High-level expression of anti FLAG tag antibody in plants / 生物工程学报

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.

Optimized biotransformation of acid-treated water melon peel hydrolyzate into ethanol / Biotransformação otimizada de hidrolisado de casca de melancia tratada com ácido em etanol

Today, global focus of research is to explore the solution of energy crisis and environmental pollution. Like other agricultural countries, bulk quantities of watermelon peels (WMP) are disposed-off in environment as waste in Pakistan and appropriate management of this waste is the need of hour to save environment from pollution. The work emphasizes the role of ethanologenic yeasts to utilize significant sugars present in WMP for low-cost bioethanol fermentation. Dilute hydrochloric acid hydrolysis of WMP was carried out on optimized conditions employing RSM (response surface methodology) following central composite design (CCD). This experimental design is based on optimization of ethanologenesis involving some key independent parameters such as WMP hydrolysate and synthetic media ratio (X1), incubation temperature (X2) and incubation temperature (X3) for maximal ethanol yield exploiting standard (Saccharomyces cerevisiae K7) as well as experimental (Metchnikowia cibodasensisY34) yeasts. The results revealed that maximal ethanol yields obtained from S. cerevisiae K7 was 0.36±0.02 g/g of reducing sugars whereas M. cibodasensisY34, yielded 0.40±0.01 g ethanol/g of reducing sugars. The yeast isolate M. cibodasensisY34 appeared as promising ethanologen and embodies prospective potential for fermentative valorization of WMP-to-bioethanol.

Hoje, o foco global da pesquisa é explorar a solução da crise energética e da poluição ambiental. Como em outros países agrícolas, grandes quantidades de cascas de melancia (WMP) são descartadas como resíduos no meio ambiente no Paquistão, mas a gestão adequada desses resíduos é a mais recente solução para salvar o meio ambiente da poluição. O trabalho enfatiza o papel das leveduras etanologênicas para utilizar açúcares significativos presentes no WMP para fermentação de bioetanol de baixo custo. A hidrólise de ácido clorídrico diluído de WMP foi realizada em condições otimizadas empregando RSM (metodologia de superfície de resposta) e seguindo o projeto de composto central (CCD). Este projeto experimental é baseado na otimização da etanologenesis envolvendo alguns parâmetros independentes importantes, como hidrolisado de WMP e razão de meio sintético (X1), temperatura de incubação (X2) e temperatura de incubação (X3) para rendimento máximo de etanol explorando o padrão (Saccharomyces cerevisiae K7) também como leveduras experimentais (Metchnikowia cibodasensis Y34). Os resultados revelaram que os rendimentos máximos de etanol obtidos a partir de S. cerevisiae K7 foi de 0,36 ± 0,02 g / g de açúcares redutores, enquanto M. cibodasensis Y34 rendeu 0,40 ± 0,01 g de etanol / g de açúcares redutores. O isolado de levedura M. cibodasensis Y34 apareceu como um etanologeno promissor e incorpora um potencial prospectivo para a valorização fermentativa de WMP em bioetanol.

Denitrifying phosphate accumulating organisms and its mechanism of nitrogen and phosphorus removal / 生物工程学报

Water eutrophication poses great threats to protection of water environment. Microbial remediation of water eutrophication has shown high efficiency, low consumption and no secondary pollution, thus becoming an important approach for ecological remediation. In recent years, researches on denitrifying phosphate accumulating organisms and their application in wastewater treatment processes have received increasing attention. Different from the traditional nitrogen and phosphorus removal process conducted by denitrifying bacteria and phosphate accumulating organisms, the denitrifying phosphate accumulating organisms can simultaneously remove nitrogen and phosphorus under alternated anaerobic and anoxic/aerobic conditions. It is worth noting that microorganisms capable of simultaneously removing nitrogen and phosphorus absolutely under aerobic conditions have been reported in recent years, but the mechanisms remain unclear. This review summarizes the species and characteristics of denitrifying phosphate accumulating organisms and the microorganisms capable of performing simultaneous nitrification-denitrification and phosphorous removal. Moreover, this review analyzes the relationship between nitrogen removal and phosphorus removal and the underlying mechanisms, discusses the challenges of denitrifying phosphorus removal, and prospects future research directions, with the aim to facilitate process improvement of denitrifying phosphate accumulating organisms.

