Functional Genomics Reveals Synthetic Lethality between Phosphogluconate Dehydrogenase and Oxidative Phosphorylation

1. Download high-res image (203KB)
2. Download full-size image

     

合成生物学与专利微生物菌种保藏

微生物菌种是一个“活的工厂”,是企业的技术核心,尤其是专利程序中涉及的微生物菌种因能产生巨大的经济效益受到社会各方面的关注。本文拟从专利微生物寄存的历史,专利法中对技术充分公开的要求出发,阐述在合成生物学背景下,部分生物技术领域的发明也能像机械领域发明一样,仅依靠说明书及相关附图记载支持就可重复和再现发明,专利申请并不需要前置程序的专利微生物菌种保藏。但作为人类文明的成果,发明人有义务适时将合成的微生物菌种进行遗传稳定性保藏,以利后续科学研究和社会发展的需要。     

Engineering an EGFR-binding Gp2 domain for increased hydrophilicity

The Gp2 domain is a 45 amino-acid scaffold that has been evolved for specific, high-affinity binding towards multiple targets and was proven useful in molecular imaging and biological antagonism. It was hypothesized that Gp2 may benefit from increased hydrophilicity for improved physiological distribution as well as for physicochemical robustness. We identified seven exposed hydrophobic sites for hydrophilic mutations and experimentally evaluated single mutants, which yielded six mutations that do not substantially hinder expression, binding affinity or specificity (to epidermal growth factor receptor), and thermal stability. Eight combinations of these mutations improved hydrophilicity relative to the parental Gp2 clone as assessed by reverse-phase high-performance liquid chromatography (p < 0.05). Secondary structures and refolding abilities of the selected single mutants and all multimutants were unchanged relative to the parental ligand. A variant with five hydrophobic-to-hydrophilic mutations was identified with enhanced solubility as well as reasonable binding affinity ( K _d = 53–63 nM), recombinant yield (1.3 ± 0.8 mg/L), and thermal stability ( T _m = 53 ± 3°C). An alternative variant with a cluster of three leucine-to-hydrophilic mutations was identified with increased solubility, nominally increased binding affinity ( K _d = 13–28 nM) and reasonable thermal stability ( T _m = 54.0 ± 0.6°C) but reduced yield (0.4 ± 0.3 mg/L). In addition, a ≥7°C increase in the midpoint of thermal denaturation was observed in one of the single mutants (T21N). These mutants highlight the physicochemical tradeoffs associated with hydrophobic-to-hydrophilic mutation within a small protein, improve the solubility and hydrophilicity of an existent molecular imaging probe, and provide a more hydrophilic starting point for discovery of new Gp2 ligands towards additional targets.     

Population‐dependent reproducible deviation from natural bread wheat genome in synthetic hexaploid wheat

Sequence elimination is one of the main mechanisms that increases the divergence among homoeologous chromosomes after allopolyploidization to enhance the stability of recently established lineages, but it can cause a loss of some economically important genes. Synthetic hexaploid wheat (SHW) is an important source of genetic variation to the natural hexaploid wheat (NHW) genepool that has low genetic diversity. Here, we investigated the change between SHW and NHW genomes by utilizing a large germplasm set of primary synthetics and synthetic derivatives. Reproducible segment elimination (RSE) was declared if a large chromosomal chunk (>5 cM) produced no aligned reads in more than five SHWs. RSE in five genomic regions was the major source of variation between SHW and NHW. One RSE eliminated almost the complete short arm of chromosome 1B, which contains major genes for flour quality, disease resistance and different enzymes. The occurrence of RSE was highly dependent on the choice of diploid and tetraploid parental lines, their ancestral subpopulation and admixture, e.g. SHWs derived from Triticum dicoccon or from one of two Aegilops tauschii subpopulations were almost free of RSE, while highly admixed parents had higher RSE rates. The rate of RSE in synthetic derivatives was almost double that in primary synthetics. Genome‐wide association analysis detected four loci with minor effects on the occurrence of RSE, indicating that both parental lines and genetic factors were affecting the occurrence of RSE. Therefore, pre‐pre‐breeding strategies should be applied before introducing SHW into pre‐breeding programs to ensure genomic stability and avoid undesirable gene loss.     

Crop genome‐wide association study: a harvest of biological relevance

With the advent of rapid genotyping and next‐generation sequencing technologies, genome‐wide association study (GWAS) has become a routine strategy for decoding genotype–phenotype associations in many species. More than 1000 such studies over the last decade have revealed substantial genotype–phenotype associations in crops and provided unparalleled opportunities to probe functional genomics. Beyond the many ‘hits’ obtained, this review summarizes recent efforts to increase our understanding of the genetic architecture of complex traits by focusing on non‐main effects including epistasis, pleiotropy, and phenotypic plasticity. We also discuss how these achievements and the remaining gaps in our knowledge will guide future studies. Synthetic association is highlighted as leading to false causality, which is prevalent but largely underestimated. Furthermore, validation evidence is appealing for future GWAS, especially in the context of emerging genome‐editing technologies.     

