RESUMO
The synthesis of vitamin D3 precursor 7-dehydrocholesterol (7-DHC) by microbial fermentation has much attracted attention owing to its advantages of environmental protection. In this study, Saccharomyces cerevisiae was engineered for a de novo biosynthesis of 7-DHC. First, seven essential genes (six endogenous genes and one heterologous gene) were overexpressed, and the ROX1 gene (heme-dependent repressor of hypoxic genes) was knocked out. The resulting strain produced 82.6 mg/L 7-DHC from glucose. Then, we predicted five gene knockout targets for 7-DHC overproduction by the reconstruction of genome-scale metabolic model. GDH1 gene knockout increased the 7-DHC titer from 82.6 to 101.5 mg/L, and the specific growth rate of the ΔGDH1 mutant was also increased by 28%. Next, Ty1 transposon in S. cerevisiae was applied to increase the copies of the ERG1 gene and DHCR24 gene, resulting in a 120% increase in 7-DHC titer to 223.3 mg/L. Besides, to optimize the metabolic flux distribution, Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system was used to dynamically inhibit the competitive pathway, and the best binding site of ERG6 (delta (24)-sterol C-methyltransferase) promoter was screened out. The OD600 value of ERG6 regulated cells increased by 43% than knocking out ERG6 directly, and 7-DHC titer increased to 365.5 mg/L in a shake flask. Finally, the 7-DHC titer reached 1328 mg/L in 3-L bioreactor and the specific titer of 7-DHC reached up to 114.7 mg/g dry cell weight). Overall, this study constructed a yeast chassis for the highly efficient production of 7-DHC by systems metabolic engineering.
Assuntos
Desidrocolesteróis , Saccharomyces cerevisiae , Desidrocolesteróis/metabolismo , Fermentação , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Enzymatic secretion of immune cells (leukocytes) plays a dominant role in host immune responses to a myriad of biological triggers, including infections, cancers, and cardiovascular diseases. Current tools to probe these leukocytes inadequately profile these vital biomarkers; the need for sample preprocessing steps of cell lysis, labeling, washing, and pipetting inevitably triggers the cells, changes its basal state, and dilutes the individual cell secretion in bulk assays. Using a fully integrated system for multiplexed profiling of native immune single-cell enzyme secretion from 50 µL of undiluted blood, we eliminate sample handling. With a total analysis time of 60 min, the integrated platform performs six tasks of leukocyte extraction, cell washing, fluorescent enzyme substrate mixing, single-cell droplet making, droplet incubation, and real-time readout for leukocyte secretion profiling of neutrophil elastase, granzyme B, and metalloproteinase. We calibrated the device, optimized the protocols, and tested the leukocyte secretion of acute heart failure (AHF) patients at admission and predischarge. This paper highlights the presence of single-cell enzymatic immune phenotypes independent of CD marker labeling, which could potentially elucidate the innate immune response states. We found that patients recovering from AHF showed a corresponding reduction in immune-cell enzymatic secretion levels and donor-specific enzymatic signatures were observed, which suggests patient-to-patient heterogeneous immune response. This platform presents opportunities to elucidate the complexities of the immune response from a single drop of blood and bridge the current technological, biological, and medical gap in understanding immune response and biological triggers.
Assuntos
Leucócitos , Biomarcadores , HumanosRESUMO
In our previous study, one-step pyruvate and D-alanine production from D,L-alanine by a whole-cell biocatalyst Escherichia coli expressing L-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (kcat/Km) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20 g/L with an L-alanine conversion rate of 77.62% and a D-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and D-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.
