. Because these plant natural products have strong physiological activities on human body, they have been widely used in the fields of medicine and health. According to the report on China's drug market (2012), a blue book on medicine of the Chinese Academy of Social Sciences, the total size of China's drug market in 2012 was 926.1 billion yuan; It is estimated that by 2020, the scale of China's pharmaceutical market will reach 2.3 trillion yuan^[1]Plant natural products have always been an important source of drugs, health products and cosmetics, such as morphine, artemisinin, ephedrine, paclitaxel and other drugs with significant efficacy are plant derived drugs; Hormone drugs with annual sales of more than 40billion US dollars are mainly produced from plant saponins such as diosgenin, papain and sisal saponin^[2, 3]In addition, plant natural products also have a wide range of needs and applications in pesticides, veterinary drugs and other industrial and agricultural production fields closely related to human life. For example, berberine and other alkaloid antibiotics in Coptis chinensis have good safety in animal husbandry, and have become ideal alternatives to feed antibiotics. At the same time, high-quality plant natural products, such as ginsenosides, rose essential oil, lycopene, anthocyanins, new sweeteners siraitin and stevia, are the main raw materials of human cosmetics, health products and flavoring agents, with broad market prospects^[3]。

Plant extraction is the main production mode of plant natural products at present. This traditional production mode has many disadvantages, such as low content and large difference of natural products, long plant growth cycle, complex analogues that lead to difficult product purification, and serious damage to biological resources, especially wild plant resources^[3-5]With the increasing market demand, the original plants of wild rare traditional Chinese medicines Ganoderma lucidum, ginseng and Panax notoginseng have been endangered or extinct in the wild, and the current resource supply has been unsustainable^[3]In chemical synthesis, due to the complex structure and more chiral centers of most natural products, inactive or even toxic optical isomers, which are difficult to separate, are easily formed in the synthesis process; ^[3]However, the operation of plant tissue and cell culture method is more complex, the cycle is long, and because the production cost is too high, it is not easy to realize industrialization. Comparatively speaking, using the similar principle of brewing beer with Saccharomyces cerevisiae to produce plant natural products through fermentation has the advantages of short production cycle, no restriction of season and raw material supply, relatively simple fermentation products, easy separation and purification, and easy to achieve large-scale industrial production.

Based on the principle of synthetic biology, the design and creation of artificial synthetic cell fermentation to produce plant natural products can not only effectively control the supply of raw materials, but also protect natural resources and the environment. As a green and efficient new production mode, it has been recognized by the scientific and industrial circles^[3]In recent years, with the rapid development of synthetic biology, the types of plant derived natural products synthesized by microorganisms are increasing, and the output is also increasing year by year^[6-8]Starting from the research route, this paper will introduce the research status of the production of plant derived natural products by synthetic cells in the production and application cases of terpenoids, phenylpropanoids and alkaloids.

In the process of realizing the fermentation production of plant natural products, the mining and optimization of characteristic gene elements, the optimization of biosynthetic pathways, and the improvement of cell factory performance constitute the basic content of plant natural product synthesis biology research.

In addition to promoters, terminators and other gene elements that mainly control gene expression, identifying and optimizing key gene elements in the biosynthetic pathway of plant natural products is the core and source of applying synthetic biology technology to innovate the production mode of natural products.

At present, the analysis of biosynthetic pathways of plant natural products mainly adopts the methods of genome or transcriptome heterologous reconstruction. In recent years, based on the development of gene sequencing technology and bioinformatics, morphine^[9-12], glycyrrhizic acid^[13]、Ginsenosides^[5], Tanshinone^[7, 14], cucurbitacin B^[15], momordoside V^[16], etoposide^[17]And vinblastine^[18]Breakthroughs have been made in the analysis of biosynthetic pathways of important plant natural products. A number of important types of gene functional elements have been mined and identified, among which the catalytic mechanism of cytochrome P450 enzyme, known as "universal biocatalyst", has been thoroughly studied^[19-21]However, there are tens of thousands of valuable plant natural products (such as artemisinin and morphine) in nature. Due to various constraints, in such a rich group, only a few molecular biosynthetic mechanisms have been resolved. Therefore, developing an efficient, reliable and low-cost method platform to analyze the molecular basis and process mechanism of large-scale synthesis of plant natural products is of great strategic significance for the systematic protection and effective development of plant natural products, a natural treasure house.

