scholarly journals Synthetic Bacterial Cell Factory for Highly Efficient Protein Secretion and Consolidated

2019 ◽  
Author(s):  
Shangxian Xie ◽  
Su Sun ◽  
Xiaoyu Zhang ◽  
Joshua S. Yuan
Keyword(s):  
Protein Secretion ◽  
Bacterial Cell ◽  
Cell Factory ◽  
Highly Efficient

scholarly journals Implication of gene distribution in the bacterial chromosome for the bacterial cell factory

2000 ◽  
Vol 78 (3) ◽  
pp. 209-219 ◽  
Author(s):  
Eduardo P.C. Rocha ◽  
Pascale Guerdoux-Jamet ◽  
Ivan Moszer ◽  
Alain Viari ◽  
Antoine Danchin
Keyword(s):  
Bacterial Cell ◽  
Bacterial Chromosome ◽  
Cell Factory ◽  
Gene Distribution

Bacterial Cell-Free System for Highly Efficient Protein Synthesis

2008 ◽  
pp. 83-97 ◽  
Author(s):  
Takanori Kigawa ◽  
Takayoshi Matsuda ◽  
Takashi Yabuki ◽  
Shigeyuki Yokoyama
Keyword(s):  
Protein Synthesis ◽  
Bacterial Cell ◽  
Free System ◽  
Cell Free System ◽  
Highly Efficient

scholarly journals Moderate Expression of SEC16 Increases Protein Secretion by Saccharomyces cerevisiae

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Jichen Bao ◽  
Mingtao Huang ◽  
Dina Petranovic ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Secretion ◽  
Recombinant Proteins ◽  
Secretory Pathway ◽  
Cell Factory ◽  
Amylase Secretion ◽  
Content Type ◽  
Cell Factories ◽  
A Genome

ABSTRACT The yeast Saccharomyces cerevisiae is widely used to produce biopharmaceutical proteins. However, the limited capacity of the secretory pathway may reduce its productivity. Here, we increased the secretion of a heterologous α-amylase, a model protein used for studying the protein secretory pathway in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased α-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species resulting from the heterologous α-amylase production was reduced. A genome-wide expression analysis indicated decreased endoplasmic reticulum stress in the strain that moderately overexpressed SEC16, which was consistent with a decreased volume of the endoplasmic reticulum. Additionally, fewer mitochondria were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-α-glucosidase, indicating that this mechanism is of general relevance. IMPORTANCE There is an increasing demand for recombinant proteins to be used as enzymes and pharmaceuticals. The yeast Saccharomyces cerevisiae is a cell factory that is widely used to produce recombinant proteins. Our study revealed that moderate overexpression of SEC16 increased recombinant protein secretion in S. cerevisiae. This new strategy can be combined with other targets to engineer cell factories to efficiently produce protein in the future.

scholarly journals Boosting Secretion of Extracellular Protein byEscherichia colivia Cell Wall Perturbation

2018 ◽  
Vol 84 (20) ◽  
Author(s):  
Haiquan Yang ◽  
Xiao Lu ◽  
Jinyuan Hu ◽  
Yuan Chen ◽  
Wei Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Protein Secretion ◽  
Extracellular Protein ◽  
Deletion Mutants ◽  
Cell Factory ◽  
Content Type ◽  
E Coli ◽  
Cell Wall Peptidoglycan ◽  
Extracellular Secretion