Recent advances in the structure and function of microbial community in anaerobic granular sludge / 生物工程学报

Anaerobic granular sludge (AnGS), a self-immobilized aggregate containing various functional microorganisms, is considered as a promising green process for wastewater treatment. AnGS has the advantages of high volume loading rate, simple process and low excess sludge generation, thus shows great technological and economical potentials. This review systematically summarizes the recent advances of the microbial community structure and function of anaerobic granular sludge, and discusses the factors affecting the formation and stability of anaerobic granular sludge from the perspective of microbiology. Moreover, future research directions of AnGS are prospected. This review is expected to facilitate the research and engineering application of AnGS.

Dynamic regulation using a quorum-sensing circuit enhances the production of tyrosol by Escherichia coli / 生物工程学报

Tyrosol is a natural polyphenolic product that is widely used in chemical, pharmaceutical and food industries. Currently, the de novo synthesis of tyrosol by Escherichia coli suffers from issues such as low cell density and poor yield. Therefore, the phenylpyruvate decarboxylase mutant ARO10F138L/D218G obtained in our previous study was fused with an alcohol dehydrogenase from different microorganisms for fusion expression, and the optimal ARO10F138L/D218G-L-YahK produced 1.09 g/L tyrosol in shake flasks. In order to further improve tyrosol production, feaB, a key gene in the competing pathway of 4-hydroxyphenylacetic acid, was knocked out, and the resulted strain produced 1.26 g/L tyrosol with an increase of 21.15% compared to that of the control. To overcome the low cell density in tyrosol fermentation, the quorum-sensing circuit was used to dynamically regulate the tyrosol synthesis pathway, so as to alleviate the toxic effect of tyrosol on chassis cells and relieve the growth inhibition. Using this strategy, the yield of tyrosol was increased to 1.74 g/L, a 33.82% increase. In a 2 L fermenter, the production of tyrosol in the engineered strain TRFQ5 dynamically regulated by quorum-sensing reached 4.22 g/L with an OD600 of 42.88. Compared with those in the engineered strain TRF5 statically regulated by induced expression, the yield was increased by 38.58% and the OD600 was enhanced by 43.62%. The combination of blocking the competing pathway using gene knockout technology, and reducing the inhibitory effect of tyrosol toxicity on chassis cells through quorum-sensing dynamic regulation increased the production of tyrosol. This study may facilitate the biosynthesis of other chemicals with high toxicity.

Hyperosmotic stress and perfusion culture strategies increase the yield of recombinant adenoviral vector produced by HEK 293 cells / 生物工程学报

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×1010 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

Highly efficient production of L-valine by multiplex metabolic engineering of Corynebacterium glutamicum / 生物工程学报

As a branched chain amino acid, L-valine is widely used in the medicine and feed sectors. In this study, a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies. First, precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products. Subsequently, the key enzyme in the L-valine synthesis pathway, acetylhydroxylate synthase, was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain. Moreover, promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway. Furthermore, cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH. The engineered strain C. glutamicum K020 showed a significant increase in L-valine titer, yield and productivity in 5 L fed-batch bioreactor, up to 110 g/L, 0.51 g/g and 2.29 g/(L‧h), respectively.