RAP-1 and the RAL-1/exocyst pathway coordinate hypodermal cell organization in Caenorhabditis elegans

The small Ras-like GTPase Rap1 has been identified as a regulator of integrin activation and cadherin-mediated cell-cell contacts. Surprisingly, null mutants of RAP-1 in Caenorhabditis elegans are viable and fertile. In a synthetic lethal RNAi screen with C. elegans rap-1 mutants, the Ras-like GTPase ral-1 emerged as one of seven genes specifically required for viability. Depletion of exoc-8 and sec-5, encoding two putative RAL-1 effectors and members of the exocyst complex, also caused lethality of rap-1 mutants, but did not affect wild-type worms. The RAP-1 and the RAL-1/exocyst pathway appear to coordinate hypodermal cell movement and elongation during embryonic development. They mediate their effect in part through targeting the alpha-catenin homologue HMP-1 to the lateral membrane. Genetic interactions show that the RAP-1 and RAL-1/exocyst pathway also act in parallel during larval stages. Together these data provide in vivo evidence for the exocyst complex as a downstream RAL-1 effector in cell migration.     

Sugar-linked biodegradable polymers: Regio-specific ester bonds of glucose hydroxyls in their reaction with maleic anhydride functionalized polystyrene and elucidation of the polymer structures formed

In the development of sugar-linked synthetic polymers as biodegradable polymers, it is imperative to know the variety of polymer structures formed by the reaction of a multi-functional sugar molecule with the functionalized synthetic polymer on which the sugar is to be anchored. Enzymes produced by the microorganisms causing the polymer to biodegrade can be sensitive to the particular type of sugar hydroxyl utilized (such as anomeric, primary, or secondary hydroxyl group) for getting anchored to the polymer. In this paper, we present synthesis of regio-specific ester derivatives of glucose with anhydride, functionalized polymers, i.e., ester formation specifically with the anomeric, primary or secondary hydroxyls of glucose. Characterization of these different esters groups was done using FTIR spectroscopy; each ester peak was further deconvoluted to yield its different components. For this purpose, we studied the reactions of d-glucose, 6-O-trityl glucose, methyl glucoside, 1,2-5,6-diisopropylidene-d-glucose, and 1,2,3,4-tetraacetyl-d-glucose with maleic anhydride functionalized polystyrene (PSMAH). In this study, the primary hydroxyl of glucose was found to be even more reactive than the anomeric hydroxyl. The peaks at 1716, 1725, and 1729–1737 cm⁻¹ were assigned to the ester carbonyl of the anomeric, primary, and secondary hydroxyls of glucose (C2, C3, and C4), respectively. An attempt was made to quantify the extent to which the different polymer structures are formed in a particular reaction by taking ratios of non-variable reference peaks (polystyrene peak at 1493 cm⁻¹) and variable peaks caused by the reaction (the residual anhydride carbonyl at 1780 cm⁻¹).     

Detection of Pseudomonas aeruginosa in water samples using a novel synthetic medium and impedimetric technology

AIMS: To investigate whether the use of a novel synthetic medium in conjunction with impedimetric technology could provide a rapid and automated detection of Pseudomonas aeruginosa in water samples. METHODS AND RESULTS: A selective synthetic medium (Z-broth) in which the only carbon and nitrogen source is acetamide was applied in direct impedimetric examination for the selective isolation of P. aeruginosa. A total of 1036 tap-water, well-water, swimming-pool water and dialysis water samples were investigated, and any P. aeruginosa contamination was detected in 7-24 h. Neither false-negative nor false-positive results were observed. CONCLUSIONS: The results of the present evaluation demonstrate that impedance measurement with the use of Z-broth is suitable for the rapid and automatic detection of P. aeruginosa in water. SIGNIFICANCE AND IMPACT OF THE STUDY: The main advantages of the method: 240 samples can be examined in one step, the procedure is fully automated, the results are obtained quickly and the labour and media requirements are low.     

Comparative analysis of biofilm levels in complete upper and lower dentures after brushing associated with specific denture paste and neutral soap

Objectives: The objective of this study was to compare and correlate biofilm levels in complete upper and lower prosthesis after brushing, associated with specific paste and soap, by means of computerised methodology. Materials and methods: Forty‐five complete denture wearers were selected and instructed to brush their prostheses (Soft Oral B 40) three times a day for 3 weeks with water (Control), specific paste for complete dentures (Corega Brite) (Experiment 1) and neutral soap (Experiment 2). The study was based on a cross‐over model and a wash‐out period was not included. For biofilm quantification, the internal surfaces were dyed (neutral red 1%), photographed (Canon EOS Digital) and the disclosed biofilm was measured with the Image Tool 2.0 software. The products were assessed by means of a questionnaire regarding their hygiene properties and acceptance. Results: The variance analysis indicated that the lower prostheses exhibited a mean biofilm percentage, significantly higher than the upper prostheses and that brushing with paste (Experiment 1) was more effective than soap (Experiment 2) and, in turn, this was more effective than water (Control). There was a high biofilm correlation (Pearson correlation) between both prostheses. Both products were well accepted by the patients, but the most favoured one was the paste. Conclusions: This was effective in controlling the biofilm and can be used preventatively in the maintenance of oral health by wearers of complete dentures. This is important where the lower prosthesis can harbour microorganisms which may act as a reservoir for other areas of the mouth and thus enhance the importance of proper hygiene.     

Exploring Performance Parameters of Artificial Allosteric Protein Switches

Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera’s activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen–deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.