Assuntos
Alanina/metabolismo , Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Mutação , Proteus mirabilis/enzimologia , Ácido Pirúvico/metabolismo , Amidoidrolases/genética , Proteínas de Bactérias/genética , Biocatálise , Escherichia coli/genética , Mutagênese Sítio-Dirigida , Engenharia de ProteínasRESUMO
Stem cell regenerative medicine strategy requires selecting functional cells to trigger repair processes. Stem cell secretion measurement is important to evaluate cellular activities for functional cell sorting. At present, to determine single cell secretions, mixing chemical sensors and cells together in a chamber is a standard procedure. However, toxic chemical sensors, such as albumin assay kits, are used during this process, causing low viability (64%) and low functionality (30%). It is especially important for stem cell profiling, as the toxicity of chemical sensors such as albumin permanently changes stem cell phenotypes, leading to unwanted analysis outcomes. Moreover, because of the sensor toxicity, the challenge of culturing sorted cells remain. In this study, an integrative synchronized droplet screen system was developed to separate a large droplet with cell encapsulation into two daughter droplets: one droplet containing cell secretions and the other droplet containing a single cell. These two daughter droplets moved along the channels at the same speed in synchronization. By injecting toxic chemical sensors into one daughter droplet, the single-cell secretions were determined without affecting the cells in the corresponding droplet. Based on the daughter droplet synchronization, the cells without mixing toxicity sensors were sorted for cell culturing. For example, to identify hepatocytes, the albumin secretion of undifferentiated HepaRG stem cells was measured in daughter droplets by injecting a toxic albumin assay kit for functional stem cell sorting. With synchronized sorting, functional hepatocytes were collected without exposure to toxic chemical sensors, showing high viability (78%) and active functionality (89%).
Assuntos
Separação Celular , Dispositivos Lab-On-A-Chip , Células-Tronco/citologia , Albuminas/química , Albuminas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Imagem Óptica , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
In the context of tumor analysis, the implementation of precision medicine requires on-time clinical measurements, which requires rapid large-scale single-cell screening that obtains cell population distributions and functions in tumors to determine disease progression for therapeutics. In this study, a high-throughput screening (HTS) platform integrating optical fluorescence detectors and a computational method was developed as a droplet-based microfluidic flow cytometer (Droplet-µFC) to comprehensively analyze multiple proteolytic activities of a patient-derived tumor (with â¼0.5-2 M cells) at single-cell resolution within 2 h. The data-driven analytical method identified distinct cell types and status through protease profiling with high precision. Multiple protease activities of single cells harvested from a tumor were thus determined with a throughput of â¼100 cells per second. This platform was used to screen protease activities of a wide range of cell types, forming a library. With the development of advanced computational clustering and cell mapping, rapid quantitative tumor profiling with a comprehensive description of cell population distributions and functions could be obtained for clinical treatments.
Assuntos
Citometria de Fluxo/métodos , Técnicas Analíticas Microfluídicas/métodos , Neoplasias/enzimologia , Peptídeo Hidrolases/análise , Animais , Antineoplásicos , Linhagem Celular Tumoral , Bases de Dados Factuais , Cloridrato de Erlotinib/farmacologia , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Dispositivos Lab-On-A-Chip , Camundongos , Neoplasias/patologia , Oligopeptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Análise de Célula Única/métodosRESUMO
Numerous biological disciplines rely on high-throughput cell sorting. Flow cytometry, the current gold standard, is capable of ultrahigh-throughput cell sorting, but measurements are primarily limited to cell size and surface marker. Droplet sorting technology is gaining increasing attention with the ability to provide an individual environment for the analysis of single-cell secretion. Although various droplet detecting methods, such as fluorescence, absorbance, mass spectrum, imaging analysis, have been developed for droplet sorting, it remains challenging to establish high-throughput sorting methods for numerous analytes. We aim to develop a high-throughput sorting system based on the glucosamine (GlcN) measurement for the directed evolution of diacetylchitobiose deacetylase (Dac), the key enzyme for GlcN production. To overcome the limitation that no high-throughput sorting system existed for GlcN, we designed a novel bacteria-based biosensor capable of converting GlcN to a positively correlated fluorescence signal. Through characterization and optimization, it was possible to detect GlcN in droplets for high-throughput droplet sorting. We recovered the best Dac mutant S60I/R157T/F168S after sorting â¼0.2 million Dac mutants; its activity was 48.6 ± 1.5 U/mL, which was 1.8-times that of our previously discovered Dac mutant R157T (27.2 ± 1.8 U/mL). This result successfully demonstrated the combination of high-throughput droplet sorting technology and a bacteria-based biosensor, which could facilitate the industrial production of GlcN and serve as a model for similar droplet sorting applications.