Enhanced nitrogen removal by bioelectrochemical coupling anammox and characteristics of microbial communities / 生物工程学报

To investigate the bioelectrochemical enhanced anaerobic ammonia oxidation (anammox) nitrogen removal process, a bioelectrochemical system with coupled anammox cathode was constructed using a dual-chamber microbial electrolysis cell (MEC). Specifically, a dark incubation batch experiment was conducted at 30 ℃ with different influent total nitrogen concentrations under an applied voltage of 0.2 V, and the enhanced denitrification mechanism was investigated by combining various characterization methods such as cyclic voltammetry, electrochemical impedance spectroscopy and high-throughput sequencing methods. The results showed that the total nitrogen removal rates of 96.9%±0.3%, 97.3%±0.4% and 99.0%±0.3% were obtained when the initial total nitrogen concentration was 200, 300 and 400 mg/L, respectively. In addition, the cathode electrode biofilm showed good electrochemical activity. High-throughput sequencing results showed that the applied voltage enriched other denitrifying functional groups, including Denitratisoma, Limnobacter, and ammonia oxidizing bacteria SM1A02 and Anaerolineaceae, Nitrosomonas europaea and Nitrospira, besides the anammox bacteria. These electrochemically active microorganisms comprised of ammonium oxidizing exoelectrogens (AOE) and denitrifying electrotrophs (DNE). Together with anammox bacteria Candidatus Brocadia, they constituted the microbial community structure of denitrification system. Enhanced direct interspecies electron transfer between AOE and DNE was the fundamental reason for the further improvement of the total nitrogen removal rate of the system.

Metabolic engineering of Escherichia coli for adipic acid production / 生物工程学报

Adipic acid is a high-value-added dicarboxylic acid which is primarily used in the production of nylon-66 for manufacturing polyurethane foam and polyester resins. At present, the biosynthesis of adipic acid is hampered by its low production efficiency. By introducing the key enzymes of adipic acid reverse degradation pathway into a succinic acid overproducing strain Escherichia coli FMME N-2, an engineered E. coli JL00 capable of producing 0.34 g/L adipic acid was constructed. Subsequently, the expression level of the rate-limiting enzyme was optimized and the adipic acid titer in shake-flask fermentation increased to 0.87 g/L. Moreover, the supply of precursors was balanced by a combinatorial strategy consisting of deletion of sucD, over-expression of acs, and mutation of lpd, and the adipic acid titer of the resulting E. coli JL12 increased to 1.51 g/L. Finally, the fermentation process was optimized in a 5 L fermenter. After 72 h fed-batch fermentation, adipic acid titer reached 22.3 g/L with a yield of 0.25 g/g and a productivity of 0.31 g/(L·h). This work may serve as a technical reference for the biosynthesis of various dicarboxylic acids.

Modular engineering of Escherichia coli for high-level production of l-tryptophan / 生物工程学报

As an essential amino acid, l-tryptophan is widely used in food, feed and medicine sectors. Nowadays, microbial l-tryptophan production suffers from low productivity and yield. Here we construct a chassis E. coli TRP3 producing 11.80 g/L l-tryptophan, which was generated by knocking out the l-tryptophan operon repressor protein (trpR) and the l-tryptophan attenuator (trpL), and introducing the feedback-resistant mutant aroGfbr. On this basis, the l-tryptophan biosynthesis pathway was divided into three modules, including the central metabolic pathway module, the shikimic acid pathway to chorismate module and the chorismate to tryptophan module. Then we used promoter engineering approach to balance the three modules and obtained an engineered E. coli TRP9. After fed-batch cultures in a 5 L fermentor, tryptophan titer reached to 36.08 g/L, with a yield of 18.55%, which reached 81.7% of the maximum theoretical yield. The tryptophan producing strain with high yield laid a good foundation for large-scale production of tryptophan.

Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7 / 生物工程学报

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

Optimization of the ellagic acid synthesis process at the bioreactor level using non-conventional yeasts

Abstract Ellagic acid (EA) is a phenolic biomolecule. For its biosynthesis, a source of ellagitannins is required, such as strawberries and yeasts, as precursors of the tannase and ß-glucosidase enzymes responsible for hydrolysis of ellagitannins. Two experimental mixture designs were applied., varying the yeast concentration and the number of ellagitannins in the culture medium, evaluating the enzymatic activity and ellagic acid biosynthesis. Aiming to find the optimal compositions of the non-conventional yeasts assessed in the research to biosynthesize ellagic acid feasibly and efficiently using a response surface performing the statistical analysis in the StatGraphics® program for obtaining a higher yield and optimizing the ellagic acid synthesis process, the results indicate that the strains Candida parapsilosis ITM LB33 and Debaryomyces hansenii ISA 1510 have a positive effect on the synthesis of ellagic acid, since as its concentration increases in the mixture the concentration of ellagic acid in the medium also increases; on the other hand, the addition of Candida utilis ITM LB02 causes a negative effect, resulting in the compositions of 0.516876, 0.483124 and 2.58687E-9 respectively, for a treatment under the same conditions, an optimal value of ellagic acid production would be obtained. With an approximate value of 7.33036 mg/mL

