High-level Production Research Articles

Systematic Engineering for High-level Production of β-nicotinamide mononucleotide from NAM and Ribose

Systematic Engineering for High-level Production of β-nicotinamide mononucleotide from NAM and Ribose

The relationship between body mass index and dysmenorrhea in adolescents: A Literature Review

Introduction: Dysmenorrhea is defined as menstrual cramps which are considered as one of the most common gynecological disorders in women of childbearing age, especially adolescents, dysmenorrhea occurs due to the production of high levels of prostaglandins. Not a few women with dysmenorrhea experience the impact of decreased daily activities to the use of painkillers because the pain is unbearable. Around 70-90% of cases of menstrual pain occur during adolescence so that it will affect academic, social and sports activities. Previous studies have shown that BMI can affect the risk of dysmenorrhea. Further research is needed to better understand how these factors can influence each other. Method: This study is a literature review, drawing from sources in Google Scholar, PUBMED, and Science Direct, focusing on research published between 2019 and 2024. The study included only original research articles in English or Indonesian with all the required components. Result and Discussion: From the literature search, 10 studies met the inclusion criteria. Among them, 9 studies found a correlation between BMI and dysmenorrhea, while 1 study found no correlation. Conclusion: According to reviews, BMI is associated with the risk of dysmenorrhea in adolescents, although some studies have not shown an association.

Increased accumulation of fatty acids in engineered Saccharomyces cerevisiae by co-overexpression of interorganelle tethering protein and lipases

Fatty acids (FAs) and their derivatives are versatile chemicals widely used in various industries. Synthetic biology, using microbial cell factories, emerges as a promising alternative technology for FA production. To enhance the production capacity of these microbial chassis, additional engineering strategies are imperative. Based on the comparison of the morphological changes of lipid droplets (LDs) between oleaginous and non-oleaginous yeasts, we developed a new engineering strategy to increase the accumulation of FAs in Saccharomyces cerevisiae through manipulation of regulation factor and lipases related to LD. The increased biogenesis of LDs, achieved by overexpressing the interorganelle tethering protein Mdm1, coupled with the accelerated degradation of LDs through upregulated lipases, resulted in a 10.70-fold increase in total FAs production. Co-overexpression of Mdm1 and selected lipases significantly improved the biosynthesis of FAs and linoleic acid in the engineered S. cerevisiae. The efficient LD-based metabolic engineering strategy presented in this study holds the potential to advance the high-level production of FAs and their derivatives in microbial cell factories.

In silicon desinging of RANKL-targeting vaccine for protection of osteoporosis based on the epitope of Denosumab

BackgroundLife quality of osteoporosis patients is affected significantly due to the severely complications of fracture and pain. RANKL, indicated as the key mediator of osteoporosis, plays a pathogenic role of osteoclasts induction. To target this program, two medications, bisphosphonate and Denosumab, were developed and achieved remarkable advantages in clinics. Unfortunately, fracture-related side-effects always emerge unavoidably, after either long-term administration of bisphosphonates or Denosumab withdrawing. To address these challenges, vaccine-based approach has been adopted to achieve sustainable protection through induction and maintenance of effective antibodies in mild level over decades. MethodsA Denosumab binding peptide was firstly identified as the basic component of vaccine. This peptide was then fused with diphtheria toxin T domain, a widely used adjuvant protein. Its capabilities to penetrate the autologous tolerance and induce the immune responses was then demonstrated with in-silicon evaluation. Finally, the efficacy of the DR3 vaccine was assessed through immunization on the human RANKL transgenic mice model of osteoporosis. ResultsThe DTT-RANKL(220–245)3 vaccine, termed as DR3, were predicted as highly antigenic and non-allergenicity. This molecule was comprised of 46.5 % of helix, 8.5 % strand and 45.1 % coil, the optimized Z-value of the tertiary structure was 6.39, and the favored area in the Ramachandran plot was 96.1 % after refinement. Molecular docking showed a tight binding of DR3 vaccine to TLR2 (−9.2 kcal/mol) and TLR4 (−9.5 kcal/mol). In addition, the immune stimulation indicated robust responses post administration of DR3 vaccine, including high level production of of antibodies and cytokines, activated T and B lymphocytes, and the long-last immune memory. In agree with the simulation, vaccinated mice generated high titers anti-hRANKL antibodies and elevated levels of IL-4 and IL-10 at 7th week post immunization. ConclusionDR3 vaccine was aroused to benefit the prevention and treatment of osteoporosis, and other bone-resorptive diseases potentially.

