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1.
Artigo em Inglês | MEDLINE | ID: mdl-32043188

RESUMO

In the present work, we used systematic engineering at transport and transcription levels to significantly enhance alkaline α-amylase production in Bacillus subtilis 168M. Signal peptide YwbN' proved to be optimal. Alkaline α-amylase production was elevated by deleting a putative peptide segment of YwbN'. Insertion of arginine (R) between residues 5 and 6 of YwbN'∆p further increased the protein yield. Enhancing positive charges at sites 4 and 10 and decreasing the hydrophobicity of the H-region of YwbN'∆p were critical for improving alkaline α-amylase production in B. subtilis 168M. PHpaII was the optimal promoter, and deleting - 27T or - 31A from PHpaII enhanced the transcription of the target gene. Using a single-pulse feeding-based fed-batch system, alkaline α-amylase activity of B. subtilis 168M P∆-27T was increased by 250.6-fold, compared with B. subtilis 168M A1.

2.
Biotechnol Bioeng ; 117(2): 531-542, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31654413

RESUMO

Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of ß-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.

3.
Opt Lett ; 44(19): 4901-4904, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31568471

RESUMO

We present a method for realizing a solid-state volumetric display based on the chromatic dispersion properties of a 150 mm diffractive lens that images a series of planar patterns presented by a digital micro-mirror device projector. The projector is driven by a narrowband polychromatic source, where the position of each image plane is defined by a distinct wavelength of light. The volumetric display system achieves 20 image planes at a volume refresh rate of 20 Hz, creating a volume of 17.4 cm3 with 13 mm of depth and a field of view of 10° floating 145 mm above the lens in real space.

4.
Opt Lett ; 44(18): 4436-4438, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31517902

RESUMO

Polarization measurement is crucial for many optical applications in science and technology. Geometric metasurfaces have been used to develop polarization-sensitive holograms, providing a new opportunity for polarization measurement. We propose and experimentally demonstrate a hologram method to measure the polarization state of light. A reflective-type metasurface hologram is used to generate holographic images of graphene pattern. The ellipticity and helicity of the incident light are measured based on the intensities of the neighboring light spots, corresponding to two opposite circular polarization states. Benefiting from the advantages of reflective geometric metasurfaces, this device can operate in broadband.

5.
Sensors (Basel) ; 19(16)2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398946

RESUMO

The stockline, which describes the measured depth of the blast furnace (BF) burden surface with time, is significant to the operator executing an optimized charging operation. For the harsh BF environment, noise interferences and aberrant measurements are the main challenges of stockline detection. In this paper, a novel encoder-decoder architecture that consists of a convolution neural network (CNN) and a long short-term memory (LSTM) network is proposed, which suppresses the noise interferences, classifies the distorted signals, and regresses the stockline in a learning way. By leveraging the LSTM, we are able to model the longer historical measurements for robust stockline tracking. Compared to traditional hand-crafted denoising processing, the time and efforts could be greatly saved. Experiments are conducted on an actual eight-radar array system in a blast furnace, and the effectiveness of the proposed method is demonstrated on the real recorded data.

6.
Opt Express ; 27(9): 13252-13262, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31052853

RESUMO

A refractive index sensor can provide refractive index measurement and continuously monitor a dynamic process. Plasmonic nanostructure based sensors suffer from severe metal losses in the optical range, leading to the performance degradation. We design and numerically analyze a high-performance refractive index sensor based on the Fano resonance generated by a dielectric metasurface. The figure of merit (FOM) and the maximum quality factor (Q-factor) of the sensor are 721 and 5126, respectively. The maximum modulation depth can exceed 99% and the enhancement factor of the electric field amplitude can reach a high value of about 100. The uniqueness of the proposed sensor is polarization insensitivity. The transmittance spectra for various polarization states of the incident light can perfectly coincide, which is a rare phenomenon in Fano resonance based sensors and can facilitate experimental measurement.

