Metabolic pathway assembly using docking domains from type I cis-AT polyketide synthases

Sun, Xiaopeng; Yuan, Yujie; Chen, Qitong; Nie, Shiqi; Guo, Jianhua; Ou, Zutian; Huang, Min; Deng, Zixin; Liu, Tiangang; Ma, Tian

doi:10.1038/s41467-022-33272-2

Cited by 13 publications

(20 citation statements)

References 72 publications

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“…Further overexpression of asm11 and asm10 genes assisted the conversion of 8 to maytansinol but failed to result in significant improvement of maytansinol production, probably due to affecting the overall metabolic state of this strain. On the basis of the docking domains (DDs) of 6-deoxyerythronolide B synthase (DEBS), the mimic DEBS enzyme assembly line strategy was recently developed to achieve the ordered assembly of key cascade enzymes, enhancing their collaborative catalytic efficiency . Consequently, this strategy was employed to assemble SmAstC M , Asm11, and Asm10, aimed at reducing the accumulation of intermediate metabolites and accelerating the conversion toward maytansinol (Figures B and S22).…”

Section: Resultsmentioning

confidence: 99%

“…On the basis of the docking domains (DDs) of 6-deoxyerythronolide B synthase (DEBS), the mimic DEBS enzyme assembly line strategy was recently developed to achieve the ordered assembly of key cascade enzymes, enhancing their collaborative catalytic efficiency. 45 Consequently, this strategy was employed to assemble SmAstC M , Asm11, and Asm10, aimed at reducing the accumulation of intermediate metabolites and accelerating the conversion toward maytansinol (Figures 6B and S22). The resulted HGFS08 strain exhibited a significant increase in maytansinol production through solid-state fermentation, reaching 27 ± 1 mg/L (Figures 6C and S23).…”

Section: Introduction Of the Smastc Gene Into Strain Hgf052 Led To Th...mentioning

confidence: 99%

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Functional Application of the Single-Module NRPS-like d-Alanyltransferase in Maytansinol Biosynthesis

Li,

Zhu,

et al. 2024

ACS Catal.

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Single-module nonribosomal peptide synthetases (NRPSs) have nonclassical domain compositions and play versatile roles in the biosynthesis of natural products. AstC, a prototypical single-module NRPS-like D-alanyltransferase with the architecture of adenylation-thiolation-thioesterase (A-T-TE) tridomain, was identified as the catalyst in the D-alanylation process during the post-polyketide synthase (PKS) modifications of ansatrienin biosynthesis. In this study, the function of the TE domain of AstC was elucidated in intermolecular esterification, and its substrate promiscuity was revealed for both acyl donors and polyketide acceptors. Through genome mining, a newly characterized AstC homolog was identified, SmAstC, and it was shown that SmAstC exhibited the highest catalytic activity for Dalanylation of N-desmethyl-4,5-desepoxymaytansinol (DDM), a precursor of the antitumor agent ansamitocins, obtained from Actinosynnema pretiosum HGF052. Harnessing the broad substrate selectivity of the TE domains and the spontaneous hydrolysis propensity of the D-alanylated products, the post-PKS modification of ansamitocin biosynthesis was reprogrammed through introducing the gene encoding SmAstC into A. pretiosum HGF052. The ansamitocin biosynthetic pathway in this engineered strain was switched toward the production of maytansinol that was a critical intermediate of maytansine-derived antibody-drug conjugates. The SmAstC-catalyzed D-alanylation of DDM interrupted the stringent post-PKS modification steps within the original biosynthesis of ansamitocins, which produced 3-O-D-alanyl maytansinol that readily underwent spontaneous hydrolysis to afford maytansinol. The innovative application of the single-module NRPS-like Dalanyltransferases combined with rational strain engineering has surmounted the longtime hurdle in converting DDM to maytansinol in vivo, enabling the biomanufacturing of maytansinol without precedent.

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Section: Resultsmentioning

confidence: 99%

Section: Introduction Of the Smastc Gene Into Strain Hgf052 Led To Th...mentioning

confidence: 99%

Functional Application of the Single-Module NRPS-like d-Alanyltransferase in Maytansinol Biosynthesis

Li,

Zhu,

et al. 2024

ACS Catal.

