Search

Abstract Background Overproduction and activation of osteoclasts result in various bone diseases, such as osteoporosis, Paget's disease, and rheumatoid arthritis. Thus, inhibiting osteoclast formation and overactivation may effectively prevent osteoclast‐related bone diseases, especially osteoporosis. Madecassic acid, one of the most important active ingredients in Centella asiatica, has various biological effects, but its role in osteoclastogenesis remains unknown. Methods RAW 264.7 cells were stimulated with receptor activator of nuclear factor (NF)‐κΒ ligand (RANKL, 25 ng/mL) to differentiate into multi‐nucleated osteoclasts. Subsequently, osteoclasts were treated with or without varying concentrations of madecassic acid (1, 2.5, 5, and 10 μmol/L). Results Madecassic acid significantly inhibited RANKL‐induced osteoclastogenesis in a concentration‐dependent manner. In addition, it reduced the percentage of bone resorptive area compared with the control, confirming that madecassic acid can inhibit osteoclast function. Furthermore, luciferase reporter gene studies indicate that madecassic acid could decrease the transcriptional activity of NF of activated T cells (NFAT) and NF‐κB in a dose‐dependent manner. Quantitative real‐time polymerase chain reaction results show that madecassic acid attenuated the expression of osteoclast‐associated genes, including V‐ATPase‐d2, cathepsin K, tartrate‐resistant acid phosphatase (TRAP), NFAT cytoplasmic 1 (NFATc1). Western blot analysis shows that madecassic acid inhibited RANKL‐mediated degradation of IκBα and NFATc1 expression, as well as phosphorylation of c‐Jun N‐terminal kinase (JNK) in RAW 264.7 cells. Conclusion Madecassic acid inhibited osteoclast formation and function in vitro by suppressing NF‐κB, JNK, and NFAT signaling pathways, indicating its potential as a novel drug for the treatment of osteoclast‐related bone diseases, especially osteoporosis.

Abstract Flexible conducting films in the forms of bendability or stretchability are developed as a key component to enable soft electronics. With the requirements of miniaturization and portability of modern electronics, conducting film that can endure in‐plane shrinkage is urgently needed but still remains challenging. Here, a new type of conducting film achieving reversible in‐plane folding–unfolding function with large deformation by infusing conductive liquids into hierarchically structured polymer films consisting of both nanostructured polymer nanofibers and microstructured frames is reported. Nanostructured polymer nanofibers that can be completely wetted by the conductive liquids provide capillary forces to gain reversible in‐plane folding–unfolding property, while the microstructured frames greatly enhance the extent during folding–unfolding process. Conductivity of the produced films can be significantly improved by rationally tuning the composition of infused conductive liquids, which always keeps high values during the folding–unfolding deformation. It is believed that this work may serve as the basis for robust fabrication of flexible conducting films with reversible in‐plane folding–unfolding function, and can also put one‐step forward of modern soft electronics.

Abstract Background Synthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. However, most of synthetic biology tools have been developed for Escherichia coli. Here we provide a platform for rapid engineering of C. glutamicum, a microorganism of great industrial interest. This bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of several economically important compounds including metabolites and recombinant proteins because of its higher capacity of secretion compared to traditional bacterial hosts like E. coli. Thus, the development of modern molecular platforms may significantly contribute to establish C. glutamicum as a robust and versatile microbial factory. Results A plasmid based platform named pTGR was created where all the genetic components are flanked by unique restriction sites to both facilitate the evaluation of regulatory sequences and the assembly of constructs for the expression of multiple genes. The approach was validated by using reporter genes to test promoters, ribosome binding sites, and for the assembly of dual gene operons and gene clusters containing two transcriptional units. Combinatorial assembly of promoter (tac, cspB and sod) and RBS (lacZ, cspB and sod) elements with different strengths conferred clear differential gene expression of two reporter genes, eGFP and mCherry, thus allowing transcriptional “fine-tuning”of multiple genes. In addition, the platform allowed the rapid assembly of operons and genes clusters for co-expression of heterologous genes, a feature that may assist metabolic pathway engineering. Conclusions We anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum. The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.