7.
J Agric Food Chem ; 67(14): 3900-3908, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30873833

RESUMO

In this study, we investigated the effects of the different critical genes in the three modules on tyrosol production in Escherichia coli. Coexpression of the yahK and ARO10 genes increased the yield of tyrosol by 10% compared to that of the control. Tyrosol production by E. coli BFPT1 and E. coli BFPA1 was higher by 15.0% and 17.8% than that by the control, respectively, via coordinated expression of key genes from modules 2 and 3. The tyrosol yield of E. coli BFPE2 was 58.3% higher than that of the control (reaching 5.72 mM) when the expression levels of the key genes aroA and tyrA* from module 2 were balanced. The tyrosol yield of E. coli BFPG1 was increased by 52.6% (reaching 5.8 mM) compared to the control via coexpression of modules 1, 2, and 3. This work suggested that microbial production of tyrosol in E. coli has potential for industrial applications.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Álcool Feniletílico/análogos & derivados , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Álcool Feniletílico/metabolismo
8.
Appl Microbiol Biotechnol ; 103(2): 793-806, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30417310

RESUMO

Most recombinant proteins in Escherichia coli are not efficiently secreted to the extracellular space. Structural stabilisation of the cell wall is essential for extracellular protein production in E. coli, for which D,D-carboxypeptidases are essential. Herein, we perturbed the peptidoglycan structure of the E. coli cell wall by overexpressing D,D-carboxypeptidase genes dacA or dacB, and investigated the effect on extracellular protein production. Overexpression of dacA or dacB promoted the accumulation of intracellular soluble peptidoglycan, altered cell morphology (shape and size) and led to the formation of transparent globular structures in E. coli cells. Compared with controls (CK), extracellular production of recombinant green fluorescent protein (GFP) was increased by 1.7- and 2.3-fold upon overexpression of dacA and dacB, respectively. Similarly, extracellular production of recombinant amylase and α-galactosidase was increased by 4.5- and 2.8-fold, respectively, upon overexpression of dacA, and by 11.9- and 2.5-fold, respectively, upon overexpression of dacB. Overexpression of dacA or dacB enhanced both the outer and inner membrane permeability of E. coli. This cell wall engineering strategy opens up a new direction for enhancing extracellular protein and chemical production in E. coli.


Assuntos
Carboxipeptidases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Peptidoglicano/biossíntese , Proteínas Recombinantes/metabolismo , Carboxipeptidases/genética , Membrana Celular/fisiologia , Parede Celular/química , Escherichia coli/genética , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Permeabilidade , Proteínas Recombinantes/genética
9.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 30(9): 830-835, 2018 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-30309407

