Heterologous biomimetic synthesis of active ingredients in traditional Chinese medicine:a new mode for protection and development of traditional Chinese medicine resources / 中国中药杂志

Heterologous biomimetic synthesis of the active ingredients of traditional Chinese medicine(TCM) is a new mode of resource acquisition and has shown great potential in the protection and development of TCM resources. According to synthetic biology and by constructing biomimetic microbial cells and imitating the synthesis of active ingredients in medicinal plants and animals, the key enzymes obtained from medicinal plants and animals are scientifically designed and systematically reconstructed and optimized to realize the heterologous synthesis of the active ingredients in microorganisms. This method ensures an efficient and green acquisition of target products, and also achieves large-scale industrial production, which is conducive to the production of scarce TCM resources. Additiona-lly, the method playes a role in agricultural industrialization, and provides a new option for promoting the green and sustainable deve-lopment of TCM resources. This review systematically summarized the important progress in the heterologous biomimetic synthesis of TCM active ingredients from three research areas: biosynthesis of terpenoids, flavonoids, phenylpropanoids, alkaloids and other active ingredients, key points and difficulties in heterologous biomimetic synthesis, and biomimetic cells with complex TCM ingredients. This study facilitated the application of new generation of biotechnology and theory to the development of TCM.

Engineering microbial consortia through synthetic biology approach / 生物工程学报

There are a large number of natural microbial communities in nature. Different populations inside the consortia expand the performance boundary of a single microbial population through communication and division of labor, reducing the overall metabolic burden and increasing the environmental adaptability. Based on engineering principles, synthetic biology designs or modifies basic functional components, gene circuits, and chassis cells to purposefully reprogram the operational processes of the living cells, achieving rich and controllable biological functions. Introducing this engineering design principle to obtain structurally well-defined synthetic microbial communities can provide ideas for theoretical studies and shed light on versatile applications. This review discussed recent progresses on synthetic microbial consortia with regard to design principles, construction methods and applications, and prospected future perspectives.

Tools for large-scale genetic manipulation of yeast genome / 生物工程学报

Large-scale genetic manipulation of the genome refers to the genetic modification of large fragments of DNA using knockout, integration and translocation. Compared to small-scale gene editing, large-scale genetic manipulation of the genome allows for the simultaneous modification of more genetic information, which is important for understanding the complex mechanisms such as multigene interactions. At the same time, large-scale genetic manipulation of the genome allows for larger-scale design and reconstruction of the genome, and even the creation of entirely new genomes, with great potential in reconstructing complex functions. Yeast is an important eukaryotic model organism that is widely used because of its safety and easiness of manipulation. This paper systematically summarizes the toolkit for large-scale genetic manipulation of the yeast genome, including recombinase-mediated large-scale manipulation, nuclease-mediated large-scale manipulation, de novo synthesis of large DNA fragments and other large-scale manipulation tools, and introduces their basic working principles and typical application cases. Finally, the challenges and developments in large-scale genetic manipulation are presented.

Advances on the microbial synthesis of plant-derived diterpenoids / 生物工程学报

Natural plant-derived diterpenoids are a class of compounds with diverse structures and functions. These compounds are widely used in pharmaceuticals, cosmetics and food additives industries because of their pharmacological properties such as anticancer, anti-inflammatory and antibacterial activities. In recent years, with the gradual discovery of functional genes in the biosynthetic pathway of plant-derived diterpenoids and the development of synthetic biotechnology, great efforts have been made to construct a variety of diterpenoid microbial cell factories through metabolic engineering and synthetic biology, resulting in gram-level production of many compounds. This article summarizes the construction of plant-derived diterpenoid microbial cell factories through synthetic biotechnology, followed by introducing the metabolic engineering strategies applied to improve plant-derived diterpenoids production, with the aim to provide a reference for the construction of high-yield plant-derived diterpenoid microbial cell factories and the industrial production of diterpenoids.

Fermentative production of tetraacetyl phytosphingosine: a review / 生物工程学报

Tetraacetyl phytosphingosine (TAPS) is an excellent raw material for natural skin care products. Its deacetylation leads to the production of phytosphingosine, which can be further used for synthesizing the moisturizing skin care product ceramide. For this reason, TAPS is widely used in the skin care oriented cosmetics industry. The unconventional yeast Wickerhamomyces ciferrii is the only known microorganism that can naturally secrete TAPS, and it has become the host for the industrial production of TAPS. This review firstly introduces the discovery, functions of TAPS, and the metabolic pathway for TAPS biosynthesis is further introduced. Subsequently, the strategies for increasing the TAPS yield of W. ciferrii, including haploid screening, mutagenesis breeding and metabolic engineering, are summarized. In addition, the prospects of TAPS biomanufacturing by W. ciferrii are discussed in light of the current progresses, challenges, and trends in this field. Finally, guidelines for engineering W. ciferrii cell factory using synthetic biology tools for TAPS production are also presented.