Exploit and elucidate chaperone assisted PET hydrolase for upcycling plastics

Polyethylene terephthalate (PET) is the most abundant plastic waste in the environment. Currently, a new biocatalyst PETase, was discovered in 2016 from Ideonella sakaiensis bacteria, owned the high ability to digest PET through a mild and sustainable process. However, the high-level production of PETase in the model Escherichia coli remains a challenge and limits its application. Therefore, we employed the native molecular chaperones from Ideonella sakaiensis to improve the quality and quantity of an outstanding PETase variant, FAST-PETase (FA) at the first time. We selected GroELS from E. coli (EcG) and I. sakaiensis (IsG) using three genetic designs while the co-expressing FA with IsG chaperone increased soluble FA and elevated its activity by 25%. On the other hand, through the genome mining of I. sakaiensis, we identified a lipase secretion chaperone (IsLsC) at the upstream of native PETase. When co-expressing IsLsC and FA, the degradation efficiency toward PET film was up to 51.7 % within one day at 50 °C. More LsC-like chaperones could be explored from the sequence similarity network (SSN) with corresponding function to IsLsC. Finally, molecular docking and dynamic simulation exploited a hydrogen bond formation between FA and IsLsC to stabilizing the overall structure. The discovery of a novel chaperone offers a promising strategy for attractive PETase engaging in PET waste valorization.

Novel Less Toxic, Lymphoid Tissue-Targeted Lipid Nanoparticles Containing a Vitamin B5-Derived Ionizable Lipid for mRNA Vaccine Delivery.

Following their approval by the Food and Drug Administration, lipid nanoparticles (LNPs) have emerged as promising tools for delivering mRNA vaccines and therapeutics. Ionizable lipids are among the essential components of LNPs, as they play crucial roles in encapsulating mRNA and facilitating its release into the cytosol. In this study, 17 innovative ionizable lipids using vitamin B5 are designed as the core structure, aiming to reduce toxicity, to maintain vaccine efficiency, and to ensure synthetic feasibility. The top-performing LNP in terms of mRNA vaccine delivery in the mouse model is LNP 5097, which is generated by incorporating ionizable lipid I97. mRNA⊂LNP 5097 demonstrates favorable structural and physicochemical properties, high mRNA transfection efficiency, and long-term stability. Moreover, mRNA⊂LNP 5097 specifically delivers the mRNA to the spleen and lymph nodes in model mice, induces balanced Th1/Th2 immune responses, and elicits the production of high levels of neutralizing antibodies with low toxicity. The findings here suggest the high utility of LNP 5097, which includes novel vitamin B5-derived ionizable lipids with reduced toxicity, in mRNA vaccine research for both infectious diseases and cancer.

A protein vaccine of RBD integrated with immune evasion mutation shows broad protection against SARS-CoV-2.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge and evade immunity, resulting in breakthrough infections in vaccinated populations. There is an urgent need for the development of vaccines with broad protective effects. In this study, we selected hotspot mutations in the receptor-binding domain (RBD) that contribute to immune escape properties and integrated them into the original RBD protein to obtain a complex RBD protein (cRBD), and we found cRBDs have broad protective effects against SARS-CoV-2 variants. Three cRBDs were designed in our study. Compared with the BA.1 RBD protein, the cRBDs induced the production of higher levels of broader-spectrum neutralizing antibodies, suggesting stronger and broader protective efficacy. In viral challenge experiments, cRBDs were more effective than BA.1 RBD in attenuating lung pathologic injury. Among the three constructs, cRBD3 showed optimal broad-spectrum and protective effects and is a promising candidate for a broad-spectrum SARS-CoV-2 vaccine. In conclusion, immunization with cRBDs triggered immunity against a wide range of variants, including those that emerged after we had completed designing the cRBDs. This study preliminarily explores and validates the feasibility of incorporating hotspot mutations that contribute to immune evasion into the RBD to expand the activity spectrum of antigen-induced antibodies.

