Advances in Biochemical Engineering/biotechnology

Metalloproteins, proteins containing metal atoms or clusters within their structures, are critical for various biological functions across all domains of life. More than hundreds of different types have been discovered, which conduct various roles such as transportation of O2 , catalyzing chemical reactions, sensing environmental changes, and relaying electrons. Metalloprotein molecules incorporate a variety of metal atoms, coordinated to specific amino acid residues that affect their conformation and functionality...

Technical advances in biotechnology have greatly accelerated the development of bottom-up synthetic biology. Unlike top-down approaches, bottom-up synthetic biology focuses on the construction of a minimal cell from scratch and the application of these principles to solve challenges. Cell-free protein synthesis (CFPS) systems provide minimal machinery for transcription and translation, from either a fractionated cell lysate or individual purified protein elements, thus speeding up the development of synthetic cell projects...

The human uses of mushrooms and cultured mycelium products for nutrition and medicine are detailed and supported by available human studies, which in many cases are clinical trials published in peer-reviewed journals. The major medically active immunomodulating compounds in the cell walls-chitin, beta-glucans, and glycoproteins, as well as lower weight molecules-nitrogen-containing compounds, phenolics, and terpenes-are discussed in relation to their current clinical uses. The nutritional content and foods derived from mushrooms, particularly related to their medical benefits, are discussed...

In vitro biotransformation (ivBT) refers to the use of an artificial biological reaction system that employs purified enzymes for the one-pot conversion of low-cost materials into biocommodities such as ethanol, organic acids, and amino acids. Unshackled from cell growth and metabolism, ivBT exhibits distinct advantages compared with metabolic engineering, including but not limited to high engineering flexibility, ease of operation, fast reaction rate, high product yields, and good scalability. These characteristics position ivBT as a promising next-generation biomanufacturing platform...

Organisms from across the tree of life have evolved highly efficient mechanisms for sensing molecules of interest using biomolecular machinery that can in turn be quite valuable for the development of biosensors. However, purification of such machinery for use in in vitro biosensors is costly, while the use of whole cells as in vivo biosensors often leads to long sensor response times and unacceptable sensitivity to the chemical makeup of the sample. Cell-free expression systems overcome these weaknesses by removing the requirements associated with maintaining living sensor cells, allowing for increased function in toxic environments and rapid sensor readout at a production cost that is often more reasonable than purification...

The use of cell-free production systems in droplet microfluidic devices has gained significant interest during the last decade. Encapsulating DNA replication, RNA transcription, and protein expression systems in water-in-oil drops allows for the interrogation of unique molecules and high-throughput screening of libraries of industrial and biomedical interest. Furthermore, the use of such systems in closed compartments enables the evaluation of various properties of novel synthetic or minimal cells. In this chapter, we review the latest advances in the usage of the cell-free macromolecule production toolbox in droplets, with a special emphasis on new on-chip technologies for the amplification, transcription, expression, screening, and directed evolution of biomolecules...

Cell-free systems for the in vitro production of proteins have revolutionized the synthetic biology field. In the last decade, this technology is gaining momentum in molecular biology, biotechnology, biomedicine and even education. Materials science has burst into the field of in vitro protein synthesis to empower the value of existing tools and expand its applications. In this sense, the combination of solid materials (normally functionalized with different biomacromolecules) together with cell-free components has made this technology more versatile and robust...

Biosynthesis involving multi-enzymatic reactions is usually an efficient and economic method to produce plentiful important molecules. To increase the product yield in biosynthesis, the involved enzymes can be immobilized to carriers for enhancing enzyme stability, increasing synthesis efficiency and improving enzyme recyclability. Hydrogels with three-dimensional porous structures and versatile functional groups are promising carriers for enzyme immobilization. Herein, we review the recent advances of the hydrogel-based multi-enzymatic system for biosynthesis...

The eCell technology is a recently introduced, specialized protein production platform with uses in a multitude of biotechnological applications. This chapter summarizes the use of eCell technology in four selected application areas. Firstly, for detecting heavy metal ions, specifically mercury, in an in vitro protein expression system. Results show improved sensitivity and lower limit of detection compared to comparable in vivo systems. Secondly, eCells are semipermeable, stable, and can be stored for extended periods of time, making them a portable and accessible technology for bioremediation of toxicants in extreme environments...

One of the grand challenges in bottom-up synthetic biology is the design and construction of synthetic cellular systems. One strategy toward this goal is the systematic reconstitution of biological processes using purified or non-living molecular components to recreate specific cellular functions such as metabolism, intercellular communication, signal transduction, and growth and division. Cell-free expression systems (CFES) are in vitro reconstitutions of the transcription and translation machinery found in cells and are a key technology for bottom-up synthetic biology...

