Biosynthesis of C4-C8 3-Hydroxycarboxylic Acids from Glucose through the Inverted Fatty Acid ß-Oxidation by Metabolically Engineered Escherichia coli.

Inverted fatty acid ß-oxidation represents a versatile biochemical platform for biosynthesis by the engineered microbial strains of numerous value-added chemicals from convenient and abundant renewable carbon sources, including biomass-derived sugars. Although, in recent years, significant progress has been made in the production through this pathway of n-alcohols, 1,3-diols, and carboxylic acids and its 2,3-unsaturated derivatives, the potential of the pathway for the biosynthesis of 3-hydroxycarboxylic acids remained almost undisclosed. In this study, we demonstrate the microaerobic production of even-chain-length C4-C8 3-hydroxycarboxylic acids from glucose through the inverted fatty acid ß-oxidation by engineered E. coli strains. The notable accumulation of target compounds was achieved upon the strong constitutive expression of the genes atoB, fadA, fadB, fadE/fabI, and tesB, which code for the key enzymes catalysing reactions of aerobic fatty acid ß-oxidation and thioesterase II, in strains devoid of mixed-acid fermentation pathways and lacking nonspecific thioesterase YciA. The best performing recombinants were able to synthesise up to 14.5 mM of 3-hydroxycarboxylic acids from glucose with a total yield of 0.34 mol/mol and a C4/C6/C8 ratio averaging approximately 63/28/9. The results provide a framework for the development of highly efficient strains and processes for the bio-based production of valuable 3-hydroxycarboxylates from renewable raw materials.

Composite Coatings Based on Recombinant Spidroins and Peptides with Motifs of the Extracellular Matrix Proteins Enhance Neuronal Differentiation of Neural Precursor Cells Derived from Human Induced Pluripotent Stem Cells.

The production and transplantation of functionally active human neurons is a promising approach to cell therapy. Biocompatible and biodegradable matrices that effectively promote the growth and directed differentiation of neural precursor cells (NPCs) into the desired neuronal types are very important. The aim of this study was to evaluate the suitability of novel composite coatings (CCs) containing recombinant spidroins (RSs) rS1/9 and rS2/12 in combination with recombinant fused proteins (FP) carrying bioactive motifs (BAP) of the extracellular matrix (ECM) proteins for the growth of NPCs derived from human induced pluripotent stem cells (iPSC) and their differentiation into neurons. NPCs were produced by the directed differentiation of human iPSCs. The growth and differentiation of NPCs cultured on different CC variants were compared with a Matrigel (MG) coating using qPCR analysis, immunocytochemical staining, and ELISA. An investigation revealed that the use of CCs consisting of a mixture of two RSs and FPs with different peptide motifs of ECMs increased the efficiency of obtaining neurons differentiated from iPSCs compared to Matrigel. CC consisting of two RSs and FPs with Arg-Gly-Asp-Ser (RGDS) and heparin binding peptide (HBP) is the most effective for the support of NPCs and their neuronal differentiation.

Nonwoven spidroin materials as scaffolds for ex vivo cultivation of aortic fragments and dorsal root ganglia.

Recombinant spidroins (RS; the analogues of silk proteins of spider's web) have multiple properties beneficial for bioengineering, including their suitability for electrospinning and thus, for production of materials with oriented fibers. This makes RS-based matrices potentially effective in stimulating regeneration of peripheral nerves. The restoration of injured nerves also depends on prompt regrowth of blood vessels. Therefore, prospective scaffold materials for neuro-regenerative therapy should positively affect both the nerves and the blood vessels. Currently, the experimental models suitable for culturing and quantitative assessment of the vascular and neuronal cells on the same material are lacking. Here, we assessed the suitability of electrospun RS-based matrices for cultivation of the mouse aorta and dorsal root ganglia (DRG) explants. We also quantified the effects of matrix topography upon both types of tissues. The RS-based materials have effectively supported aortic explants survival and sprouting. The cumulative length of endothelial sprouts on rS1/9-coated inserts was significantly higher as compared to type I collagen coatings, suggesting stimulatory effects on angiogenesis in vitro. In contrast to matrices with random fibers, on matrices with parallel fibers the migration of both smooth muscle and endothelial cells was highly oriented. Furthermore, alignment of RS fibers effectively directs the growth of axons and the migration of Schwann cells from DRGs. Thus, the electrospun RS matrices are highly suitable to culture both, the DRGs and aortic explants and to study the effects of matrix topography on cell migration. This model has a high potential for further endeavor into interactions of nerve and vascular cells and tissues.