Preface for special issue on chemical bioproduction / 生物工程学报

Engineering efficient enzymes or microbial cell factories should help to establish green bio-manufacturing process for chemical overproduction. The rapid advances and development in synthetic biology, systems biology and enzymatic engineering accerleate the establishing feasbile bioprocess for chemical biosynthesis, including expanding the chemical kingdom and improving the productivity. To consolidate the latest advances in chemical biosynthesis and promote green bio-manufaturing, we organized a special issue on chemical bioproduction that including review or original research papers about enzymatic biosynthesis, cell factory, one-carbon based biorefinery and feasible strategies. These papers comprehensively discussed the latest advaces, the challenges as well as the possible solutions in chemical biomanufacturing.

The degradation of plastics and the production of polyhydroxyalkanoates (PHA) / 生物工程学报

In recent years, the petroleum-based plastic pollution problem has been causing global attention. The idea of "degradation and up-cycling of plastics" was proposed for solving the environmental pollution caused by non-degradable plastics. Following this idea, plastics would be firstly degraded and then reconstructed. Polyhydroxyalkanoates (PHA) can be produced from the degraded plastic monomers as a choice to recycle among various plastics. PHA, a family of biopolyesters synthesized by many microbes, have attracted great interest in industrial, agricultural and medical sectors due to its biodegradability, biocompatibility, thermoplasticity and carbon neutrality. Moreover, the regulations on PHA monomer compositions, processing technology, and modification methods may further improve the material properties, making PHA a promising alternative to traditional plastics. Furthermore, the application of the "next-generation industrial biotechnology (NGIB)" utilizing extremophiles for PHA production is expected to enhance the PHA market competitiveness, promoting this environmentally friendly bio-based material to partially replace petroleum-based products, and achieve sustainable development with carbon-neutrality. This review summarizes the basic material properties, plastic upcycling via PHA biosynthesis, processing and modification methods of PHA, and biosynthesis of novel PHA.

Characterization and application of several lysis cassettes / 生物工程学报

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S105, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD600, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S105, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.

Biomanufacturing driven by engineered organisms (2022) / 生物工程学报

This article summarizes the reviews and original research papers published in Chinese Journaol of Biotechnology in the area of biomanufacturing driven by engineered organisms in the year of 2022. The enabling technologies including DNA sequencing, DNA synthesis, and DNA editing as well as regulation of gene expression and in silico cell modeling were highlighted. This was followed by discussing the biomanufacturing of biocatalytics products, amino acids and its derivatives, organic acids, natural products, antibiotics and active peptides, functional polysaccharides, and functional proteins. Lastly, the technologies for utilizing C1 compounds and biomass as well as synthetic microbial consortia were discussed. The aim of this article was to help the readers to gain insights into this rapidly developing field from the journal point of view.

Application of live biotherapeutic products and perspective in the treatment of inherited metabolic disease / 生物工程学报

Live biotherapeutic products (LBPs) refer to the living bacteria derived from human body intestinal gut or in nature that can be used to treat the human disease. However, the naturally screened living bacteria have some disadvantages, such as deficient therapeutic effect and great divergence, which fall short of the personalized diagnosis and treatment needs. In recent years, with the development of synthetic biology, researchers have designed and constructed several engineered strains that can respond to external complex environmental signals, which speeded up the process of development and application of LBPs. Recombinant LBPs modified by gene editing can have therapeutic effect on specific diseases. Inherited metabolic disease is a type of disease that causes a series of clinical symptoms due to the genetic defect of some enzymes in the body, which may cause abnormal metabolism the corresponding metabolites. Therefore, the use of synthetic biology to design LBPs targeting specific defective enzymes will be promising for the treatment of inherited metabolic defects in the future. This review summarizes the clinic applications of LBPs and its potential for the treatment of inherited metabolic defects.

Construction of cell factories for production of valencene in Saccharomyces cerevisiae / 药学学报

Valencene, a kind of sesquiterpenoid with a citrus flavor, is mainly found in Valencia orange and is commonly used in cosmetics and food additives, as well as industrial synthetic nootkatone. In this study, synthetic biology was used to create a Saccharomyces cerevisiae cell factory to produce valencene. Fistly, valencene synthase gene (CnVS) from Callitropsis nootkatensis was inserted into the chromosome of the chassis strain YTT-T5. The resulting strain VAL-01 could produce 1.1 mg·L-1 valencene. Protein fusion technique was used, different valencene synthases were compared and the copy number of key genes was adjusted, yielding valencene to 436.4 mg·L-1. Then, knocking-out the transcription factor ROX1 resulted in valencene improvement by 17.4%. Moreover, the induction system of galactose was regulated, transcription factor PDR3 and INO2 were overexpressed. The engineered strain VAL-10 could produce 2 798.6 mg·L-1 valencene by high cell density fermentation method (nearly 2 500 times higher than VAL-01). This study provides a basis for green production of valencene.