Expansion of cord blood stem cells in fibronectin-coated microfluidic bioreactor

ABSTRACT Background: Hematopoietic stem/progenitor cell transplantation is the main treatment option for hematological malignancies and disorders. One strategy to solve the problem of low stem cell doses used in transplantation is pre-transplant expansion. We hypothesized that using fibronectin-coated microfluidic channels would expand HSPCs and keep self-renewal potential in a three-dimensional environment, compared to the conventional method. We also compared stem cell homing factors expression in microfluidic to conventional cultures. Materials and methods: A microfluidic device was created and characterized by scanning electron microscopy. The CD133+ cells were collected from cord blood and purified. They were subsequently cultured in 24-well plates and microfluidic bioreactor systems using the StemSpan serum-free medium. Eventually, we analyzed cell surface expression levels of the CXCR4 molecule and CXCR4 mRNA expression in CD133+ cells cultured in different systems. Results: The expansion results showed significant improvement in CD133+ cell expansion in the microfluidic system than the conventional method. The median expression of the CXCR4 in the expanded cell was lower in the conventional system than in the microfluidic system. The CXCR4 gene expression up-regulated in the microfluidic system. Conclusion: Utilizing microfluidic systems to expand desired cells effectively is the next step in cell culture. Comparative gene expression profiling provides a glimpse of the effects of culture microenvironments on the genetic program of HSCs grown in different systems.

Rational design of a 500 m3 fermenter for erythromycin production by Saccharopolyspora erythraea / 生物工程学报

Erythromycin is a macrolide antibiotic produced by Saccharopolyspora erythraea. Its yield is greatly affected by the fermentation conditions and the bioreactor configurations. In this study, a novel scale-up method for erythromycin fermentation was developed based on computational fluid dynamics (CFD) and time constant analysis. Firstly, the dissolved oxygen (DO) was determined as a key parameter according to the physiological properties of S. erythraea cultivated in a 50 L bioreactor. It was found that the time constant of oxygen supply (tmt) in a 500 m3 bioreactor should be less than 6.25 s in order to satisfy the organism's oxygen uptake rate (OUR). Subsequently, a 500 m3 bioreactor was designed using the time constant method combined with empirical correlations. The impeller combination with one BDT8 impeller at bottom and two MSX4 impellers at upper part was determined, and then validated by numerical simulation. The results indicated that the tmt of the bioreactor (< 6.25 s) and the fluid properties, including gas hold-up, shear stress and fluid vector, met the requirements of erythromycin fermentation. Finally, the industrial production of erythromycin in the 500 m3 showed the design method was applicable in large scale fermentation.

Construction of an l-cysteine hyper-producing strain of Escherichia coli based on a balanced carbon and sulfur module strategy / 生物工程学报

l-cysteine is an important sulfur-containing α-amino acid. It exhibits multiple physiological functions with diverse applications in pharmaceutical cosmetics and food industry. Here, a strategy of coordinated gene expression between carbon and sulfur modules in Escherichia coli was proposed and conducted for the production of l-cysteine. Initially, the titer of l-cysteine was improved to (0.38±0.02) g/L from zero by enhancing the biosynthesis of l-serine module (serAf, serB and serCCg) and overexpression of CysB. Then, promotion of l-cysteine transporter, increased assimilation of sulfur, reduction or deletion of l-cysteine and l-serine degradation pathway and enhanced expression of cysEf (encoding serine acetyltransferase) and cysBSt (encoding transcriptional dual regulator CysB) were achieved, resulting in an improved l-cysteine titer (3.82±0.01) g/L. Subsequently, expressions of cysM, nrdH, cysK and cysIJ genes that were involved in sulfur module were regulated synergistically with carbon module combined with utilization of sulfate and thiosulfate, resulting in a strain producing (4.17±0.07) g/L l-cysteine in flask shake and (11.94±0.1) g/L l-cysteine in 2 L bioreactor. Our results indicated that efficient biosynthesis of l-cysteine could be achieved by a proportional supply of sulfur and carbon in vivo. This study would facilitate the commercial bioproduction of l-cysteine.

