Terpenoids are a large class of secondary metabolites known for their remarkable diverse biological activities, making them widely utilized in the pharmaceutical, food, cosmetic, biofuel and agricultural fields. However, the current production of terpenoids heavily relies on plant extraction and chemical synthesis, which brings about concerns regarding infield, environmental and ecological issues. With the advancements in metabolic engineering and emerging synthetic biology tools, it is now possible to sustainably produce these high value-added terpenoids using microbial chassis. Among them, yeast has emerged as a promising candidate for the heterologous biosynthesis of terpenoids due to its inherent advantages, including robustness, safety, and the availability of sufficient precursor. This review focuses on the diverse strategies employed to enable terpenoids production in yeasts. These strategies encompass metabolic engineering approaches to optimize the mevalonate pathway, protein engineering techniques to improve terpenoid biosynthesis, the applications of organelles compartmentalization, high throughput screening and global approaches for the development of efficient cell factories. Furthermore, this review discusses the future prospects and challenges associated with yeast-based terpenoid production, while also emphasizing guidelines for future studies in this field.
Uroporphyrin (UP) is a porphyrin compound with medical applications and a key precursor for heme biosynthesis. However, there is no biosynthetic strategy for UP production. In this study, we present a novel bioprocess for enhanced production of UP in engineered Escherichia coli. We first implemented the Shemin/C4 pathway heterologously in an E. coli strain with an enlarged intracellular pool of succinyl-CoA. Using a plasmid with the trc promoter regulating the expression of a synthesized gene operon, the effects of key pathway genes, including hemA, hemB, hemC, and hemD, on UP biosynthesis were characterized. By cultivating the resulting engineered E. coli strains in a batch bioreactor with 30 g/L glycerol under aerobic conditions, up to 901.9 mg/L UP was produced. Most of the synthesized UP was extracellularly secreted with a high purity more than 80 wt%, facilitating its downstream purification. The study paves the way for large-scale bio-based production of UP using synthetic biology and metabolic engineering strategies.
Photofermentative hydrogen production with non-sulfur purple bacteria like Cereibacter sphaeroides (formerly Rhodobacter sphaeroides) is a promising and sustainable process to convert organic waste into the energy carrier hydrogen gas. However, this conversion is inhibited by elevated organic nitrogen concentrations in the substrate, which limits its applicability to nitrogen-poor organic waste. We present genomic and transcriptomic insights into a substrain of Cereibacter sphaeroides strain 2.4.1 that shows unexpected high levels of photofermentative hydrogen evolution when fed with glutamate. Genome sequencing revealed 222 single nucleotide variances (SNVs) between the reference genome of C. sphaeroides strain 2.4.1 and the analyzed substrain H2. These affect 61 protein coding genes. A leucine-proline exchange is present in the σ54 factor (rpoN2 gene), a global hydrogen and nitrogen metabolism regulator. We propose a model how this mutation alters DNA-binding properties that explain the unexpected organic nitrogen tolerance of hydrogen production. Transcriptomic analyses under varying glutamate concentrations support this finding. Thus, we present the first thorough genomic and transcriptomic analysis of a Cereibacter strain that shows promising metabolic characteristics for biotechnological hydrogen gas production from organic waste. These results suggest a potential target for strain optimization. Possibly, our key finding can be transferred to other hydrogen producing microorganisms.
Pattern formation is a fundamental process in biological development, enabling the transformation of initially uniform or random states into spatially ordered structures. A comprehensive understanding of the formation and function of these patterns is crucial for unraveling the underlying principles of biological design and engineering. In recent years, synthetic biology has emerged as a powerful discipline for investigating and manipulating pattern formation in biological systems, involving the design and construction of novel biological components, circuits, and networks with specific functionalities. The integration of computational simulations (in silico) and experimental techniques (wet lab) in synthetic biology has significantly advanced our knowledge of pattern formation and its implications in biological design and engineering. This review provides an overview of the computational simulations employed in studying pattern formation and introduces the representative and cutting-edge experimental methods utilized in wet labs.