RESUMO
Owing to its ability to isolate single cells and perform high-throughput sorting, droplet sorting has been widely applied in several research fields. Compared with flow cytometry, droplet allows the encapsulation of single cells for cell secretion or lysate analysis. With the rapid development of this technology in the past decade, various droplet sorting devices with high throughput and accuracy have been developed. A droplet sorter with the highest sorting throughput of 30,000 droplets per second was developed in 2015. Since then, increased attention has been paid to expanding the possibilities of droplet sorting technology and strengthening its advantages over flow cytometry. This review aimed to summarize the recent progress in droplet sorting technology from the perspectives of device design, detection signal, actuating force, and applications. Technical details for improving droplet sorting through various approaches are introduced and discussed. Finally, we discuss the current limitations of droplet sorting for single-cell studies along with the existing gap between the laboratory and industry and provide our insights for future development of droplet sorters.
Assuntos
Técnicas Biossensoriais , Técnicas Analíticas Microfluídicas , Citometria de Fluxo , Ensaios de Triagem em Larga EscalaRESUMO
Microscopy imaging of living cells is becoming a pivotal, noninvasive, and highly specific tool in metabolic engineering to visualize molecular dynamics in industrial microorganisms. This review describes the different microscopy methods, from fluorescence to super resolution, with application in microbial bioengineering. Firstly, the role and importance of microscopy imaging is analyzed in the context of strain design. Then, the advantages and disadvantages of different microscopy technologies are discussed, including confocal laser scanning microscopy (CLSM), spatial light interference microscopy (SLIM), and super-resolution microscopy, followed by their applications in synthetic biology. Finally, the future perspectives of live-cell imaging and their potential to transform microbial systems are analyzed. This review provides theoretical guidance and highlights the importance of microscopy in understanding and engineering microbial metabolism.
Assuntos
Engenharia Metabólica , Microscopia Confocal , Microscopia de Fluorescência/métodosRESUMO
In this study, semi-rational design based on site-directed saturation mutagenesis and surface charge modification was used to improve the catalytic efficiency of the diacetylchitobiose deacetylase derived from Pyrococcus horikoshii (PhDac). PhDac mutant M14, which was screened by site-directed saturation mutagenesis, showed a ~ 2.21 -fold enhanced catalytic efficiency (kcat/Km) and the specific activity was improved by 70.02%. To keep the stability of glucosamine (GlcN), we reduced the optimal pH of M14 by modifying the surface charge from -35 to -59 to obtain mutant M20, whose specific activity reached 2 -fold of the wild-type. The conversion rate of N-acetylglucosamine (GlcNAc) to GlcN catalyzed by M20 reached 94.3%. Moreover, the decline of GlcN production was slowed down by the reduction of pH when temperature was higher than 50 â. Our results would accelerate the process of industrial production of GlcN by biocatalysis.
RESUMO
Menaquinone-4 (MK-4) plays a significant role in bone health and cardiovascular therapy. Although many strategies have been adopted to increase the yield of MK-4 in Bacillus subtilis 168, the effectiveness of MK-4 is still low due to the inherent limitations of metabolic pathways. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. Nevertheless, in most reports, QS systems depend on down-regulated expression rather than up-regulated expression, which greatly limit their potential as molecular switches to control metabolic flux. To address this challenge, a modular PhrQ-RapQ-ComA QS system is developed based on promoter PA11, which is up-regulated by phosphorylated ComA (ComA-P). In this paper, firstly we analyzed the ComA-based gene expression regulation system in Bacillus subtilis 168. We constructed a promoter library of diï¬ ;erent abilities, selected best promoters from a library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrQ-RapQ-ComA QS system to dynamically control the synthesis of MK-4 in B. subtilis 168. Cell growth and eï¬cient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of MK-4 in shake flask from 120.1 ± 0.6 to 178.9 ± 2.8 mg/L, and reached 217 ± 4.1 mg/L in a 3-L bioreactor, which verified the effectiveness of this strategy. In summary, PhrQ-RapQ-ComA QS system can realize dynamic pathway regulation in B. subtilis 168, which can be stretched to a great deal of microorganisms to fine-tune gene expression and enhance the production of metabolites.