In terms of functional element optimization, rational design method can be used for enzymes with clear protein crystal structure and catalytic mechanism. For example, based on the structure of Candida cellulosae xylose reductase (XR), the amino acid sites such as lys274 and asn276 were mutated, and it was found that the preference of XR for the cofactor NADPH increased by 170 times^[3, 22]However, the types of functional enzymes derived from the synthesis pathway of plant natural products are complex, and the vast majority of crystal structures have not been resolved. At present, the modification of this kind of enzyme generally uses random mutation and other methods to further enrich the positive mutations, so that the activity, thermal stability, affinity and preference for substrates of the enzyme are greatly improved^[3]For example, by using the method of random mutation, the directed evolution transformation of Rhodobacter sphaeroides synthases has doubled the yield of lycopene engineering strains^[23]。

In addition, through the methods of functional enzyme fusion and protein scaffold construction, each enzyme in the metabolic pathway can form a controllable complex, improve the effective concentration of substrate and reduce the accumulation of toxic intermediates, so as to achieve the effect of improving the substrate conversion rate^[3]For example, fusion of farnesyl pyrophosphate synthase and farnesyl pyrophosphate synthase in Saccharomyces cerevisiae can significantly improve the synthesis ability of farnesyl pyrophosphate in Saccharomyces cerevisiae^[7]Through the method of building a protein scaffold, the proportion and spatial layout of the three mevalonate synthetases were optimized to increase the production of mevalonate, while reducing the cell load, and the final concentration of mevalonate was increased by 77 times^[24]。

. At present, some strategies for optimizing biosynthetic pathways have been developed at home and abroad.

(1) Material flow distribution control.The distribution proportion of target compounds in the material flow supply network is controlled by regulating the expression of key node genes. For example, during the construction of engineering strains of artemisinic acid, by increasing the expression of upstream genes in biosynthetic pathways to increase precursor supply and inhibiting the expression of genes in branching pathways to reduce the substrate competition material flow control scheme, the ability of engineering strains to produce artemisinic acid by fermentation was significantly improved^[3, 25]。

(2) Precise control of synthetic pathways.Through the establishment of promoter library to accurately control the expression of genes, make the coordinated expression of genes in the synthesis pathway, reduce the accumulation of intermediate metabolites, reduce cell load, and ultimately improve the fermentation performance of Engineering Strains^[3]For example, the co expression of fatty acid synthesis pathway and heterologous betulinic acid synthesis pathway using promoters with different intensities revealed that the yield of betulinic acid of the engineered strain could vary within a 200 fold range^[26]。

(3) Dynamic control of synthetic pathways.A very important step in the optimization of microbial synthesis pathway is the dynamic quantification and monitoring of metabolites in the pathway. . Based on this property, biosensors can be applied to build dynamic metabolite regulation circuits to increase the yield of target compounds^[3]For example, a biosensor that can respond to propionyl COA is used to monitor the intracellular accumulation of propionyl COA, thereby feedback down regulating the expression of acetyl CoA carboxylase to reduce cytotoxicity^[27]The farnesyl pyrophosphate biosensor identified by RNA micro matrix method can be used to balance metabolic flux and increase the yield of sophoridine^[28]。

The improvement of the comprehensive efficiency of cell factories is affected by many factors, including efficient utilization of raw materials, storage capacity of products, excellent fermentation performance and many other aspects. .

