scholarly journals Towards a Synthetic Biology Toolset for Metallocluster Enzymes in Biosynthetic Pathways: What We Know and What We Need

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6930
Author(s):  
Helena Shomar ◽  
Gregory Bokinsky
Keyword(s):  
Synthetic Biology ◽  
Protein Interactions ◽  
Enzyme Stability ◽  
Functional Expression ◽  
Specific Protein ◽  
Live Cells ◽  
Proper Function ◽  
Biosynthetic Pathways ◽  
Renewable Materials ◽  
Cell Factories

Microbes are routinely engineered to synthesize high-value chemicals from renewable materials through synthetic biology and metabolic engineering. Microbial biosynthesis often relies on expression of heterologous biosynthetic pathways, i.e., enzymes transplanted from foreign organisms. Metallocluster enzymes are one of the most ubiquitous family of enzymes involved in natural product biosynthesis and are of great biotechnological importance. However, the functional expression of recombinant metallocluster enzymes in live cells is often challenging and represents a major bottleneck. The activity of metallocluster enzymes requires essential supporting pathways, involved in protein maturation, electron supply, and/or enzyme stability. Proper function of these supporting pathways involves specific protein–protein interactions that remain poorly characterized and are often overlooked by traditional synthetic biology approaches. Consequently, engineering approaches that focus on enzymatic expression and carbon flux alone often overlook the particular needs of metallocluster enzymes. This review highlights the biotechnological relevance of metallocluster enzymes and discusses novel synthetic biology strategies to advance their industrial application, with a particular focus on iron-sulfur cluster enzymes. Strategies to enable functional heterologous expression and enhance recombinant metallocluster enzyme activity in industrial hosts include: (1) optimizing specific maturation pathways; (2) improving catalytic stability; and (3) enhancing electron transfer. In addition, we suggest future directions for developing microbial cell factories that rely on metallocluster enzyme catalysis.

scholarly journals PCR & Go: A Pre-installed Expression Chassis for Facile Integration of Multi-Gene Biosynthetic Pathways

2021 ◽  
Vol 8 ◽  
Author(s):  
Mingming Qi ◽  
Bei Zhang ◽  
Lihong Jiang ◽  
Saijuan Xu ◽  
Chang Dong ◽  
...  
Keyword(s):  
High Efficiency ◽  
Functional Expression ◽  
Single Step ◽  
Yeast Genome ◽  
Biosynthetic Pathways ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Plasmid Library ◽  
Yeast Chromosome ◽  
Β Carotene

The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.

LUCIFERASES FOR THE STUDY OF PROTEIN–PROTEIN INTERACTIONS IN LIVE CELLS AND ANIMALS

Nano LIFE ◽  
2010 ◽  
Vol 01 (01n02) ◽  
pp. 79-87 ◽  
Author(s):  
A. K. M. KAFI ◽  
MITSURU HATTORI ◽  
TAKEAKI OZAWA
Keyword(s):  
Transcriptional Regulation ◽  
Protein Interactions ◽  
Protein Modification ◽  
Specific Protein ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Recent Developments ◽  
Split Luciferase Complementation ◽  
Spatio Temporal ◽  
Split Luciferase

Many imaging technologies based on luminescent proteins have proven useful for detecting protein–protein interactions, tracking cells in mice, and monitoring transcriptional regulation of specific genes. Especially, novel bioluminescent proteins have advanced the study of induced protein interactions and protein modification in live cells and animals. This review focuses on recent developments of bioluminescent probes for quantitative evaluation of specific protein–protein interactions and their spatio-temporal imaging by means of split luciferase complementation techniques. From the comparison between fluorescent and bioluminescent proteins, advantages and drawbacks of the bioluminescence techniques are described.

