scholarly journals Directed Evolution of Enzymes based on in vitro Programmable Self-Replication

2021 ◽  
Author(s):  
Adèle Dramé-Maigné ◽  
Rocio Espada ◽  
Giselle McCallum ◽  
Rémi Sieskind ◽  
Yannick Rondelez
Keyword(s):  
Directed Evolution ◽  
Feedback Loop ◽  
Enzyme Engineering ◽  
Molecular Networks ◽  
Individual Variant ◽  
Activity Screening ◽  
Nicking Enzyme ◽  
Self Replication

High-throughput directed evolution, implemented in well-controlled in vitro conditions, provides a powerful route for enzyme engineering. Most existing technologies are based on activity screening and require the sequential observation and sorting of each individual variant. By contrast, approaches based on autonomous feedback loops, linking phenotype to genotype replication, enable autonomous selection without screening. However, these approaches are only possible in vivo, or applicable to very specific activities, such as polymerases or ligases. Here, we leverage synthetic molecular networks to create a programmable in vitro feedback loop linking a target enzymatic activity to gene amplification. After encapsulation and lysis of up to 10^7 transformed variants, the genes present in each droplet are amplified according to the activity of the encoded enzyme, resulting in the autonomous enrichment of interesting sequences. Applied to a nicking enzyme with thermal or kinetic selection pressures, this method reveals detailed mutational landscapes and provides improved variants.

scholarly journals Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

Life ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 179
Author(s):  
Jorge D. García-García ◽  
Jaya Joshi ◽  
Jenelle A. Patterson ◽  
Lidimarie Trujillo-Rodriguez ◽  
Christopher R. Reisch ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Target Gene ◽  
Enzyme Engineering ◽  
Plant Evolution ◽  
Kinetic Properties ◽  
Continuous Systems ◽  
Yield And Quality ◽  
Plant Enzyme

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

scholarly journals Potential For Applying Continuous Directed Evolution To Plant Enzymes

2020 ◽  
Author(s):  
Jorge D. García-García ◽  
Jaya Joshi ◽  
Jenelle A. Patterson ◽  
Lidimarie Trujillo-Rodriguez ◽  
Christopher R. Reisch ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Target Gene ◽  
Enzyme Engineering ◽  
Plant Evolution ◽  
Kinetic Properties ◽  
Continuous Systems ◽  
Yield And Quality ◽  
Plant Enzyme

SUMMARYPlant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized faster than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

scholarly journals LncRNA MIR17HG promotes colorectal cancer liver metastasis by mediating a glycolysis-associated positive feedback circuit

Oncogene ◽  
2021 ◽  
Author(s):  
Senlin Zhao ◽  
Bingjie Guan ◽  
Yushuai Mi ◽  
Debing Shi ◽  
Ping Wei ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Liver Metastasis ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Metastatic Tumor ◽  
Feedback Circuit ◽  
Colorectal Cancer Liver Metastasis ◽  
Positive Feedback Circuit

AbstractGlycolysis plays a crucial role in reprogramming the metastatic tumor microenvironment. A series of lncRNAs have been identified to function as oncogenic molecules by regulating glycolysis. However, the roles of glycolysis-related lncRNAs in regulating colorectal cancer liver metastasis (CRLM) remain poorly understood. In the present study, the expression of the glycolysis-related lncRNA MIR17HG gradually increased from adjacent normal to CRC to the paired liver metastatic tissues, and high MIR17HG expression predicted poor survival, especially in patients with liver metastasis. Functionally, MIR17HG promoted glycolysis in CRC cells and enhanced their invasion and liver metastasis in vitro and in vivo. Mechanistically, MIR17HG functioned as a ceRNA to regulate HK1 expression by sponging miR-138-5p, resulting in glycolysis in CRC cells and leading to their invasion and liver metastasis. More interestingly, lactate accumulated via glycolysis activated the p38/Elk-1 signaling pathway to promote the transcriptional expression of MIR17HG in CRC cells, forming a positive feedback loop, which eventually resulted in persistent glycolysis and the invasion and liver metastasis of CRC cells. In conclusion, the present study indicates that the lactate-responsive lncRNA MIR17HG, acting as a ceRNA, promotes CRLM through a glycolysis-mediated positive feedback circuit and might be a novel biomarker and therapeutic target for CRLM.

scholarly journals CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jin-Chun Qi ◽  
Zhan Yang ◽  
Tao Lin ◽  
Long Ma ◽  
Ya-Xuan Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Activation ◽  
Positive Feedback ◽  
Feedback Loop ◽  
Biological Effects ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Positive Feedback Loop

