14 results on '"Koffas, Mattheos A. G."'
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2. Improved Butanol Production Using FASII Pathway in E. coli.
- Author
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Jawed, Kamran, Abdelaal, Ali Samy, Koffas, Mattheos A. G., and Yazdani, Syed Shams
- Published
- 2020
- Full Text
- View/download PDF
3. Design and Characterization of Biosensors for the Screening of Modular Assembled Naringenin Biosynthetic Library in Saccharomyces cerevisiae.
- Author
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Wang, Rufeng, Cress, Brady F., Yang, Zheng, Hordines III, John C., Zhao, Shujuan, Jung, Gyoo Yeol, Wang, Zhengtao, and Koffas, Mattheos A. G.
- Published
- 2019
- Full Text
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4. De novo Biosynthesis of Salvianolic Acid B in Saccharomyces cerevisiae Engineered with the Rosmarinic Acid Biosynthetic Pathway.
- Author
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Xu Y, Geng L, Zhang Y, Jones JA, Zhang M, Chen Y, Tan R, Koffas MAG, Wang Z, and Zhao S
- Subjects
- Benzofurans, Biosynthetic Pathways genetics, Cinnamates metabolism, Depsides, Rosmarinic Acid, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Salvia miltiorrhiza genetics, Salvia miltiorrhiza metabolism
- Abstract
Salvianolic acid B (SAB), also named lithospermic acid B, belongs to a class of water-soluble phenolic acids, originating from plants such as Salvia miltiorrhiza . SAB exhibits a variety of biological activities and has been clinically used to treat cardio- and cerebrovascular diseases and also has great potential as a health care product and medicine for other disorders. However, its biosynthetic pathway has not been completely elucidated. Here, we report the de novo biosynthesis of SAB in Saccharomyces cerevisiae engineered with the heterologous rosmarinic acid (RA) biosynthetic pathway. The created pathway contains seven genes divided into three modules on separate plasmids, pRS424-FjTAL-Sm4CL2, pRS425-SmTAT-SmHPPR or pRS425-SmTAT-CbHPPR, and pRS426-SmRAS-CbCYP-CbCPR. These three modules were cotransformed into S. cerevisiae , resulting in the recombinant strains YW-44 and YW-45. Incubation of the recombinant strains in a basic medium without supplementing any substrates yielded 34 and 30 μg/L of SAB. The findings in this study indicate that the created heterologous RA pathway cooperates with the native metabolism of S. cerevisiae to enable the de novo biosynthesis of SAB. This provides a novel insight into a biosynthesis mechanism of SAB and also lays the foundation for the production of SAB using microbial cell factories.
- Published
- 2022
- Full Text
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5. In Vitro Naringenin Biosynthesis from p -Coumaric Acid Using Recombinant Enzymes.
- Author
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Zang Y, Zha J, Wu X, Zheng Z, Ouyang J, and Koffas MAG
- Subjects
- Acyltransferases genetics, Acyltransferases metabolism, Biocatalysis, Coenzyme A Ligases genetics, Coenzyme A Ligases metabolism, Escherichia coli genetics, Escherichia coli metabolism, Intramolecular Lyases genetics, Intramolecular Lyases metabolism, Metabolic Engineering, Oryza enzymology, Plant Proteins chemistry, Plant Proteins genetics, Plant Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Acyltransferases chemistry, Coenzyme A Ligases chemistry, Coumaric Acids chemistry, Flavanones chemistry, Intramolecular Lyases chemistry
- Abstract
Naringenin is an important precursor for the production of a wide spectrum of flavonoids, and its production is of great interest in metabolic engineering. However, in cellular systems, identification of rate-limiting factors is often difficult because of complex regulatory networks. Cell-free catalytic systems emerge as a promising method to address this issue. Here, we explored the cell-free biosystem for naringenin production by combining different sources of 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). After systematic analysis of enzyme levels, substrate concentrations, and cofactors, 4CL and CHS were found to be crucial to the reaction. The best loading ratio of 4CL/CHS/CHI was 10:10:1, and malonyl-CoA was the limiting factor, as identified previously in fermentation. For the first time, we successfully constructed the system for naringenin production in vitro . Our study will deepen our understanding of the key factors in naringenin production and guide further engineering.