Engineering Escherichia coli for efficient aerobic conversion of glucose to fumaric acid.

Escherichia coli was engineered for efficient aerobic conversion of glucose to fumaric acid. A novel design for biosynthesis of the target product through the modified TCA cycle rather than via glyoxylate shunt, implying oxaloacetate formation from pyruvate and artificial channelling of 2-ketoglutarate towards succinic acid via succinate semialdehyde formation, was implemented. The main fumarases were inactivated in the core strain MSG1.0 (∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PL-glk, Ptac-galP) by the deletion of the fumA, fumB, and fumC genes. The Bacillus subtilis pycA gene was expressed in the strain to ensure pyruvate to oxaloacetate conversion. The Mycobacterium tuberculosis kgd gene was expressed to enable succinate semialdehyde formation. The resulting strain was able to convert glucose to fumaric acid with a yield of 0.86 mol/mol, amounting to 86% of the theoretical maximum. The results demonstrated the high potential of the implemented strategy for development of efficient strains for bio-based fumaric acid production.

Effects of Recombinant Spidroin rS1/9 on Brain Neural Progenitors After Photothrombosis-Induced Ischemia.

The existence of niches of stem cells residence in the ventricular-subventricular zone and the subgranular zone in the adult brain is well-known. These zones are the sites of restoration of brain function after injury. Bioengineered scaffolds introduced in the damaged loci were shown to support neurogenesis to the injury area, thus representing a strategy to treat acute neurodegeneration. In this study, we explored the neuroprotective activity of the recombinant analog of Nephila clavipes spidroin 1 rS1/9 after its introduction into the ischemia-damaged brain. We used nestin-green fluorescent protein (GFP) transgenic reporter mouse line, in which neural stem/progenitor cells are easily visualized and quantified by the expression of GFP, to determine the alterations in the dentate gyrus (DG) after focal ischemia in the prefrontal cortex. Changes in the proliferation of neural stem/progenitor cells during the first weeks following photothrombosis-induced brain ischemia and in vitro effects of spidroin rS1/9 in rat primary neuronal cultures were the subject of the study. The introduction of microparticles of the recombinant protein rS1/9 into the area of ischemic damage to the prefrontal cortex leads to a higher proliferation rate and increased survival of progenitor cells in the DG of the hippocampus which functions as a niche of brain stem cells located at a distance from the injury zone. rS1/9 also increased the levels of a mitochondrial probe in DG cells, which may report on either an increased number of mitochondria and/or of the mitochondrial membrane potential in progenitor cells. Apparently, the stimulation of progenitor cells was caused by formed biologically active products stemming from rS1/9 biodegradation which can also have an effect upon the growth of primary cortical neurons, their adhesion, neurite growth, and the formation of a neuronal network. The high biological activity of rS1/9 suggests it as an excellent material for therapeutic usage aimed at enhancing brain plasticity by interacting with stem cell niches. Substances formed from rS1/9 can also be used to enhance primary neuroprotection resulting in reduced cell death in the injury area.

Akt and Src mediate the photocrosslinked fibroin-induced neural differentiation.

Neural transplantation is a promising modality for treatment of neurodegenerative diseases, traumatic brain injury and stroke. Biocompatible scaffolds with optimized properties improve the survival of transplanted neural cells and differentiation of progenitor cells into the desired types of neurons. Silk fibroin is a biocompatible material for tissue engineering. Here, we describe thin-film scaffolds based on photocrosslinked methacrylated silk fibroin (FBMA). These scaffolds exhibit an increased mechanical stiffness and improved water stability. Photocrosslinking of fibroin increased its rigidity from 25 to 480 kPa and the contact angle from 59.7 to 70.8, the properties important for differentiation of neural cells. Differentiation of SH-SY5Y neuroblastoma cells on FBMA increased the length of neurites as well as the levels of neural differentiation markers MAP2 and ßIII-tubulin. Growth of SH-SY5Y cells on the unmodified fibroin and FBMA substrates led to a spontaneous phosphorylation of Src and Akt protein kinases critical for neuronal differentiation; this effect was paralleled by neural cell adhesion molecule elevation. Thus, FBMA is an easily manufactured, cytocompatible material with improved and sustainable properties applicable for neural tissue engineering.

