transient-gene-expression-tobacco-using-gibson-assembly-gene

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Harvard University

Harvard Medical School

Nuclear transport and cancer: from mechanism to intervention.

Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.

Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond.

The design of artificial biological systems and the understanding of their natural counterparts are key objectives of the emerging discipline of synthetic biology. Toward both ends, research in synthetic biology has primarily focused on the construction of simple devices, such as transcription-based oscillators and switches. Construction of such devices should provide us with insight on the design of natural systems, indicating whether our understanding is complete or whether there are still gaps in our knowledge. Construction of simple biological systems may also lay the groundwork for the construction of more complex systems that have practical utility. To realize its full potential, biological systems design borrows from the allied fields of protein design and metabolic engineering. In this review, we describe the scientific accomplishments in this field, as well as its forays into biological part standardization and education of future biological designers.

Designing biological systems.

The presence of erythropoietin (Epo) receptors on cells besides red blood cell precursors, such as cancer cells or megakaryocyte precursors, can lead to side effects during Epo therapy including enhanced tumor growth and platelet production. It would be ideal if the action of Epo could be limited to erythroid precursors. To address this issue, we constructed single-chain variable fragment (scFv)-Epo fusion proteins that used the anti-glycophorin 10F7 scFv amino-terminal to Epo analogues that would have minimal activity alone. We introduced the Epo mutations N147A, R150A and R150E, which progressively weakened receptor affinity in the context of Epo alone, as defined by cell proliferation assays using TF-1 or UT-7 cells. Fusion of these mutant proteins to the 10F7 scFv significantly rescued the activity of the mutant proteins, but had a relatively small effect on wild-type Epo. For example, fusion to the 10F7 scFv enhanced the activity of Epo(R150A) by 10- to 27-fold, while a corresponding fusion to wild-type Epo enhanced its activity only up to 2.7-fold. When glycophorin was blocked by antibody competition or reduced by siRNA-mediated inhibition of expression, the activity of 10F7 scFv-Epo(R150A) was correspondingly reduced, while such inhibition had essentially no effect on the activity of 10F7 scFv-Epo(wild-type). In addition, potent stimulation of Epo receptors by 10F7 scFv-Epo(R150A) was observed in long-term proliferation and viability assays. Taken together, these results indicate that a combination of targeting and affinity modulation can be used to engineer forms of Epo with enhanced cell-type specificity.

Anti-glycophorin single-chain Fv fusion to low-affinity mutant erythropoietin improves red blood cell-lineage specificity.

The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued.

Insulation of a synthetic hydrogen metabolism circuit in bacteria.

Metabolic engineering of cyanobacteria has the advantage that sunlight and CO(2) are the sole source of energy and carbon for these organisms. However, as photoautotrophs, cyanobacteria generally lack transporters to move hydrophilic primary metabolites across membranes. To address whether cyanobacteria could be engineered to produce and secrete organic primary metabolites, Synechococcus elongatus PCC7942 was engineered to express genes encoding an invertase and a glucose facilitator, which mediated secretion of glucose and fructose. Similarly, expression of lactate dehydrogenase- and lactate transporter-encoding genes allowed lactate accumulation in the extracellular medium. Expression of the relevant transporter was essential for secretion. Production of these molecules was further improved by expression of additional heterologous enzymes. Sugars secreted by the engineered cyanobacteria could be used to support Escherichia coli growth in the absence of additional nutrient sources. These results indicate that cyanobacteria can be engineered to produce and secrete high-value hydrophilic products.

Engineering cyanobacteria to synthesize and export hydrophilic products.

The principles of DNA nanotechnology and protein engineering have been combined to generate a new class of artificial extracellular matrices. The potential of this material for ex vivo cellular scaffolding was demonstrated using experiments in which human cervical cancer cells were found to adhere strongly, stay alive, and grow with high migration rates. The use of DNA in our DNA/protein-based matrices makes these structures inherently amenable to structural tunability. By engineering single-stranded domains into the DNA portions, we were able to fine-tune the scaffold's persistence length and stiffness as perceived by cells. This was used to direct the outcome of the cell's cytoskeletal arrangement and overall shape, the status of its signal transduction protein p-FAK, and the localization of its intracellular transcription factors FOXO1a. This contribution lays the groundwork for the facile and modular construction of programmable extracellular matrices that can bring about the systematic study and replication of the naturally occurring extracellular niche.

A structurally tunable DNA-based extracellular matrix.

