Browsing by Author "Marsafari, Monireh"
Now showing 1 - 8 of 8
Results Per Page
Sort Options
Item Biotechnological production of flavonoids: an update on plant metabolic engineering, microbial host selection and genetically encoded biosensorsMarsafari, Monireh; Samizadeh, Habibollah; Rabiei, Babak; Mehrabi, Ali Ashraf; Koffas, Mattheos; Xu, PengFlavonoids represent a diversified family of phenylpropanoid-derived plant secondary metabolites. They are widely found in fruits, vegetables and medicinal herbs and plants. There has been increasing interest on flavonoids because of their proven bioactivity associated with anti-obesity, anti-cancer, anti-inflammatory, anti-diabetic activity and the prevention of aging-related chronic conditions, such as nervous and cardiovascular disease. Low bioavailability of flavonoids is a major challenge restricting their wide applications. Due to safety and economic issues, traditional plant extraction or chemical synthesis could not provide a scalable route for large-scale production of flavonoids. Alternatively, reconstruction of biosynthetic gene clusters in plants and industrially relevant microbes offer significant promise for discovery and scalable synthesis of flavonoids. This review provides an update on biotechnological production of flavonoids. We summarized the recent advances on plant metabolic engineering, microbial host and genetically encoded biosensors. Plant metabolic engineering holds the promise to improve the yield of specific flavonoids and expand the chemical space of novel flavonoids. The choice of microbial host provides the cellular chassis that could be tailored for various stereo- or regio-selective chemistries that are crucial for their bioactivities. When coupled with transcriptional biosensing, genetically encoded biosensors could be welded into cellular metabolism to achieve high throughput screening or dynamic carbon flux re-allocation to deliver efficient and robust microbial workhorse. The convergence of these technologies will translate the vast majority of plant genetic resources into valuable flavonoids with pharmaceutical/nutraceutical values in the foreseeable future.Item Debottlenecking mevalonate pathway for antimalarial drug precursor amorphadiene biosynthesis in Yarrowia lipolytica(Elsevier B.V., 2020-06-10) Marsafari, Monireh; Xu, PengWorld Health Organization reports that half of the population in developing countries are at risk of malaria infection. Artemisinin, the most potent anti-malaria drug, is a sesquiterpene endoperoxide extracted from the plant Artemisia annua. Due to scalability and economics issues, plant extraction or chemical synthesis could not provide a sustainable route for large-scale manufacturing of artemisinin. The price of artemisinin has been fluctuating from 200$/Kg to 1100$/Kg, due to geopolitical and climate factors. Microbial fermentation was considered as a promising method to stabilize the artemisinin supply chain. Yarrowia lipolytica, is an oleaginous yeast with proven capacity to produce large quantity of lipids and oleochemicals. In this report, the lipogenic acetyl-CoA pathways and the endogenous mevalonate pathway of Y. lipolytica were harnessed for amorphadiene production. Gene overexpression indicate that HMG-CoA and acetyl-CoA supply are two limiting bottlenecks for amorphadiene production. We have identified the optimal HMG-CoA reductase and determined the optimal gene copy number for the precursor pathways. Amorphadiene production was improved further by either inhibiting fatty acids synthase or activating the fatty acid degradation pathway. With co-expression of mevalonate kinase (encoded by Erg12), a push-and-pull strategy enabled the engineered strain to produce 171.5 mg/L of amorphadiene in shake flasks. These results demonstrate that balancing carbon flux and manipulation of precursor competing pathways are key factors to improve amorphadiene biosynthesis in oleaginous yeast; and Y. lipolytica is a promising microbial host to expand nature’s biosynthetic capacity, allowing us to quickly access antimalarial drug precursors.Item Debottlenecking mevalonate pathway for antimalarial drug precursor amorphadiene biosynthesis in Yarrowia lipolytica(Elsevier, 2020-01-02) Marsafari, Monireh; Xu, PengWorld Health Organization reports that half of the population in developing countries are at risk of malaria infection. Artemisinin, the most potent anti-malaria drug, is a sesquiterpene endoperoxide extracted from the plant Artemisia annua. Due to scalability and economics issues, plant extraction or chemical synthesis could not provide a sustainable route for large-scale manufacturing of artemisinin. The price of artemisinin has been fluctuating from 200$/Kg to 1100$/Kg, due to geopolitical and climate factors. Microbial fermentation was considered as a promising method to stabilize the artemisinin supply chain. Yarrowia lipolytica, is an oleaginous yeast with proven capacity to produce large quantity of lipids and oleochemicals. In