Expression of nano-engineered RNA organelles in bacteria.

BackgroundAdvanced DNA synthesis, biosensor assembly, and genetic circuit development in synthetic biology and metabolic engineering have reinforced the application of filamentous bacteria, yeasts, and fungi as promising chassis cells for chemical production, but their industrial application remains a major challenge that needs to be solved.ResultsAs important chassis strains, filamentous microorganisms can synthesize important enzymes, chemicals, and niche pharmaceutical products through microbial fermentation. With the aid of metabolic engineering and synthetic biology, filamentous bacteria, yeasts, and fungi can be developed into efficient microbial cell factories through genome engineering, pathway engineering, tolerance engineering, and microbial engineering. Mutant screening and metabolic engineering can be used in filamentous bacteria, filamentous yeasts (Candida glabrata, Candida utilis), and filamentous fungi (Aspergillus sp., Rhizopus sp.) to greatly increase their capacity for chemical production. This review highlights the potential of using biotechnology to further develop filamentous bacteria, yeasts, and fungi as alternative chassis strains.ConclusionsIn this review, we recapitulate the recent progress in the application of filamentous bacteria, yeasts, and fungi as microbial cell factories. Furthermore, emphasis on metabolic engineering strategies involved in cellular tolerance, metabolic engineering, and screening are discussed. Finally, we offer an outlook on advanced techniques for the engineering of filamentous bacteria, yeasts, and fungi.

Chapter 1 - Systems biology, synthetic biology, and metabolic engineering

3rd congress on applied synthetic biology in Europe (Costa da Caparica, Portugal, February 2016)

Metabolic engineering is defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. Cellular engineering, a more inclusive term, is defined as the purposeful modification of cell properties using the same techniques. Examples of cellular and metabolic engineering are divided into five categories: 1. Improved production of chemicals already produced by the host organism; 2. Extended substrate range for growth and product formation; 3. Addition of new catabolic activities for degradation of toxic chemicals; 4. Production of chemicals new to the host organism; and 5. Modification of cell properties. Over 100 examples of cellular and metabolic engineering are summarized. Several molecular biological, analytical chemistry, and mathematical and computational tools of relevance to cellular and metabolic engineering are reviewed. The importance of host selection and gene selection is emphasized. Finally, some future directions and emerging areas are presented.

BiotecVisions 2012, April

Condensation of RNA and proteins is central to cellular functions, and the ability to program it would be valuable in synthetic biology and synthetic cell science. Here we introduce a modular platform for engineering synthetic RNA condensates from tailor-made, branched RNA nanostructures that fold and assemble co-transcriptionally. Up to three orthogonal condensates can form simultaneously and selectively accumulate fluorophores through embedded fluorescent light-up aptamers. The RNA condensates can be expressed within synthetic cells to produce membrane-less organelles with a controlled number and relative size, and showing the ability to capture proteins using selective protein-binding aptamers. The affinity between otherwise orthogonal nanostructures can be modulated by introducing dedicated linker constructs, enabling the production of bi-phasic RNA condensates with a prescribed degree of interphase mixing and diverse morphologies. The in situ expression of programmable RNA condensates could underpin the spatial organization of functionalities in both biological and synthetic cells.

Living organisms in analogy with chemical factories use simple molecules such as sugars to produce a variety of compounds which are necessary for sustaining life and some of which are also commercially valuable. The metabolisms of simple (such as bacteria) and higher organisms (such as plants) alike can be exploited to convert low value inputs into high value outputs. Unlike conventional chemical factories, microbial production chassis are not necessarily tuned for a single product overproduction. Despite the same end goal, metabolic and industrial engineers rely on different techniques for achieving productivity goals. Metabolic engineers cannot affect reaction rates by manipulating pressure and temperature, instead they have at their disposal a range of enzymes and transcriptional and translational processes to optimize accordingly. In this review, we first highlight how various analytical approaches used in metabolic engineering and synthetic biology are related to concepts developed in systems and control engineering. Specifically, how algorithmic concepts derived in operations research can help explain the structure and organization of metabolic networks. Finally, we consider the future directions and challenges faced by the field of metabolic network modeling and the possible contributions of concepts drawn from the classical fields of chemical and control engineering. The aim of the review is to offer a current perspective of metabolic engineering and all that it entails without requiring specialized knowledge of bioinformatics or systems biology.