RESUMO

OBJECTIVE: To determine the effect of bone mesenchymal stem cells (BMSCs) in transplantation therapy for lipopolysaccharide (LPS)-induced coagulation disorder and the underlying mechanism of high mobility group protein B1-receptors for advanced glycation end products/Toll-like receptors-nuclear factor-κB (HMGB1-RAGE/TLRs-NF-κB) signaling pathway. METHODS: BMSCs of female Sprague-Dawley (SD) rats ageing 4-5 weeks old were extracted and cultivated in vitro, and the fourth-passaged BMSCs phenotype was identified by flow cytometry for transplantation in the following experimental study. The rats were randomly divided into normal saline (NS) control group, LPS group, and BMSC group according to the random number table with 15 rats in each group. Coagulation disorders model was reproduced by injection of 1 mg/kg LPS via saphenous vein, and the rats in the NS control group was injected with equal volume NS. Those in the BMSC group were infused BMSC 0.5 mL containing 1×106 cells via tail vein at 2 hours after LPS injection, and the rats in other groups were injected with equal volume NS. Abdominal aorta blood was collected at 1, 3 and 7 days post operation. Coagulation indexes such as platelet count (PLT), platelet volume distribution width (PDW), mean platelet volume (MPV), plateletcrit (PCT), platelet large cell ratio (P-LCR), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), international normalized ratio (INR), and fibrinogen (FIB) were determined. The mRNA levels and contents of HMGB1, RAGE, TLR2/4 and NF-κB were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: (1) The cells cultured in vitro were spindle shaped or flat. The fourth-passaged BMSCs phenotype was successfully identified by flow cytometry technology. (2) Coagulation indexes: compared with NS control group, PLT, PCT and FIB in LPS group were significantly decreased, PDW, MPV, P-LCP, and INR were significantly increased, and APTT, PT, and TT were significantly prolonged from the first day. Furthermore, those in LPS group were gradually ameliorated with prolongation of LPS induction time. The coagulation function abnormality induced by LPS was reversed by BMSCs with significant difference at 1 day as compared with LPS group [PLT (×109/L): 398.8±17.9 vs. 239.1±15.8, PCT (%): 0.35±0.04 vs. 0.23±0.06, FIB (g/L): 1.7±0.6 vs. 0.8±0.1, PDW (%): 12.4±1.6 vs. 16.2±1.5, MPV (fl): 11.0±1.6 vs. 13.7±1.1, P-LCP (%): 13.0±2.1 vs. 15.3±2.7, INR: 1.52±0.17 vs. 1.82±0.19, APTT (s): 66.3±4.1 vs. 89.5±4.5, PT (s): 18.3±0.7 vs. 25.1±1.9, TT (s): 87.5±7.8 vs. 115.0±9.7, all P < 0.05], till 7 days. (3) HMGB1-RAGE/TLRs-NF-κB signaling pathway related molecules: compared with NS control group, the mRNA expressions and contents of HMGB1, RAGE, TLR2/4 and NF-κB were significantly increased in LPS group from the first day. However, the mRNA expressions and contents of the molecules in LPS group were gradually decreased with prolongation of LPS induction time. After BMSC intervention, the mRNA expressions and contents of molecules at 1 day were significantly lower than those of LPS group [HMGB1 mRNA (2-ΔΔCt): 10.77±0.04 vs. 24.51±3.69, HMGB1 content (µg/L): 0.48±0.01 vs. 0.95±0.06; RAGE mRNA (2-ΔΔCt): 11.57±1.11 vs. 18.08±0.29, RAGE content (µg/L): 0.73±0.04 vs. 1.37±0.06; TLR2 mRNA (2-ΔΔCt): 2.60±0.22 vs. 12.61±0.27, TLR2 content (µg/L): 0.81±0.03 vs. 1.59±0.09; TLR4 mRNA (2-ΔΔCt): 2.95±0.52 vs. 4.06±0.11, TLR4 content (µg/L): 0.80±0.09 vs. 1.18±0.11; NF-κB mRNA (2-ΔΔCt): 1.29±0.06 vs. 7.79±0.25, NF-κB content (µg/L): 1.22±0.24 vs. 2.42±0.26, all P < 0.05], till 7 days. CONCLUSIONS: BMSCs administration could ameliorate the coagulation function in LPS-induced coagulation disorder rats and these might be associated with HMGB1-RAGE/TLRs-NF-κB signaling pathway inhibition.


Assuntos
Transtornos da Coagulação Sanguínea/terapia , Transplante de Células-Tronco Mesenquimais , Transdução de Sinais , Animais , Transtornos da Coagulação Sanguínea/etiologia , Células da Medula Óssea , Feminino , Proteína HMGB1 , Lipopolissacarídeos/toxicidade , Células-Tronco Mesenquimais , NF-kappa B , Ratos , Ratos Sprague-Dawley , Receptor para Produtos Finais de Glicação Avançada , Receptor 2 Toll-Like , Receptor 4 Toll-Like
10.
Metab Eng ; 47: 374-382, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29702277

RESUMO

N-acetyl-d-neuraminic acid (Neu5Ac) is a valuable resource that has seen increasing demand in both medicine and biotechnology. Although enzymatic systems and whole-cell biocatalysts have been developed for the synthesis of Neu5Ac, low yield and productivity still hamper the use of these methods on larger scales. We report the creation of an Escherichia coli biocatalyst for the efficient synthesis of Neu5Ac using a metabolic and protein engineering strategy. Expression of the two enzymes, N-acetyl-D-glucosamine 2-epimerase (AGE) and Neu5Ac lyase (NAL), was balanced using promoter engineering. Genes encoding competing pathways and GlcNAc catabolism were deleted, and then a structure-guided process was used to identify a more efficient NAL and an AGE mutant with a higher rate of Neu5Ac synthesis. The resulting biocatalyst produced 351.8 mM Neu5Ac with a yield of 58.6% from GlcNAc. This work exemplifies the use of rational design and protein engineering to construct a complex bacterial biocatalyst that can serve as a platform for the large-scale synthesis of a useful biological material.