ABSTRACTEscherichia coliis one of the most widely used host microorganisms for recombinant protein expression and metabolic engineering, but it cannot efficiently secrete recombinant proteins to extracellular space. Here, extracellular protein secretion was enhanced inE. coliby deleting twod,d-carboxypeptidase genes (dacAanddacB, single and double deletions) to perturb the cell wall peptidoglycan network. Deletion ofdacAanddacBenhanced the accumulation of intracellular soluble peptidoglycan inE. coliand affected cell morphology, resulting in a more irregular cell shape and the appearance of transparent bulges. Deletion ofdacAanddacBappears to disrupt the normal rigid structure, presumably due to perturbation and destruction of the cell wall peptidoglycan network. The extracellular green fluorescent protein (GFP) fluorescence intensity of deletion mutants was increased by >2.0-fold compared with that of control cells, and that of the double deletion mutant was increased by 2.7-fold. Extracellular recombinant fibroblast growth factor receptor 2 (FGFR2) and collagen E4 secretion in deletion mutants was also enhanced compared with that in the control cells. Additionally, the extracellular recombinant amylase activity of single-deletion mutants BL21 ΔdacApETDuet-amykand BL21 ΔdacBpETDuet-amykwas increased 2.5- and 3.1-fold, respectively. The extracellular distribution of α-galactosidase by deletion mutants was also increased by >2.0-fold. Deletion ofdacAanddacBincreased outer membrane permeability, which could explain the enhanced extracellular protein secretion.IMPORTANCECell surface structure stabilization is important for extracellular secretion of proteins inEscherichia coli. As the main constituent of the cell wall, peptidoglycan contributes to cell structure robustness and stability. Here, we perturbed the peptidoglycan network by deletingdacAanddacBgenes encodingd,d-carboxypeptidase enzymes to improve extracellular protein secretion. This new strategy could enhance the capacity ofE. colias a microbial cell factory for extracellular secretion of proteins and chemicals.

scholarly journals RNAi expression tuning, microfluidic screening, and genome recombineering for improved protein production inSaccharomyces cerevisiae

2019 ◽  
Vol 116 (19) ◽  
pp. 9324-9332 ◽  
Author(s):  
Guokun Wang ◽  
Sara M. Björk ◽  
Mingtao Huang ◽  
Quanli Liu ◽  
Kate Campbell ◽  
...  
Keyword(s):  
Protein Secretion ◽  
High Throughput ◽  
Protein Modification ◽  
Protein Production ◽  
Enrichment Analysis ◽  
Cell Factory ◽  
Cellular Processes ◽  
Cellular Machinery ◽  
Specific Proteins

The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeastSaccharomyces cerevisiaethat allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1,AAD4,ADE8, andSDH1), protein modification and degradation (VPS73,KTR2,CNL1, andSSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.

scholarly journals Construction and application of a CRISPR/Cas9-assisted genomic editing system for Corynebacterium glutamicum

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chengzhen Yao ◽  
Xiaoqing Hu ◽  
Xiaoyuan Wang
Keyword(s):  
Corynebacterium Glutamicum ◽  
Cell Factory ◽  
Temperature Sensitive ◽  
Microbial Cell Factory ◽  
Time Saving ◽  
Editing Efficiency ◽  
Highly Efficient ◽  
Native Promoter ◽  
Insertion And Deletion ◽  
Genomic Editing

AbstractCorynebacterium glutamicum is widely used as microbial cell factory for various bioproducts, but its genomic editing efficiency needs to be improved. In this study, a highly efficient CRISPR/Cas9-assisted genomic editing system for C. glutamicum was constructed. This system mainly involves a plasmid and can be used for both gene insertion and deletion in the chromosome of C. glutamicum. The recombinant plasmid for the target gene containing all the editing elements, and first constructed it in E. coli, then purified and transformed it into C. glutamicum. This temperature-sensitive plasmid was cured at high temperature after the genomic editing was completed in C. glutamicum. Using this genetic editing system, the genetic editing efficiency in C. glutamicum ATCC 13032 could reach 95%. The whole work of editing could be done in 8–9 days and showed most time-saving compared to the reported. Using this system, the native promoter of gdhA1 in ATCC 13032 has been replaced with the strong promoter PtacM, and more than 10 genes in ATCC 13032 have been deleted. The results demonstrate that this CRISPR/Cas9-assisted system is highly efficient and very suitable for genome editing in C. glutamicum.

scholarly journals Mechanism‐Guided Design of Highly Efficient Protein Secretion and Lipid Conversion for Biomanufacturing and Biorefining

2019 ◽  
pp. 1801980 ◽  
Author(s):  
Shangxian Xie ◽  
Su Sun ◽  
Furong Lin ◽  
Muzi Li ◽  
Yunqiao Pu ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Highly Efficient

scholarly journals Recombinant protein secretion by Bacillus subtilis and Lactococcus lactis: pathways, applications, and innovation potential