Serine integrases are emerging as one of the powerful tools for synthetic biology. They have been widely developed across genome engineering, biological part construction, genetic circuit design, and in vitro DNA assembly. However, the strategy of in vivo DNA assembly by serine integrases has not yet been reported. To address this opportunity, here we developed a serine integrase-based in vivo DNA (plasmid) assembly approach. First, we demonstrated that the engineered “Acceptor” plasmids could be assembled with diverse “Donor” plasmids by serine integrases (Bxb1 and phiC31) in Escherichia coli (E. coli). Then, by programming the “Donor” plasmids and the host E. coli cells, we established an assembly cascade procedure and finally constructed plasmids that could constitutively express three different fluorescent proteins. Moreover, we used this approach to assemble different chromoprotein genes and generated colored E. coli cells. We anticipate that this approach will enrich the serine integrase-based biotechnology toolbox, and accelerate multiple plasmid assembly for synthetic biology with broad applications.
Fast, flexible and non-randomized modification, production and screening of proteins in fully automated system are of high interest in biological research and applications. The conventional methods for protein engineering and screening, especially for mutations of multiple residues. are time consuming and often unreliable. We demonstrate here a new, fast and flexible protein production and screening method which combines linear expression template (LET) based cell free protein synthesis (CFPS) with specific screening methods. This approach is demonstrated using green fluorescence protein, phosphoserine aminotransferase (serC) and aspartokinase III (AKIII) as model systems. The results show that mutants with changes in different protein properties upon multiple point mutations can be produced and screened within 6 to 15 h. This method can be used further to generate mutants of enzymes and multi-enzyme complexes and be implemented within the workflow of a feedback-guided protein optimization and screening system.
Five-carbon (C5) and six-carbon (C6) chemicals are essential components in the manufacturing of a variety of pharmaceuticals, fuels, polymers, and other materials. However, the predominant reliance on chemical synthesis methods and unsustainable feedstock sources has placed significant strain on Earth’s finite fossil resources and the environment. To address this challenge and promote sustainability, significant efforts have been undertaken to re-program microorganisms through metabolic engineering and synthetic biology approaches allowing for bio-based manufacturing of these compounds. This review provides a comprehensive overview of the advancements in microbial production of commercially significant non-natural C5 chemicals, including 1-pentanol, 1,5-pentanediol, cadaverine, δ-valerolactam, glutaric acid, glutaconic acid, and 5-hydroxyvaleric acid, as well as C6 chemicals, including cis, cis-muconic acid, adipic acid, 1,6-hexamethylenediamine, 6-aminocaproic acid, β-methyl-δ-valerolactone, 1-hexanol, ε-caprolactone, 6-hydroxyhexanoic acid, and 1,6-hexanediol.
In this article, we provided an overview of the current state of the SynBio industry in China with a focus on its research and technology, its main applications, and major players. We also discussed future prospects including the challenges and advantages of the SynBio industry in China.
Great needs always motivate the birth and development of new disciplines and tools. Here we propose in vitro BioTransformation (ivBT) as a new biomanufacturing platform, between the two dominant platforms—microbial fermentation and enzymatic biocatalysis. ivBT mediated by in vitro synthetic enzymatic biosystems (ivSEBs) is an emerging biomanufacturing platform for the production of biocommodities (i.e., low-value and bulk biochemicals). ivSEB is the in vitro reconstruction of artificial (non-natural) enzymatic pathways with numerous natural enzymes, artificial enzymes, and/or (biomimetic or natural) coenzymes without living cell’s constraints, such as cell duplication, basic metabolisms, complicated regulation, bioenergetics, and so on. The two great needs (i.e., food security and the carbon-neutral renewable energy system) have motivated the birth and development of ivBT. Food security could be addressed by making artificial food from nonfood lignocellulose and artificial photosynthesis for starch synthesis from CO2. The carbon-neutral renewable energy system could be addressed by the construction of the electricity-hydrogen-carbohydrate cycle, where starch could be a high density of hydrogen carrier (up to 14.8% H2 wt/wt) and an electricity storage compound (greater than 3000 Wh/kg). Also, ivBT can make a number of biocommodities, such as inositol, healthy sweeteners (e.g., D-allulose, D-tagatose, D-mannose), advanced biofuels, polymer precursors, organic acids, and so on. The industrial biomanufacturing of the first several biocommodities (e.g., myo-inositol, D-tagatose, D-mannose, and cellulosic starch) would wipe off any prejudice and doubt on ivBT. Huge potential markets of biocommodities with more than tens of trillions of Chinese Yuan would motivate scientists and engineers to address the remaining technical challenges and develop new tools within the next decade.