Although many microorganisms have been used for the bioindustrial generation of valuable metabolites, the productive potential of cyanobacterial species has remained largely unexplored. Cyanobacteria possess several advantages as organisms for bioindustrial processes, including simple input requirements, tolerance of marginal agricultural environments, rapid genetics, and carbon-neutral applications that could be leveraged to address global climate change concerns. Here, we review recent research involving the engineering of cyanobacterial species for the production of valuable bioindustrial compounds, including natural cyanobacterial products (e.g. sugars and isoprene), biofuels (e.g. alcohols, alkanes and hydrogen), and other commodity chemicals. Biological and economic obstacles to scaled cyanobacterial production are highlighted, and methods for increasing cyanobacterial production efficiencies are discussed.

Engineering cyanobacteria to generate high-value products.

The evolution of eukaryotic cells is widely agreed to have proceeded through a series of endosymbiotic events between larger cells and proteobacteria or cyanobacteria, leading to the formation of mitochondria or chloroplasts, respectively. Engineered endosymbiotic relationships between different species of cells are a valuable tool for synthetic biology, where engineered pathways based on two species could take advantage of the unique abilities of each mutualistic partner.

Towards a synthetic chloroplast.

The bioindustrial production of fuels, chemicals, and therapeutics typically relies upon carbohydrate inputs derived from agricultural plants, resulting in the entanglement of food and chemical commodity markets. We demonstrate the efficient production of sucrose from a cyanobacterial species, Synechococcus elongatus, heterologously expressing a symporter of protons and sucrose (cscB). cscB-expressing cyanobacteria export sucrose irreversibly to concentrations of >10 mM without culture toxicity. Moreover, sucrose-exporting cyanobacteria exhibit increased biomass production rates relative to wild-type strains, accompanied by enhanced photosystem II activity, carbon fixation, and chlorophyll content. The genetic modification of sucrose biosynthesis pathways to minimize competing glucose- or sucrose-consuming reactions can further improve sucrose production, allowing the export of sucrose at rates of up to 36.1 mg liter(-1) h illumination(-1). This rate of production exceeds that of previous reports of targeted, photobiological production from microbes. Engineered S. elongatus produces sucrose in sufficient quantities (up to ∼80% of total biomass) such that it may be a viable alternative to sugar synthesis from terrestrial plants, including sugarcane.

Rerouting carbon flux to enhance photosynthetic productivity.

The 3-hydroxypropionate (3-HPA) bicycle is unique among CO2-fixing systems in that none of its enzymes appear to be affected by oxygen. Moreover, the bicycle includes a number of enzymes that produce novel intermediates of biotechnological interest, and the CO2-fixing steps in this pathway are relatively rapid. We expressed portions of the 3-HPA bicycle in a heterologous organism, E. coli K12. We subdivided the 3-HPA bicycle into four sub-pathways: (1) synthesis of propionyl-CoA from acetyl-CoA, (2) synthesis of succinate from propionyl-CoA, (3) glyoxylate production and regeneration of acetyl-CoA, and (4) assimilation of glyoxylate and propionyl-CoA to form pyruvate and regenerate acetyl-CoA. We expressed the novel enzymes of the 3-HPA bicycle in operon form and used phenotypic tests for activity. Sub-pathway 1 activated a propionate-specific biosensor. Sub-pathway 2, found in non-CO2-fixing bacteria, was reassembled in E. coli using genes from diverse sources. Sub-pathway 3, operating in reverse, generated succinyl-CoA sufficient to rescue a sucAD(-) double mutant of its diaminopimelic acid (DAP) auxotrophy. Sub-pathway 4 was able to reduce the toxicity of propionate and allow propionate to contribute to cell biomass in a prpC(-)(2 methylcitrate synthase) mutant strain. These results indicate that all of the sub-pathways of the 3-HPA bicycle can function to some extent in vivo in a heterologous organism, as indicated by growth tests. Overexpression of certain enzymes was deleterious to cell growth, and, in particular, expression of MMC-CoA lyase caused a mucoid phenotype. These results have implications for metabolic engineering and for bacterial evolution through horizontal gene transfer.

Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth.

