Spatial organization of heterologous metabolic system in vivo based on TALE

Zhu, Lv‐yun; Zhu, Lingyun; Wu, Xiaomin; Yuan, Zhang; Zhu, Qianhui; Fan, Dong-Yu; Zhu, Chushu; Zhang, Dongyi

doi:10.1038/srep26065

Cited by 16 publications

(12 citation statements)

References 35 publications

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“…Lee et al exhibited the great potential of cytoscaffold for enzyme organization [ 11 ]. Furthermore, in addition to protein scaffolds, DNA scaffolds [ 12 , 13 ] and lipid-containing scaffolds [ 14 ] are also applied to improve the pathway efficiency. In addition to scaffolds, enzymes were also located to bacterial microcompartments (MCPs), such as ethanolamine utilization (Eut) [ 15 ] and 1,2-propanediol utilization (Pdu) MCPs for similar purposes [ 16 ].…”

Section: Introductionmentioning

confidence: 99%

Optimizing the localization of astaxanthin enzymes for improved productivity

Zhu

et al. 2018

Biotechnol Biofuels

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BackgroundOne important metabolic engineering strategy is to localize the enzymes close to their substrates for improved catalytic efficiency. However, localization configurations become more complex the greater the number of enzymes and substrates is involved. Indeed, optimizing synthetic pathways by localizing multiple enzymes remains a challenge. Terpenes are one of the most valuable and abundant natural product groups. Phytoene, lycopene and β-carotene serve as common intermediates for the synthesis of many carotenoids and derivative compounds, which are hydrophobic long-chain terpenoids, insoluble in water and usually accumulate in membrane compartments.ResultsWhile β-ionone synthesis by β-carotene cleavage dioxygenase PhCCD1 and astaxanthin synthesis by β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ) differ in complexity (single and multiple step pathways), the productivity of both pathways benefited from controlling enzyme localization to the E. coli cell membrane via a GlpF protein fusion. Especially, the astaxanthin synthesis pathway comprises both CrtW and CrtZ, which perform four interchangeable reactions initiated from β-carotene. Up to four localization strategies of CrtW and CrtZ were exhaustively discussed in this work, and the optimal positioning strategy was achieved. CrtW and CrtZ were linked using a flexible linker and localized to the membrane via a GlpF protein fusion. Enzymes in the optimal localization configuration allowed a 215.4% astaxanthin production increase.ConclusionsThis work exploits a localization situation involving membrane-bound substrates, intermediates and multiple enzymes for the first time, and provides a workable positioning strategy to solve problems in similar circumstances.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1270-1) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Optimizing the localization of astaxanthin enzymes for improved productivity

Zhu

et al. 2018

Biotechnol Biofuels

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“…Using plasmid DNA as a scaffold, enzymes were proved to be colocalized around the plasmid. An approximately tenfold improvement on the productivity of an indole-3-acetic synthetic pathway in E. coli was also reported [ 41 ]. Compared to the ZFN approach, the TALEN technique provides a more efficient and practical approach for the clustering of multienzymes in vivo given its simpler design, higher specificity and lower toxicity [ 42 ].…”

Section: Genome-editing Techniques: New Hints For Developing Spatial mentioning

confidence: 99%

Spatial organization of enzymes to enhance synthetic pathways in microbial chassis: a systematic review

et al. 2018

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For years, microbes have been widely applied as chassis in the construction of synthetic metabolic pathways. However, the lack of in vivo enzyme clustering of heterologous metabolic pathways in these organisms often results in low local concentrations of enzymes and substrates, leading to a low productive efficacy. In recent years, multiple methods have been applied to the construction of small metabolic clusters by spatial organization of heterologous metabolic enzymes. These methods mainly focused on using engineered molecules to bring the enzymes into close proximity via different interaction mechanisms among proteins and nucleotides and have been applied in various heterologous pathways with different degrees of success while facing numerous challenges. In this paper, we mainly reviewed some of those notable advances in designing and creating approaches to achieve spatial organization using different intermolecular interactions. Current challenges and future aspects in the further application of such approaches are also discussed in this paper.

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“…Determining how to apply the in vitro model of DNA scaffolds to the internal environment is a new challenge for researchers. So far, researchers have begun to use the characteristics of specific bindings of nucleic acid binding proteins (such as ZFPs and TALEs) and nucleotide sequences to realize the assembly of pathway enzymes [81][82][83][84][85][86][87]. For example, through fusing glucosamine-6-phosphate synthase (GlmS) and GlcNAc (N-acetylglucosamine)-6-phosphate N-acetyltransferase (GNA1) with the zinc finger proteins ADB3 and ADB2 by pHT plasmid, respectively, Liu and co-workers successfully constructed a DNA-guided scaffold system to modulate the activities of GlmS and GNA1 ( Figure 2), and they found that the co-location and control of the stoichiometric ratios of GlmS and GNA1 significantly increased the GlcNAc titer from 1.83 to 4.55 g/L in a shake flask [84].…”

Section: Nucleic Acid Scaffoldmentioning

confidence: 99%

Enzyme Assembly for Compartmentalized Metabolic Flux Control

Cui

et al. 2020

Metabolites

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Enzyme assembly by ligand binding or physically sequestrating enzymes, substrates, or metabolites into isolated compartments can bring key molecules closer to enhance the flux of a metabolic pathway. The emergence of enzyme assembly has provided both opportunities and challenges for metabolic engineering. At present, with the development of synthetic biology and systems biology, a variety of enzyme assembly strategies have been proposed, from the initial direct enzyme fusion to scaffold-free assembly, as well as artificial scaffolds, such as nucleic acid/protein scaffolds, and even some more complex physical compartments. These assembly strategies have been explored and applied to the synthesis of various important bio-based products, and have achieved different degrees of success. Despite some achievements, enzyme assembly, especially in vivo, still has many problems that have attracted significant attention from researchers. Here, we focus on some selected examples to review recent research on scaffold-free strategies, synthetic artificial scaffolds, and physical compartments for enzyme assembly or pathway sequestration, and we discuss their notable advances. In addition, the potential applications and challenges in the applications are highlighted.

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Spatial organization of heterologous metabolic system in vivo based on TALE

Cited by 16 publications

References 35 publications

Optimizing the localization of astaxanthin enzymes for improved productivity

Optimizing the localization of astaxanthin enzymes for improved productivity

Spatial organization of enzymes to enhance synthetic pathways in microbial chassis: a systematic review

Enzyme Assembly for Compartmentalized Metabolic Flux Control

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