2021 ◽  
Author(s):  
Jolanda Neef ◽  
Jan Maarten van Dijl ◽  
Girbe Buist
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Lactococcus Lactis ◽  
Protein Secretion ◽  
Protein Production ◽  
Cell Factory ◽  
Efficient Production ◽  
High Quality ◽  
Gram Positive ◽  
Secretion Pathway

Abstract Secreted recombinant proteins are of great significance for industry, healthcare and a sustainable bio-based economy. Consequently, there is an ever-increasing need for efficient production platforms to deliver such proteins in high amounts and high quality. Gram-positive bacteria, particularly bacilli such as Bacillus subtilis, are favored for the production of secreted industrial enzymes. Nevertheless, recombinant protein production in the B. subtilis cell factory can be very challenging due to bottlenecks in the general (Sec) secretion pathway as well as this bacterium’s intrinsic capability to secrete a cocktail of highly potent proteases. This has placed another Gram-positive bacterium, Lactococcus lactis, in the focus of attention as an alternative, non-proteolytic, cell factory for secreted proteins. Here we review our current understanding of the secretion pathways exploited in B. subtilis and L. lactis to deliver proteins from their site of synthesis, the cytoplasm, into the fermentation broth. An advantage of this cell factory comparison is that it identifies opportunities for protein secretion pathway engineering to remove or bypass current production bottlenecks. Noteworthy new developments in cell factory engineering are the mini-Bacillus concept, highlighting potential advantages of massive genome minimization, and the application of thus far untapped ‘non-classical’ protein secretion routes. Altogether, it is foreseen that engineered lactococci will find future applications in the production of high-quality proteins at the relatively small pilot scale, while engineered bacilli will remain a favored choice for protein production in bulk.

scholarly journals FlbA-Regulated GenerpnRIs Involved in Stress Resistance and Impacts Protein Secretion whenAspergillus nigerIs Grown on Xylose

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
David Aerts ◽  
Stijn G. van den Bergh ◽  
Harm Post ◽  
Maarten A. F. Altelaar ◽  
Mark Arentshorst ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Niger ◽  
Protein Secretion ◽  
Stress Resistance ◽  
Ribosomal Subunit ◽  
Biofuel Production ◽  
Cell Factory ◽  
Content Type ◽  
Proteotoxic Stress ◽  
Transcription Factor Genes

ABSTRACTProteins are secreted throughout the mycelium ofAspergillus nigerexcept for the sporulating zone. A link between sporulation and repression of protein secretion was underlined by the finding that inactivation of the sporulation geneflbAresults in mycelial colonies that secrete proteins throughout the colony. However, ΔflbAstrain hyphae also lyse and have thinner cell walls. This pleiotropic phenotype is associated with differential expression of 36 predicted transcription factor genes, one of which,rpnR, was inactivated in this study. Sporulation, biomass, and secretome complexity were not affected in the ΔrpnRdeletion strain of the fungus. In contrast, ribosomal subunit expression and protein secretion into the medium were reduced whenA. nigerwas grown on xylose. Moreover, the ΔrpnRstrain showed decreased resistance to H2O2and the proteotoxic stress-inducing agent dithiothreitol. Taking the data together, RpnR is involved in proteotoxic stress resistance and impacts protein secretion whenA. nigeris grown on xylose.IMPORTANCEAspergillus nigersecretes a large amount and diversity of industrially relevant enzymes into the culture medium. This makes the fungus a widely used industrial cell factory. For instance, carbohydrate-active enzymes ofA. nigerare used in biofuel production from lignocellulosic feedstock. These enzymes represent a major cost factor in this process. Higher production yields could substantially reduce these costs and therefore contribute to a more sustainable economy and less dependence on fossil fuels. Enzyme secretion is inhibited inA. nigerby asexual reproduction. The sporulation protein FlbA is involved in this process by impacting the expression of 36 predicted transcription factor genes. Here, we show that one of these predicted transcriptional regulators, RpnR, regulates protein secretion and proteotoxic stress resistance. The gene is thus an interesting target to improve enzyme production inA. niger.

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