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“…These interrelated modules interact with each other through docking domains (DDs) mediated by folding regions at the C-and N-termini (Weissman, 2016). Sun et al (2022) utilized the DDs of type I cis-AT-PKS as mediators to develop a multi-enzyme assembly strategy named mimic PKS enzyme assembly line (mPKSeal), which mimics the assembly line of PKS enzymes (Figure 1B). This strategy was applied in engineered E. coli to enhance astaxanthin production and possesses the ability to co-locate enzymes within the cell, enabling the assembly of two or three enzyme units in different cellular environments (Sun et al, 2022).…”

Section: Peptide-peptide Pairmentioning

confidence: 99%

Application of artificial scaffold systems in microbial metabolic engineering

Liu,

Dong,

Yang

et al. 2023

Front. Bioeng. Biotechnol.

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In nature, metabolic pathways are often organized into complex structures such as multienzyme complexes, enzyme molecular scaffolds, or reaction microcompartments. These structures help facilitate multi-step metabolic reactions. However, engineered metabolic pathways in microbial cell factories do not possess inherent metabolic regulatory mechanisms, which can result in metabolic imbalance. Taking inspiration from nature, scientists have successfully developed synthetic scaffolds to enhance the performance of engineered metabolic pathways in microbial cell factories. By recruiting enzymes, synthetic scaffolds facilitate the formation of multi-enzyme complexes, leading to the modulation of enzyme spatial distribution, increased enzyme activity, and a reduction in the loss of intermediate products and the toxicity associated with harmful intermediates within cells. In recent years, scaffolds based on proteins, nucleic acids, and various organelles have been developed and employed to facilitate multiple metabolic pathways. Despite varying degrees of success, synthetic scaffolds still encounter numerous challenges. The objective of this review is to provide a comprehensive introduction to these synthetic scaffolds and discuss their latest research advancements and challenges.

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“…Recently, researchers were inspired by the type I modular polyketide synthases (PKSs) and developed a strategy named mimic PKS enzyme assembly line (mPKSeal), through which sequential pathway enzymes were assembled into multienzyme complexes. Specifically, docking domains from type I cis -AT PKS were tagged to astaxanthin biosynthetic pathway enzymes for multienzyme assembly, resulting in the improvement of astaxanthin production by 2.4-fold …”

Section: Introductionmentioning

confidence: 99%

“…Specifically, docking domains from type I cis-AT PKS were tagged to astaxanthin biosynthetic pathway enzymes for multienzyme assembly, resulting in the improvement of astaxanthin production by 2.4-fold. 7 Given that scaffolds mentioned above are generally soluble and dispersive, protein assembly mediated by scaffold engineering has certain limitations. For example, it is difficult to recruit large numbers of various enzymes and spatially sequester native proteins to prevent unwanted crosstalk.…”

Section: Introductionmentioning

confidence: 99%

Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli

et al. 2023

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Extensive research efforts have been focused on spatial organization of biocatalytic cascades or catalytic networks in confined cellular environments. Inspired by the natural metabolic systems that spatially regulate pathways via sequestration into subcellular compartments, formation of artificial membraneless organelles through expressing intrinsically disordered proteins in host strains has been proven to be a feasible strategy. Here we report the engineering of a synthetic membraneless organelle platform, which can be used to extend compartmentalization and spatially organize pathway sequential enzymes. We show that heterologous overexpression of the RGG domain derived from the disordered P granule protein LAF-1 in an Escherichia coli strain can form intracellular protein condensates via liquid−liquid phase separation. We further demonstrate that different clients can be recruited to the synthetic compartments via directly fusing with the RGG domain or cooperating with different protein interaction motifs. Using the 2′-fucosyllactose de novo biosynthesis pathway as a model system, we show that clustering sequential enzymes into synthetic compartments can effectively increase the titer and yield of the target product compared to strains with free-floating pathway enzymes. The synthetic membraneless organelle system constructed here gives a promising approach in the development of microbial cell factories, wherein it could be used for the compartmentalization of pathway enzymes to streamline metabolic flux.

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Metabolic pathway assembly using docking domains from type I cis-AT polyketide synthases

Cited by 13 publications

References 72 publications

Functional Application of the Single-Module NRPS-like d-Alanyltransferase in Maytansinol Biosynthesis

Functional Application of the Single-Module NRPS-like d-Alanyltransferase in Maytansinol Biosynthesis

Application of artificial scaffold systems in microbial metabolic engineering

Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli

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