Recent advances in bacterial therapeutics based on sense and response

Intelligent drug delivery is a promising strategy for cancer therapies. In recent years, with the rapid development of synthetic biology, some properties of bacteria, such as gene operability, excellent tumor colonization ability, and host-independent structure, make them ideal intelligent drug carriers and have attracted extensive attention. By implanting condition-responsive elements or gene circuits into bacteria, they can synthesize or release drugs by sensing stimuli. Therefore, compared with traditional drug delivery, the usage of bacteria for drug loading has better targeting ability and controllability, and can cope with the complex delivery environment of the body to achieve the intelligent delivery of drugs. This review mainly introduces the development of bacterial-based drug delivery carriers, including mechanisms of bacterial targeting to tumor colonization, gene deletions or mutations, environment-responsive elements, and gene circuits. Meanwhile, we summarize the challenges and prospects faced by bacteria in clinical research, and hope to provide ideas for clinical translation.

Metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3β,20S-Di-O-Glc-DM

Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity. Traditional approaches for producing them rely on direct extraction from Panax ginseng, enzymatic catalysis or chemical synthesis, all of which result in low yield and high cost. Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products. Hence we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-O-Glc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titers of F2 and 3β,20S-Di-O-Glc-DM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level, respectively, by multistep metabolic engineering strategies. Additionally, pharmacological evaluation showed that both F2 and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-Glc-DM was even better. Furthermore, the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor. To our knowledge, this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM, and achieving their de novo biosynthesis by the engineered yeasts. Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass, which lays a foundation for drug research and development.

Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient's body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.

Advances of synthetic biology of flavonoids / 药学学报

Flavonoids is one of the biggest families of the plant-derived secondary metabolites with structural diversity. Until now, over 10 000 kinds of flavonoids with distinct structures have been purified and identified from plants, and some of them possess a range of important pharmacological effects, such as anticancer, anti-inflammatory and so on. So far, a number of genes and enzymes responsible for the biosynthesis of flavonoids have been reported, especially, a great of progress has been achieved in the synthetic biology of flavonoids in the recent years. Herein, based upon a brief introduction on the biosynthesis of flavonoids, this review summarizes the research advances in synthetic biology of flavonoids in the past two decades (2001-2021), highlighting the cell factories construction of the representative flavonoids. And, a brief discussion and prospects of the relevant metabolic bottlenecks and optimizing strategies are proposed.

Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae / 中国中药杂志

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Design and implementation of the course on Synthetic Biology based on the concept of general education / 生物工程学报

As an emerging branch of biology, Synthetic Biology has seen rapid development with great potential in theoretical research and application. With a lot of brand-new concepts and research methods, it brings challenges to university teachers, and little experience is available in China on the teaching of Synthetic Biology. In this study, we discussed the general education-based development and application of the course on Synthetic Biology (a discipline in "liberal arts" in Zhejiang University) from the background, design, implementation, outcome, and problems of the course, hoping to provide a reference for the optimization of the course and the design of similar courses in other universities in China.

Cultivation of college students' innovative and entrepreneurial thinking and ability based on Synthetic Biology and iGEM / 生物工程学报

Synthetic Biology is one of the most promising fields of modern Biology and a frontier interdisciplinary subject in the 21st century. With the rapid development of synthetic biology, the International Genetically Engineered Machine (iGEM) competition has emerged. The iGEM competition, based on the subject foundation of Synthetic Biology, intends to solve the biological problems in our daily life by applying modern biological technology. In recent years, with the continuous increase of participating teams, the iGEM competition has received extensive attention and achieved great progress. On the basis of the development of Synthetic Biology, we analyzed the 2018-2020 award-winning projects of the iGEM competition and illustrated the role and significance of the iGEM competition in cultivating college students' innovative thinking and ability with the participation experience of the iGEM team of Southwest Jiaotong University as an example.

Application and population control strategy of microbial modular co-culture engineering / 生物工程学报

Traditional methods of microbial synthesis usually rely on a single engineered strain to synthesize the target product through metabolic engineering. The key cofactors, precursors and energy are produced by the introduced complex synthetic pathways. This would increase the physiological burden of engineering strains, resulting in a decrease in the yield of target products. The modular co-culture engineering has become an attractive solution for effective heterologous biosynthesis, where product yield can be greatly improved. In the modular co-culture engineering, the coordination between the population of different modules is essential for increasing the production efficiency. This article summarized recent advances in the application of modular co-culture engineering and population control strategies.

Production of carboxylic acids by metabolically engineered Yarrowia lipolytica: a review / 生物工程学报

Yarrowia lipolytica is a non-conventional yeast with unique physiological and metabolic characteristics. It is suitable for production of various products due to its natural ability to utilize a variety of inexpensive carbon sources, excellent tolerance to low pH, and strong ability to secrete metabolites. Currently, Y. lipolytica has been demonstrated to produce a wide range of carboxylic acids with high efficiency. This article summarized the progress in engineering Y. lipolytica to produce various carboxylic acids by using metabolic engineering and synthetic biology approaches. The current bottlenecks and solutions for high-level production of carboxylic acids by engineered Y. lipolytica were also discussed, with the aim to provide useful information for relevant studies in this field.