(1) .Mitochondria, Golgi apparatus, endoplasmic reticulum and other organelles in cells can provide a catalytic environment for biological reactions. ^[29]By anchoring the C-terminus of related enzymes in the morphine biosynthesis pathway to the endoplasmic reticulum, the production rate of morphine can be increased^[30]。

(2) Cell membrane engineering.A considerable part of plant natural products are hydrophobic compounds, which are generally retained in cells, but due to the limited cell space, the production of target products is affected. Scientists from Tianjin Institute of industrial biotechnology, Chinese Academy of Sciences recently studied the effect of E. coli cell membrane modification on improving the synthesis ability of terpenoids. ^[31]。

(3) .Providing more catalytic sites for functional enzymes is a new method developed recently, such as by knocking down thePHA1Gene to increase the content of endoplasmic reticulum in yeast, greatly increasing the attachment space of cytochrome P450 enzyme located in the endoplasmic reticulum membrane, and increasing the content of enzyme and the yield of catalytic product^[32]。

(4) Atypical nutrient utilization.In the large-scale fermentation process of engineering bacteria, the pollution of miscellaneous bacteria is more common, which seriously affects the production efficiency. Scientists from novogy and the Massachusetts Institute of technology first transformed the utilization of microbial nutrient elements such as Saccharomyces cerevisiae, Yarrowia lipolytica and Escherichia coli. By using nutrient elements that cannot be used by miscellaneous bacteria in the fermentation process to establish the fermentation process, the anti pollution ability of Engineering microbial cells can be significantly improved^[33]。

In the past 10 years, research on synthetic biology of plant natural products has developed rapidly. A series of synthetic cell factories of plant natural products such as terpenoids, phenylpropanoids and alkaloids have been successfully established.

Artemisinin, the first-line antimalarial drug, is a sesquiterpene compound found in Artemisia annua, a traditional Chinese herbal medicine, by Tu youyou and her research team from the Institute of traditional Chinese medicine, Chinese Academy of traditional Chinese medicine in the 1970s^[3]In the past, the production method was direct extraction from Artemisia annua. Scientists at the University of california,berkeley in the United States spent 10 years to realize the production of artemisinic acid by fermentation in a yeast cell factory, and developed a process for the synthesis of artemisinin through simple chemical reactions^[6]After calculation, it is less than 100 m³The annual production of artemisinin in the fermentation workshop can reach 35 t, which is equivalent to the planting output of nearly 50000 mu of arable land in China. This work is considered as a milestone in the research field of using synthetic cells to produce plant terpenoids^[3]。

In the production of analgesic drugs, scientists at Stanford University in the United States combined with functional element mining and optimization, synthesis pathway construction and optimization and other means to introduce a series of functional genes from bacteria, plants, animals and yeast into yeast, achieving a breakthrough in the production of opioid alkaloids such as morphine by fermentation^[34]。

In terms of the production of anticancer drugs, the research team of Massachusetts Institute of technology in the United States introduced the taxadiene synthase biosynthesis of the diterpene anticancer drug paclitaxel into Escherichia coli, and the upstreamMEPFunctional modules and downstream terpene synthesis functional modules were precisely regulated, and finally a cell factory with a yield of 1 g/l of taxadiene was obtained^[35]。

In terms of the production of long-chain fatty acids, such as DHA and EPA, commonly known as "brain gold", DuPont's R & D personnel introduced 21 foreign genes encoding five different enzymes into Yarrowia lipolytica, which has a fatty acid content of 40% of cell dry weight. The engineered strain can produce long-chain fatty acid EPA, which accounts for 15% of cell dry weight^[36]。

. A complete set of technology system for synthetic biology production of plant natural products has been established, including gene element mining technology of plant natural products, high-throughput automated cloning technology, synthetic pathway creation and precise regulation technology, fermentation and separation extraction technology. Also created a series of artificial synthetic cells of plant natural products including β - elemene, carotenoids, ginsenosides, gastrodin, stevioside, triterpenoid acid, salidroside, siraitin, scutellarin, as detailed below.

Terpenoids widely exist in nature. At present, more than 50000 kinds of terpenoids have been found, most of which are effective ingredients in medicinal plants^[37]Artemisinin, anticancer drug paclitaxel, Ginsenosides with health effects and carotenoids as antioxidants belong to terpenoids^[3]。