- Published
- 2019
- Full Text
- View/download PDF
6. Engineering Bacillus megaterium Strains To Secrete Cellulases for Synergistic Cellulose Degradation in a Microbial Community.
- Author
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Kalbarczyk KZ, Mazeau EJ, Rapp KM, Marchand N, Koffas MAG, and Collins CH
- Subjects
- Bacillus megaterium growth & development, Cellulases genetics, Plasmids genetics, Plasmids metabolism, Protein Sorting Signals genetics, Bacillus megaterium metabolism, Cellulases metabolism, Cellulose metabolism, Metabolic Engineering methods
- Abstract
Recent environmental concerns have intensified the need to develop systems to degrade waste biomass for use as an inexpensive carbon source for microbial chemical production. Current approaches to biomass utilization rely on pretreatment processes that include expensive enzymatic purification steps for the requisite cellulases. We aimed to engineer a synthetic microbial community to synergistically degrade cellulose by compartmentalizing the system with multiple specialized Bacillus megaterium strains. EGI1, an endoglucanase, and Cel9AT, a multimodular cellulase, were targeted for secretion from B. megaterium. A small library of signal peptides (SPs) with five amino acid linkers was selected to tag each cellulase for secretion from B. megaterium. Cellulase activity against amorphous cellulose was confirmed through a series of bioassays, and the most active SP constructs were identified as EGI1 with the LipA SP and Cel9AT with the YngK SP. The activity of the optimized cellulase secretion strains was characterized individually and in tandem to assess synergistic cellulolytic activity. The combination of EGI1 and Cel9AT yielded higher activity than either single cellulase. A coculture of EGI1 and Cel9AT secreting B. megaterium strains demonstrated synergistic behavior with higher activity than either monoculture. This cellulose degradation module can be further integrated with bioproduct synthesis modules to build complex systems for the production of high value molecules.
- Published
- 2018
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7. Production of Deuterated Cyanidin 3- O -Glucoside from Recombinant Escherichia coli .
- Author
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Gupta M, Zha J, Zhang X, Jung GY, Linhardt RJ, and Koffas MAG
- Abstract
Anthocyanins are plant secondary metabolites that, despite their chemical instability, have found many applications as natural food colorants. They are also known for their beneficial health effects because of their antioxidant and anticancer properties. More stable versions of these molecules, particularly at neutral pH conditions, are required to study the anthocyanin pharmacokinetic properties and obtain effective therapeutic results. In the present report, a cost-effective technique was developed to prepare the deuterated anthocyanin using recombinant Escherichia coli as a production host and deuterated glycerol and D
2 O in the culture media. This approach resulted in the formation of endogenous deuterated uridine 5'-diphosphate-glucose that was further incorporated by the recombinant anthocyanin pathway, resulting in the formation of deuterated cyanidin 3- O -glucoside (C3G). The deuterium exchange of O-D and C-D were studied by liquid chromatography (LC)-mass spectrometry and NMR analysis. The labeled C3G, purified by high-performance LC showed a stable nature at pH 7.0 as compared to nondeuterated C3G., Competing Interests: The authors declare no competing financial interest.- Published
- 2018
- Full Text
- View/download PDF
8. Development of Artificial Riboswitches for Monitoring of Naringenin In Vivo.
- Author
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Jang S, Jang S, Xiu Y, Kang TJ, Lee SH, Koffas MAG, and Jung GY
- Subjects
- Biosensing Techniques methods, Escherichia coli genetics, Escherichia coli metabolism, Flavanones analysis, Flavanones pharmacology, Gene Expression Regulation, Bacterial drug effects, Riboswitch, SELEX Aptamer Technique
- Abstract
Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist. Unlike the protein-based trans-regulating controllers, riboswitches can respond to their ligands faster and eliminate off-target effects. Here, we developed artificial riboswitches that activate gene expression in response to naringenin, an important flavonoid. RNA aptamers for naringenin were developed using SELEX and cloned upstream of a dual selectable marker gene to construct a riboswitch library. Two in vivo selection routes were applied separately to the library by supplementing naringenin at two different concentrations during enrichments to modulate the operational ranges of the riboswitches. The selected riboswitches were responsive to naringenin and activated gene expression up to 2.91-fold. Operational ranges of the riboswitches were distinguished on the basis of their selection route. Additionally, the specificity of the riboswitches was assessed, and their applicability as dynamic regulators was confirmed. Collectively, the naringenin riboswitches reported in this work will be valuable tools in metabolic engineering of microorganisms for the production of flavonoids.
