facilitate high-throughput screening (HTS) of targeted enzymes, expanding enzyme discovery and engineering strategies with advances in systems and synthetic biology. This review examines biosensor-guided HTS systems and highlights studies that have utilized these tools to discover enzymes in diverse areas and engineer enzymes to enhance their properties, such as catalytic efficiency, specificity, and stability.

Endolysins are bacteriophage-encoded enzymes that can specifically degrade the peptidoglycan layer of bacterial cell wall, making them an attractive tool for the development of novel antibacterial agents. The use of genetic engineering techniques for the production and modification of endolysins offers the opportunity to customize their properties and activity against specific bacterial targets, paving the way for the development of personalized therapies for bacterial infections. Gram-negative bacteria possess an outer membrane that can hinder the action of recombinantly produced endolysins. However, certain endolysins are capable of crossing the outer membrane by virtue of segments that share properties resembling those of cationic peptides. These regions increase the affinity of the endolysin towards the bacterial surface and assist in the permeabilization of the membrane. In order to improve the bactericidal effectiveness of endolysins, approaches have been implemented to increase their net charge, including the development of Artilysins containing positively charged amino acids at one end. At present, there are no specific guidelines outlining the steps for implementing these modifications. There is an ongoing debate surrounding the optimal location of positive charge, the need for a linker region, and the specific amino acid composition of peptides for modifying endolysins. The aim of this study is to provide clarity on these topics by analyzing and comparing the most effective modifications found in previous literature.

Cereal crops, including triticeae species (barley, wheat, rye), as well as edible cereals (wheat, corn, rice, oat, rye, sorghum), are significant suppliers for human consumption, livestock feed, and breweries. Over the past half-century, modern varieties of cereal crops with increased yields have contributed to global food security. However, presently cultivated elite crop varieties were developed mainly for optimal environmental conditions. Thus, it has become evident that taking into account the ongoing climate changes, currently a priority should be given to developing new stress-tolerant cereal cultivars. It is necessary to enhance the accuracy of methods and time required to generate new cereal cultivars with the desired features to adapt to climate change and keep up with the world population expansion. The CRISPR/Cas9 system has been developed as a powerful and versatile genome editing tool to achieve desirable traits, such as developing high-yielding, stress-tolerant, and disease-resistant transgene-free lines in major cereals. Despite recent advances, the CRISPR/Cas9 application in cereals faces several challenges, including a significant amount of time required to develop transgene-free lines, laboriousness, and a limited number of genotypes that may be used for the transformation and in vitro regeneration. Additionally, developing elite lines through genome editing has been restricted in many countries, especially Europe and New Zealand, due to a lack of flexibility in GMO regulations. This review provides a comprehensive update to researchers interested in improving cereals using gene-editing technologies, such as CRISPR/Cas9. We will review some critical and recent studies on crop improvements and their contributing factors to superior cereals through gene-editing technologies.

Sulfide ions are regarded to be toxic to microorganisms in engineered methanogenic systems (EMS), where organic substances are anaerobically converted to products such as methane, hydrogen, alcohols, and carboxylic acids. A vast body of research has addressed solutions to mitigate process disturbances associated with high sulfide levels, yet the established paradigm has drawn the attention away from the multifaceted sulfide interactions with minerals, organics, microbial interfaces and their implications for performance of EMS. This brief review brings forward sulfide-derived pathways other than toxicity and with potential significance for anaerobic organic matter degradation. Available evidence on sulfide reactions with organic matter, interventions with key microbial metabolisms, and interspecies electron transfer are critically synthesized as a guidance for comprehending the sulfide effects on EMS apart from the microbial toxicity. The outcomes identify existing knowledge gaps and specify future research needs as a step forward towards realizing the potential of sulfide-derived mechanisms in diversifying and optimizing EMS applications.

