Plant Salvia Miltiorrhiza Research Articles

Synthesis of Tanshinone IIA and Related Terpenes via a C-H Functionalization Strategy.

The total synthesis of tanshinone IIA and related bioactive diterpenes isolated from the Chinese plant Salvia miltiorrhiza was completed from a common tetralin building block. The synthetic route highlights a 3,4-disubstituted furan synthesis and various regioselective C-H functionalization reactions, including a Pd catalyzed iodination and an Ir catalyzed borylation, along with an intramolecular stanna-Brook type reaction to construct the ortho-quninone ring of the target molecule.

3D bioprinting of Salvianolic acid B-sodium alginate-gelatin skin scaffolds promotes diabetic wound repair via antioxidant, anti-inflammatory, and proangiogenic effects

In patients with diabetic wounds, wound healing is impaired due to the presence of persistent oxidative stress, an altered inflammatory response, and impaired angiogenesis and epithelization. Salvianolic acid B (SAB), which is derived from the Chinese medicinal plant Salvia miltiorrhiza, has been found to exhibit antioxidant, anti-inflammatory, and proangiogenic effects. Previous studies have used 3D bioprinting technology incorporating sodium alginate (SA) and gelatin (Gel) as basic biomaterials to successfully produce artificial skin. In the current study, 3D bioprinting technology was used to incorporate SAB into SA-Gel to form a novel SAB-SA-Gel composite porous scaffold. The morphological characteristics, physicochemical characteristics, biocompatibility, and SAB release profile of the SAB-SA-Gel scaffolds were evaluated in vitro. In addition, the antioxidant, anti-inflammatory, and proangiogenic abilities of the SAB-SA-Gel scaffolds were evaluated in cells and in a rat model. Analysis demonstrated that 1.0 wt% (the percentage of SAB in the total weight of the solution containing SA and Gel) SAB-SA-Gel scaffolds had strong antioxidant, anti-inflammatory, and proangiogenic properties both in cells and in the rat model. The 1.0% SAB-SA-Gel scaffold reduced the expression of tumor necrosis factor-α, interleukin-6, and interluekin-1β and increased the expression of transforming growth factor-β. In addition, this scaffold removed excessive reactive oxygen species by increasing the expression of superoxide dismutase, thereby protecting fibroblasts from injury. The scaffold increased the expression of vascular endothelial growth factor and platelet/endothelial cell adhesion molecule-1, accelerated granulation tissue regeneration and collagen deposition, and promoted wound healing. These findings suggest that this innovative scaffold may have promise as a simple and efficient approach to managing diabetic wound repair.

Miltiradiene Production by Cytoplasmic Metabolic Engineering in Nicotiana benthamiana.

Plant natural products are important sources of innovative drugs, but the extraction and isolation of medicinal natural products from plants is challenging as these compounds have complex structures that are difficult to synthesize chemically. Therefore, utilizing heterologous expression systems to produce medicinal natural products in plants is a novel, environmentally friendly, and sustainable method. In this study, Nicotiana benthamiana was used as the plant platform to successfully produce miltiradiene, the key intermediate of tanshinones, which are the bioactive constituents of the Chinese medicinal plant Salvia miltiorrhiza. The yield of miltiradiene was increased through cytoplasmic engineering strategies combined with the enhancement of isoprenoid precursors. Additionally, we discovered that overexpressing SmHMGR alone accelerated apoptosis in tobacco leaves. Due to the richer membrane systems and cofactors in tobacco compared to yeast, tobacco is more conducive to the expression of plant enzymes. Therefore, this study lays the foundation for dissecting the tanshinone biosynthetic pathway in tobacco, which is essential for subsequent research. Additionally, it highlights the potential of N. benthamiana as an alternative platform for the production of natural products in plants.