- Published
- 2017
- Full Text
- View/download PDF
9. Effect of Genomic Integration Location on Heterologous Protein Expression and Metabolic Engineering in E. coli.
- Author
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Englaender JA, Jones JA, Cress BF, Kuhlman TE, Linhardt RJ, and Koffas MAG
- Subjects
- Ammonia-Lyases metabolism, Chromatography, High Pressure Liquid, Chromosomes, Bacterial genetics, Chromosomes, Bacterial metabolism, Cinnamates analysis, Cinnamates metabolism, Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Genetic Loci, Indoles analysis, Indoles metabolism, Lac Operon genetics, Luminescent Proteins genetics, Methyltransferases genetics, Plasmids genetics, Plasmids metabolism, Rec A Recombinases genetics, Red Fluorescent Protein, Escherichia coli metabolism, Luminescent Proteins metabolism, Metabolic Engineering
- Abstract
Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E. coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E. coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.
- Published
- 2017
- Full Text
- View/download PDF
10. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli.
- Author
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Cress BF, Toparlak ÖD, Guleria S, Lebovich M, Stieglitz JT, Englaender JA, Jones JA, Linhardt RJ, and Koffas MA
- Subjects
- Cloning, Molecular, Clustered Regularly Interspaced Short Palindromic Repeats, Disaccharides metabolism, Down-Regulation, Flavanones biosynthesis, Metabolic Engineering, Plasmids, Promoter Regions, Genetic, CRISPR-Cas Systems, Epigenetic Repression, Escherichia coli genetics, Transcriptional Activation
- Abstract
Programmable control over an addressable global regulator would enable simultaneous repression of multiple genes and would have tremendous impact on the field of synthetic biology. It has recently been established that CRISPR/Cas systems can be engineered to repress gene transcription at nearly any desired location in a sequence-specific manner, but there remain only a handful of applications described to date. In this work, we report development of a vector possessing a CRISPathBrick feature, enabling rapid modular assembly of natural type II-A CRISPR arrays capable of simultaneously repressing multiple target genes in Escherichia coli. Iterative incorporation of spacers into this CRISPathBrick feature facilitates the combinatorial construction of arrays, from a small number of DNA parts, which can be utilized to generate a suite of complex phenotypes corresponding to an encoded genetic program. We show that CRISPathBrick can be used to tune expression of plasmid-based genes and repress chromosomal targets in probiotic, virulent, and commonly engineered E. coli strains. Furthermore, we describe development of pCRISPReporter, a fluorescent reporter plasmid utilized to quantify dCas9-mediated repression from endogenous promoters. Finally, we demonstrate that dCas9-mediated repression can be harnessed to assess the effect of downregulating both novel and computationally predicted metabolic engineering targets, improving the yield of a heterologous phytochemical through repression of endogenous genes. These tools provide a platform for rapid evaluation of multiplex metabolic engineering interventions.
- Published
- 2015
- Full Text
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11. Design and kinetic analysis of a hybrid promoter-regulator system for malonyl-CoA sensing in Escherichia coli.
- Author
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Xu P, Wang W, Li L, Bhan N, Zhang F, and Koffas MA
- Subjects
- Bacillus subtilis genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cerulenin metabolism, Escherichia coli chemistry, Kinetics, Malonyl Coenzyme A metabolism, Models, Molecular, Biosensing Techniques methods, Escherichia coli genetics, Escherichia coli metabolism, Malonyl Coenzyme A analysis, Promoter Regions, Genetic
- Abstract
Malonyl-CoA is the rate-limiting precursor involved in the chain elongation reaction of a range of value-added pharmaceuticals and biofuels. Development of malonyl-CoA responsive sensors holds great promise in overcoming critical pathway limitations and optimizing production titers and yields. By incorporating the Bacillus subtilis trans-regulatory protein FapR and the cis-regulatory element fapO, we constructed a hybrid promoter-regulatory system that responds to a broad range of intracellular malonyl-CoA concentrations (from 0.1 to 1.1 nmol/mgDW) in Escherichia coli. Elimination of regulatory protein and nonspecific DNA cross-communication leads to a sensor construct that exhibits malonyl-CoA-dependent linear phase kinetics with increased dynamic response range. The sensors reported in this study could potentially control and optimize carbon flux leading to robust biosynthetic pathways for the production of malonyl-CoA-derived compounds.