Hyaluronic acid (HA) is a high value glycosaminoglycan mostly used in health and cosmetic applications. Commercial HA is produced from animal tissues or in toxigenic bacteria of the genus Streptococcus grown in complex media, which are expensive and raise environmental concerns due to the disposal of large amounts of broth with high organic loads. Other microorganisms were proposed as hosts for the heterologous production of HA, but the methods are still costly. The extraordinary capacity of this biopolymer to bind and retain water attracts interest for large-scale applications where biodegradable materials are needed, but its high cost and safety concerns are barriers for its adoption. Bacillus subtilis 3NA strain is prototrophic, amenable for genetic manipulation, GRAS, and can rapidly reach high cell densities in salt-based media. These phenotypic traits were exploited to create a platform for biomolecule production using HA as a proof of concept. First, the 3NA strain was engineered to produce HA; second, a chemically defined medium was formulated using commodity-priced inorganic salts combined at the stoichiometric ratios needed to build the necessary quantities of biomass and HA; and third, a scalable fermentation process, where HA can be produced at the maximum volumetric productivity (VP), was designed. A comparative economic analysis against other methods indicates that the new process may increase the operating profit of a manufacturing plant by more than 100%. The host, the culture medium, and the rationale employed to develop the fermentation process described here, introduce an IP-free platform that could be adaptable for production of other biomolecules. KEY POINTS: • A biomolecule production platform based on B. subtilis 3NA strain and a synthetic medium was tested for hyaluronic acid biosynthesis • A fermentation process with the maximum volumetric productivity was designed • A techno-economic analysis forecasts a significant reduction in the manufacturing cost compared to the current methods.

The growing demand for food and biofuels urges the vegetable oil processing industry to adopt cleaner technologies to mitigate the environmental pollution caused by chemical refining processes. Over the past decade, several enzymatic methods have proven to be efficient at reducing the generated waste, but improving the benefit-cost ratio is still necessary for the widespread adoption of this technology. In this work, we show that lecithin:cholesterol acyltransferase from Aeromonas enteropelogenes (LCAT AE ) provides a higher extra-yield of soybean oil than a type A1 phospholipase (PLA) enzyme currently commercialized for soybean oil deep degumming. Our model indicates that crude soybean oil treated with the new enzyme generates 87% more neutral oil from phospholipids than the widely used PLA, with the corresponding reduction in waste and byproducts generation. The refined oil retains the phytosterols naturally present in crude oil, enriching its nutritional value. The results presented here position LCAT AE as a promising candidate to provide the green solutions needed by the industrial oil processing sector. Key points • Selected LCAT gene candidates were expressed in E. coli. • Aeromonas enteropelogenes LCAT hydrolyzes all the phospholipids present in crude soybean oil. • The LCAT enzyme provides a higher yield of neutral oil than commercial PLA enzymes and generates less waste. • The degummed oil retains sterols with high nutritional value.

Phospholipids play a central role in all living organisms. Phospholipases, the enzymes aimed at modifying phospholipids, are consequently widespread in nature and play diverse roles, from lipid metabolism and cellular signaling in eukaryotes to virulence and nutrient acquisition in microbes. Phospholipases catalyze the hydrolysis of one or more ester or phosphodiester bonds of glycerophospholipids. The use of phospholipases with industrial purposes has constantly increased over the last 30 years. This demand is rapidly growing given the ongoing improvements in protein engineering and the reduction of enzymes manufacturing costs, making them suitable for industrial use. Here, a general overview of phopholipases A, B, C, and D and their industrial application is presented along with potential new uses for these enzymes. We draw attention to commercial phospholipases used to improve the emulsifying properties of products in the baking, egg, and dairy industries. On the other hand, the improvement of oil degumming by phospholipases is thoroughly analyzed. Moreover, recent developments in enzymatic biodiesel production and the use of phospholipases for the synthesis of phospholipids with pharmaceutical or nutritional value are reviewed.

βγ-crystallin has emerged as a superfamily of structurally homologous proteins with representatives across all domains of life. A major portion of this superfamily is constituted by microbial members. This superfamily has also been recognized as a novel group of Ca 2+ -binding proteins with a large diversity and variable properties in Ca 2+ binding and stability. We have recently described a new phosphatidylinositol phospholipase C from Lysinibacillus sphaericus (LS-PIPLC) which was shown to efficiently remove phosphatidylinositol from crude vegetable oil. Here, the role of the C-terminal βγ-crystallin domain of LS-PIPLC was analyzed in the context of the whole protein. A truncated protein in which the C-terminal βγ-crystallin domain was deleted (LS-PIPLC ΔCRY ) is catalytically as efficient as the full-length protein (LS-PIPLC). However, the thermal and chemical stability of LS-PIPLC ΔCRY are highly affected, demonstrating a stabilizing role for this domain. It is also shown that the presence of Ca 2+ increases the thermal and chemical stability of the protein both in aqueous media and in oil, making LS-PIPLC an excellent candidate for use in industrial soybean oil degumming.

Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent "silent" nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.

We have synthesized new derivatives of the macrolide antibiotics erythromycin and azithromycin. Novel deoxysugar moieties were attached to these standard antibiotics by biotransformation using a heterologous host. The resulting compounds were tested against several standard laboratory and clinically isolated bacterial strains. In addition, they were also tested in vitro against standard and drug-resistant strains of human malaria parasites (Plasmodium falciparum) and the liver stages of the rodent malaria parasite (Plasmodium berghei). Antibacterial activity of modified erythromycin and azithromycin showed no improvement over the unmodified macrolides, but the modified compounds showed a 10-fold increase in effectiveness after a short-term exposure against blood stages of malaria. The new compounds also remained active against azithromycin-resistant strains of P. falciparum and inhibited growth of liver-stage parasites at concentrations similar to those used for primaquine. Our findings show that malaria parasites have two distinct responses to macrolide antibiotics, one reflecting the prokaryotic origin of the apicoplast and a second, as-yet uncharacterized response that we attribute to the eukaryotic nature of the parasite. This is the first report for macrolides that target two different functions in the Plasmodium parasites.

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V(max) in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V(max) of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.

In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.

Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6-deoxyerythronolide B (6-dEB) or 3-α-mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60- and 25-fold more erythromycin D (EryD) than the original strain K207-3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars., (© 2008 The Authors. Journal compilation © 2008 Society for Applied Microbiology and Blackwell Publishing Ltd.)

In vivo reconstitution of the dTDP-D-desosamine pathway of the megalomicin gene cluster from Micromonospora megalomicea was achieved by expression of the genes in Escherichia coli. LC/MS/MS analysis of the dTDP-sugar intermediates produced by operons containing different sets of genes showed that production of dTDP-D-desosamine from dtdp-4-keto-6-deoxy-D-glucose requires only four biosynthetic steps, catalysed by MegCIV, MegCV, MegDII and MegDIII, and that MegCII is not involved. Instead, bioconversion studies demonstrated that MegCII is needed together with MegCIII to catalyse transfer of D-desosamine to 3-alpha-mycarosylerythronolide B.

An Escherichia coli strain capable of producing the potent antibiotic erythromycin C (Ery C) was developed by expressing 17 new heterologous genes in a 6-deoxyerythronolide B (6dEB) producer strain. The megalomicin gene cluster was used as the source for the construction of two artificial operons that contained the genes encoding the deoxysugar biosynthetic and tailoring enzymes necessary to convert 6dEB to Ery C. The reconstructed mycarose operon contained the seven genes coding for the enzymes that convert glucose-1-phosphate (G-1-P) to TDP-L-mycarose, a 6dEB mycarosyl transferase, and a 6dEB 6-hydroxylase. The activity of the pathway was confirmed by demonstrating conversion of exogenous 6dEB to 3-O-alpha-mycarosylerythronolide B (MEB). The reconstructed desosamine operon contained the six genes necessary to convert TDP-4-keto-6-deoxyglucose, an intermediate formed in the mycarose pathway, to TDP-D-desosamine, a desosamine transferase, a 6dEB 12-hydroxylase, and the rRNA methyltransferase ErmE; the last was required to confer resistance to the host cell upon production of mature macrolide antibiotics. The activity of this pathway was demonstrated by conversion of MEB to Ery C. When the mycarose and desosamine operons were expressed in an E. coli strain engineered to synthesize 6dEB, Ery C and Ery D were produced. The successful production of Ery C in E. coli shows the potentiality of this model microorganism to synthesize novel 6-deoxysugars and to produce bioactive glycosylated compounds and also establishes the basis for the future use of E. coli both in the production of new glycosylated polyketides and for the generation of novel bioactive compounds through combinatorial biosynthesis.

Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.

The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms. CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the Km value for oxaloacetate was 0.52+/-0.03 mM. However, no malic activity was found for this enzyme. Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram-positive bacteria. Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.

Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.