Scylla. I. A Pure-parallel, Multiwavelength Imaging Survey of the ULLYSES Fields in the LMC and SMC

Scylla is a deep Hubble Space Telescope (HST) survey of the stellar populations, interstellar medium, and star formation in the LMC and SMC. As a pure-parallel complement to the Ultraviolet Legacy Library of Young Stars as Essential Standards (ULLYSES) survey, Scylla obtained 342 orbits of ultraviolet (UV) through near-IR imaging of the LMC and SMC with Wide Field Camera 3. In this paper, we describe the science objectives, observing strategy, data reduction procedure, and initial results from our photometric analysis of 96 observed fields. Although our observations were constrained by ULLYSES primary exposures, we imaged all fields in at least two filters (F475W and F814W) and 64% of fields in at least three and as many as seven WFC3 filters spanning the UV to IR. Overall, we reach average 50% completeness of m F225W = 26.0, m F275W = 26.2, m F336W = 26.9, m F475W = 27.8, m F814W = 25.5, m F110W = 24.7, and m F160W = 24.0 Vega mag in our photometric catalogs, which is faintward of the ancient main-sequence turnoff in all filters. The primary science goals of Scylla include characterizing the structure and properties of dust in the MCs, as well as their spatially resolved star formation and chemical enrichment histories. Our images and photometric catalogs, which represent the widest-area coverage of MCs with HST photometry to date, are available as a high-level science product at the Barbara A. Mikulski Archive for Space Telescopes.

Microbial production of 5-epi-jinkoheremol, a plant-derived antifungal sesquiterpene.

Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from Catharanthus roseus, 5-epi-jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and m6A writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5-epi-jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5-epi-jinkoheremol synthase TPS18, resulting in the variant TPS18I21P/T414S, with the most improved properties. Finally, the titer of 5-epi-jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5-epi-jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5-epi-jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5-epi-jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5-epi-jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.

Retraction Note: Systems engineering of Escherichia coli for high-level glutarate production from glucose

Retraction Note: Systems engineering of Escherichia coli for high-level glutarate production from glucose

Synthesis of β-ionone from xylose and lignocellulosic hydrolysate in genetically engineered oleaginous yeast Yarrowia lipolytica.

β-ionone, an apocarotenoid derived from a C40 terpenoid has an intense, woody smell and a low odor threshold that has been widely used in as an ingredient in food and cosmetics. Yarrowia lipolytica is a promising host for β-ionone production because of its oleaginous nature, its ability to produce high levels of acetyl-CoA (an important precursor for terpenoids), and the availability of synthetic biology tools to engineer the organism. In this study, β-carotene-producing Y. lipolytica strain XK17 was employed for β-ionone biosynthesis. First, we explored the effect of different sources of carotenoid cleavage dioxygenase (CCD) genes on β-ionone production. A high-yielding strain rUinO-D14 with 122mg/L of β-ionone was obtained by screening promoters combined with rDNA mediated multi-round iterative transformations to optimize the expression of the CCD gene of Osmanthus fragrans. Second, to further develop a high-level production strain for β-ionone, we optimized key genes in the mevalonate pathway by multi-round iterative transformations mediated by non-homologous end joining, combined with a protein tagging strategy. Finally, the introduction of a heterologous oxidoreductase pathway enabled the engineered Y. lipolytica strain to use xylose as a sole carbon source and produce β-ionone. In addition, the potential for use of lignocellulosic hydrolysate as the carbon source for β-ionone production showed that the NHA-A31 strain had a high β-ionone productivity level. This study demonstrates that engineered Y. lipolytica can be used for the efficient, green and sustainable production of β-ionone.