Psychedelic mushrooms containing psilocybin and related tryptamines have long been used for ethnomycological purposes, but emerging evidence points to the potential therapeutic value of these mushrooms to address modern neurological, psychiatric health, and related disorders. As a result, psilocybin containing mushrooms represent a re-emerging frontier for mycological, biochemical, neuroscience, and pharmacology research. This work presents crucial information related to traditional use of psychedelic mushrooms, as well as research trends and knowledge gaps related to their diversity and distribution, technologies for quantification of tryptamines and other tryptophan-derived metabolites, as well as biosynthetic mechanisms for their production within mushrooms. In addition, we explore the current state of knowledge for how psilocybin and related tryptamines are metabolized in humans and their pharmacological effects, including beneficial and hazardous human health implications. Finally, we describe opportunities and challenges for investigating the production of psychedelic mushrooms and metabolic engineering approaches to alter secondary metabolite profiles using biotechnology integrated with machine learning. Ultimately, this critical review of all aspects related to psychedelic mushrooms represents a roadmap for future research efforts that will pave the way to new applications and refined protocols.

Carbohydrates are chemically and structurally diverse biomolecules, serving numerous and varied roles in agricultural ecosystems. Crops and horticulture products are inherent sources of carbohydrates that are consumed by humans and non-human animals alike; however carbohydrates are also present in other agricultural materials, such as soil and compost, human and animal tissues, milk and dairy products, and honey. The biosynthesis, modification, and flow of carbohydrates within and between agricultural ecosystems is intimately related with microbial communities that colonize and thrive within these environments. Recent advances in -omics techniques have ushered in a new era for microbial ecology by illuminating the functional potential for carbohydrate metabolism encoded within microbial genomes, while agricultural glycomics is providing fresh perspective on carbohydrate-microbe interactions and how they influence the flow of functionalized carbon. Indeed, carbohydrates and carbohydrate-active enzymes are interventions with unrealized potential for improving carbon sequestration, soil fertility and stability, developing alternatives to antimicrobials, and circular production systems. In this manner, glycomics represents a new frontier for carbohydrate-based biotechnological solutions for agricultural systems facing escalating challenges, such as the changing climate.

The development of novel, sustainable, and robust food production technologies represents one of the major pillars to address the most significant challenges humanity is going to face on earth in the upcoming decades - climate change, population growth, and resource depletion. The implementation of microfoods, i.e., foods formulated with ingredients from microbial cultivation, into the food supply chain has a huge potential to contribute towards energy-efficient and nutritious food manufacturing and represents a means to sustainably feed a growing world population. This review recapitulates and assesses the current state in the establishment and usage of gas fermenting bacteria as an innovative feedstock for protein production. In particular, we focus on the most promising representatives of this taxon: the hydrogen-oxidizing bacteria (hydrogenotrophs) and the methane-oxidizing bacteria (methanotrophs). These unicellular microorganisms can aerobically metabolize gaseous hydrogen and methane, respectively, to provide the required energy for building up cell material. A protein yield over 70% in the dry matter cell mass can be reached with no need for arable land and organic substrates making it a promising alternative to plant- and animal-based protein sources. We illuminate the holistic approach to incorporate protein extracts obtained from the cultivation of gas fermenting bacteria into microfoods. Herein, the fundamental properties of the bacteria, cultivation methods, downstream processing, and potential food applications are discussed. Moreover, this review covers existing and future challenges as well as sustainability aspects associated with the production of microbial protein through gas fermentation.

3D bioprinting is a new 3D manufacturing technology, that can be used to accurately distribute and load microorganisms to form microbial active materials with multiple complex functions. Based on the 3D printing of human cells in tissue engineering, 3D bioprinting technology has been developed. Although 3D bioprinting technology is still immature, it shows great potential in the environmental field. Due to the precise programming control and multi-printing pathway, 3D bioprinting technology provides a high-throughput method based on micron-level patterning for a wide range of environmental microbiological engineering applications, which makes it an on-demand, multi-functional manufacturing technology. To date, 3D bioprinting technology has been employed in microbial fuel cells, biofilm material preparation, microbial catalysts and 4D bioprinting with time dimension functions. Nevertheless, current 3D bioprinting technology faces technical challenges in improving the mechanical properties of materials, developing specific bioinks to adapt to different strains, and exploring 4D bioprinting for intelligent applications. Hence, this review systematically analyzes the basic technical principles of 3D bioprinting, bioinks materials and their applications in the environmental field, and proposes the challenges and future prospects of 3D bioprinting in the environmental field. Combined with the current development of microbial enhancement technology in the environmental field, 3D bioprinting will be developed into an enabling platform for multifunctional microorganisms and facilitate greater control of in situ directional reactions.