Abstract Background Both E2F transcription factor and cyclin-dependent kinases (CDKs), which increase or decrease E2F activity by phosphorylating E2F or its partner, are involved in the control of cell proliferation, and some circRNAs and miRNAs regulate the expression of E2F and CDKs. However, little is known about whether dysregulation among E2Fs, CDKs, circRNAs and miRNAs occurs in human PCa. Methods The expression levels of CDK13 in PCa tissues and different cell lines were determined by quantitative real-time PCR and Western blot analysis. In vitro and in vivo assays were preformed to explore the biological effects of CDK13 in PCa cells. Co-immunoprecipitation anlysis coupled with mass spectrometry was used to identify E2F5 interaction with CDK13. A CRISPR-Cas9 complex was used to activate endogenous CDK13 and circCDK13 expression. Furthermore, the mechanism of circCDK13 was investigated by using loss-of-function and gain-of-function assays in vitro and in vivo. Results Here we show that CDK13 is significantly upregulated in human PCa tissues. CDK13 depletion and overexpression in PCa cells decrease and increase, respectively, cell proliferation, and the pro-proliferation effect of CDK13 is strengthened by its interaction with E2F5. Mechanistically, transcriptional activation of endogenous CDK13, but not the forced expression of CDK13 by its expression vector, remarkably promotes E2F5 protein expression by facilitating circCDK13 formation. Further, the upregulation of E2F5 enhances CDK13 transcription and promotes circCDK13 biogenesis, which in turn sponges miR-212-5p/449a and thus relieves their repression of the E2F5 expression, subsequently leading to the upregulation of E2F5 expression and PCa cell proliferation. Conclusions These findings suggest that CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 is responsible for PCa development. Targeting this newly identified regulatory axis may provide therapeutic benefit against PCa progression and drug resistance.

scholarly journals MDM2 Suppresses p73 Function without Promoting p73 Degradation

1999 ◽  
Vol 19 (5) ◽  
pp. 3257-3266 ◽  
Author(s):  
Xiaoya Zeng ◽  
Lihong Chen ◽  
Christine A. Jost ◽  
Ruth Maya ◽  
David Keller ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Feedback Loop ◽  
Human Lung ◽  
Activation Function ◽  
Wild Type ◽  
Mdm2 Protein ◽  
Induced Apoptosis ◽  
P53 Inactivation

ABSTRACT The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73α and p73β stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

scholarly journals PUMA amplifies necroptosis signaling by activating cytosolic DNA sensors

2018 ◽  
Vol 115 (15) ◽  
pp. 3930-3935 ◽  
Author(s):  
Dongshi Chen ◽  
Jingshan Tong ◽  
Liheng Yang ◽  
Liang Wei ◽  
Donna B. Stolz ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Dependent Manner ◽  
Dna Sensors ◽  
Necrotic Cell ◽  
Developmental Defects ◽  
Tnf Α ◽  
Amplification Mechanism ◽  
Signaling Process

Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-α and enhanced NF-κB activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo.

scholarly journals Disruption of TP63-miR-27a* Feedback Loop by Mutant TP53 in Head and Neck Cancer

2019 ◽  
Vol 112 (3) ◽  
pp. 266-277 ◽  
Author(s):  
Nikhil S Chari ◽  
Cristina Ivan ◽  
Xiandong Le ◽  
Jinzhong Li ◽  
Ainiwaer Mijiti ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Oral Cavity ◽  
Head And Neck ◽  
Feedback Loop ◽  
Growth Factor Receptor ◽  
Epidermal Growth

Abstract Background Alterations in the epidermal growth factor receptor and PI3K pathways in head and neck squamous cell carcinomas (HNSCCs) are frequent events that promote tumor progression. Ectopic expression of the epidermal growth factor receptor–targeting microRNA (miR), miR-27a* (miR-27a-5p), inhibits tumor growth. We sought to identify mechanisms mediating repression of miR-27a* in HNSCC, which have not been previously identified. Methods We quantified miR-27a* in 47 oral cavity squamous cell carcinoma patient samples along with analysis of miR-27a* in 73 oropharyngeal and 66 human papillomavirus–positive (HPV+) samples from The Cancer Genome Atlas. In vivo and in vitro TP53 models engineered to express mutant TP53, along with promoter analysis using chromatin immunoprecipitation and luciferase assays, were used to identify the role of TP53 and TP63 in miR-27a* transcription. An HNSCC cell line engineered to conditionally express miR-27a* was used in vitro to determine effects of miR-27a* on target genes and tumor cells. Results miR-27a* expression was repressed in 47 oral cavity tumor samples vs matched normal tissue (mean log2 difference = −0.023, 95% confidence interval = −0.044 to −0.002; two-sided paired t test, P = .03), and low miR-27a* levels were associated with poor survival in HPV+ and oropharyngeal HNSCC samples. Binding of ΔNp63α to the promoter led to an upregulation of miR-27a*. In vitro and in vivo findings showed that mutant TP53 represses the miR-27a* promoter, downregulating miR-27a* levels. ΔNp63α and nucleoporin 62, a protein involved in ΔNP63α transport, were validated as novel targets of miR-27a*. Conclusion Our results characterize a negative feedback loop between TP63 and miR-27a*. Genetic alterations in TP53, a frequent event in HNSCC, disrupt this regulatory loop by repressing miR-27a* expression, promoting tumor survival.