Recombinant Spidroins as the Basis for New Materials.

Spider web proteins are unique materials created by nature that, considering the combination of their properties, do not have analogues among natural or human-created materials. Obtaining significant amounts of these proteins from natural sources is not feasible. Biotechnological manufacturing in heterological systems is complicated by the very high molecular weight of spidroins and their specific amino acid composition. Obtaining recombinant analogues of spidroins in heterological systems, mainly in bacteria and yeast, has become a compromise solution. Because they can self-assemble, these proteins can form various materials, such as fibers, films, 3D-foams, hydrogels, tubes, and microcapsules. The effectiveness of spidroin hydrogels in deep wound healing, as 3D scaffolds for bone tissue regeneration and as oriented fibers for axon growth and nerve tissue regeneration, was demonstrated in animal models. The possibility to use spidroin micro- and nanoparticles for drug delivery was demonstrated, including the use of modified spidroins for virus-free DNA delivery into animal cell nuclei. In the past few years, significant interest has arisen concerning the use of these materials as biocompatible and biodegradable soft optics to construct photonic crystal super lenses and fiber optics and as soft electronics to use in triboelectric nanogenerators. This review summarizes the latest achievements in the field of spidroin production, the creation of materials based on them, the study of these materials as a scaffold for the growth, proliferation, and differentiation of various types of cells, and the prospects for using these materials for medical applications (e.g., tissue engineering, drug delivery, coating medical devices), soft optics, and electronics. Accumulated data suggest the use of recombinant spidroins in medical practice in the near future.

Engineering Escherichia coli for respiro-fermentative production of pyruvate from glucose under anoxic conditions.

An Escherichia coli K-12 MG1655-derived strain was engineered for respiro-fermentative production of pyruvate from glucose under anoxic conditions, which is preferred for industrial-scale microbial synthesis of valuable chemicals. The pathways of anaerobic pyruvate dissimilation were blocked in the strain by the deletion of the ackA, pta, poxB, ldhA, adhE, and pflB genes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was substituted by an alternative ATP-dependent system resulting from the overexpression of galP and glk upon deletion of ptsG. The channelling of pyruvate towards the oxidative branch of the TCA cycle under respiratory conditions was prevented in the strain due to the deletion of aceEF genes, encoding components of pyruvate dehydrogenase, while the operation of the entire reductive branch of the TCA cycle was interrupted by knocking out frdAB and sdhAB. Reoxidation of glycolytic NADH was ensured via anaerobic respiration with nitrate serving as an external electron acceptor. To enforce anaerobic ATP hydrolysis, an ATP-consuming futile cycle of pyruvate-oxaloacetate-malate-pyruvate was established in the strain by expressing the Bacillus subtilis pycA gene, encoding pyruvate carboxylase. In the presence of sufficient amounts of an external electron acceptor and CO2 source, the engineered strain was able to efficiently utilise glucose and convert it to pyruvate anaerobically with a yield of 1.73 mol/mol, amounting to 87% of the theoretical maximum. The implemented strategy offers the potential for the development of highly efficient processes of bio-based pyruvate production.

Novel Biodegradable Polymeric Microparticles Facilitate Scarless Wound Healing by Promoting Re-epithelialization and Inhibiting Fibrosis.