The global market for flavors and fragrances is vast. Terpenoid essential oils such as sandalwood alcohol, patchouli alcohol, nerolidol, β - elemene are widely used in daily chemicals, food, medicine and other fields. Among them, β - elemene is an effective component of a national class of anticancer drugs extracted from medicinal plants such as Curcuma wenyujin and curcuma zedoary. The low content of β - elemene from natural sources and the complex composition of chemical analogues lead to its high separation cost. Tianjin Institute of industrial biotechnology, Chinese Academy of Sciences, in cooperation with the Chinese medicine resource center, Chinese Academy of traditional Chinese medicine, has used metabolic engineering and synthetic biology technology to improve the biosynthetic flux and product compatibility of terpenoids in Saccharomyces cerevisiae. On this basis, the protein engineering transformation of gemene a synthase and the creation of high-yield gemene a engineering bacteria were carried out, and the coupled process of thermal conversion of gemene a to β - elemene was successfully developed, which finally reduced the cost of obtaining high-purity β - elemene to 0.15% of plant extraction;^[38]。

In recent years, China has made important progress in the field of tanshinone synthetic cells. Tanshinone belongs to rosin type diterpenoid compounds. It is the main effective component of traditional Chinese medicine Salvia miltiorrhiza in China. It has antioxidant, antibacterial, anti-inflammatory, anti-tumor and other activities. The traditional Chinese medicine resource center of the Chinese Academy of Sciences, in cooperation with the Dalian Institute of Chemical Physics of the Chinese Academy of Sciences, Iowa State University, Institute of genetics and developmental biology of the Chinese Academy of Sciences, Shanghai Chenshan plant science research center of the Chinese Academy of Sciences, Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences and other research institutions, has discovered two functional enzymes catalyzing the formation of the basic skeleton of tanshinone, the secondary tanshinone diene^[39], a yeast engineered strain with high yield of tanshinone diene was constructed^[7,40]Further, the role of the carbon skeleton structure in tanshinone biosynthesis was determined by using C-13 isotope labeling technology. On this basis, the key enzyme gene catalyzing the production of intermediate metabolite ferruginous alcohol from secondary tanshinone diene was also discoveredCYP76AH1, and a yeast engineered strain with high production of ferruginol was constructed^[14]After that, the catalytic C-7, C-11, and C-20 sites were further discoveredP450Gene, and a yeast engineered strain capable of simultaneously producing multiple tanshinones was obtained^[41]。

Stevia is the next generation of important healthy natural sweeteners. Shanghai Institute of plant physiology and ecology, Chinese Academy of Sciences has fully mined the key enzymes of Stevia biosynthesis pathway by constructing Stevia functional gene database. The key enzymes in the stevioside biosynthesis pathway were successfully identified, and the unnatural synthesis pathway for de novo synthesis of stevioside compounds was reconstructed in E. coli chassis cells. ;^[42]On this basis, the team also analyzed the biosynthesis process of sweet tea in Rubus sweet leaves and tomorrow leaves, reported six new diterpene glycosyltransferases, and studied the mechanism of substrate recognition. Through orthogonal combinations of glycosyltransferases from different species, the efficient whole cell transformation of sweet tea catechin was achieved in microbial cells^[43]This study laid a foundation for the synthesis of important diterpenoid glycosides and provided a successful example for transforming Escherichia coli into a chassis cell for heterologous synthesis of complex terpenoids.

Ginsenosides are the effective components of the rare traditional Chinese medicine ginseng and American ginseng. They are the general name of a series of mixtures formed by protopanaxadiol, protopanaxatriol and oleanolic acid aglycones under the catalysis of glycosyltransferases. They have anti-tumor^[44, 45], hypoglycemic^[46], promoting immunity and other functions. Tianjin Institute of industrial biotechnology, Chinese Academy of Sciences, in cooperation with the Chinese medicine resource center, Chinese Academy of traditional Chinese medicine, successfully constructed the biosynthetic pathway of protopanaxadiol in Saccharomyces cerevisiae for the first time, and found that squalene epoxidase played a key role in controlling the biosynthesis of triterpenoids^[3]On this basis, the yield of protopanaxadiol was increased by 262 times by increasing the expression activity of key genes. ^[47]Recently, the cooperation team publicly reported the construction scheme to increase the triterpene synthesis flux of the cell factory to 10 g/l level, and create an efficient yeast cell factory with a production capacity of 15 g/l ginsenoside precursors^[48]In addition, the three functional modules of oleanolic acid, protopanaxadiol and protopanaxatriol were introduced into the same chassis cells to obtain the first generation of "ginseng yeast" cell factory based on multi-component concept, which can simultaneously synthesize three basic Ginsenosides of ginseng^[49]。

