Rapid and Efficient One-Step Metabolic Pathway Integration in E.coli.

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/.

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

Biological Tools for and from the Environment

Synthetic biology is built on genetic engineering and principles of design engineering, which provides control over the biological functions of interest. This chapter explores the uses, processes, and applications of genetic engineering in synthetic biology. The chapter provides a brief history and course of development of the field of synthetic biology and genetic engineering and their unbreakable association. Next, the chapter delves into materials and methods and the applications of synthetic biology. This includes discussing the generally used components of genetic engineering to design new functions into organisms and even the general steps that are part of any synthetic biology experiment. Lastly, the chapter also explains the use of the materials and methodology discussed in solving a specific problem related to amodel mentioned in the paper titled"Development of Integrase-mediated differentiation circuits to improve evolutionary stability in E. coli." It explains how by using genetic engineering a synthetic biology-related problem was solved efficiently.

BackgroundThe genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences.ResultsWe showed that, in genome-integrated E. coli expression systems that used σ70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.ConclusionsBased on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

Metabolite biosensors based on metabolite-responsive transcription factors are key synthetic biology components for sensing and precisely controlling cellular metabolism. Biosensors are often designed under laboratory conditions but are deployed in applications where cellular growth rate differs drastically from its initial characterization. Here we asked how growth rate impacts the minimum and maximum biosensor outputs and the dynamic range, which are key metrics of biosensor performance. Using LacI, TetR, and FadR-based biosensors in Escherichia coli as models, we find that the dynamic range of different biosensors have different growth rate dependencies. We developed a kinetic model to explore how tuning biosensor parameters impact the dynamic range growth rate dependence. Our modeling and experimental results revealed that the effects to dynamic range and its growth rate dependence are often coupled, and the metabolite transport mechanisms shape the dynamic range-growth rate response. This work provides a systematic understanding of biosensor performance under different growth rates, which will be useful for predicting biosensor behavior in broad synthetic biology and metabolic engineering applications.

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

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

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.

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

Synthetic biology is an interdisciplinary field that combines engineering principles to design and construct new biological components, devices, and systems for understanding and reprogramming biological functions. This field aims to create novel biological entities with specific functions or solutions to particular problems through precise manipulation of biomolecules and cells. Bladder cancer is a type of cancer that originates in the tissues of the urinary bladder and primarily affects the urothelial cells lining the bladder wall. Synthetic biology technology, while relatively new for the treatment of bladder cancer, has promising potential for providing innovative solutions for the detection, treatment, and management of bladder cancer. This article reviews the latest research progress in the field of synthetic biology applied to bladder cancer. This research focuses on the application of gene editing technologies such as CRISPR–CRISPR-associated protein 9 to precisely modify the genome of bladder cancer cells to inhibit their growth and proliferation. Additionally, it introduces methods for enhancing antitumor immune responses through the modification of immune cells, such as chimeric antigen receptor-T-cell therapy. Furthermore, this article explores the potential of the use of genetically engineered bacteria as an emerging treatment option for bladder cancer. Despite challenges such as targeting specificity, safety, and cost, synthetic biology technologies provide new perspectives and strategies for the treatment of bladder cancer. With continuous advancements in technology and strengthened interdisciplinary collaboration, the application of synthetic biology in bladder cancer treatment holds great promise, potentially offering patients new treatment options and hope.

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.

Being the green gold of the future, microalgae and cyanobacteria have recently attracted considerable interest worldwide, for their metabolites such as lipids, protein, pigments, and bioactive compounds have immense potential for sustainable energy and pharmaceutical production capabilities. In the last decades, the efforts attended to enhance the usage of microalgae and cyanobacteria by genetic manipulation, synthetic and metabolic engineering. However, the development of photoautotrophic cell factories have rarely compared to the heterotrophic counterparts due to limited tools, bioinformatics, and multi-omics database. Therefore, recent advances of their genome editing techniques by clustered regularly interspaced short palindromic repeats (CRISPR) technology, and potential applications of their metabolic engineering and regulation approaches are examined in this review. Moreover, the contemporary achievements of synthetic biology approaches of microalgae and cyanobacteria in carbon fixation and sequestration, lipid and triacylglycerol (TAG), and sustainable production of high value-added chemicals, such as carotenoids and docosahexaenoic acid (DHA), have been also discussed. From recent genomic study to trends in metabolic regulation of microalgae and cyanobacteria and a comprehensive assessment of the current challenges and opportunities for microalgae and cyanobacteria is also conducted.

SyPro Poplar: Improving Poplar Biomass Production under Abiotic Stress Conditions: an Integrated Omics, Bioinformatics, Synthetic Biology and Genetic Engineering Approach In the SyPro Poplar project, we aimed to integrate omics, bioinformatics, synthetic biology, and genetic engineering approaches to develop transgenic poplar trees with sustained photosynthetic activity and increased biomass production under individual and the simultaneous occurrence of water deficit, increased soil salinity, and elevated temperatures. Specifically, we intended to (i) study the functions of selected stress-responsive genes at tissue and cell type-specific levels, (ii) discover novel motifs and construct stress-responsive synthetic promoters, and (iii) use these promoters to drive the expression of genes shown to confer abiotic stress tolerance in a variety of crops and develop abiotic stress-tolerant poplars in a coordinated fashion.

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.