Resurrecting essential amino acid biosynthesis in mammalian cells.

Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation. 13C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.

Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond.

We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within PRE4 (YFR050C), encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the HIS2 transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.

"Perfect" designer chromosome V and behavior of a ring derivative.

Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.

Generation of genetically-altered mice producing very low levels of coagulation factorVII.

It has been shown earlier that mice with a total targeted deletion of the factorVII gene (FVII(-/-)) die perinatally, thereby precluding study of adult animals with this total deficiency. Consequently, mice producing very low levels of FVII were developed by targeted replacement of the wild-type (WT) murine FYII gene with its corresponding cDNA, under control of the tetracycline transactivator (tTA) promoter. When backcrossed into the C57BI/6 strain, unchallenged mice containing two replaced FVII(tTA) alleles (FVII(tTA/tTA) produce approximately 0.7% of WT FVII levels, but yet live to adulthood despite displaying severely downregulated overall thrombin production and spontaneously developing cardiac fibrosis at a young adult age. This genetically-altered mouse line provides an excellent animal model to study consequences of a severe FVII deficiency in unchallenged mice and in mice subjected to a variety of experimental challenges.

Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units.

We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae.

Mice with a severe deficiency of the endothelial protein C receptor gene develop, survive, and reproduce normally, and do not present with enhanced arterial thrombosis after challenge.

The endothelial cell Protein C receptor (EPCR) functions to enhance activation of anticoagulant Protein C (PC) by the thrombin/ thrombomodulin (Tm) complex on the surface of the endothelium. This overall system functions in anticoagulation, profibrinolytic, and antiinflammatory responses. Mice with a severe targeted deficiency of this receptor have been generated by integration of exogenous DNA elements into the 5'-untranslated region of the EPCR gene. Despite the retention of the entire endogenous EPCR coding sequence in the altered EPCR gene locus, only very low EPCR message contents were detected in mice by quantitative RT-PCR during embryogenesis and up to at least early adulthood. Immunohistochemical analysis of various regions of the arterial tree of mice up to 4 months of age, employing an anti-murine EPCR antibody, confirmed that undetectable levels of this protein were present in arterial regions during these periods. Despite this, these mice are not more prone to arterial thrombosis after challenge in a FeCl3 carotid artery thrombosis model. Small amounts (<10% of wild-type) of this protein were found in other tissues. Matings of mice homozygous for this deficiency led to normal births and survival of the offspring, in contrast to results by others demonstrating early embryonic lethality of a total EPCR deficiency. These data further show that minimal levels of EPCR are able to support male and female virility, as well as embryonic development, birth, and survival to adulthood.

Total synthesis of a functional designer eukaryotic chromosome.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in a-mater derivatives resulting from loss of the MATα allele on synIII. The complete design and synthesis of synIII establishes S. cerevisiae as the basis for designer eukaryotic genome biology.

Coagulation factor Xa modulates airway remodeling in a murine model of asthma.

RATIONALE: Previous studies have demonstrated that dysregulated coagulation and fibrinolysis contribute to the pathogenesis of asthma. OBJECTIVE: The role of procoagulant factor X in a murine model of ovalbumin (OVA)-induced asthma was investigated. METHODS: Biochemical, cellular, and physiologic in vivo and in vitro approaches were used to determine effects of factor X on the asthmatic response in mice. MEASUREMENTS AND MAIN RESULTS: Factor X transcript levels and factor Xa activity were increased in lungs of asthmatic mice challenged with OVA, compared with controls treated with phosphate-buffered saline. Factor X was highly expressed in bronchoalveolar lavage fluid macrophages from asthmatic mice. Treatment of mice with the factor Xa inhibitor fondaparinux during the last 4 wk of OVA challenge resulted in the attenuation of airway hyperresponsiveness but did not alter infiltration of inflammatory cells into the lung. There was a significant decrease in the thickness of the mucosal layer and in lung collagen deposition in fondaparinux-treated mice. In vitro investigations using human mucus-producing NCI-H292 cells indicated that exogenous factor Xa enhanced mucin production in a dose-dependent manner. Levels of amphiregulin, a protein that induces mucin production, were also increased in cells stimulated by factor Xa. CONCLUSIONS: The results of this study introduce a novel participant in the asthmatic response and indicate that factor Xa functions in airway remodeling in asthma by stimulating mucin production, through regulation of amphiregulin expression and collagen deposition.

A cardioprotective role for the endothelial protein C receptor in lipopolysaccharide-induced endotoxemia in the mouse.

A model of gram-negative lethal endotoxin shock, involving continuous peritoneal infusion of lipopolysaccharide (LPS), has been applied to wild-type (WT) mice and mice with a severe deficiency of endothelial protein C receptor (EPCR(delta/delta)). The survival of EPCR(delta/delta) mice was significantly diminished as compared to WT mice after administration of LPS via this route. Heart rates and central blood pressures also were significantly more depressed in EPCR(delta/delta) mice, indicating that the receptor-based protein C (PC) pathway functions in regulation of hemodynamic properties in the mouse. Further, heart muscle damage was more severe in EPCR(delta/delta) mice as compared to WT mice after endotoxin administration, as revealed by the more elevated plasma myoglobin levels in EPCR(delta/delta) mice and by microscopic examination of stained heart sections. Neutrophil infiltration was more pronounced in heart tissue of EPCR(delta/delta) mice, perhaps in response to the greatly increased expression level of the chemokine, MIP-2, which also significantly more up-regulated in the LPS-treated EPCR(delta/delta) mouse cohort. In conclusion, a severe deficiency of EPCR adversely affects survival of mice subjected to continuous infusion of endotoxin, via contributions of more responsive hemodynamic and cardiac alterations, thus suggesting that, among its other functions, the PC-based receptor system has a cardioprotective role after acute inflammatory challenge.

Enhanced in vitro proliferation of aortic endothelial cells from plasminogen activator inhibitor-1-deficient mice.

A number of studies have identified a role for plasminogen activator inhibitor-1 (PAI-1) in regulating angiogenesis, although results from these investigations have been controversial. Among key cellular components of an angiogenic vessel are endothelial cells (ECs), which are known to express several components of the fibrinolytic system, including PAI-1. Thus, alterations in expression of this protein may have direct effects on cell functions involved in vascular development. In this study, ECs were isolated from sections of murine arterial trees from wild-type and PAI-1-deficient mice, and low passage (passages 3-4) homogeneous subpopulations of these cells were obtained by immunomagnetic absorption to antibodies against CD105/CD106. The homogeneity of these cells was further assessed by immunohistochemistry and quantitative real-time reverse transcription-PCR analysis of a number of EC markers. Comparative analyses of EC proliferation (one event associated with angiogenesis) in wild-type and PAI-1-deficient ECs demonstrated enhanced rates of cell growth for PAI-1-deficient cells relative to wild-type cells. Additional studies demonstrated similar levels of both vascular endothelial growth factor (VEGF) mRNA and protein and enhanced levels of VEGF receptor-1 (Flt-1) mRNA in PAI-1-deficient cells relative to wild-type cells. Immunohistochemical analyses indicated that phosphorylation of Akt was also enhanced in PAI-1-deficient cells, implicating VEGF-induced cell signaling alterations in PAI-1-deficient cells, the result of which may contribute to alterations in cell proliferation.