Shanghai Institute of plant physiology and ecology, Chinese Academy of Sciences, in cooperation with Shanghai Institute of Materia Medica, Chinese Academy of Sciences, first cloned and identified synthetic rare ginsenoside compound KGinsenosidesRh2、GinsenosidesRg3、GinsenosidesRh1 andGinsenosidesThe key glycosyltransferase and P450 reductase pgcpr1 required by F1. ;^{[5, 50, 51]}Recently, the team further excavated more than 20 glycosyltransferases from ginseng and Panax notoginseng, and comprehensively analyzed the biosynthetic pathway of ginseng and Panax notoginseng saponins. .

The epicuticular waxes of apple, hawthorn, loquat, jujube and pear contain trace amounts of high-value triterpenoid acids, including corosolic acidHawthorn acid, malic acid, asiatic acid and other compounds. They are antiviral^[52]Diabetes control^[53]And skin repair^[54]It is a kind of important dietary supplements. Among them, corosolic acid has a significant effect on anti diabetes^[53], which is considered as a natural plant insulin. Direct extraction from original plants is the main way to produce such compounds at present. . Using this platform, the team first screened the functional P450 enzyme maa45 that can catalyze oleanolic acid and ursolic acid to generate the 2-position α - hydroxylation products Maslinic Acid and corosolic acid from the medicinal plant Hawthorn P450 library. On this basis, a Saccharomyces cerevisiae cell factory for the efficient production of Maslinic Acid and corosolic acid was established, with the production reaching 384 mg / L and 141 mg / L, respectively;^[55, 56]。

Carotenoids have important applications in medicine, nutrition, cosmetics and food fields. β - carotene, lycopene and astaxanthin are representative carotenoids. Taking carotenoids as the research object, Tianjin Institute of industrial biotechnology, Chinese Academy of Sciences carried out research from three aspects of material metabolism regulation, energy metabolism regulation and cell physiological regulation, and systematically analyzed the regulation mechanism of efficient terpenoid synthesis by microorganisms^[57, 58]。① In terms of substance metabolism, it was found that ispg and IspH are important rate limiting steps, and these two enzymes need to be expressed cooperatively to function. Overexpression of ispg alone leads to accumulation of the toxic intermediate metabolite hmbpp, which severely inhibits cell growth and metabolism^[58]。② In terms of energy metabolism,Through the multi-scale modular regulation of the central metabolic pathways (pentose phosphate and TCA), it was found that the metabolic flux of the TCA pathway was the most important limiting factor for the aerobic synthesis of NADPH by Escherichia coli, which solved the problem of the imbalance of reducing power in the synthesis and metabolism of terpenoids^[57]。③ In terms of cell physiological regulation,The limiting factors of the storage capacity of E. coli cell membrane were systematically studied. It was found that the morphology of cell membrane and the synthesis ability of cell membrane components were the key factors that restricted the storage capacity of E. coli cell membrane. The introduction of exogenous membrane folding protein can change the cell membrane morphology of E. coli and form the inward fold of the cell membrane. Enhancing glycerophospholipid synthesis ability can further increase the inward fold of cell membrane, thus further enhancing the storage capacity of cell membrane and significantly increasing the yield of terpenoids^[31]On this basis, a series of microbial cell factories for efficient production of carotenoids were constructed. The pilot test of lycopene was completed in a 200 L fermentor, and the lycopene yield reached 7 g / L after 48 hours of fermentation.

Phenylpropanoids are naturally occurring compounds composed of benzene rings and three straight chain carbons, such as anthocyanins, resveratrol and caffeic acid. They have significant pharmacological activities in antioxidation, cardiovascular health care, antiviral and coagulation. Recent breakthroughs have also been made in the fermentation production of these compounds. The resveratrol production technology developed by evolva has entered the industrialization stage. Chinese scientists have made breakthroughs in gastrodin, scutellarin, salidroside and danshensu.