Effects of Potassium Application on Growth and Root Metabolism of Salvia miltiorrhiza under Drought Stress

Potassium application can effectively mitigate the effects of drought stress on plant growth, and few studies have reported its application to the medicinal plant Salvia miltiorrhiza (S. miltiorrhiza). Four experimental treatments were used, including a control (Z1K0), non-drought potassium application (Z1K3), drought treatments (Z2K0), and drought-stress potassium application (Z2K3). The findings revealed that, in contrast to Z2K0, Z2K3 promoted the absorption of potassium in S. miltiorrhiza, elicited the aggregation of osmoregulatory compounds such as soluble protein and proline, and mitigated membrane impairment as a defense against the deleterious consequences of drought stress. Additionally, we extended our investigation to encompass comprehensive metabolomics analysis of the roots. Interestingly, subsequent root metabolomics analyses demonstrated that the drought application of potassium not only significantly reduced the amino acid content, but also increased the amount of terpenoids and phenolic acids in the roots. Nonetheless, the application of a particular amount of K under moderate drought conditions promoted the growth and yield of S. miltiorrhiza, but proved to be detrimental to its active ingredients. Indeed, the findings of this study offer valuable insights and recommendations for the application of potassium to mitigate the impact of drought stress in S. miltiorrhiza and other medicinal plants.

5-Azacytidine increases tanshinone production in Salvia miltiorrhiza hairy roots through epigenetic modulation

Recent studies have indicated strong connections between epigenetic modulation and secondary metabolites in plants. It is vital to understand the roles of epigenetics in the production of secondary metabolites. In this study, the inhibitor of DNA methylation 5-azacytidine (5-Az) was used on the hairy roots of the medicinal plant Salvia miltiorrhiza to investigate its effect on secondary metabolite production, gene expression, methylation levels in genomic DNA and promoter regions. Our results showed that the contents of tanshinones in S. miltiorrhiza hairy roots increased by 1.5–5 times, and some genes in the biosynthesis pathway showed an upward trend. According to our NGS analysis, the methylation pattern in the promotor of the gene encoding copalyl diphosphate synthase (CPS) was altered, and 51 out of 145 cytosines were demethylated during 5-Az treatment. A total of 36 putative transcription factors (TFs) binding cites were identified in these demethylation sites. Among these TFs binding cites, cis-regulatory elements for the binding of NF-Y and MYB were frequently found in our results. This is the first report to demonstrate a possible mechanism of DNA methylation participating in tanshinone biosynthesis in S. miltiorrhiza hairy roots by modulating the CPS promoter and TFs binding sites.

Overexpression of SmSCR1 Promotes Tanshinone Accumulation and Hairy Root Growth in Salvia miltiorrhiza.

Lipid-soluble tanshinone is one of the main bioactive substances in the medicinal plant Salvia miltiorrhiza, and its medicinal demand is growing rapidly. Yeast extract (YE) modulates the tanshinone biosynthesis, but the underlying regulatory network remains obscure. In this study, a YE-responsive transcriptional factor Scarecrow1 (SCR1) was identified in S. miltiorrhiza from the YE-induced transcriptome dataset. SmSCR1 is located in the nucleus. Overexpression of SmSCR1 in S. miltiorrhiza roots resulted in a significantly higher accumulation of tanshinone than the control, with the highest 1.49-fold increase. We also detected upregulation of tanshinone biosynthetic genes, SmSCR1 and SmHMGR1, and distinct alteration of growth and development of the hairy roots in the overexpression lines compared to the control. An inverse phenotype was observed in SmSCR1-SRDX suppression expression lines. We found that SmSCR1 can bind to the promoter of SmCPS1 to induce its expression. This study provides new insight into the regulatory mechanism on the growth and development of hairy roots, tanshinone accumulation, and the metabolic engineering of bioactive compounds in S. miltiorrhiza.

Dihydrotanshinone-I modulates Epithelial Mesenchymal Transition (EMT) Thereby Impairing Migration and Clonogenicity of Triple Negative Breast Cancer Cells.