this report, the lipogenic acetyl-CoA pathways and the endogenous mevalonate pathway of Y. lipolytica were harnessed for amorphadiene production. Gene overexpression indicate that HMG-CoA and acetyl-CoA supply are two limiting bottlenecks for amorphadiene production. We have identified the optimal HMG-CoA reductase and determined the optimal gene copy number for the precursor pathways. Amorphadiene production was improved further by either inhibiting fatty acids synthase or activating the fatty acid degradation pathway. With co-expression of mevalonate kinase (encoded by Erg12), a push-and-pull strategy enabled the engineered strain to produce 171.5 mg/L of amorphadiene in shake flasks. These results demonstrate that balancing carbon flux and manipulation of precursor competing pathways are key factors to improve amorphadiene biosynthesis in oleaginous yeast; and Y. lipolytica is a promising microbial host to expand nature’s biosynthetic capacity, allowing us to quickly access antimalarial drug precursors.Item Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: current state and perspectives(BioMed Central Ltd, 2019-03-26) Wan, Xia; Marsafari, Monireh; Xu, PengNature has evolved exquisite sensing mechanisms to detect cellular and environmental signals surrounding living organisms. These biosensors have been widely used to sense small molecules, detect environmental cues and diagnose disease markers. Metabolic engineers and synthetic biologists have been able to exploit metabolites-responsive transcriptional factors (MRTFs) as basic tools to rewire cell metabolism, reprogram cellular activity as well as boost cell’s productivity. This is commonly achieved by integrating sensor-actuator systems with biocatalytic functions and dynamically allocating cellular resources to drive carbon flux toward the target pathway. Up to date, most of identified MRTFs are derived from bacteria. As an endeavor to advance intelligent biomanufacturing in yeast cell factory, we will summarize the opportunities and challenges to transfer the bacteria-derived MRTFs to expand the small-molecule sensing capability in eukaryotic cells. We will discuss the design principles underlying MRTF-based biosensors in eukaryotic cells, including the choice of reliable reporters and the characterization tools to minimize background noise, strategies to tune the sensor dynamic range, sensitivity and specificity, as well as the criteria to engineer activator and repressor-based biosensors. Due to the physical separation of transcription and protein expression in eukaryotes, we argue that nuclear import/export mechanism of MRTFs across the nuclear membrane plays a critical role in regulating the MRTF sensor dynamics. Precisely-controlled MRTF response will allow us to repurpose the vast majority of transcriptional factors as molecular switches to achieve temporal or spatial gene expression in eukaryotes. Uncovering this knowledge will inform us fundamental design principles to deliver robust cell factories and enable the design of reprogrammable and predictable biological systems for intelligent biomanufacturing, smart therapeutics or precision medicine in the foreseeable future.Item Modular Co-Culture Engineering of Yarrowia Lipolytica for Amorphadiene Biosynthesis(2022-03-02) Marsafari, Monireh; Azi, Fidelis; Xu, PengAmorphadiene is an intermediate product of the first dedicated step to artemisinin production. It has attracted wide research interest as an antimalaria drug precursor in recent years. The efficient conversion of renewable carbon sources and redirection of metabolic flux toward a metabolite of interest has become a fascinating strategy for plant secondary metabolite overproduction. A modular pathway that divides the hosts' labor, a co-culture system has shown great biosynthetic potential and can be leveraged to achieve cost-effective bioproduction of natural products. Using a co-culture system of Y. lipolytica Po1f and Po1g strains, subcellular localization of ADS gene into the endoplasmic reticulum, co-utilization of mixed carbon source, and enlargement of the endoplasmic reticulum size were investigated to improve amorphadiene producton in this work. Using Po1g/PPtM and Po1f/AaADSERₓ₃/iGFMPDU strains and co-utilization of 5 µM sodium acetate with 20 g/L glucose in YPD media, amorphadiene titer increased to 65.094 mg/L. The enlargement of the Endoplasmic reticulum membrane caused by the deletion of the PAH1 gene provided more subcellular space for the action of the ADS-tagged gene. It further increased the amorphadiene production to 71.74 mg/L. The results demonstrated that manipulating metabolic flux in the co-culture of Y. lipolytica can be efficient over a single culture for the bioproduction of many value-added metabolites in a whole or large biosynthetic pathway.Item The optimization of Naringenin biosynthesis pathway using Yarrowia lipolitica cell culture(Kharazmi University, 2020-06-30) Marsafari, Monireh; Lahiji, Habibollah Samizadeh; Rabiei, Babak; Mehrabi, Ali