Nucleic acid-based therapies such as messenger RNA have the potential to revolutionize modern medicine and enhance the performance of existing pharmaceuticals. The key challenges of mRNA-based therapies are delivering the mRNA safely and effectively to the target tissues and cells and controlling its release from the delivery vehicle. Lipid nanoparticles (LNPs) have been widely studied as drug carriers and are considered to be state-of-the-art technology for nucleic acid delivery. In this review, we begin by presenting the advantages and mechanisms of action of mRNA therapeutics. Then we discuss the design of LNP platforms based on ionizable lipids and the applications of mRNA-LNP vaccines for prevention of infectious diseases and for treatment of cancer and various genetic diseases. Finally, we describe the challenges and future prospects of mRNA-LNP therapeutics.

Considering an ever-growing global population, which hit 8 billion people in the fall of 2022, it is essential to find solutions to avoid the competition between human food and animal feed for croplands. Agricultural co-products have become important components of the circular economy with their use in animal feed. Their implementation was made possible by the addition of exogenous enzymes in the diet, especially carbohydrate-active enzymes (CAZymes). In this review, we describe the diversity and versatility of microbial CAZymes targeting non-starch polysaccharides to improve the nutritional potential of diets containing cereals and protein meals. We focused our attention on cellulases, hemicellulases, pectinases which were often found to be crucial in vivo. We also highlight the performance and health benefits brought by the exogenous addition of enzymatic cocktails containing CAZymes in the diets of monogastric animals. Taking the example of the well-studied commercial cocktail Rovabio™, we discuss the evolution, constraints and future challenges faced by feed enzymes suppliers. We hope that this review will promote the use and development of enzyme solutions for industries to sustainably feed humans in the future.

Different from other aerobic microorganisms that oxidise carbon sources to water and carbon dioxide, Gluconobacter catalyses the incomplete oxidation of various substrates with regio- and stereoselectivity. This ability, as well as its capacity to release the resulting products into the reaction media, place Gluconobacter as a privileged member of a non-model microorganism class that may boost industrial biotechnology. Knowledge of new technologies applied to Gluconobacter has been piling up in recent years. Advancements in its genetic modification, application of immobilisation tools and careful designs of the transformations, have improved productivities and stabilities of Gluconobacter strains or enabled new bioconversions for the production of valuable marketable chemicals. In this work, the latest advancements applied to Gluconobacter-catalysed biotransformations are summarised with a special focus on recent available tools to improve them. From genetic and metabolic engineering to bioreactor design, the most recent works on the topic are analysed in depth to provide a comprehensive resource not only for scientists and technologists working on/with Gluconobacter, but for the general biotechnologist.

Biophotovoltaics (BPV) is a clean power generation technology that uses self-renewing photosynthetic microorganisms to capture solar energy and generate electrical current. Although the internal quantum efficiency of charge separation in photosynthetic microorganisms is very high, the inefficient electron transfer from photosystems to the extracellular electrodes hampered the electrical outputs of BPV systems. This review summarizes the approaches that have been taken to increase the electrical outputs of BPV systems in recent years. These mainly include redirecting intracellular electron transfer, broadening available photosynthetic microorganisms, reinforcing interfacial electron transfer and design high-performance devices with different configurations. Furthermore, three strategies developed to extract photosynthetic electrons were discussed. Among them, the strategy of using synthetic microbial consortia could circumvent the weak exoelectrogenic activity of photosynthetic microorganisms and the cytotoxicity of exogenous electron mediators, thus show great potential in enhancing the power output and prolonging the lifetime of BPV systems. Lastly, we prospected how to facilitate electron extraction and further improve the performance of BPV systems.