Assuntos
Biocatálise , Escherichia coli , Microrganismos Geneticamente Modificados , Ácido N-Acetilneuramínico , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Ácido N-Acetilneuramínico/biossíntese , Ácido N-Acetilneuramínico/genética , Engenharia de Proteínas/métodos
11.
Adv Mater ; 30(21): e1707499, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29603423

RESUMO

Optical metasurfaces have shown unprecedented capabilities in the local manipulation of the light's phase, intensity, and polarization profiles, and represent a new viable technology for applications such as high-density optical storage, holography and display. Here, a novel metasurface platform is demonstrated for simultaneously encoding color and intensity information into the wavelength-dependent polarization profile of a light beam. Unlike typical metasurface devices in which images are encoded by phase or amplitude modulation, the color image here is multiplexed into several sets of polarization profiles, each corresponding to a distinct color, which further allows polarization modulation-induced additive color mixing. This unique approach features the combination of wavelength selectivity and arbitrary polarization control down to a single subwavelength pixel level. The encoding approach for polarization and color may open a new avenue for novel, effective color display elements with fine control over both brightness and contrast, and may have significant impact for high-density data storage, information security, and anticounterfeiting.

12.
Ann Clin Lab Sci ; 48(6): 743-750, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30610044

RESUMO

Bone marrow mesenchymal stromal cells (BMSCs) have positive therapeutic effects on inflammation associated diseases. However, the underlying mechanism is largely unknown. This study was conducted to investigate whether BMSCs could alleviate the inflammation reaction in lipopolysaccaride (LPS)-induced acute kidney injury (septic-AKI) of rats via inhibition of toll-like receptors (TLR4)-nuclear factor-kappa B (NF-κB) signaling pathway. The septic-AKI rat model was established by injecting the 1ml/mg LPS through the femoral vein. Based on this model, rats were subjected to BMSC transplantation, PDTC (a kind of NF-κB inhibitor) administration alone, and combined treatment of the first two together. Results showed that LPS treatment caused the increases of the concentration of blood urea nitrogen (BUN) and serum creatinine (SCr), accompanied by tissue injury and the up-regulation of TLR4 and NF-κB, that was its key downstream signaling molecule, in both mRNA and protein level. Notably, it has been found that BMSCs transplantation significantly reversed the already upregulated concentration of BUN and SCr, dramatically attenuated the event of the tissue injury, and prominently reduced mortality after AKI. These were paralleled by down-regulation of the level of TLR4 and NF-κB. These effects of BMSCs transplantation were similar to those of PDTC treatment. Importantly, the effects in the combination therapy of BMSCs transplantation and PDTC group were much stronger than those of either BMSCs or PDTC used alone. These findings suggest that BMSCs transplantation contributes to therapeutic effects in LPS-induced AKI rat model, and that the most obvious effects occurred in the combined treatment group, with BMSCs and PDTC together, which was tightly associated with inhibition of the TLR4-NF-κB signaling pathway.


Assuntos
Lesão Renal Aguda/complicações , Inflamação/etiologia , Inflamação/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Transdução de Sinais/fisiologia , Lesão Renal Aguda/induzido quimicamente , Análise de Variância , Animais , Antioxidantes/uso terapêutico , Nitrogênio da Ureia Sanguínea , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/fisiologia , Lipopolissacarídeos/toxicidade , Células-Tronco Mesenquimais/fisiologia , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Prolina/análogos & derivados , Prolina/uso terapêutico , RNA Mensageiro/metabolismo , Ratos , Tiocarbamatos/uso terapêutico , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
13.
Light Sci Appl ; 7: 17129, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30839643

RESUMO

Images perceived by human eyes or recorded by cameras are usually optical patterns with spatially varying intensity or color profiles. In addition to the intensity and color, the information of an image can be encoded in a spatially varying distribution of phase or polarization state. Interestingly, such images might not be able to be directly viewed by human eyes or cameras because they may exhibit highly uniform intensity profiles. Here, we propose and experimentally demonstrate an approach to hide a high-resolution grayscale image in a square laser beam with a size of less than half a millimeter. An image with a pixel size of 300 × 300 nm is encoded into the spatially variant polarization states of the laser beam, which can be revealed after passing through a linear polarizer. This unique technology for hiding grayscale images and polarization manipulation provides new opportunities for various applications, including encryption, imaging, optical communications, quantum science and fundamental physics.

