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.
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•Modular co-culture engineering for the production of natural products.•Advantages of modular co-culture engineering in the field of biosynthesis.•Designs and strategies for ...engineering microbial co-cultures.•Novel approaches are helping to improve properties of modular co-culture system.
The microbial production of natural products has been traditionally accomplished in a single organism engineered to accommodate target biosynthetic pathways. Often times, such approaches result in large metabolic burdens as key cofactors, precursor metabolites and energy are channeled to pathways of structurally complex chemicals. Recently, modular co-culture engineering has emerged as a new approach to efficiently conduct heterologous biosynthesis and greatly enhance the production of natural products. This review highlights recent advances that leverage Escherichia coli-based modular co-culture engineering for making natural products. Potential future perspectives for studies in this promising field are addressed as well.
Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) ...in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo.
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.
Malonyl-coenzyme A is an important precursor metabolite for the biosynthesis of polyketides, flavonoids and biofuels. However, malonyl-CoA naturally synthesized in microorganisms is consumed for the ...production of fatty acids and phospholipids leaving only a small amount available for the production of other metabolic targets in recombinant biosynthesis. Here we present an integrated computational and experimental approach aimed at improving the intracellular availability of malonyl-CoA in
Escherichia coli. We used a customized version of the recently developed OptForce methodology to predict a minimal set of genetic interventions that guarantee a prespecified yield of malonyl-CoA in
E. coli strain BL21 Star™. In order to validate the model predictions, we have successfully constructed an
E. coli recombinant strain that exhibits a 4-fold increase in the levels of intracellular malonyl-CoA compared to the wild type strain. Furthermore, we demonstrate the potential of this
E. coli strain for the production of plant-specific secondary metabolites naringenin (474
mg/L) with the highest yield ever achieved in a lab-scale fermentation process. Combined effect of the genetic interventions was found to be synergistic based on a developed analysis method that correlates genetic modification to cell phenotype, specifically the identified knockout targets (Δ
fumC and Δ
sucC) and overexpression targets (ACC, PGK, GAPD and PDH) can cooperatively force carbon flux towards malonyl-CoA. The presented strategy can also be readily expanded for the production of other malonyl-CoA-derived compounds like polyketides and biofuels.
► Metabolic network modeling identified minimal set of genetic interventions leading to improved intracellular malonyl-CoA. ► Engineered strain exhibits 5.6-fold increase in flavanone production. ► Combined effect of the genetic interventions was found to be synergistic based on an analysis correlating genetic modifications to cell phenotype. ► Same strategy can be applied to the production of other malonyl-CoA-derived compounds.
Bacteriolytic enzymes (cell lytic enzymes) are promising alternatives to antibiotics especially in killing drug‐resistant bacteria. However, some bacteria slowly become resistant to various classes ...of peptidoglycan hydrolases, for reasons not well studied, in the presence of growth‐supporting nutrients, which are prevalent at sites of infection. Here, we show that Staphylococcus aureus, a human and animal pathogen, while susceptible to the potent staphylolytic enzyme lysostaphin (Lst) in buffered saline, is highly resistant in the rich medium tryptic soy broth (TSB). Through a series of biochemical analysis, we identified that the resistance was due to prevention of Lst‐cell binding mediated by the wall teichoic acids (WTAs) present on the cell surface. Inhibition or deletion of the gene tarO responsible for the first step of WTA biosynthesis greatly reduced S. aureus resistance to Lst in TSB. To overcome the resistance, we took advantage of the gene regulation potential of CRISPR‐dCas9 and demonstrated that downregulation of tarO, tarH, and/or tarG gene expression, the latter two encoding enzymes that anchor WTAs in the outer layer of cell wall peptidoglycan, sensitized S. aureus to Lst and enabled eradication of the bacterium in TSB in 24 hr. As a result, we elucidate a key mechanism of Lst resistance in metabolically active S. aureus and provide a potential approach for treating life‐threatening or hard‐to‐treat infections caused by Gram‐positive pathogens.
Lysostaphin (Lst) is a potent bacteriolytic enzyme against the pathogenic bacterium Staphylococcus aureus. However, in nutrient‐rich environment, the bacterial cell surface glycopolymer wall teichoic acid (WTA) blocks the binding of Lst to S. aureus and helps the cells to resist the action of Lst. By using CRISPR‐dCas9 targeting key steps in WTA biosynthesis, Lst can interact with and degrade S. aureus cell wall peptidoglycan, thus causing cell lysis and death.
