FMM: a web server for metabolic pathway reconstruction and comparative analysis

Direct Pathway Cloning (DiPaC) to unlock natural product biosynthetic potential

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

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

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.

Synthetic biology is an emerging technology that uses the principles of physics, chemistry, mathematics, and biology. Synthetic biology is the technology enabler that could be applied across all fields such as agricultural, food, health, chemical, and industrial manufacturing sectors. In this chapter, several applications of synthetic biology across all disciplines are delineated, and challenges associated with the implementation of synthetic biology technologies are addressed in detail.

Biological Tools for and from the Environment

Catalytic RNA, ribozyme, and its applications in synthetic biology

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.

T7 system is a commonly used in protein expression and the highest transcription activity of T7 RNAP usually caused the instability of T7 system. In order to apply T7 system extensively, it is essential to characterize T7 RNAP activity. In the present paper, an assay method for T7 RNAP activity was developed with a transcription-translation (TX-TL) system. After the optimization of TX-TL system, the operating parameters were determined as 34°C, 60min with 20ng/μl of plasmid DNA template. The standard curve of TX-TL assay method indicated an excellent correlation (r = 0.998), and the sensitivity was better than that of western blotting method. The precision investigation indicated a mean-relative error of 2.58% and a standard-relative error of 7.01%. Moreover, the cell lysate could be added directly to the optimized TX-TL system without affecting T7 RNAP activity assay. The feasibility of present method was further confirmed by characterizing T7 RNAP activity in cell lysate of five strains of Escherichia coli (E. coli) DH5α with different T7 RNAP activities and seven commercial strains of E. coli (DE3). The present assay method for T7 RNAP activity would have a great application in synthetic biology, metabolic engineering, enzyme engineering and biomedicine.

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.

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.

Methods for importing heterologous genes into genetically tractable hosts are among the most desired tools of synthetic biology. Easy plug-and-play construction methods to rapidly test genes and pathways stably in the host genome would expedite synthetic biology and metabolic engineering applications. Here, we describe a CRISPR-based strategy that allows highly efficient, single step integration of large pathways in Escherichia coli. This strategy allows high efficiency integration in a broad range of homology arm sizes and genomic positions, with efficiencies ranging from 70 to 100% in 7 distinct loci. To demonstrate the large size capability, we integrated a 10 kb construct to implement isobutanol production in a single day. The ability to efficiently integrate entire metabolic pathways in a rapid and markerless manner will facilitate testing and engineering of novel pathways using the E.coli genome as a stable testing platform.

As a core discipline for the engineering modification of biological systems, synthetic biology is currently confronted with key challenges such as poor functional stability of synthetic communities, insufficient metabolic pathway adaptability of chassis strains, and difficulties in simulating interactions in natural microecosystems. Traditional single technologies fail to achieve precise design and regulation of synthetic biological systems. However, microbiomics, which integrates multi-omics, spatial omics, and culturomics, provides a full-chain solution for synthetic biology covering "functional prediction-dynamic regulation-functional verification-community construction" by decoding multidimensional molecular information, spatial distribution patterns, and cultivation interaction mechanisms of biological systems. This chapter systematically elaborates on how integrated multi-omics guides the optimization of metabolic pathways in synthetic biological systems, how spatial omics deciphers the spatial interaction mechanisms of synthetic communities, and how innovations in culturomics break through the cultivation bottlenecks of synthetic strains. Ultimately, it realizes the in-depth integration of omics technologies and synthetic biology, thereby promoting the industrial application of synthetic biological systems in fields such as biomanufacturing, environmental remediation, and medical health.

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.