Ute A Hoffmann, Anna Z Schuppe, Axel Knave, Emil Sporre, Hjalmar Brismar, Elias Englund, Per-Olof Syrén, Elton P Hudson
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引用次数: 0
摘要
Rubisco是无机碳进入生物圈的主要入口,也是全球碳系统的核心角色。相对较低的比活性和接受O2作为底物的倾向使Rubisco成为酶工程的一个有吸引力但具有挑战性的目标。我们开发了一种酶工程和Rubisco筛选平台,使用模型蓝藻Synechocystis sp. PCC 6803。从Gallionella的Form II Rubisco开始,我们首先证明了该酶可以取代Synechocystis中天然的Form I Rubisco,并且生长速度对CO2和O2水平变得敏感。我们通过将系统发育引导模型EV突变与“硅进化”相结合,解决了在没有事先实验知识的情况下设计Gallionella Rubisco零射击输入库的挑战。为了比较Rubisco变体,我们在不同的气体饲料中竞争生长,并结合深度测序,建立了聚囊藻(n = 16)的多位点突变文库。我们发现了一种氨基酸交换,增加了Gallionella Rubisco的热稳定性,并传递了对其他有害氨基酸交换的弹性。该平台是高通量筛选聚胞菌中Rubisco变体和创建优化酶变体以加速蓝藻和叶绿体中的Calvin-Benson-Bassham循环的第一步。
A Cyanobacterial Screening Platform for Rubisco Mutant Variants.
Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. The relatively low specific activity and tendency to accept O2 as a substrate have made Rubisco an attractive but challenging target for enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO2 and O2 levels. We address the challenge of designing a zero-shot input library of the Gallionella Rubisco, without prior experimental knowledge, by coupling the phylogenetically guided model EV mutation with "in silico evolution". This multisite mutagenesis library of Synechocystis (n = 16) was subjected to competitive growth in different gas feeds coupled to deep sequencing, in order to compare Rubisco variants. We identified an amino acid exchange that increased the thermostability of Gallionella Rubisco and conveyed resilience to otherwise detrimental amino acid exchanges. The platform is a first step toward high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts.
期刊介绍:
The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism.
Topics may include, but are not limited to:
Design and optimization of genetic systems
Genetic circuit design and their principles for their organization into programs
Computational methods to aid the design of genetic systems
Experimental methods to quantify genetic parts, circuits, and metabolic fluxes
Genetic parts libraries: their creation, analysis, and ontological representation
Protein engineering including computational design
Metabolic engineering and cellular manufacturing, including biomass conversion
Natural product access, engineering, and production
Creative and innovative applications of cellular programming
Medical applications, tissue engineering, and the programming of therapeutic cells
Minimal cell design and construction
Genomics and genome replacement strategies
Viral engineering
Automated and robotic assembly platforms for synthetic biology
DNA synthesis methodologies
Metagenomics and synthetic metagenomic analysis
Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction
Gene optimization
Methods for genome-scale measurements of transcription and metabolomics
Systems biology and methods to integrate multiple data sources
in vitro and cell-free synthetic biology and molecular programming
Nucleic acid engineering.