L-Serine has wide and increasing applications in industries with fast-growing market demand. Although strategies for achieving and improving L-serine production in Corynebacterium glutamicum (C. ...glutamicum) have focused on inhibiting its degradation and enhancing its biosynthetic pathway, L-serine yield has remained relatively low. Exporters play an essential role in the fermentative production of amino acids. To achieve higher L-serine yield, L-serine export from the cell should be improved. In C. glutamicum, ThrE, which can export L-threonine and L-serine, is the only identified L-serine exporter so far.
In this study, a novel L-serine exporter NCgl0580 was identified and characterized in C. glutamicum ΔSSAAI (SSAAI), and named as SerE (encoded by serE). Deletion of serE in SSAAI led to a 56.5% decrease in L-serine titer, whereas overexpression of serE compensated for the lack of serE with respect to L-serine titer. A fusion protein with SerE and enhanced green fluorescent protein (EGFP) was constructed to confirm that SerE localized at the plasma membrane. The function of SerE was studied by peptide feeding approaches, and the results showed that SerE is a novel exporter for L-serine and L-threonine in C. glutamicum. Subsequently, the interaction of a known L-serine exporter ThrE and SerE was studied, and the results suggested that SerE is more important than ThrE in L-serine export in SSAAI. In addition, probe plasmid and electrophoretic mobility shift assays (EMSA) revealed NCgl0581 as the transcriptional regulator of SerE. Comparative transcriptomics between SSAAI and the NCgl0581 deletion strain showed that NCgl0581 is a positive regulator of NCgl0580. Finally, by overexpressing the novel exporter SerE, combined with L-serine synthetic pathway key enzyme serAΔ197, serC, and serB, the resulting strain presented an L-serine titer of 43.9 g/L with a yield of 0.44 g/g sucrose, which is the highest L-serine titer and yield reported so far in C. glutamicum.
This study provides a novel target for L-serine and L-threonine export engineering as well as a new global transcriptional regulator NCgl0581 in C. glutamicum.
Plants are the source of various natural compounds with pharmaceutical and nutraceutical importance which have shown numerous health benefits with relatively fewer side effects. However, extraction ...of these compounds from native producers cannot meet the ever-increasing demands of the growing population due to, among other things, the limited production of the active compound(s). Their production depends upon the metabolic demands of the plant and is also subjected to environmental conditions, abundance of crop species and seasonal variations. Moreover, their extraction from plants requires complex downstream processing and can also lead to the extinction of many useful plant varieties. Microbial engineering is one of the alternative approaches which can meet the global demand for natural products in an eco-friendly manner. Metabolic engineering of microbes or pathway reconstruction using synthetic biology tools and novel enzymes lead to the generation of a diversity of compounds (like flavonoids, stilbenes, anthocyanins etc.) and their natural and non-natural derivatives. Strain and pathway optimization, pathway regulation and tolerance engineering have produced microbial cell factories into which the metabolic pathway of plants can be introduced for the production of compounds of interest on an industrial scale in an economical and eco-friendly way. While microbial production of phytochemicals needs to further increase product titer if it is ever to become a commercial success. The present review covers the advancements made for the improvement of microbial cell factories in order to increase the product titer of recombinant polyphenolic compounds.
Plant natural products (NPs) not only serve many functions in an organism's survivability but also demonstrate important pharmacological activities. Isolation of NPs from native sources is frequently ...limited by low abundance and environmental, seasonal, and regional variation while total chemical synthesis of what are often complex structures is typically commercially infeasible. Reconstruction of biosynthetic pathways in heterologous microorganisms offers significant promise for a scalable means to provide sufficient quantities of a desired NP while using inexpensive renewable resources. To this end, metabolic engineering provides the technological platform for enhancing NP production in these engineered heterologous hosts. Recent advancements in the production of isoprenoids, phenylpropanoids, and alkaloids were made possible by utilizing a variety of techniques including combinatorial biosynthesis, codon optimization, expression of regulatory elements, and protein engineering of P450s.
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