B-152 Analysis of carbonyl compounds in exhaled breath for detection of COVID-19

Abstract Background COVID-19 pandemic has caused enormous loss of lives and economic disruption. The global crisis has resulted in more than 700 million infections and 7 million deaths worldwide. Rapid screening and detection of highly infectious respiratory diseases is a key for preventing and curbing future epidemic and pandemic. The objective of this work is to develop a novel breath analysis technique for detection of COVID-19 using chemoselective capture of carbonyl compounds in exhaled breath for analysis by gas chromatography-mass spectrometry (GC-MS). Respiratory viral infection causes oxidative stress and inflammation in its host body which leads to production of higher levels of some carbonyl compounds through oxidation of lipids and could be detected in exhaled breath. This breath analysis technique can be rapidly adapted for screening infectious respiratory diseases for future epidemic and potential pandemic. Methods A total of 237 subjects were enrolled in this study. Of these, 142 were COVID-19 positive and 95 were negative. One liter exhaled breath in Tedlar bags from both COVID-19 positive and negative subjects confirmed by reverse transcriptase polymerase chain reaction between April 2022 and September 2023. The collected one liter breath samples were completely evacuated through a cartridge packed with silica particles in 2-3 minutes. Silica particles were coated with a reactive aminooxy agent for capture of carbonyl compounds in the breath samples through oximation reactions. The captured carbonyl compounds were eluted by methanol and the eluted solutions were analyzed by GC-MS for calculation of each compound concentration in the exhaled breath samples. Biostatistics was used to identify significant carbonyl compounds (defined as p value < 0.05) as markers of CPOVID-19 infection. A machine learning algorithm was implemented with the captured carbonyl compounds as input features for differentiation of COVID-19 positive from negative subjects. Results A total of more than 30 carbonyl compounds were detected in exhaled breath samples of all subjects. 10 features including 3 individual carbonyl compounds and 7 compound ratios show significant differences (p value <0.05) between COVID-19 positive and negative groups. The developed machine learning and artificial intelligence (AI) algorithms successfully separated COVOD-19 positive from negative with an accuracy of 95.4%, sensitivity of 96.5%, specificity of 93.7%, and AUROC=0.974. Conclusions The analysis of carbonyl compounds in exhaled breath by GC-MS has great potential for non-invasive detection of COVID-19. The method could be rapidly adapted for detection of other respiratory infectious diseases with a small sample size and data set for training of the machine learning and AI algorithms.

High-level production of patatin in Pichia pastoris and characterization of N-glycosylation modification in food processing properties

Patatin is an acidic protein found in potatoes that is commonly used in food and pharmaceutical industries due to its excellent emulsifying and gelation abilities. Pichia pastoris is widely used as a host for recombinant protein production because it can incorporate post-translational modifications. In this study, a patatin titre of 2189.8 mg/L was achieved in a 5 L bioreactor using P. pastoris GS115 with signal peptide mutation, dual promoter construction, co-expression of chaperone proteins and optimised fermentation. To enhance the application of recombinant patatin in the food processing field, the level of N-glycosylation was elevated by genetic engineering. Properties of natural patatin, recombinant patatin and patatinL109T (N-glycosylated modified patatin) were investigated including foaming, hydrophobicity and emulsifying abilities. The functional properties of recombinant patatin were enhanced by introducing N-glycosylation, which also improved the water-holding capacity of its gel. The patatinL109T gel exhibited superior elasticity and water retention properties. The findings provide valuable insight and serve as a reference for the utilisation of recombinant patatin. The established enhancement strategy could be applied to other recombinant proteins.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of l-Pipecolic Acid from Glucose.

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain Saccharomyces cerevisiae BY4743. Initially, an optimized biosynthetic pathway was constructed within S. cerevisiae, converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask in vivo. Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.

PHYSICOCHEMICAL CHARACTERISTICS OF BANANA PSEUDO-STEM FERMENTED WITH PLEUROTUS TUBER-REGIUM

The study was aimed at investigating the physicochemical changes in banana pseudo-stem (BPS) during a 20-day solid-state fermentation with Pleurotus tuber-regium . The experiment which was carried out in a completely randomized design (CRD), comprised of two treatments (T1 and T2) replicated thrice. One of the portions of the dried and ground BPS was composted and inoculated with P. tuber-regium as T2 while the untreated portion referred to as T1 (control). The physicochemical characteristics were analyzed for both fermented and unfermented BPS. Data generated were analyzed using the student t-test to determine the statistical effects of the treatment. The mushroom failed to colonize the composted BPS due to the production of high levels of ammonia gas in acidic pH (5.9–6.7) of the substrate. The results revealed significant reductions (p<0.05) in Water holding capacity (WHC), oil absorption capacity (OAC), swelling capacity (SWC), crude protein (CP), Gross Energy (GE) and metabolizable energy (ME) values due to the degradation process. Similarly, the fiber fraction results showed significant reductions (p<0.05) in the neutral detergent fiber (NDF), acid detergent fiber (ADF), and hemicellulose contents of the fermented BPS. It was concluded that banana pseudo-stem may not be suited for king tuber mushroom mycelia colonization using the method described in this study. It is recommended that the composting method adopted for the banana pseudo-stem be reviewed either by extending composting period or blending with other substrates.