14.
Sci Rep ; 7(1): 11440, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28900301

RESUMO

An optical illusion, such as "Rubin's vase", is caused by the information gathered by the eye, which is processed in the brain to give a perception that does not tally with a physical measurement of the stimulus source. Metasurfaces are metamaterials of reduced dimensionality which have opened up new avenues for flat optics. The recent advancement in spin-controlled metasurface holograms has attracted considerate attention, providing a new method to realize optical illusions. We propose and experimentally demonstrate a metasurface device to generate an optical illusion. The metasurface device is designed to display two asymmetrically distributed off-axis images of "Rubin faces" with high fidelity, high efficiency and broadband operation that are interchangeable by controlling the helicity of the incident light. Upon the illumination of a linearly polarized light beam, the optical illusion of a 'vase' is perceived. Our result provides an intuitive demonstration of the figure-ground distinction that our brains make during the visual perception. The alliance between geometric metasurface and the optical illusion opens a pathway for new applications related to encryption, optical patterning, and information processing.

15.
Sci Rep ; 7(1): 4520, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28674409

RESUMO

Metasurface holograms are typically fabricated on rigid substrates. Here we experimentally demonstrate broadband, flexible, conformable, helicity multiplexed metasurface holograms operating in the visible range, offering increased potential for real life out-of-the-lab applications. Two symmetrically distributed holographic images are obtained when circularly polarized light impinges on the reflective-type metasurface positioned on non-planar targets. The two off-axis images with high fidelity are interchangeable by controlling the helicity of incident light. Our metasurface features the arrangement of spatially varying gold nanorods on a flexible, conformable epoxy resist membrane to realize a Pancharatnam-Berry phase profile. These results pave the way to practical applications including polarization manipulation, beam steering, novel lenses, and holographic displays.

16.
J Agric Food Chem ; 65(23): 4708-4714, 2017 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-28530096

RESUMO

Tyrosol is a phenolic compound found in olive oil and wines. The health benefits of tyrosol have attracted considerable attention. Because the tyrosol extraction from plants poses a major obstacle, biosynthesizing this compound using microbial hosts is of interest. In this study, the phenylpyruvate decarboxylase gene ARO10 and the aromatic amino acid aminotransferase gene ARO8 were introduced into Escherichia coli to generate two recombinant tyrosol producers. Deleting the prephenate dehydratase gene pheA and the phenylacetaldehyde dehydrogenase gene feaB improved the tyrosol production. Under the optimal fermentation conditions, a recombinant strain overexpressing ARO10 gene produced 4.15 mM tyrosol from 1% (w/v) glucose during a 48 h shake flask cultivation. Furthermore, when tyrosine was used as the substrate, the recombinant strain co-overexpressing ARO8 and ARO10 genes displayed a higher tyrosol yield, in which 8.71 mM tyrosol was produced from 10 mM tyrosine. This investigation suggests that microbial tyrosol production has application potential.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Álcool Feniletílico/análogos & derivados , Fermentação , Glucose/metabolismo , Engenharia Metabólica , Álcool Feniletílico/metabolismo , Tirosina/metabolismo
17.
Adv Mater ; 29(15)2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28207164

RESUMO

A facile metasurface approach is shown to realize polarization-controllable multichannel superpositions of orbital angular momentum (OAM) states with various topological charges. By manipulating the polarization state of the incident light, four kinds of superpositions of OAM states are realized using a single metasurface consisting of space-variant arrays of gold nanoantennas.

18.
Bioengineered ; 8(2): 115-119, 2017 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-27459271

RESUMO

The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.


Assuntos
Etanol/metabolismo , Microbiologia Industrial/métodos , 6-Fitase/metabolismo , Biocombustíveis/microbiologia , Fermentação/fisiologia , Saccharomyces cerevisiae/metabolismo
19.
J Biol Eng ; 10: 13, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27777616

RESUMO

BACKGROUND: Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168. RESULTS: The activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#. CONCLUSION: The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis. Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell factories.

20.
Appl Microbiol Biotechnol ; 100(22): 9567-9580, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27522195

RESUMO

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura+ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10-8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.


Assuntos
Candida tropicalis/genética , Marcação de Genes/métodos , Genética Microbiana/métodos , Genes Reporter , Proteínas de Fluorescência Verde/genética , Orotidina-5'-Fosfato Descarboxilase/deficiência
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