Assuntos
Bacillus subtilis , Percepção de Quorum , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Engenharia Metabólica , Vitamina K 2/análogos & derivadosRESUMO
Diacetylchitobiose deacetylase (Dac) from Pyrococcus horikoshii can realize the one-step production of glucosamine (GlcN). The efficient expression and secretion of Dac play a central role in the green production of GlcN. In this study, Bacillus subtilis WB600 was used as the expression host. Firstly, we screened 12 signal peptides, among which signal peptide NprB had the strongest ability of guiding Dac secretion. Further optimization of the functional region showed that the extracellular Dac activity of NprB mutant was increased to 3682.2 U/mL. Next, the extracellular Dac activity was increased to 4807.6 U/mL by RBS sequence optimization. Then we got a new recombinant B. subtilis C6 for plasmid-free expression of Dac by integrating comK gene and silencing bpr, nprB, aprE, mpr and nprE genes. Finally, the extracellular Dac activity of genome-integrating strain reached 6357.38 U/mL, which was the highest level reported so far.
Assuntos
Bacillus subtilis , Glucosamina , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Plasmídeos/genéticaRESUMO
Bioprocess, a biocatalysis-based technology, is becoming popular in many research fields and widely applied in industrial manufacturing. However, low bioconversion, low productivity, and high costs during industrial processes are usually the limitation in bioprocess. Therefore, many biocatalyst strategies have been developed to meet these challenges in recent years. In this review, we firstly discuss protein engineering strategies, which are emerged for improving the biocatalysis activity of biocatalysts. Then, we summarize metabolic engineering strategies that are promoting the development of microbial cell factories. Next, we illustrate the necessity of using the combining strategy of protein engineering and metabolic engineering for efficient biocatalysts. Lastly, future perspectives about the development and application of novel biocatalyst strategies are discussed. This review provides theoretical guidance for the development of efficient, sustainable, and economical bioprocesses mediated by novel biocatalysts.
Assuntos
Engenharia Metabólica , Engenharia de Proteínas , Biocatálise , IndústriasRESUMO
Tumor blood vessels are chaotic and abundantly distributed, owing to their heterogeneity. Therefore, imaging techniques which reveal abnormalities of tumor vasculature play significant roles in both mechanistic and clinical diagnostic tumor studies. Photoacoustic (PA) imaging uses the intrinsic characteristics of hemoglobin, to acquire tumor hemodynamic information, while ultrasound (US) imaging provides information about tumoral vessel structures and blood flow. To improve the imaging contrast performance, hydrogel-based microdroplets were designed for both US blood flow and PA imaging in this study. The microdroplets served as carriers for PA contrast agent solution in the innermost part while oil and hydrogel formed the inner and outer layers of the droplets. In vitro experiments firstly demonstrated the dual modality contrast effects of the microdroplets on US flow determination and PA imaging. In vivo experiments were then carried out in both healthy nude mice and nude mice with subcutaneous tumor to validate the contrast effects and to monitor the duration of contrast effects in animals. Using the dual-modality microdroplets, we were able to obtain distinct edges of tumor and blood flow mapping of the tumor microvascular with improved sensitivity up to 11.09 dB for PA and 6.69 dB for US flow. Besides, the in vivo evaluation with microdroplets showed US flow enhancement for more than 60 min. Therefore, the microdroplets are able to provide the contrast effects for both US flow and PA in a relative long duration and have potential to be applied in the tumor related diagnoses and studies.
Assuntos
Meios de Contraste/química , Nanopartículas/química , Técnicas Fotoacústicas/métodos , Ultrassonografia/métodos , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos NusRESUMO
Cell nucleocytoplasmic profiles of microRNAs (miRNAs) are critical to determining a single cell's essential functionalities, such as cellular transcription, nucleus export and degradation, which gives a comprehensive view of cellular processes. Despite the importance of addressing nucleocytoplasmic heterogeneity, the challenge of high-throughput screening remains. Although a droplet-based approach was developed for single-cell miRNA assays, the challenge of quantifying miRNA with high sensitivity to indicate nucleocytoplasmic heterogeneity remains. In this study, a nanoplasmon-enhanced droplet screening platform was developed to quantify single-cell nucleocytoplasmic heterogeneity with the high sensitivity of 0.1 nM. Droplet screening and multiplexed plasmonic assays are synergistic: droplet screening is used to isolate single cells for high-throughput screening, while enhanced nanoplasmonic assays are conducted to precisely determine different types of miRNAs, addressing the cell nucleocytoplasmic profile. Here, two nucleic acid-functionalized plasmonic nanosensors, silver nanoparticles functionalized with designed sequences to target miRNAs, are synthesized. After the targets are bound, competitive formation of sensor-target hybrids interferes with plasmonic coupling between the nanoparticles, decreasing a fluorescence signal and thus enabling high-sensitivity single-cell miRNA quantification. Using the fluorescence signal change as a readout allows continuous-flow measurement to provide a single-cell nucleocytoplasmic profile in a high-throughput manner (â¼100 cells per minute) for effective quantitative cell biology.