Despite decades of research, the goal of achieving scarless wound healing remains elusive. One of the approaches, treatment with polymeric microcarriers, was shown to promote tissue regeneration in various in vitro models of wound healing. The in vivo effects of such an approach are attributed to transferred cells with polymeric microparticles functioning merely as inert scaffolds. We aimed to establish a bioactive biopolymer carrier that would promote would healing and inhibit scar formation in the murine model of deep skin wounds. Here we characterize two candidate types of microparticles based on fibroin/gelatin or spidroin and show that both types increase re-epithelialization rate and inhibit scar formation during skin wound healing. Interestingly, the effects of these microparticles on inflammatory gene expression and cytokine production by macrophages, fibroblasts, and keratinocytes are distinct. Both types of microparticles, as well as their soluble derivatives, fibroin and spidroin, significantly reduced the expression of profibrotic factors Fgf2 and Ctgf in mouse embryonic fibroblasts. However, only fibroin/gelatin microparticles induced transient inflammatory gene expression and cytokine production leading to an influx of inflammatory Ly6C+ myeloid cells to the injection site. The ability of microparticle carriers of equal proregenerative potential to induce inflammatory response may allow their subsequent adaptation to treatment of wounds with different bioburden and fibrotic content.

Biosynthesis of enantiopure (S)-3-hydroxybutyrate from glucose through the inverted fatty acid ß-oxidation pathway by metabolically engineered Escherichia coli.

Enantiomers of 3-hydroxybutyric acid (3-HB) can be used as the chiral precursors for the production of various optically active fine chemicals, including drugs, perfumes, and pheromones. In this study, Escherichia coli was engineered to produce (S)-3-HB from glucose through the inverted reactions of the native aerobic fatty acid ß-oxidation pathway. Expression of only specific genes encoding enzymes responsible for the conversion of acetyl-CoA to acetoacetyl-CoA, reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA and subsequent hydrolysis of 3-hydroxybutyryl-CoA to 3-HB was directly upregulated in an engineered strain. The operation of multiple turns of the inverted fatty acid ß-oxidation was precluded by the deletion of gene encoding enzyme that catalyse the terminal stage of the respective cycle. While the overexpression of the C-acetyltransferase gene enabled 3-HB biosynthesis through the inverted fatty acid ß-oxidation, the efficient conversion of glucose to the target product was achieved resulting from the additional overexpression of the gene encoding appropriate termination thioesterase II. The engineered strain synthesised the (S)-stereoisomer of 3-HB with an enantiomeric excess of more than 99%. Under microaerobic conditions, up to 9.58g/L of enantiopure (S)-3-HB was produced from glucose, with a yield of 66% of the theoretical maximum.

Manipulating pyruvate to acetyl-CoA conversion in Escherichia coli for anaerobic succinate biosynthesis from glucose with the yield close to the stoichiometric maximum.

Efficient succinate production in Escherichia coli is attained during anaerobic glucose fermentation in biosynthetic processes combining the reductive branch of the TCA cycle and the glyoxylate bypass. Pyruvate dehydrogenase (PDH) or pyruvate formate lyase (PFL) serves in E. coli as a source of acetyl-CoA, a substrate for the glyoxylate bypass. Depending on enzymes responsible for acetyl-CoA generation, the contribution of the glyoxylate bypass to the anaerobic succinate biosynthesis may vary to support redox balance resulting in diverse maximum achievable yield values. Anaerobic succinate biosynthesis from glucose was studied using E. coli strains with altered expression of genes encoding PFL and PDH. For acetyl-CoA formation by PFL, the yield of 1.32 mol succinate per mole of glucose was achieved with the theoretical value of 1.6 mol/mol. Involvement of PDH in anaerobic acetyl-CoA synthesis increased succinate yield up to 1.49 mol/mol, which is 89.8% of the predicted maximum (1.6(6) mol/mol). The maximum yield of 1.69 mol succinate per mol glucose, amounting to 98.8% of the stoichiometric maximum (1.71 mol/mol), was achieved with the strain possessing PDH as the primary anaerobic source of acetyl-CoA. During high cell density fermentation, the best engineered strain produced high amounts of succinate (570.7 mM) and only small quantities of acetate (11.9 mM).

Comparison of different approaches to activate the glyoxylate bypass in Escherichia coli K-12 for succinate biosynthesis during dual-phase fermentation in minimal glucose media.