Biopolymers, such as proteins and RNA, are integral components of living organisms and have evolved through a process of repeated mutation and selection. The technique of "cell-free in vitro evolution" is a powerful experimental approach for developing biopolymers with desired functions and structural properties. Since Spiegelman's pioneering work over 50 years ago, biopolymers with a wide range of functions have been developed using in vitro evolution in cell-free systems. The use of cell-free systems offers several advantages, including the ability to synthesize a wider range of proteins without the limitations imposed by cytotoxicity, and the capacity for higher throughput and larger library sizes than cell-based evolutionary experiments...

Microarrays are widely utilized in bioanalysis. Electrochemical biosensing techniques are often applied in microarray-based assays because of their simplicity, low cost, and high sensitivity. In such systems, the electrodes and sensing elements are arranged in arrays, and the target analytes are detected electrochemically. These sensors can be utilized for high-throughput bioanalysis and the electrochemical imaging of biosamples, including proteins, oligonucleotides, and cells. In this chapter, we summarize recent progress on these topics...

Cell-free protein synthesis (CFPS) with flexibility and controllability can provide a powerful platform for high-throughput screening of biomolecules, especially in the evolution of peptides or proteins. In this chapter, the emerging strategies for enhancing the protein expression level using different source strains, energy systems, and template designs in constructing CFPS systems are summarized and discussed in detail. In addition, we provide an overview of the ribosome display, mRNA display, cDNA display, and CIS display in vitro display technologies, which can couple genotype and phenotype by forming fusion complexes...

Cofactors, such as adenosine triphosphate, nicotinamide adenine dinucleotide, and coenzyme A, are involved in nearly 50% of enzymatic reactions and widely used in biocatalytic production of useful chemicals. Although commercial production of cofactors has been mostly dependent on extraction from microbial cells, this approach has a theoretical limitation to achieve a high-titer, high-yield production of cofactors owing to the tight regulation of cofactor biosynthesis in living cells. Besides the cofactor production, their regeneration is also a key challenge to enable continuous use of costly cofactors and improve the feasibility of enzymatic chemical manufacturing...

Cyanobacteria are promising microbial cell factories for the direct production of biochemicals and biofuels from CO2 . Through genetic and metabolic engineering, they can be modified to produce a variety of both natural and non-natural compounds. To enhance the yield of these products, various design strategies have been developed. In this chapter, strategies used to enhance metabolic fluxes towards common precursors used in biosynthesis, including pyruvate, acetyl-CoA, malonyl-CoA, TCA cycle intermediates, and aromatics, are discussed...

Hydrogen gas (H2 ) is one of the potential future sustainable and clean energy carriers that may substitute the use of fossil resources including fuels since it has a high energy content (heating value of 141.65 MJ/kg) when compared to traditional hydrocarbon fuels [1]. Water is a primary product of combustion being a most significant advantage of H2 being environmentally friendly with the capacity to reduce global greenhouse gas emissions. H2 is used in various applications. It generates electricity in fuel cells, including applications in transportation, and can be applied as fuel in rocket engines [2]...

Cyanobacteria are highly interesting microbes with the capacity for oxygenic photosynthesis. They fulfill an important purpose in nature but are also potent biocatalysts. This chapter gives a brief overview of this diverse phylum and shortly addresses the functions these organisms have in the natural ecosystems. Further, it introduces the main topics covered in this volume, which is dealing with the development and application of cyanobacteria as solar cell factories for the production of chemicals including potential fuels...

Cyanobacteria, the evolutionary originators of oxygenic photosynthesis, have the capability to convert CO2 , water, and minerals into biomass using solar energy. This process is driven by intricate bioenergetic mechanisms that consist of interconnected photosynthetic and respiratory electron transport chains coupled. Over the last few decades, advances in physiochemical analysis, molecular genetics, and structural analysis have enabled us to gain a more comprehensive understanding of cyanobacterial bioenergetics...

Fatty acids and their derivatives are highly valuable chemicals that can be produced through chemical or enzymatic processes using plant lipids. This may compete with human food sources. Therefore, there has been an urge to create a new method for synthesizing these chemicals. One approach is to use microbial cells, specifically cyanobacteria, as a factory platform. Engineering may need to be implemented in order to allow a cost-competitive production and to enable a production of a variety of different fatty acids and derivatives...

Great interest for large-scale production of medicinal mushroom biomass and various pharmaceutically active compounds production dictates the development of comprehensive technologies. Solid state and submerged cultivations in bioreactors represent the most promising technologies for fast and large amount production of medicinal fungi biomass and pharmaceutically active products for human and veterinary need. There are many stages from shaking culture studies to large-scale industrial production. Pilot-scale studies represent the bridge and the balance between the gap of laboratory and industrial scale...