Medium-chain fatty acids (MCFAs, 4-12 carbons) are valuable as precursors to industrial chemicals and biofuels, but are not canonical products of microbial fatty acid synthesis. We engineered microbial production of the full range of even- and odd-chain-length MCFAs and found that MCFA production is limited by rapid, irreversible elongation of their acyl-ACP precursors. To address this limitation, we programmed an essential ketoacyl synthase to degrade in response to a chemical inducer, thereby slowing acyl-ACP elongation and redirecting flux from phospholipid synthesis to MCFA production. Our results show that induced protein degradation can be used to dynamically alter metabolic flux, and thereby increase the yield of a desired compound. The strategy reported herein should be widely useful in a range of metabolic engineering applications in which essential enzymes divert flux away from a desired product, as well as in the production of polyketides, bioplastics, and other recursively synthesized hydrocarbons for which chain-length control is desired.

Tailored fatty acid synthesis via dynamic control of fatty acid elongation.

Plant bioengineers require simple genetic devices for predictable localization of heterologous proteins to multiple subcellular compartments.

Targeting a heterologous protein to multiple plant organelles via rationally designed 5' mRNA tags.

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly.

The mammalian gut is a dynamic community of symbiotic microbes that interact with the host to impact health, disease, and metabolism. We constructed engineered bacteria that survive in the mammalian gut and sense, remember, and report on their experiences. Based on previous genetic memory systems, we constructed a two-part system with a "trigger element" in which the lambda Cro gene is transcribed from a tetracycline-inducible promoter, and a "memory element" derived from the cI/Cro region of phage lambda. The memory element has an extremely stable cI state and a Cro state that is stable for many cell divisions. When Escherichia coli bearing the memory system are administered to mice treated with anhydrotetracycline, the recovered bacteria all have switched to the Cro state, whereas those administered to untreated mice remain in the cI state. The trigger and memory elements were transferred from E. coli K12 to a newly isolated murine E. coli strain; the stability and switching properties of the memory element were essentially identical in vitro and during passage through mice, but the engineered murine E. coli was more stably established in the mouse gut. This work lays a foundation for the use of synthetic genetic circuits as monitoring systems in complex, ill-defined environments, and may lead to the development of living diagnostics and therapeutics.

Programmable bacteria detect and record an environmental signal in the mammalian gut.

Synthetic biology seeks to extend approaches from engineering and computation to redesign of biology, with goals such as generating new chemicals, improving human health, and addressing environmental issues. Early on, several guiding principles of synthetic biology were articulated, including design according to specification, separation of design from fabrication, use of standardized biological parts and organisms, and abstraction. We review the utility of these principles over the past decade in light of the field's accomplishments in building complex systems based on microbial transcription and metabolism and describe the progress in mammalian cell engineering.

Article	Year
Nuclear transport and cancer: from mechanism to intervention. Nature reviews. Cancer\| PubMed ID: 14732865	2004
Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond. Protein engineering, design & selection : PEDS\| PubMed ID: 15820978	2005
Designing biological systems. Genes & development\| PubMed ID: 17289915	2007
Anti-glycophorin single-chain Fv fusion to low-affinity mutant erythropoietin improves red blood cell-lineage specificity. Protein engineering, design & selection : PEDS\| PubMed ID: 20083493	2010
Insulation of a synthetic hydrogen metabolism circuit in bacteria. Journal of biological engineering\| PubMed ID: 20184755	2010
Engineering cyanobacteria to synthesize and export hydrophilic products. Applied and environmental microbiology\| PubMed ID: 20363793	2010
A structurally tunable DNA-based extracellular matrix. Journal of the American Chemical Society\| PubMed ID: 20925350	2010
Engineering cyanobacteria to generate high-value products. Trends in biotechnology\| PubMed ID: 21211860	2011
Towards a synthetic chloroplast. PloS one\| PubMed ID: 21533097	2011
Rerouting carbon flux to enhance photosynthetic productivity. Applied and environmental microbiology\| PubMed ID: 22307292	2012
Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: Toward horizontal transfer of autotrophic growth. Metabolic engineering\| PubMed ID: 23376595	2013
Tailored fatty acid synthesis via dynamic control of fatty acid elongation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23798438	2013
Targeting a heterologous protein to multiple plant organelles via rationally designed 5' mRNA tags. Journal of biological engineering\| PubMed ID: 24011257	2013
Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research\| PubMed ID: 24078086	2014
Programmable bacteria detect and record an environmental signal in the mammalian gut. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24639514	2014
Integrating Biological Redesign: Where Synthetic Biology Came From and Where It Needs to Go. Cell\| PubMed ID: 24679533	2014

Jeffrey C. Way

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