Background: Salvia miltiorrhiza Bunge (Danshen), has been used for its therapeutic value in Traditional Chinese Medicine (TCM), for almost a thousand years. Dihydrotanshinone-I (DHTS) is a lipophilic compound isolated from the plant Salvia miltiorrhiza that has been shown to induce anti-proliferative and apoptotic effects on breast cancer cells. In the present study, we investigated the anti-migratory effect of DHTS on TNBC cell lines by studying the Epithelial Mesenchymal Transition (EMT) changes. Methods:IC50 values for DHTS in TNBC breast cancer cells were either discovered by literature search or by performing MTT assay. DHTS effect on EMT markers (viz. CD44, E-cadherin, Vimentin, N-cadherin, and active β-catenin) was studied using western blotting. Association between EMT and migration was further carried out in DHTS treated TNBC cells by wound healing assay. Cancer stemness and proliferation potential were further accessed using colony formation assay.Results:MTT assay revealed IC50 of MDA-MB-468 cells at 2 µM for 24 h. Subsequently, DHTS treatment in TNBC cell lines (MDA-MB-468 and MDA-MB-231) led to decrease in mesenchymal markers i.e. vimentin, N-cadherin and, active β-catenin. DHTS treated MDA-MB-468 cells showed a decrease in adhesion protein CD44 and an increase in epithelial protein E-cadherin. Additionally, a decrease in EMT potential was positively associated with the inhibition of migration and clonogenic potential in DHTS treated TNBC cells. Conclusion:In this study, we have demonstrated for the first time that DHTS has the potential to inhibit the migration and clonogenicity of highly aggressive TNBC cells by obstructing Epithelial to Mesenchymal Transition.

A Skin Lipidomics Study Reveals the Therapeutic Effects of Tanshinones in a Rat Model of Acne.

Tanshinone (TAN), a class of bioactive components in traditional Chinese medicinal plant Salvia miltiorrhiza, has antibacterial and anti-inflammatory effects, can enhance blood circulation, remove blood stasis, and promote wound healing. For these reasons it has been developed as a drug to treat acne. The purpose of this study was to evaluate the therapeutic effects of TAN in rats with oleic acid-induced acne and to explore its possible mechanisms of action through the identification of potential lipid biomarkers. In this study, a rat model of acne was established by applying 0.5 ml of 80% oleic acid to rats’ back skin. The potential metabolites and targets involved in the anti-acne effects of TAN were predicted using lipidomics. The results indicate that TAN has therapeutic efficacy for acne, as supported by the results of the histological analyses and biochemical index assays for interleukin (IL)-8, IL-6, IL-β and tumor necrosis factor alpha. The orthogonal projection of latent structure discriminant analysis score was used to analyze the lipidomic profiles between control and acne rats. Ninety-six potential biomarkers were identified in the skin samples of the acne rats. These biomarkers were mainly related to glycerophospholipid and sphingolipid metabolism, and the regulation of their dysfunction is thought to be a possible therapeutic mechanism of action of TAN on acne.

Genome-wide identification of the Tubby-Like Protein (TLPs) family in medicinal model plant Salvia miltiorrhiza.

Tubby-Like Proteins (TLPs) are important transcription factors with many functions and are found in both animals and plants. In plants, TLPs are thought to be involved in the abiotic stress response. To reveal the potential function of TLPs in the medicinal model plant Salvia miltiorrhiza, we identified 12 S. miltiorrhiza TLPs (SmTLPs) and conducted a comprehensive analysis. We examined SmTLP gene structure, protein structure, phylogenetics, and expression analysis. Our results show that all SmTLPs, except SmTLP11, have a complete typical Tub domain. Promoter analysis revealed that most SmTLPs are involved in hormone and abiotic stress responses. Expression analysis revealed that the 12 SmTLPs could be divided into three categories: those specifically expressed in roots, those specifically expressed in stems, and those specifically expressed in leaves. Additional studies have shown that SmTLP10 may play an important role in the plant cold resistance, while SmTLP12 may be involved in the S. miltiorrhiza ABA metabolic pathway. Our study represents the first comprehensive investigation of TLPs in S. miltiorrhiza. These data may provide useful clues for future studies and may support the hypotheses regarding the role of TLPs in plant abiotic stress process. All in all, we may provide a reference for improving S. miltiorrhiza quality using genetic engineering technology.

SmKFB5 protein regulates phenolic acid biosynthesis by controlling the degradation of phenylalanine ammonia-lyase in Salvia miltiorrhiza.

