Metabolic engineering tools for Saccharomyces cerevisiae

Increased human population and the rapid decline of fossil fuels resulted in a global tendency to look for alternative fuel sources. Environmental concerns about fossil fuel combustion led to a sharp move towards renewable and environmentally friendly biofuels. Ethanol has been the primary fossil fuel alternative due to its low carbon emission rates, high octane content and comparatively facile microbial production processes. In parallel to the increased use of bioethanol in various fields such as transportation, heating and power generation, improvements in ethanol production processes turned out to be a global hot topic. Ethanol is by far the leading yeast output amongst a broad spectrum of bio-based industries. Thus, as a well-known platform microorganism and native ethanol producer, baker’s yeast Saccharomyces cerevisiae has been the primary subject of interest for both academic and industrial perspectives in terms of enhanced ethanol production processes. Metabolic engineering strategies have been primarily adopted for direct manipulation of genes of interest responsible in mainstreams of ethanol metabolism. To overcome limitations of rational metabolic engineering, an alternative bottom-up strategy called inverse metabolic engineering has been widely used. In this context, evolutionary engineering, also known as adaptive laboratory evolution (ALE), which is based on random mutagenesis and systematic selection, is a powerful strategy to improve bioethanol production of S. cerevisiae. In this review, we focus on key examples of metabolic and evolutionary engineering for improved first- and second-generation S. cerevisiae bioethanol production processes. We delve into the current state of the field and show that metabolic and evolutionary engineering strategies are intertwined and many metabolically engineered strains for bioethanol production can be further improved by powerful evolutionary engineering strategies. We also discuss potential future directions that involve recent advancements in directed genome evolution, including CRISPR-Cas9 technology.

Synthetic biology and metabolic engineering strategies in identifying and producing plant natural products; with emphasis on the CRISPR/Cas systems

This book chapter addresses the recent advancements made in metabolic engineering for the heterologous production of flavonoids (both natural and non-natural) and also throws light on their biosynthesis and significance. Flavonoids represent the phenolic family of compounds. They are produced naturally as a product of plant secondary metabolism and have numerous health promoting benefits that make them valuable in food and pharmaceutical industries. But their limited production in plants cannot meet the globally increasing demand. The introduction of flavonoid biosynthesis pathways in microbial hosts by the application of metabolic engineering is a promising alternative for the large-scale production of flavonoids in an environmentally friendly manner. The modern innovations of synthetic/system biology in combination with metabolic engineering have been applied for the improvement of existing pathways as well as for the design, construction and optimization of the artificial pathways for the industrial scale production of flavonoids. Various rational and computational engineering strategies are available for optimizing the carbon flux towards the biosynthesis of flavonoids in a balanced manner without compromising the cell viability. Moreover, the emerging tools in metabolic engineering like computational and regulatory tools have increased the product titer by removing the bottlenecks from the pathway with minimum genetic perturbations. Also, the amalgamation of protein engineering and metabolic engineering have created novel enzymes as well as improved existing enzymes for the production of natural and non-natural flavonoids.

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.

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

Recent advances in systems metabolic engineering tools and strategies

Transplastomic approaches for metabolic engineering

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

Amorpha-4,11-diene synthase (ADS) catalyzes the first committed step in the artemisinin biosynthetic pathway, which is the first catalytic reaction enzymatically and genetically characterized in artemisinin biosynthesis. The advent of ADS in Artemisia annua is considered crucial for the emergence of the specialized artemisinin biosynthetic pathway in the species. Microbial production of amorpha-4,11-diene is a breakthrough in metabolic engineering and synthetic biology. Recently, numerous new techniques have been used in ADS engineering; for example, assessing the substrate promiscuity of ADS to chemoenzymatically produce artemisinin. In this review, we discuss the discovery and catalytic mechanism of ADS, its application in metabolic engineering and synthetic biology, as well as the role of sesquiterpene synthases in the evolutionary origin of artemisinin.

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.

Customized design of host-independent T7 expression system (HITES) for a broad host range.

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

Biological Tools for and from the Environment

Synthetic Biology, a multidisciplinary field, is growing rapidly. Improving the understanding of biological systems through mimicry and producing bio-orthogonal systems with new functions are two complementary pursuits in this field. A web server called FMM (From Metabolite to Metabolite) was developed for this purpose. FMM can reconstruct metabolic pathways form one metabolite to another metabolite among different species, based mainly on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and other integrated biological databases. Novel presentation for connecting different KEGG maps is newly provided. Both local and global graphical views of the metabolic pathways are designed. FMM has many applications in Synthetic Biology and Metabolic Engineering. For example, the reconstruction of metabolic pathways to produce valuable metabolites or secondary metabolites in bacteria or yeast is a promising strategy for drug production. FMM provides a highly effective way to elucidate the genes from which species should be cloned into those microorganisms based on FMM pathway comparative analysis. Consequently, FMM is an effective tool for applications in synthetic biology to produce both drugs and biofuels. This novel and innovative resource is now freely available at http://FMM.mbc.nctu.edu.tw/.

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.