Since the inception of the first electrochemical devices on paper substrates, many different reports of microfluidic paper-based electroanalytical devices (μPEDs), innovative hydrophobic barriers and electrode fabrication processes have allowed the incorporation of diverse materials, resulting in different applications and a boost in performance. These advancements have led to the creation of paper-based devices with comparable performance to many standard conventional devices, with the added benefits of pumpless fluidic transport, component separation and reagent storage that can be exploited to automate and handle sample preprocessing. Herein, we review μPEDs, summarize the characteristics and functionalities of μPEDs, such as separation, fluid flow control and storage, and outline the conventional and emerging fabrication and modification approaches for μPEDs. We also examine the recent application of μPEDs in biomedicine, the environment, and food and water safety, as well as some limitations and challenges that must be addressed.

Synthetic biology transforms the way we perceive biological systems. Emerging technologies in this field affect many disciplines of science and engineering. Traditionally, synthetic biology approaches were commonly aimed at developing cost-effective microbial cell factories to produce chemicals from renewable sources. Based on this, the immediate beneficial impact of synthetic biology on the environment came from reducing our oil dependency. However, synthetic biology is starting to play a more direct role in environmental protection. Toxic chemicals released by industries and agriculture endanger the environment, disrupting ecosystem balance and biodiversity loss. This review highlights synthetic biology approaches that can help environmental protection by providing remediation systems capable of sensing and responding to specific pollutants. Remediation strategies based on genetically engineered microbes and plants are discussed. Further, an overview of computational approaches that facilitate the design and application of synthetic biology tools in environmental protection is presented.

The last decade has witnessed great progress in the field of adoptive cell therapies, with the authorization of Kymriah (tisagenlecleucel) in 2017 by the Food and Drug Administration (FDA) as a crucial stepstone. Since then, five more CAR-T therapies have been approved for the treatment of hematological malignancies. While this is a great step forward to treating several types of blood cancers, CAR-T cell therapies are still associated with severe side-effects such as Graft-versus-Host Disease (GvHD), cytokine release syndrome (CRS) and neurotoxicity. Because of this, there has been continued interest in Natural Killer cells which avoid these side-effects while offering the possibility to generate allogeneic cell therapies. Similar to T-cells, NK cells can be genetically modified to improve their therapeutic efficacy in a variety of ways. In contrast to T cells, viral transduction of NK cells remains inefficient and induces cytotoxic effects. Viral vectors also require a lengthy and expensive product development process and are accompanied by certain risks such as insertional mutagenesis. Therefore, non-viral transfection technologies are avidly being developed aimed at addressing these shortcomings of viral vectors. In this review we will present an overview of the potential of NK cells in cancer immunotherapies and the non-viral transfection technologies that have been explored to engineer them.

Polyphenolic compounds (such as quercetin and resveratrol) possess potential medicinal values due to their various bioactivities, but poor water solubility hinders their health benefits to humankind. Glycosylation is a well-known post-modification method to biosynthesize natural product glycosides with improved hydrophilicity. Glycosylation has profound effects on decreasing toxicity, increasing bioavailability and stability, together with changing bioactivity of polyphenolic compounds. Therefore, polyphenolic glycosides can be used as food additives, therapeutics, and nutraceuticals. Engineered biosynthesis provides an environmentally friendly and cost-effective approach to generate polyphenolic glycosides through the use of various glycosyltransferases (GTs) and sugar biosynthetic enzymes. GTs transfer the sugar moieties from nucleotide-activated diphosphate sugar (NDP-sugar) donors to sugar acceptors such as polyphenolic compounds. In this review, we systematically review and summarize the representative polyphenolic O-glycosides with various bioactivities and their engineered biosynthesis in microbes with different biotechnological strategies. We also review the major routes towards NDP-sugar formation in microbes, which is significant for producing unusual or novel glycosides. Finally, we discuss the trends in NDP-sugar based glycosylation research to promote the development of prodrugs that positively impact human health and wellness.

Arylsulfatase is a subset of sulfatase which catalyzes the hydrolysis of aryl sulfate ester. Arylsulfatase is widely distributed among microorganisms, mammals and green algae, but the arylsulfatase-encoding gene has not yet been found in the genomes of higher plants so far. Arylsulfatase plays an important role in the sulfur flows between nature and organisms. In this review, we present the maturation and catalytic mechanism of arylsulfatase, and the recent literature on the expression and production of arylsulfatase in wild-type and engineered microorganisms, as well as the modification of arylsulfatase by genetic engineering are summarized. We focus on arylsulfatases from microbial origin and give an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the researches about arylsulfatase application on the field of agar desulfation, soil sulfur cycle and soil evaluation are also discussed. Finally, the perspectives concerning the future research on arylsulfatase are prospected.

