دانلود رایگان مقاله رونویسی ژن نوترکیب برای زیست شناسی مصنوعی سلول CHO

Abstract

The next generation of mammalian cell factories for biopharmaceutical production will be genetically engineered to possess both generic and product-specific manufacturing capabilities that may not exist naturally. Introduction of entirely new combinations of synthetic functions (e.g. novel metabolic or stress-response pathways), and retro-engineering of existing functional cell modules will drive disruptive change in cellular manufacturing performance. However, before we can apply the core concepts underpinning synthetic biology (design, build, test) to CHO cell engineering we must first develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components we simultaneously introduce into the host cell factory. In this review we discuss how this can be achieved by design of engineered promoters that enable concerted control of recombinant gene transcription. We describe the specific mechanisms of transcriptional regulation that affect promoter function during bioproduction processes, and detail the highly-specific promoter design criteria that are required in the context of CHO cell engineering. The relative applicability of diverse promoter development strategies are discussed, including re-engineering of natural sequences, design of synthetic transcription factor-based systems, and construction of synthetic promoters. This review highlights the potential of promoter engineering to achieve precision transcriptional control for CHO cell synthetic biology.

5. Concluding remarks and future perspectives: utilizing promoter engineering strategies to enable CHO cell synthetic biology

In summary, there are three divergent routes available to engineer promoters for use in CHO cell synthetic biology. New promoter technology can be developed by i) optimizing endogenous promoters that have been identified by transcriptomics profiling, ii) designing transcriptional control systems that use synthetic TFs to regulate the activity of target synthetic promoters, or iii) constructing synthetic promoters from characterized building-blocks. Each of these strategies could be used to design promoters that have the specific functionalities required in the context of CHO cell engineering. For example, transcriptional control over a broad dynamic range in CHO cells could be achieved by engineering endogenous promoters of genes that have varying expression levels, modifying promoter architecture and/or protein domains in synthetic TF-based systems, or constructing libraries of synthetic promoters with varying building-block compositions. Similarly, the timing of gene expression could be controlled by engineering endogenous promoters of genes with appropriate spatiotemporal expression profiles, designing trigger-inducible synthetic TFs, or constructing synthetic promoters from TFRE blocks that have variable activity during bioproduction processes. Homologous recombination-mediated silencing in multigene circuits could be avoided by engineering endogenous promoters that have minimal sequence homology or constructing libraries of synthetic promoters with unique TFRE-complements. This would also minimize the risk of altering the host cell transcriptome via TF-titration, although of course the ultimate defence against this potential problem would be achieved by designing synthetic TF-based systems. Finally, protection against heterochromatin silencing may be improved by (re)designing endogenous and synthetic sequences in silico to optimize both their CpG content and nucleosome affinity (Curran et al., Li et al., 2014). By rationally utilizing the available design space in this way, promoters that exhibit all desired functionalities for CHO cell engineering can be constructed.