Assuntos
Nanopartículas Metálicas , MicroRNAs , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , MicroRNAs/genética , PrataRESUMO
Bacterial cellulose (BC), a renewable type of cellulose, has been used in the manufacture of foods, cosmetics, and biomedical products. To produce BC, a high-throughput single-bacterium measurement is necessary to identify the functional bacteria that can produce BC with sufficient amount and desirable morphology. In this study, a continuous-flow intelligent optofluidic device was developed to enable high-throughput single-bacterium profiling of BC. Single bacteria were incubated in agarose hydrogel particles to produce BC with varied densities and structures. An intelligent convolutional neural network (CNN) computational method was developed to analyze the scattering patterns of BC. The BC production and morphology were determined with a throughput of â¼35 bacteria per second. A total of â¼105 single-bacterium BC samples were characterized within 3 hours. The high flexibility of this approach facilitates high-throughput comprehensive single-cell production analysis for a range of applications in engineering biology.
Assuntos
Acetobacteraceae/química , Materiais Biocompatíveis/metabolismo , Celulose/biossíntese , Gluconacetobacter xylinus/química , Dispositivos Lab-On-A-Chip , Acetobacteraceae/metabolismo , Materiais Biocompatíveis/química , Celulose/química , Gluconacetobacter xylinus/metabolismo , Fenômenos ÓpticosRESUMO
The recent maturation of continuous-flow microfluidic technologies has coincided with transformative new methods to profile single cells, including their genetic types, protein expression and enzyme activities. Continuous-flow high-throughput single-cell screening and sorting can reveal relationships across cellular phenotypes (e.g., enzyme activity and secretion) and genetic fingerprints. This technology provides unique opportunities, as well as experimental and computational challenges, for integrative approaches that can process large amounts of single-cell data. In this chapter, we discuss recent advances in integrated continuous-flow microfluidic approaches with a focus on measurements and statistical analysis of single-cell enzyme activity and their applications in quantitative biology, synthetic biology, and diagnosis.
Assuntos
Ensaios Enzimáticos/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação , Animais , Linhagem Celular , Linhagem Celular Tumoral , Desenho de Equipamento , Transferência Ressonante de Energia de Fluorescência/instrumentação , Humanos , Camundongos Nus , Neoplasias/enzimologia , ProteóliseRESUMO
Analysis of microRNA (miRNA), a pivotal primary regulator of fundamental cellular processes, at the single-cell level is essential to elucidate regulated gene expression precisely. Most single-cell gene sequencing methods use the polymerase chain reaction (PCR) to increase the concentration of the target gene for detection, thus requiring a barcoding process for cell identification and creating a challenge for real-time, large-scale screening of sequences in cells to rapidly profile physiological samples. In this study, a rapid, PCR-free, single-cell miRNA assay is developed from a continuous-flow microfluidic process employing a DNA hybridization chain reaction to amplify the target miRNA signal. Individual cells are encapsulated with DNA amplifiers in water-in-oil droplets and then lysed. The released target miRNA interacts with the DNA amplifiers to trigger hybridization reactions, producing fluorescence signals. Afterward, the target sequences are recycled to trigger a cyclic cascade reaction and significantly amplify the fluorescence signals without using PCR thermal cycling. Multiple DNA amplifiers with distinct fluorescence signals can be encapsulated simultaneously in a droplet to measure multiple miRNAs from a single cell simultaneously. Moreover, this process converts the lab bench PCR assay to a real-time droplet assay with the post-reaction fluorescence signal as a readout to allow flow cytometry-like continuous-flow measurement of sequences in a single cell with an ultrahigh throughput (300-500 cells per minute) for rapid biomedical identification.