Synthetic biology and organelle genomics: A rubisco library as a case study in evolutionary landscapes and organellar engineering
合成生物学和细胞器基因组学:rubisco 文库作为进化景观和细胞器工程的案例研究
基本信息
- 批准号:10441273
- 负责人:
- 金额:$ 9.39万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-07-12 至 2024-06-30
- 项目状态:已结题
- 来源:
- 关键词:AddressBasic ScienceBiochemicalBiochemistryBiological AssayBiological ModelsBiologyCarbonCareer MobilityCase StudyChloroplastsDataEngineeringEnvironmentEnzymesEscherichia coliEvolutionFood SupplyFutureGene ExpressionGene LibraryGene MutationGenerationsGenesGenomeGenomic LibraryGenomicsGoalsGrowthHealthHumanIn VitroIndividualInstitutesKnowledgeLaboratoriesLibrariesLinkMapsMentorsMetabolic ControlMetabolic PathwayModelingMolecular EvolutionMolecular TargetMutateNuclearNutritionalOrganellesOutcomePhasePhotosynthesisPlant ModelPlantsPopulationProcessProtein IsoformsProteinsResearch PersonnelRibulose-Bisphosphate CarboxylaseScienceSeriesTechnologyTestingTobaccoTrainingTransgenic OrganismsTransgenic PlantsTranslatingVariantWorkcarbon fixationcareerclimate changedesignexperimental studyfood securitygenetic selectiongenetic variantgenome editinghomologous recombinationimprovedin vivoinnovationinsightknockout genemachine learning modelmetabolic engineeringmutantmutation screeningnovelnucleaseplant growth/developmentpreferencepromotersynthetic biologytechnology developmenttranscription activator-like effector nucleases
项目摘要
Food security is a critical issue facing human health in the 21st century as the global population
approaches 10 billion and climate change threatens the food supply. Photosynthetic engineering has
been successful in improving crop yields but the central enzyme of carbon fixation, rubisco, has
remained intractable as a target of molecular evolution. The few plants tested with heterologous
rubiscos grow uniformly slower than their wild-type counterparts. The goal of this proposal is to
generate a chloroplast genome library in plants to explore the limits of rubisco function and
organellar genome organization.
In Aim 1 of this proposal I will explore the sequence-function landscape of a model, homodimeric
rubisco enzyme and a novel, linked variant I have generated. I have generated a deep-mutational
scan (OMS: all possible point-mutants) library and will assay their function in a uniquely suited
rubisco-dependent E. coli strain. I have shown that these linked rubiscos function in vivo and in
vitro and have already constructed the mutant library; in the K99 phase of this project I will
perform selections on this library and analyze the data with a machine learning model. In the R00
phase I will generate further libraries to explore regions of the sequence landscape predicted to
be rich in improved rubiscos with the goal of testing them in plants.
In Aim 2 I will improve chloroplast transformation technology in order to enable the generation of
chloroplast genome libraries of unprecedented size in the chloroplast-editing model plant N.
tabacum. To do this I will generate sequence-specific TALEN nucleases that will be expressed from
the nuclear genome and trafficked to the chloroplast where they will cut and disrupt the genome,
preferencing homologous recombination of transgenic donor ONA. After demonstrating TALEN-cutting in
the K99 phase I will develop this technology into an intracellular gene drive which will accelerate
chloroplast genome editing.
In Aim 3, with the technology developed in Aim 2 and the rubisco variants discovered in Aim 1, I
will generate libraries of plants with altered rubisco genes and promoters in order to demonstrate
the potential of rubisco engineering to improve plant growth. In addition, in the R00 phase, I will
produce a comprehensive chloroplast gene knockout library in order to answer fundamental questions
about the evolutionary flow of genes from organellar genomes to nuclear genomes.
My goal as an independent investigator is to study organellar biology in model plants in order to
explore the limits of engineering in individual proteins and metabolic pathways. My training in
organellar transformation, protein library generation and laboratory management during the K99
phase will prepare me well for the R00 phase. The Innovative Genomics Institute at UC Berkeley and
the laboratories of Ors. Oavid Savage and Brian Staskawicz in particular are the ideal environment
for my career transition, combining precisely the necessary mixture of expertise - protein
evolution and plant transformation, respectively.
作为全球人口,粮食安全是21世纪人类健康面临的关键问题
接近100亿,气候变化威胁到粮食供应。光合作用工程有
在提高作物产量方面取得了成功,但固碳的中心酶Rubisco
作为分子进化的目标仍然难以处理。用异源植物测试的为数不多的植物
红宝石的生长速度都比野生红宝石慢得多。这项提议的目标是
在植物中建立叶绿体基因组文库,以探索Rubisco功能的极限和
细胞器基因组组织。
在本提案的目标1中,我将探索同源二聚体模型的序列功能图景
Rubisco酶和我产生的一种新的连锁变体。我已经产生了一种深度突变
扫描(OMS:所有可能的点突变)文库,并将在唯一适合的情况下分析它们的功能
依赖Rubisco的大肠杆菌菌株。我已经证明了这些相连的Ruiscos在体内和体内发挥作用
已经构建了突变体库;在这个项目的K99阶段,我将
对该库执行选择,并使用机器学习模型分析数据。在R00中
第一阶段将生成更多的文库,以探索预计将
富含改良的红宝石,目标是在植物中测试它们。
在目标2中,我将改进叶绿体转化技术,以便能够产生
在叶绿体编辑模式植物N。
烟草属。为了做到这一点,我将产生序列特异性的TALEN核酸酶,它将从
核基因组并被运送到叶绿体,在那里它们将切割和破坏基因组,
倾向于转基因供体ONA的同源重组。在演示了TALEN-切入
K99第一阶段将把这项技术发展成细胞内的基因驱动,它将加速
叶绿体基因组编辑。
在目标3中,随着目标2中开发的技术和目标1中发现的Rubisco变体,我
将生成具有改变的Rubisco基因和启动子的植物文库,以便证明
Rubisco工程在改善植物生长方面的潜力。此外,在R00阶段,我将
制作全面的叶绿体基因敲除文库,以回答基本问题
关于基因从细胞器基因组到核基因组的进化流动。
作为一名独立研究员,我的目标是研究模式植物的细胞器生物学,以便
探索单个蛋白质和代谢途径的工程学极限。我受过的培训
K99期间细胞器的转化、蛋白质文库的生成和实验室管理
阶段将使我为R00阶段做好准备。加州大学伯克利分校创新基因组研究所和
奥斯的实验室。Oavid Savage和Brian Staskawicz尤其是理想的环境
在我的职业生涯过渡中,精确地结合了必要的专业知识-蛋白质
分别是进化和植物转化。
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Noam Prywes其他文献
Noam Prywes的其他文献
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{{ truncateString('Noam Prywes', 18)}}的其他基金
Synthetic biology and organelle genomics: A rubisco library as a case study in evolutionary landscapes and organellar engineering
合成生物学和细胞器基因组学:rubisco 文库作为进化景观和细胞器工程的案例研究
- 批准号:
10191932 - 财政年份:2021
- 资助金额:
$ 9.39万 - 项目类别:
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