Nucleolus activity-dependent recruitment and biomolecular condensation by pH sensing

Prospects and progress in the production of valuable carotenoids: Insights from metabolic engineering, synthetic biology, and computational approaches

Hemoglobin is an essential protein to the human body as it transports oxygen to organs and tissues through the bloodstream (Looker et al., 1992). In recent years, there has been an increasing concern regarding the global supply of this vital protein, as blood availability cannot currently meet the high demands in many developing countries. There are, in addition, several risks associated with conventional blood transfusions such as the presence of blood-borne viruses like HIV and Hepatitis. These risks along with some limitations are presented in Figure 1 (Kim and Greenburg, 2013; Martínez et al., 2015). As an alternative, producing hemoglobin recombinantly will eliminate the obstacles, since hemoglobin-based oxygen carriers are pathogen-free, have a longer shelf-life, are universally compatible and the supply can be adjusted to meet the demands (Chakane, 2017). A stable, safe, and most importantly affordable production, will lead to high availability of blood to the world population, and hence reduce global inequality, which is a focus point of the World Health Organization for the millennium (WHO, 2018). Synthetic biology and metabolic engineering have created a unique opportunity to construct promising candidates for hemoglobin production (Liu et al., 2014; Martínez et al., 2016). This review sets out to describe the recent advances in recombinant hemoglobin production, the societal and the economic impact along with the challenges that researchers will face in the coming years, such as low productivity, degradation, and difficulties in scale-up. The challenges are diverse and complex but with the powerful tools provided by synthetic biology and metabolic engineering, they are no longer insurmountable. An efficient production of cell-free recombinant hemoglobin poses tremendous challenges while having even greater potential, therefore some possible future directions are suggested in this review.

The ability of biomolecular condensates to reversibly dissolve and reform is crucial for maintaining cellular stability and functions. In the context of cell physiology and disease, they can serve as a metastable phase mediating the liquid-to-solid transition of disease proteins or rapidly assemble/disassemble as a mechanism for stress response. However, as metabolic rates decline with aging, the protein-rich condensates persist longer, therefore increasing the propensity of undergoing irreversible liquid-to-solid transitions. Temperature, as a physical stimulus, plays a key role in controlling the condensate formation, dissolution, and material properties. In this study, we explore how the reversibility of short peptide biomolecular condensates (z-FF) can be modulated by a temperature change. Our findings reveal that aged condensates exhibit reduced responsiveness to external temperature stimuli. By using thermal cycling experiments to simulate repeated heat stress, we found that the time taken for irreversible fiber formation could be delayed up to 4.7-fold compared to that of condensates without thermal cycles. We also found that the dissolution rate of condensates progressively slows as they age but remains more stable with thermal cycles. Importantly, our results indicate that continuous cycles of liquid-liquid phase separation and dissolution act as a reset mechanism, preserving the biomolecular condensates from further liquid-to-solid transition. These findings provide valuable insights into how aging impacts condensate behavior and highlight potential strategies to preserve cellular function through controlled phase transitions.

Metabolic engineering, a core pillar of synthetic biology, enables the precise reprogramming of cellular metabolism for efficient production of valuable compounds. This review outlines recent advances in genome editing, pathway construction, and dynamic regulation strategies that underpin modern metabolic engineering. Emerging tools such as CRISPR-derived editors, machine learning-guided design, and automated high-throughput workflows have accelerated the design–build–test–learn cycle and enabled predictive strain optimization. Applications span microbial cell factories, mammalian and plant hosts, cell-free biosynthesis systems, synthetic consortia, and non-traditional chassis such as photosynthetic microbes, extremophiles, and filamentous fungi. These engineered systems have facilitated the biosynthesis of fuels, pharmaceuticals, specialty chemicals, and nutraceuticals, often under environmentally sustainable conditions. Challenges remain in scaling processes, overcoming metabolic bottlenecks, and ensuring biosafety in complex systems. Nevertheless, the integration of systems biology, artificial intelligence, and synthetic design is ushering in a new era of intelligent and modular biomanufacturing. Continued innovation in chassis development, global metabolic rewiring, and closed-loop automation will further expand the capabilities and impact of metabolic engineering across biotechnology and industrial applications.

Metabolic engineering tools for Saccharomyces cerevisiae

Efficient production of chemicals from microorganism by metabolic engineering and synthetic biology

Proteins with chemically regulatable phase separation are of great interest in the fields of biomolecular condensates and synthetic biology. Intrinsically disordered proteins (IDPs) are the dominating building blocks of biomolecular condensates which often lack orthogonality and small-molecule regulation desired to create synthetic biomolecular condensates or membraneless organelles (MLOs). Here, we discover a well-folded globular protein, lipoate-protein ligase A (LplA) from E. coli involved in lipoylation of enzymes essential for one-carbon and energy metabolisms, that exhibits structural homomeric oligomerization and a rare LCST-type reversible phase separation in vitro. In both E. coli and human U2OS cells, LplA can form orthogonal condensates, which can be specifically dissolved by its natural substrate, the small molecule lipoic acid and its analogue lipoamide. The study of LplA phase behavior and its regulatability expands our understanding and toolkit of small-molecule regulatable protein phase behavior with impacts on biomedicine and synthetic biology.