Ashraf; Lv, Yongkun; Xu, PengYarrowia lipolytica, as a good cell factory to speed up the production of plant pharmaceutical components, has been considered to be one of the most important and attractive micro-organisms in recent years, due to its high secretion capacity, limited glycosylation, large range of genetic markers and molecular tools. Naringenin, as a central core of flavonoids production, plays important roles both in plants and in the treatment of different types of human diseases. For this purpose, specific naringenin biosynthesis genes from different origins were selected and introduced after comparative expression profiling in Y. lipolytica. This research indicated that chs plays the main role in the production of naringenin, so the increase copy number of this gene in each construct was investigated. The HPLC results confirmed that the construct with 5 copy numbers of chs resulted in 7.14 fold increase of naringenin extracellular titer to 90.16 mg/L in shake flask cultures. The results reported in this study demonstrated that sufficient knowledge of genes involved in the specific biosynthesis pathway, synthetic gene pathway and using Y. lipolytica as a capable and cheap host could help bioengineers to produce significant amounts of pharmaceutical components.Item Rapid ultra-sensitive and high-throughput bioburden detection: Microfluidics and instrumentation(ACS, 2022-06-06) Hasan, Md Sadique; Marsafari, Monireh; Tolosa, Michael; Andar, Abhay; Ramamurthy, Sai Sathish; Ge, Xudong; Kostov, Yordan; Rao, GovindContamination detection often requires lengthy culturing steps to detect low-level bioburden. To increase the rate of detection and decrease the limit of detection (LOD), a system featuring microfluidics and a multichannel fluorometer has been developed. The eight-channel fluorometer enables parallel testing of multiple samples with the LOD as low as <1 cfu/mL. This low-cost system utilizes the slope of fluorescence intensity that serves as the criterion for bioburden detection. The redox indicator dye resazurin is used to monitor the presence of viable cells in this study and is reduced to resorufin with a high quantum yield at 585 nm. The sample under investigation is spiked with resazurin and loaded in a special-design microfluidic cassette, and the rate of change is observed via the fluorometer. The method was validated using primary Escherichia coli culture in comparison with a spectrophotometer which served as the gold standard. An optimized assay based on Luria–Bertani medium was developed. The impact on the assay sensitivity based on incubation and filtration steps was also explored. The assay is shown to pick up inadvertent contamination from test tubes and pipette tips showing its applicability in real-world settings. The data analysis demonstrated a comparable performance of the multichannel fluorometer vis-a-vis the conventional plate reader. The multichannel system is shown to detect bioburden presence in as low as 20 s for bacterial concentrations ≥5 cfu/mL after 6 h of incubation. Considering its portability, low cost, simplicity of operation, and relevant assay sensitivity, the system is well positioned to detect low-level bioburden in the laboratory, pharmaceutical, and field settings.Item Understanding lipogenesis by dynamically profiling transcriptional activity of lipogenic promoters in Yarrowia lipolytica(Springer Nature Switzerland AG., 2019-02-07) Liu, Huan; Marsafari, Monireh; Deng, Li; Xu, PengLipogenesis is a complicated process involving global transcriptional reprogramming of lipogenic pathways. It is commonly believed that nitrogen starvation triggers a metabolic shift that reroutes carbon flux from Krebs cycles to lipogenesis. In this study, we systematically surveyed and dynamically profiled the transcriptional activity of 22 lipogenic promoters aiming to delineate a picture how nitrogen starvation regulates lipogenesis in Y. lipolytica. These lipogenic promoters drive the expression of critical pathways that are responsible for the generation of reducing equivalents (NADPH), carbon backbones (acetyl-CoA, malonyl-CoA, DHAP, etc.), synthesis and degradation of fatty acids. Specifically, our investigated promoters span across an array of metabolic pathways, including glycolysis, Krebs cycle, pentose phosphate pathway, mannitol cycle, glutamine–GABA cycle, fatty acid and lipid synthesis, glyoxylate, β-oxidation, and POM (pyruvate–oxaloacetate–malate) cycle. Our work provides evidences that mannitol cycle, glutamine–GABA cycle and amino acid degradation, pyruvate oxidation, and acetate assimilation pathways are lipogenesis-related steps involved in generating cytosolic NADPH and acetyl-CoA precursors. This systematic investigation and dynamic profiling of lipogenic promoters may help us better understand lipogenesis, facilitate the formulation of structure-based kinetic models, as well as develop efficient cell factories for fuels and chemical production in oleaginous species.