scholarly journals Soluble Heparin and Heparan Sulfate Glycosaminoglycans Interfere with Sonic Hedgehog Solubilization and Receptor Binding

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1607 ◽  
Author(s):  
Manikowski ◽  
Jakobs ◽  
Jboor ◽  
Grobe
Keyword(s):  
Heparan Sulfate ◽  
Sonic Hedgehog ◽  
Receptor Binding ◽  
Feedback Loop ◽  
Cancer Cell Growth ◽  
Shh Signaling ◽  
Epithelial Cancers ◽  
And Function

Sonic hedgehog (Shh) signaling plays a tumor-promoting role in many epithelial cancers. Cancer cells produce soluble a Shh that signals to distant stromal cells that express the receptor Patched (Ptc). These receiving cells respond by producing other soluble factors that promote cancer cell growth, generating a positive feedback loop. To interfere with reinforced Shh signaling, we examined the potential of defined heparin and heparan sulfate (HS) polysaccharides to block Shh solubilization and Ptc receptor binding. We confirm in vitro and in vivo that proteolytic cleavage of the N-terminal Cardin–Weintraub (CW) amino acid motif is a prerequisite for Shh solubilization and function. Consistent with the established binding of soluble heparin or HS to the Shh CW target motif, both polysaccharides impaired proteolytic Shh processing and release from source cells. We also show that HS and heparin bind to, and block, another set of basic amino acids required for unimpaired Shh binding to Ptc receptors on receiving cells. Both modes of Shh activity downregulation depend more on HS size and overall charge than on specific HS sulfation modifications. We conclude that heparin oligosaccharide interference in the physiological roles of HS in Shh release and reception may be used to expand the field of investigation to pharmaceutical intervention of tumor-promoting Shh functions.

scholarly journals Creating Hybrid Genes by Homologous Recombination

2000 ◽  
Vol 16 (1-2) ◽  
pp. 3-13 ◽  
Author(s):  
Peter L. Wang
Keyword(s):  
Directed Evolution ◽  
High Efficiency ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Strand Breaks ◽  
E Coli ◽  
Hybrid Genes ◽  
Distinct Features

Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution.In vitrorecombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties.In vivorecombination in bothE. coliand yeast is greatly enhanced by double-strand breaks; forE. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombinationIn vivohave distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recentlyin vivorecombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.

scholarly journals Interaction of Apoptotic Cells with Macrophages Upregulates COX-2/PGE2and HGF Expression via a Positive Feedback Loop

2014 ◽  
Vol 2014 ◽  
pp. 1-17 ◽  
Author(s):  
Ji Yeon Byun ◽  
Young-So Youn ◽  
Ye-Ji Lee ◽  
Youn-Hee Choi ◽  
So-Yeon Woo ◽  
...  
Keyword(s):  
Positive Feedback ◽  
Tissue Repair ◽  
Feedback Loop ◽  
Apoptotic Cell ◽  
Raw 264.7 Cells ◽  
Apoptotic Cells ◽  
Positive Feedback Loop ◽  
Cox 2

Recognition of apoptotic cells by macrophages is crucial for resolution of inflammation, immune tolerance, and tissue repair. Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) play important roles in the tissue repair process. We investigated the characteristics of macrophage COX-2 and PGE2expression mediated by apoptotic cells and then determined how macrophages exposed to apoptotic cellsin vitroandin vivoorchestrate the interaction between COX-2/PGE2and HGF signaling pathways. Exposure of RAW 264.7 cells and primary peritoneal macrophages to apoptotic cells resulted in induction of COX-2 and PGE2. The COX-2 inhibitor NS-398 suppressed apoptotic cell-induced PGE2production. Both NS-398 and COX-2-siRNA, as well as the PGE2receptor EP2 antagonist, blocked HGF expression in response to apoptotic cells. In addition, the HGF receptor antagonist suppressed increases in COX-2 and PGE2induction. Thein vivorelevance of the interaction between the COX-2/PGE2and HGF pathways through a positive feedback loop was shown in cultured alveolar macrophages followingin vivoexposure of bleomycin-stimulated lungs to apoptotic cells. Our results demonstrate that upregulation of the COX-2/PGE2and HGF in macrophages following exposure to apoptotic cells represents a mechanism for mediating the anti-inflammatory and antifibrotic consequences of apoptotic cell recognition.

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