Phenolic acids are the major secondary metabolites and significant bioactive constituents of the medicinal plant Salvia miltiorrhiza. Many enzyme-encoding genes and transcription factors involved in the biosynthesis of phenolic acids have been identified, but the underlying post-translational regulatory mechanisms are poorly understood. Here, we demonstrate that the S. miltiorrhiza Kelch repeat F-box protein SmKFB5 physically interacts with three phenylalanine ammonia-lyase (PAL) isozymes and mediates their proteolytic turnover via the ubiquitin-26S proteasome pathway. Disturbing the expression of SmKFB5 reciprocally affected the abundance of SmPAL protein and the accumulation of phenolic acids, suggesting that SmKFB5 is a post-translational regulator responsible for the turnover of PAL and negatively controlling phenolic acids. Furthermore, we discovered that treatment of the hairy root of S. miltiorrhiza with methyl jasmonate suppressed the expression of SmKFB5 while inducing the transcription of SmPAL1 and SmPAL3. These data suggested that methyl jasmonate consolidated both transcriptional and post-translational regulation mechanisms to enhance phenolic acid biosynthesis. Taken together, our results provide insights into the molecular mechanisms by which SmKFB5 mediates the regulation of phenolic acid biosynthesis by jasmonic acid, and suggest valuable targets for plant breeders in tailoring new cultivars.

Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism

BackgroundThe contribution of mitogen-activated protein kinase (MAPK) cascades to plant growth and development has been widely studied, but this knowledge has not yet been extended to the medicinal plant Salvia miltiorrhiza, which produces a number of pharmacologically active secondary metabolites.ResultsIn this study, we performed a genome-wide survey and identified six MAPKKK kinases (MAPKKKKs), 83 MAPKK kinases (MAPKKKs), nine MAPK kinases (MAPKKs) and 18 MAPKs in the S. miltiorrhiza genome. Within each class of genes, a small number of subfamilies were recognized. A transcriptional analysis revealed differences in the genes’ behaviour with respect to both their site of transcription and their inducibility by elicitors and phytohormones. Two genes were identified as strong candidates for playing roles in phytohormone signalling. A gene-to-metabolite network was constructed based on correlation analysis, highlighting the likely involvement of two of the cascades in the synthesis of two key groups of pharmacologically active secondary metabolites: phenolic acids and tanshinones.ConclusionThe data provide insight into the functional diversification and conservation of MAPK cascades in S. miltiorrhiza.

Differential distribution of characteristic constituents in root, stem and leaf tissues of Salvia miltiorrhiza using MALDI mass spectrometry imaging

Segmentation-quantification is the most commonly used method for studying the tissue distribution of bioactive constituents in plant, but this method would bring uncontrollable pollution, compound migration and denaturation. Mass spectrometry imaging (MSI), as a new method developed in the past 20 years, has high sensitivity, high spatial resolution, high degree of visualization, and low risk of contamination and degeneration when studying tissue distribution of compounds. For the first time we applied matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to tissue distribution of characteristic constituents of the medicinal plant Salvia miltiorrhiza. From the collected data, we found the regional differences in root, stem, and leaf tissues, and the ion information with differential distribution characteristics. We also identified 18 bioactive constituents in S. miltiorrhiza with their spatial distribution information. In addition, the plant was divided into five parts, and the identified compounds were analyzed for differences between tissues using LC-MS, which results verified those found from the MSI. It is figured out that MALDI-MSI can be reliably applied to the differential distribution of salvianolic acids and tanshinones.

Parasitic microorganisms related to photosynthetic algae - biomedical and biotechnological point of view

D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding.

Simplify: A Mass Spectrometry Metabolomics Approach to Identify Additives and Synergists from Complex Mixtures.

In fields ranging from environmental toxicology to drug discovery, it is critical to identify how multiple chemical compounds interact to perturb biological systems. Isolation-based approaches fail to incorporate multiconstituent interactions, such as synergy. We have developed an approach called "Simplify", which identifies mixture constituents that interact to achieve biological effects. Simplify combines biological and mass spectrometric data sets and uses an "activity index" to predict mixture interactions. Using the plant Salvia miltiorrhiza as a case study, we employed Simplify to identify four individual constituents that contribute to antimicrobial activity, three additives and one synergist. Our study is the first to enable identification of unknown synergists prior to isolating them, demonstrating the ability of the Simplify workflow to predict key contributors to the biological effect of a complex mixture. While utilized for natural products discovery in this study, this approach is expected to prove useful across multiple disciplines that rely on mixture analysis.