Mechanism of trehalose-enhanced metabolism of heterotrophic nitrification-aerobic denitrification community under high-salt stress / 生物工程学报

Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria are aerobic microorganisms that can remove nitrogen under high-salt conditions, but their performance in practical applications are not satisfactory. As a compatible solute, trehalose helps microorganisms to cope with high salt stress by participating in the regulation of cellular osmotic pressure, and plays an important role in promoting the nitrogen removal efficiency of microbial populations in the high-salt environment. We investigated the mechanism of exogenous-trehalose-enhanced metabolism of HN-AD community under high-salt stress by starting up a membrane aerobic biofilm reactor (MABR) to enrich HN-AD bacteria, and designed a C150 experimental group with 150 μmol/L trehalose addition and a C0 control group without trehalose. The reactor performance and the community structure showed that NH4+-N, total nitrogen (TN) and chemical oxygen demand (COD) removal efficiency were increased by 29.7%, 28.0% and 29.1%, respectively. The total relative abundance of salt-tolerant HN-AD bacteria (with Acinetobacter and Pseudofulvimonas as the dominant genus) in the C150 group reached 66.8%, an 18.2% increase compared with that of the C0 group. This demonstrated that trehalose addition promoted the enrichment of salt-tolerant HN-AD bacteria in the high-salt environment to enhance the nitrogen removal performance of the system. In-depth metabolomics analysis showed that the exogenous trehalose was utilized by microorganisms to improve proline synthesis to increase resistance to high-salt stress. By regulating the activity of cell proliferation signaling pathways (cGMP-PKG, PI3K-Akt), phospholipid metabolism pathway and aminoacyl-tRNA synthesis pathway, the abundances of phosphoethanolamine, which was one of the glycerophospholipid metabolites, and purine and pyrimidine were up-regulated to stimulate bacterial aggregation and cell proliferation to promote the growth of HN-AD bacteria in the high-salt environment. Meanwhile, the addition of trehalose accelerated the tricarboxylic acid (TCA) cycle, which might provide more electron donors and energy to the carbon and nitrogen metabolisms of HN-AD bacteria and promote the nitrogen removal performance of the system. These results may facilitate using HN-AD bacteria in the treatment of high-salt and high-nitrogen wastewater.

The relationship between methane production metabolic flux and microorganisms in a microbial electrolytic cell coupled anaerobic digestion / 生物工程学报

In this study, voltage was used as a disturbance factor to investigate the relationship between microbial community and methane (CH4) production flux in a microbial electrolytic cell coupled anaerobic digestion (MEC-AD). Metabolic flux analysis (MFA) was used to explore the relationship between the CH4 metabolic flux produced and the microbes. The results showed that both methane production flux and hydrogen production flux changed significantly upon voltage disturbance, while the voltage disturbance had little effect on acetic acid production flux. The maximum CH4 production flux under 0.6 V disturbance was 0.522±0.051, which increased by 77% and 32%, respectively, compared with that of the control group under 1.0 V (0.295±0.013) and under 1.4 V (0.395±0.029). In addition, an average of 15.7%±2.9% of H2 (flux) was used to reduce CO2 to produce CH4 and acetic acid, and an average of 27.7%±6.9% of acetic acid (flux) was converted to CH4. Moreover, the abundance of Lachnospiraceae significantly affected the flux of acetic acid. The flux of CH4 production is positively correlated with the abundances of Petrimonas, Syntrophomonas, Blvii28, and Acinetobacter, and negatively correlated with the abundances of Tuzzerella and Sphaerochaeta. The species that affected the flux of H2 and CH4 were similar, mostly belonging to Bacteroides, Clostridium, Pseudomonas and Firmicutes. Furthermore, the interspecies interaction is also an important factor affecting the MEC-AD methanogenesis flux.