How to Make My Bug Bounty Cost-Effective? A Game-Theoretical Model

A bug bounty program (BBP) is an innovative crowdsourcing security solution increasingly adopted by organizations. We use a game-theoretical model to analyze how key characteristics impact BBPs and offer practical insights into managing a BBP as part of an organization’s vulnerability management for better cost-effectiveness. Our findings indicate that organizations with high patching complexity should announce lower bounties, especially if they face limited security resources. BBPs should complement, not substitute, an organization’s security characteristics. Evaluating patching complexity and security posture is crucial when designing a BBP. Furthermore, security researchers drive BBP performance. Higher productivity in researchers doesn’t always require higher bounties even with high postdiscovery costs. Novice productivity can increase total costs if unit postdiscovery costs are high, whereas expert productivity consistently reduces costs. Organizations should disclose high-level product and information technology (IT) features to increase expert productivity. The number of security researchers in a BBP is important, but increasing their numbers doesn’t always necessitate higher bounties. A larger crowd may not always be cost-effective. Lastly, enhanced legal protection for security researchers might not increase organizational risks, especially in organizations with robust security or less sophisticated IT infrastructure. Industrial associations and policymakers should consider these factors in standards and legal frameworks.

Metabolic engineering of Escherichia coli for high-level production of benzyl acetate from glucose

BackgroundBenzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate.ResultsTwo biosynthetic routes based on the CoA-dependent β-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent β-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation.ConclusionsWe were able to establish the whole pathway for benzyl acetate based on the CoA-dependent β-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms.Graphical abstract

Engineering yeast for high-level production of β-farnesene from sole methanol

Methanol, a rich one-carbon feedstock, can be massively produced from CO2 by the liquid sunshine route, which is helpful to realize carbon neutrality. β-Farnesene is widely used in the production of polymers, surfactants, lubricants, and also serves as a suitable substitute for jet fuel. Constructing an efficient cell factory is a feasible approach for β-farnesene production through methanol biotransformation. Here, we extensively engineered the methylotrophic yeast Ogataea polymorpha for the efficient bio-production of β-farnesene using methanol as the sole carbon source. Our study demonstrated that sufficient supply of precursor acetyl-CoA and cofactor NADPH in an excellent yeast chassis had a 1.3-fold higher β-farnesene production than that of wild-type background strain. Further optimization of the mevalonate pathway and enhancement of acetyl-CoA supply led to a 7-fold increase in β-farnesene accumulation, achieving the highest reported sesquiterpenoids production (14.7 g/L with a yield of 46 mg/g methanol) from one-carbon feedstock under fed-batch fermentation in bioreactor. This study demonstrates the great potential of engineering O. polymorpha for high-level terpenoid production from methanol.

Engineering a non-oxidative glycolysis pathway in escherichia coli for high-level citramalate production

BackgroundMethyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation.ResultsThis study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively.ConclusionThis is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.

Metabolic engineering of artificially modified transcription factor SmMYB36-VP16 for high-level production of tanshinones and phenolic acids

Tanshinones and phenolic acids are the two main chemical constituents in Salvia miltiorrhiza, which are used clinically for the treatment of hypertension, coronary heart disease, atherosclerosis, and many other diseases, and have broad medicinal value. The efficient synthesis of the target products of these two metabolites in isolated plant tissues cannot be achieved without the regulation and optimization of metabolic pathways, and transcription factors play an important role as common regulatory elements in plant tissue metabolic engineering. However, most of the regulatory effects are specific to one class of metabolites, or an opposing regulation of two classes of metabolites exists. In this study, an artificially modified transcription factor, SmMYB36-VP16, was constructed to enhance tanshinones and phenolic acids in Salvia miltiorrhiza hair roots simultaneously. Further in combination with the elicitors dual-screening technique, by applying the optimal elicitors screened, the tanshinones content in the transgenic hairy roots of Salvia miltiorrhiza reached 6.44 mg/g DW, which was theoretically 6.08-fold that of the controls without any treatment, and the content of phenolic acids reached 141.03 mg/g DW, which was theoretically 5.05-fold that of the controls without any treatment. The combination of artificially modified transcriptional regulatory and elicitors dual-screening techniques has facilitated the ability of plant isolated tissue cell factories to produce targeted medicinal metabolites. This strategy could be applied to other species, laying the foundation for the production of potential natural products for the medicinal industry.