One-pot, three-step cascade synthesis of D-danshensu using engineered Escherichia coli whole cells

D-danshensu (D-DSS), extracted from the plant Salvia miltiorrhiza (Danshen), is widely used to treat cardiovascular and cerebrovascular diseases. Here we engineered Escherichia coli strains to produce D-DSS from catechol, pyruvate and ammonia by one-pot biotransformation. Tyrosin-phenol lyase (TPL), L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD) and glucose dehydrogenase (gdh) genes were overexpressed in Escherichia coli strain. First, the expression of genes was regulated by different copy number plasmids combination, the result of E. coli TALG6, with strong overexpression of TPL, aadL, ldhD and moderate overexpression of gdh, exhibited 253.7% increase D-DSS production compared to E. coli TALG1. Second, the optimum concentration of catechol was found to be 50 mM. Finally, a fed-batch biotransformation strategy was proposed, namely the amount of catechol was added to 50 mM every 2 h. The total production of D-DSS reached 55.35 mM within 14 h, which was 1.7 times that without feeding.

The biosynthesis of phenolic acids is positively regulated by the JA-responsive transcription factor ERF115 inSalvia miltiorrhiza

Phenolic acids are important secondary metabolites produced in the Chinese medicinal plant Salvia miltiorrhiza, but little is known about the transcription factors involved in the regulation of tanshinone and phenolic acid biosynthesis. Here, a novel AP2/ERF transcription factor SmERF115 was isolated and functionally characterized. SmERF115 was most responsive to methyl jasmonate (MeJA) treatment and was localized in the nucleus. The phenolic acid production was increased in SmERF115-overexpressing hairy roots, but with a decrease in tanshinone content. In contrast, silencing of SmERF115 reduced the phenolic acid level, but increased tanshinone content. The expression of the key biosynthetic gene SmRAS1 was up-regulated in SmERF115 overexpression lines but was down-regulated in SmERF115-RNAi lines. Yeast one-hybrid (Y1H) assay and EMSA showed that SmERF115 directly binds to the promoter of SmRAS1, while dual-luciferase assays showed that SmERF115 could activate expression of SmRAS1 in vivo. Furthermore, global transcriptomic analysis by RNA sequencing revealed that expression of other genes such as PAL3, 4CL5, TAT3, and RAS4 was also increased in the overexpression line, implying that they were potentially involved in the SmERF115-mediated pathway. Our data show that SmERF115 is a positive regulator of phenolic acid biosynthesis, and may be a potential target for further metabolic engineering of phenolic acid biosynthesis in S. miltiorrhiza.

Tanshinone IIA Attenuates Insulin Like Growth Factor 1 -Induced Cell Proliferation in PC12 Cells through the PI3K/Akt and MEK/ERK Pathways.

The insulin like growth factor 1 (IGF-1) and its receptor (IGF-1R) facilitate tumor proliferation and progression. Tanshinone IIA (TSN) is an active diterpene quinone isolated from the roots of the herbal plant Salvia miltiorrhiza. TSN inhibits the proliferation of various types of cancer cells but its role in the IGF-1R-induced proliferation of pheochromocytoma (PC12) cells and the potential mechanisms are largely unknown. This study aims to investigate the anti-proliferative effect of TSN in PC12 cells and its role on IGF-1R signaling transduction. PC12 cells were treated with IGF-1 with or without TSN, methyl thiazolytetrazolium (MTT) assay, and cell counting kit-8 and flow cytometry were used to evaluate the proliferation of PC12 cells. The role of TSN on the apoptosis of PC12 cells were detected by flow cytometry as well. The effects of TSN and IGF-1 on the phosphorylation of IGF-1R, protein kinase B (Akt), extracellular-signal related kinase 1/2 (ERK1/2) and other downstream targets were analyzed by Western blotting analysis. Our results showed that IGF-1 promoted the growth of PC12 cells in a dose-dependent manner and increased the phosphorylation of IGF-1R, whereas TSN attenuated the effect of IGF-1. Interestingly, TSN did not induce cell apoptosis in PC12 cells. Moreover, TSN attenuated the phosphorylation of Akt and ERK1/2 induced by IGF-1, and the phosphorylation of glycogen synthase kinase-3β, forkhead box O3a (FOXO3a) and c-Raf were also inhibited by TSN. Furthermore, TSN inhibited cell growth induced by IGF-1 and blocked the activation of IGF-1R in SH-SY5Y cells. Taken together, TSN has an inhibitory effect on the proliferation of PC12 cells via down-regulation of the phosphorylated IGF-1R and its downstream signaling.