Lignin is the most affluent natural aromatic biopolymer on the earth, which is the promising renewable source for valuable products to promote the sustainability of biorefinery. Flavonoids are a class of plant polyphenolic secondary metabolites containing the benzene ring structure with various biological activities, which are largely applied in health food, pharmaceutical, and medical fields. Due to the aromatic similarity, microbial conversion of lignin derived aromatics to flavonoids could facilitate flavonoid biosynthesis and promote the lignin valorization. This review thereby prospects a novel valorization route of lignin to high-value natural products and demonstrates the potential advantages of microbial bioconversion of lignin to flavonoids. The biodegradation of lignin polymers is summarized to identify aromatic monomers as momentous precursors for flavonoid synthesis. The biosynthesis pathways of flavonoids in both plants and strains are introduced and compared. After that, the key branch points and important intermediates are clearly discussed in the biosynthesis pathways of flavonoids. Moreover, the most significant enzyme reactions including Claisen condensation, cyclization and hydroxylation are demonstrated in the biosynthesis pathways of flavonoids. Finally, current challenges and potential future strategies are also discussed for transforming lignin into various flavonoids. The holistic microbial conversion routes of lignin to flavonoids could make a sustainable production of flavonoids and improve the feasibility of lignin valorization.

Cellular membranes, predominantly described as a dynamic bilayer, are composed of different lipids, transmembrane proteins, and carbohydrates. Most research on biological membranes focuses on the identification, characterization, and mechanistic aspects of their different components. These studies provide a fundamental understanding of membrane structure, function, and dynamics, establishing a basis for the development of membrane engineering strategies. To date, approaches in this field concentrate on membrane adaptation to harsh conditions during industrial fermentation, which can be caused by temperature, osmotic, or organic solvent stress. With advances in the field of metabolic engineering and synthetic biology, recent breakthroughs include proof of concept microbial production of essential medicines, such as cannabinoids and vinblastine. However, long pathways, low yields, and host adaptation continue to pose challenges to the efficient scale up production of many important compounds. The lipid bilayer is profoundly linked to the activity of heterologous membrane-bound enzymes and transport of metabolites. Therefore, strategies for improving enzyme performance, facilitating pathway reconstruction, and enabling storage of products to increase the yields directly involve cellular membranes. At the forefront of membrane engineering research are re-emerging approaches in lipid research and synthetic biology that manipulate membrane size and composition and target lipid profiles across species. This review summarizes engineering strategies applied to cellular membranes and discusses the challenges and future perspectives, particularly with regards to their applications in host engineering and bioproduction.

Searching for renewable alternatives for fossil carbon resources to produce chemicals, fuels and materials is essential for the development of a sustainable society. Lignin, a major component of lignocellulosic biomass, is an abundant renewable source of aromatics and is currently underutilized as it is often burned as an undesired side stream in the production of paper and bioethanol. This lignin harbors great potential as source of high value aromatic chemicals and materials. Biorefinery schemes focused on lignin are currently under development with aim of acquiring added value from lignin. However, the performance of these novel lignin-focused biorefineries is closely linked with the quality of extracted lignin in terms of the level of degradation and modification. Thus, the reactivity including the degradation pathways of the native lignin contained in the plant material needs to be understood in detail to potentially achieve higher value from lignin. Undegraded native-like lignin with an as close as possible structure to native lignin contained in the lignocellulosic plant material serves as a promising model lignin to support detailed studies on the structure and reactivity of native lignin, yielding key understanding for the development of lignin-focused biorefineries. The aim of this review is to highlight the different methods to attain "native-like" lignins that can be valuable for such studies. This is done by giving a basic introduction on what is known about the native lignin structure and the techniques and methods used to analyze it followed by an overview of the fractionation and isolation methods to isolate native-like lignin. Finally, a perspective on the isolation and use of native-like lignin is provided, showing the great potential that this type of lignin brings for understanding the effect of different biomass treatments on the native lignin structure.