Two different approaches to activate the glyoxylate bypass in model Escherichia coli K-12 strains for succinate biosynthesis during dual-phase fermentation in minimal glucose media were examined. Inactivation of IclR and FadR, the transcriptional regulators of the aceBAK operon, were insufficient for the involvement of the glyoxylate bypass in anaerobic succinate biosynthesis by strains grown aerobically under glucose-abundant conditions. In contrast, the strains that constitutively expressed the aceEF-lpdA operon coding for the pyruvate dehydrogenase complex could partially synthesise succinate anaerobically via the glyoxylate bypass, even in the presence of intact regulators. The results suggest that the intensive acetyl-CoA formation in the strains constitutively expressing pyruvate dehydrogenase matches the physiological conditions that favour the activation of the glyoxylate bypass.

Tissue regeneration in vivo within recombinant spidroin 1 scaffolds.

One of the major tasks of tissue engineering is to produce tissue grafts for the replacement or regeneration of damaged tissue, and natural and recombinant silk-based polymer scaffolds are promising candidates for such grafts. Here, we compared two porous scaffolds made from different silk proteins, fibroin of Bombyx mori and a recombinant analog of Nephila clavipes spidroin 1 known as rS1/9, and their biocompatibility and degradation behavior in vitro and in vivo. The vascularization and intergrowth of the connective tissue, which was penetrated with nerve fibers, at 8 weeks after subcutaneous implantation in Balb/c mice was more profound using the rS1/9 scaffolds. Implantation of both scaffolds into bone defects in Wistar rats accelerated repair compared to controls with no implanted scaffold at 4 weeks. Based on the number of macrophages and multinuclear giant cells in the subcutaneous area and the number of osteoclasts in the bone, regeneration was determined to be more effective after the rS1/9 scaffolds were implanted. Microscopic examination of the morphology of the matrices revealed differences in their internal microstructures. In contrast to fibroin-based scaffolds, the walls of the rS1/9 scaffolds were visibly thicker and contained specific micropores. We suggest that the porous inner structure of the rS1/9 scaffolds provided a better micro-environment for the regenerating tissue, which makes the matrices derived from the recombinant rS1/9 protein favorable candidates for future in vivo applications.

Metabolic engineering of Escherichia coli for 1-butanol biosynthesis through the inverted aerobic fatty acid ß-oxidation pathway.

The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid ß-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid ß-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid ß-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI(Q) attB-P(trc-ideal-4)-SD(φ10)-adhE(Glu568Lys) attB-P(trc-ideal-4)-SD(φ10)-atoB attB-P(trc-ideal-4)-SD(φ10)-fadB attB-P(trc-ideal-4)-SD(φ10)-fadE) synthesises 0.3-1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer.

A novel model system for design of biomaterials based on recombinant analogs of spider silk proteins.

Spider dragline silk possesses impressive mechanical and biochemical properties. It is synthesized by a couple of major ampullate glands in spiders and comprises of two major structural proteins--spidroins 1 and 2. The relationship between structure and mechanical properties of spider silk is not well understood. Here, we modeled the complete process of the spider silk assembly using two new recombinant analogs of spidroins 1 and 2. The artificial genes sequence of the hydrophobic core regions of spidroin 1 and 2 have been designed using computer analysis of existing databases and mathematical modeling. Both proteins were expressed in Pichia pastoris and purified using a cation exchange chromatography. Despite the absence of hydrophilic N- and C-termini, both purified proteins spontaneously formed the nanofibrils and round micelles of about 1 microm in aqueous solutions. The electron microscopy study has revealed the helical structure of a nanofibril with a repeating motif of 40 nm. Using the electrospinning, the thin films with an antiparallel beta-sheet structure were produced. In summary, we were able to obtain artificial structures with characteristics that are perspective for further biomedical applications, such as producing three-dimensional matrices for tissue engineering and drug delivery.

The threonine story.

L-Threonine is an essential amino acid which has recently been brought into agricultural industry for balancing the livestock feed. L-Threonine is produced by microbial synthesis using glucose or sucrose as substrates. For the process to be cost-effective, the microbial strain must be capable of threonine overproduction. This paper reviews the biochemical pathways of L-threonine synthesis in bacteria and the regulation of these pathways, the principles and the techniques of constructing high-producing strains, and the most efficient strains thus developed.