- Published
- 2014
- Full Text
- View/download PDF
12. ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli.
- Author
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Xu P, Vansiri A, Bhan N, and Koffas MA
- Subjects
- Gene Expression, Genes, Bacterial, Genetic Engineering, Metabolic Networks and Pathways genetics, Multigene Family, Synthetic Biology, Escherichia coli genetics, Escherichia coli metabolism, Genetic Vectors, Metabolic Engineering methods
- Abstract
Harnessing cell factories for producing biofuel and pharmaceutical molecules has stimulated efforts to develop novel synthetic biology tools customized for modular pathway engineering and optimization. Here we report the development of a set of vectors compatible with BioBrick standards and its application in metabolic engineering. The engineered ePathBrick vectors comprise four compatible restriction enzyme sites allocated on strategic positions so that different regulatory control signals can be reused and manipulation of expression cassette can be streamlined. Specifically, these vectors allow for fine-tuning gene expression by integrating multiple transcriptional activation or repression signals into the operator region. At the same time, ePathBrick vectors support the modular assembly of pathway components and combinatorial generation of pathway diversities with three distinct configurations. We also demonstrated the functionality of a seven-gene pathway (~9 Kb) assembled on one single ePathBrick vector. The ePathBrick vectors presented here provide a versatile platform for rapid design and optimization of metabolic pathways in E. coli.
- Published
- 2012
- Full Text
- View/download PDF
13. Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids.
- Author
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Leonard E, Yan Y, Fowler ZL, Li Z, Lim CG, Lim KH, and Koffas MA
- Subjects
- Anthocyanins biosynthesis, Escherichia coli genetics, Fatty Acid Synthases antagonists & inhibitors, Malonates metabolism, Operon, Orotic Acid metabolism, Recombination, Genetic, Escherichia coli metabolism, Flavonoids biosynthesis
- Abstract
Plant flavonoid polyphenols continue to find increasing pharmaceutical and nutraceutical applications; however their isolation, especially of pure compounds, from plant material remains an underlying challenge. In the past Escherichia coli, one of the most well-characterized microorganisms, has been utilized as a recombinant host for protein expression and heterologous biosynthesis of small molecules. However, in many cases the expressed protein activities and biosynthetic efficiency are greatly limited by the host cellular properties, such as precursor and cofactor availability and protein or product tolerance. In the present work, we developed E. coli strains capable of high-level flavonoid synthesis through traditional metabolic engineering techniques. In addition to grafting the plant biosynthetic pathways, the methods included engineering of an alternative carbon assimilation pathway and the inhibition of competitive reaction pathways in order to increase intracellular flavonoid backbone precursors and cofactors. With this strategy, we report the production of plant-specific flavanones up to 700 mg/L and anthocyanins up to 113 mg/L from phenylpropanoic acid and flavan-3-ol precursors, respectively. These results demonstrated the efficient and scalable production of plant flavonoids from E. coli for pharmaceutical and nutraceutical applications.
- Published
- 2008
- Full Text
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14. Combinatorial mutasynthesis of flavonoid analogues from acrylic acids in microorganisms.
- Author
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Chemler JA, Yan Y, Leonard E, and Koffas MA
- Subjects
- Hydrogen chemistry, Molecular Structure, Acrylates chemistry, Acrylates metabolism, Flavonoids biosynthesis, Flavonoids chemistry, Saccharomyces cerevisiae metabolism
- Abstract
Flavonoids are plant secondary metabolites often used as nutraceutical supplements, but a growing number of unnatural flavonoids are being investigated as therapeutic agents. Cultures of Saccharomyces cerevisiae expressing recombinant flavonoid enzymes, including 4-coumaroyl:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone 3beta-hydroxylase (FHT), produced novel flavanones and dihydroflavonols when fed with a number of aromatic acrylic acids. The flavonoid network also exhibited broad substrate specificity by converting muconic acid into a unique polypropanoid.
- Published
- 2007
- Full Text
- View/download PDF
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