Research progress of combined anaerobic ammonium oxidation (ANAMMOX) process for nitrogen removal / 生物工程学报

Anaerobic ammonia oxidation (ANAMMOX) process is an efficient and low-cost biological nitrogen removal process. However, it still faces some challenges in mainstream applications due to the limitation of substrate types and nitrate accumulation. In recent years, the combined process of anammox has been widely studied to solve the above problems. In this paper, the combined processes of anammox developed in recent years are reviewed, and discussed from the process principle, advantages and disadvantages, influencing factors, process extensibility and the key bottlenecks existing in the promotion and application, as well as the relevant work of the subject group. Finally, we take an outlook on the development of the combined anaerobic ammonia oxidation process in municipal domestic wastewater treatment.

Valuation of angiogenesis in bovine xenografts implanted in intracorporal sites of rabbits as models of in-vivo bioreactors / Avaliação da angiogênese em xenoenxertos bovinos implantados em sítios intracorpóreos de coelhos como modelos de biorreatores in vivo

The aim of this study was to evaluate neovascularization of bovine xenografts implanted in intracorporeal sites of rabbits (bioreactors). 30 rabbits were used, divided into 6 groups, according to the evaluation time (7, 15, 30, 45, and 60 days); each animal received xenogenic implants in 3 different intracorporeal sites (A1 - omentum bag; A2 - intermuscular space of quadriceps femoris; A3 - subperiosteal of ilium bone). Histological assessments graded the presence of angiogenesis, the number of inflammatory cells, newly formed bone tissue, and the presence of giant cells. Histological analyses showed intense angiogenesis in all implanted xenografts. Presence of inflammatory infiltrate and giant cells at the A1 implant site and presence of bone neoformation at the A3 implant site were noted. Degeneration of implants and formation of a fibrous capsule were noted. When comparing the interaction of the site with the days of evaluation, statistical analysis showed a significant difference (p≤0.05) in any time of neovascularization analysis. The vascular endothelial growth factor (VEGF) and inflammatory cells of the omentum in its structure, may have contributed to the greater presence of neovessels and inflammatory cells, a fact that may indicate functionality as a possible bone substitute.(AU)

O objetivo deste estudo foi avaliar a neovascularização de xenoenxertos bovinos implantados em sítios intracorpóreos de coelhos (biorreatores). Foram utilizados 30 coelhos, os quais foram divididos em seis grupos, de acordo com o tempo de avaliação (sete, 15, 30, 45 e 60 dias); cada animal recebeu implantes xenogênicos em três diferentes sítios intracorpóreos (A1 - bolsa de omento; A2 - espaço intermuscular do quadríceps femoral; A3 - subperiosteal do osso ílio). Avaliações histológicas classificaram a presença de angiogênese, o número de células inflamatórias, de tecido ósseo neoformado e a presença de células gigantes. As análises histológicas mostraram intensa angiogênese em todos os xenoenxertos implantados. Observou-se presença de infiltrado inflamatório e células gigantes no local do implante A1 e presença de neoformação óssea no local do implante A3. Ao mesmo tempo, a degeneração dos implantes e a formação de uma cápsula fibrosa foram observadas. Ao comparar a interação do local com os dias de avaliação, a análise estatística mostrou diferença significativa (P≤0,05) em qualquer momento da análise de neovascularização. O fator de crescimento endotelial vascular (VEGF) e as células inflamatórias do omento em sua estrutura podem ter contribuído para a maior presença de neovasos e células inflamatórias, fato que pode indicar funcionalidade como possível substituto ósseo.(AU)

Media development and process parameter optimization using statistical experimental designs for the production of nonribosomal peptides in Escherichia coli

BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.