Distribution of lipid raft markers in live cells

2004 ◽  
Vol 32 (5) ◽  
pp. 673-675 ◽  
Author(s):  
O.O. Glebov ◽  
B.J. Nichols
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Interactions ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Receptor ◽  
Specific Protein ◽  
Live Cells ◽  
Jurkat T Cells

GPI (glycosylphosphatidylinositol)-anchored proteins are characteristic components of biochemically defined lipid rafts. Rafts may be involved in T-cell stimulation, but it is not clear whether molecules involved in TCR (T-cell receptor) signalling are partitioned to T-cell synapses through raft microdomains or through specific protein–protein interactions. We have used FRET (fluorescence resonance energy transfer) analysis to study the distribution of GPI-anchored fluorescent proteins in the plasma membrane of live cells. Multiple criteria suggested that FRET between different GPI-anchored fluorescent proteins in COS-7 or unstimulated Jurkat T-cells is generated by a random, unclustered distribution. Stimulation of TCR signalling in Jurkat T-cells by beads coated with antibodies against TCR subunits resulted in localized increases in fluorescence of raft markers. However, measurements of FRET and ratio imaging showed that there was no detectable clustering and no overall enrichment of raft markers in these regions.

scholarly journals Upregulation of Vitamin C Transporter Functional Expression in 5xFAD Mouse Intestine

Nutrients ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 617
Author(s):  
Trevor Teafatiller ◽  
Christopher W. Heskett ◽  
Anshu Agrawal ◽  
Jonathan S. Marchant ◽  
Janet E. Baulch ◽  
...  
Keyword(s):  
Mouse Model ◽  
Functional Expression ◽  
Specific Protein ◽  
Glutathione Peroxidase 1 ◽  
Stress Response Genes ◽  
Nuclear Rna ◽  
The Status ◽  
5Xfad Mouse ◽  
Mouse Intestine ◽  
5Xfad Mice

The process of obtaining ascorbic acid (AA) via intestinal absorption and blood circulation is carrier-mediated utilizing the AA transporters SVCT1 and SVCT2, which are expressed in the intestine and brain (SVCT2 in abundance). AA concentration is decreased in Alzheimer’s disease (AD), but information regarding the status of intestinal AA uptake in the AD is still lacking. We aimed here to understand how AA homeostasis is modulated in a transgenic mouse model (5xFAD) of AD. AA levels in serum from 5xFAD mice were markedly lower than controls. Expression of oxidative stress response genes (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1)) were significantly increased in AD mice jejunum, and this increase was mitigated by AA supplementation. Uptake of AA in the jejunum was upregulated. This increased AA transport was caused by a marked increase in SVCT1 and SVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression. A significant increase in the expression of HNF1α and specific protein 1 (Sp1), which drive SLC23A1 and SLC23A2 promoter activity, respectively, was observed. Expression of hSVCT interacting proteins GRHPR and CLSTN3 were also increased. SVCT2 protein and mRNA expression in the hippocampus of 5xFAD mice was not altered. Together, these investigations reveal adaptive up-regulation of intestinal AA uptake in the 5xFAD mouse model.

Structure, Function, Involvement in Diseases and Targeting of 14-3-3 Proteins: An Update

2018 ◽  
Vol 25 (1) ◽  
pp. 5-21 ◽  
Author(s):  
Ylenia Cau ◽  
Daniela Valensin ◽  
Mattia Mori ◽  
Sara Draghi ◽  
Maurizio Botta
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Protein Partners ◽  
High Sequence Homology ◽  
Small Molecule Modulators

14-3-3 is a class of proteins able to interact with a multitude of targets by establishing protein-protein interactions (PPIs). They are usually found in all eukaryotes with a conserved secondary structure and high sequence homology among species. 14-3-3 proteins are involved in many physiological and pathological cellular processes either by triggering or interfering with the activity of specific protein partners. In the last years, the scientific community has collected many evidences on the role played by seven human 14-3-3 isoforms in cancer or neurodegenerative diseases. Indeed, these proteins regulate the molecular mechanisms associated to these diseases by interacting with (i) oncogenic and (ii) pro-apoptotic proteins and (iii) with proteins involved in Parkinson and Alzheimer diseases. The discovery of small molecule modulators of 14-3-3 PPIs could facilitate complete understanding of the physiological role of these proteins, and might offer valuable therapeutic approaches for these critical pathological states.

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