The AP2/ERF transcription factor SmERF1L1 regulates the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza

Tanshinones and phenolic acids are two important metabolites synthesized by the traditional Chinese medicinal plant Salvia miltiorrhiza. There is increasing market demand for these compounds. Here, we isolated and functionally characterized SmERF1L1, a novel JA (Jasmonic acid)-responsive gene encoding AP2/ERF transcription factor, from Salvia miltiorrhiza. SmERF1L1 was responsive to methyl jasmonate (MJ), yeast extraction (YE), salicylic acid (SA) and ethylene treatments. Subcellular localization assay indicated that SmERF1L1 located in the nucleus. Overexpression of SmERF1L1 significantly increased tanshinones production in transgenic S. miltiorrhiza hairy roots by comprehensively upregulating tanshinone biosynthetic pathway genes, especially SmDXR. Yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) showed that SmERF1L1 binds to the GCC-box of SmDXR promoter while dual luciferase (Dual-LUC) assay showed that SmERF1L1 positively regulated the expression of SmDXR. Our study suggested that the SmERF1L1 may be a good potential target for further metabolic engineering of bioactive component biosynthesis in S. miltiorrhiza.

Cryptotanshinone induces ROS-mediated apoptosis in human gastric cancer cells.

Cryptotanshinone (CT), isolated from the plant Salvia miltiorrhiza Bunge, has been reported to have potential anticancer effects on human prostate and breast cancer cells. However, the mechanisms of action of CT on gastric cancer (GC) cells are not well understood. Here we investigated the antitumor effects of CT on GC cells and its possible molecular mechanism. We found CT suppressed viability of twelve GC cell lines in a dose-dependent manner. CT induced cell cycle arrest at the G2/M phase and mitochondrial apoptosis accompanying the accumulation of reactive oxygen species (ROS). Pretreatment with ROS inhibitor N-acetyl-L-cysteine (NAC) blocked CT-induced apoptosis. CT increased p-JNK and p-p38, and decreased p-ERK and p-STAT3 protein expression, these effects were prevented by NAC. Furthermore, a xenograft assay showed that CT significantly inhibited MKN-45 cell-induced tumor growth in vivo by increasing expression of pro-apoptotic proteins (p-JNK, p-38 and cleaved-caspase-3) and reducing expression of anti-apoptotic proteins (p-ERK and p-STAT3) without adverse effects on nude mice weight. In conclusion, CT induced apoptosis and cell cycle arrest in GC cells via ROS-mediated MAPK and AKT signaling pathways, and this CT may be a useful compound for the developing anticancer agents for GC.

Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A

BackgroundSalvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Recently, production of SAA in engineered Escherichia coli was established via the artificial biosynthetic pathway of SAA on the multiple plasmids in our previous work. However, the plasmid-mediated system required to supplement expensive inducers and antibiotics during the fermentation process, restricting scale-up production of SAA. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering.ResultsThe limited enzymatic reactions in SAA biosynthetic pathway from glucose were grouped into three modules, which were sequentially integrated into chromosome of engineered E. coli by λ Red homologous recombination method. With starting strain E. coli BAK5, in which the ptsG, pykF, pykA, pheA and tyrR genes were previously deleted, chassis strain BAK11 was constructed for constitutive production of precursor l-tyrosine by replacing the 17.7-kb mao-paa cluster with module 1 (PlacUV5-aroGfbr-tyrAfbr-aroE) and the lacI gene with module 2 (Ptrc-glk-tktA-ppsA). The synthetic 5tacs promoter demonstrated the optimal strength to drive the expression of hpaBC-d-ldhY52A in module 3, which then was inserted at the position between nupG and speC on the chromosome of strain BAK11. The final strain BKD13 produced 5.6 g/L of SAA by fed-batch fermentation in 60 h from glucose without any antibiotics and inducers supplemented.ConclusionsThe plasmid-free and inducer-free strain for SAA production was developed by targeted integration of the constitutive expression of SAA biosynthetic genes into E. coli chromosome. Our work provides the industrial potential for constitutive production of SAA by the indel microbial cell factory and also sets an example of further producing other valuable natural and unnatural products.