Gastrodia elata is one of China's valuable traditional Chinese medicines. Gastrodin, as the main active ingredient of Gastrodia elata, is widely used in the treatment of neurasthenia and neurasthenic syndrome. However, Gastrodia elata resource itself is rare, and its gastrodin content is low (only 0.4%), so the plant extraction of gastrodin is expensive. In addition, chemical synthesis methods have disadvantages of high cost and serious pollution; The biosynthetic pathway of Gastrodin in plants has not yet been clarified. Therefore, the team of Tianjin Institute of industrial biotechnology, Chinese Academy of Sciences, took the mycolic acid of shikimic acid pathway of Escherichia coli as the precursor, overexpressed the key genes from Escherichia coli, Nocardia and Bacillus subtilis, and introduced the glycosyltransferase ugt73b6 from Rhodiola, creating the gastrodin synthesis pathway of Escherichia coli for the first time in the world. . At present, the production cost of gastrodin using glucose as raw material is expected to be less than 500 yuan /kg, only 1 / 200 of that extracted from plants and 1 / 2 of that synthesized chemically^[59]Recently, Qingdao Institute of bioenergy and process, Chinese Academy of Sciences, and Hunan Normal University cooperated to produce gastrodin by biotransformation using the oxidative degradation pathway of aromatic precursor 4-cresol. ^{[20, 21, 60]}。

. Tianjin Institute of industrial biotechnology, Chinese Academy of Sciences, in cooperation with Yunnan Agricultural University, successfully identified the key genes in scutellarin biosynthesis pathway from the genome of Erigeron breviscapus by using synthetic biology and bioinformatics technology, and successfully constructed a cell factory for scutellarin synthesis in Saccharomyces cerevisiae chassis. Through metabolic engineering transformation and fermentation process optimization, the content of scutellarin produced by the cell factory has reached 100 mg level^[61]。

Tanshinol is a polyphenolic drug derived from Salvia miltiorrhiza. Tianjin University has created a new artificial alternative biosynthetic pathway by using the similarity between tanshinol and the cell's own metabolite p-hydroxyphenylpyruvate; And the artificial elements lactate dehydrogenase and hydroxylase were rationally transformed to improve the specificity with non-natural substrates, realizing the efficient synthesis of Danshensu by microbial cell factory, and the fermentation yield reached 7 g / L^[62]。

Salidroside is a promising environmental drug. Rhodiola sachalinensis grows in an alpine environment, is lack of resources, is not easy to grow, has low content of medicinal components, and contains toxic substances and impurities. The team of Tianjin Institute of industrial biotechnology, Chinese Academy of Sciences constructed the salidroside synthesis pathway in Escherichia coli by using the shikimic acid pathway, and obtained an engineering bacterium with high salidroside production through directed evolution of enzymes and metabolic regulation. ;^[59]。

In recent years, a series of achievements have been made to strengthen the confidence in the development of the field, such as the successful establishment of cell factories such as artemisinin, β - elemene, lycopene, ginsenoside and morphine, based on the principle of synthetic biology to design artificial synthetic cell factories to ferment and produce natural products of plant origin^[3]Compared with the traditional production mode, this new resource acquisition strategy has significant advantages in the sustainable utilization of resources and economic benefits, so it has emerged as an innovative mode.

According to statistics, there are tens of thousands of medicinal plants (traditional Chinese Medicine) containing bioactive components in China alone. They provide a treasure house of precursor drugs with rich targets for modern drug development, and their rich biosynthetic pathways provide us with a natural enzyme library with diverse catalytic types. However, the effective development of the "double treasure house" is a highly cross cutting frontier research field, which requires the joint efforts and collaborative research of biology, informatics, chemistry, traditional Chinese medicine, pharmacy and other disciplines.

. The research foundation of some compound types is weak, and the heterologous synthesis efficiency of engineering cells is still relatively low, resulting in the cost of fermentation method is not obvious compared with the traditional route, which needs to further strengthen the research. But in general, with the gradual maturity of artificial intelligence technology, the low-cost sequencing technology of plant genome, the improvement of high-throughput chemical synthesis gene technology, and further breakthroughs in the concept and operation of metabolic pathway optimization based on the global metabolic network, people will finally usher in a new era of artificial synthesis of plant derived natural products.