Synthetic metabolic channel by functional membrane microdomains for compartmentalized flux control

Wu, Yaokang; Tian, Rongzhen; Gu, Yang; Liu, Yanfeng; Li, Jianghua; Du, Guocheng; Ledesma-Amaro, Rodrigo; Liu, Long

doi:10.1016/j.ymben.2020.02.003

Cited by 22 publications

(18 citation statements)

References 56 publications

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“…When the three enzymes were assembled together in CYP-13 (KO-RIDD/KAH-GGGGS 3 -trCPR1-RIAD), the steviol titer was only 24.5 mg/L. To visualize whether the distance between KAH and trCPR1 was reduced by the short protein linker in CYP-11, Bimolecular Fluorescence Complementation (BiFC, a method used to directly visualize protein–protein interactions 28 ) assay was used. Yellow fluorescence was only observed when KAH was fused with trCPR1, but not when it was fused with CPR1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts

Wang

Zhang

et al. 2022

Nat Commun

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High-sugar diet causes health problems, many of which can be addressed with the use of sugar substitutes. Rubusoside and rebaudiosides are interesting molecules, considered the next generation of sugar substitutes due to their low-calorie, superior sweetness and organoleptic properties. However, their low abundance in nature makes the traditional plant extraction process neither economical nor environmental-friendly. Here we engineer baker’s yeast Saccharomyces cerevisiae as a chassis for the de novo production of rubusoside and rebaudiosides. In this process, we identify multiple issues that limit the production, including rate-liming steps, product stress on cellular fitness and unbalanced metabolic networks. We carry out a systematic engineering strategy to solve these issues, which produces rubusoside and rebaudiosides at titers of 1368.6 mg/L and 132.7 mg/L, respectively. The rubusoside chassis strain here constructed paves the way towards a sustainable, large-scale fermentation-based manufacturing of diverse rebaudiosides.

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Section: Resultsmentioning

confidence: 99%

De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts

Wang

Zhang

et al. 2022

Nat Commun

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“…Hence, FMMs have been implicated in a wide range of bacterial processes, among which motility, cell division, protein secretion, biofilm formation, sporulation and cell wall organization (Lopez and Koch, 2017 ). Notably, a recent study demonstrated that bacterial FMMs can also be exploited as spatial scaffolds in order to optimize the production of industrial relevant compounds (Lv et al ., 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Membrane composition and organization of Bacillus subtilis 168 and its genome‐reduced derivative miniBacillus PG10

Tilburg

Warmer

Heel

et al. 2021

Microbial Biotechnology

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Summary A form of lateral membrane compartmentalization in bacteria is represented by functional membrane microdomains (FMMs). FMMs are important for various cellular processes and offer application possibilities in microbial biotechnology. We designed a lipidomics method to directly measure relative abundances of lipids in detergent‐resistant and detergent‐sensitive membrane fractions of the model bacterium Bacillus subtilis 168 and the biotechnologically attractive mini Bacillus PG10 strain. Our study supports previous work suggesting that cardiolipin and prenol lipids are enriched in FMMs of B. subtilis . Additionally, structural analysis of acyl chains of major phospholipids indicated that FMMs display increased order and thickness compared with the surrounding bilayer. Despite the 36% genome reduction, membrane and FMM integrity are largely preserved in mini Bacillus PG10, as supported by analysis of membrane fluidity, flotillin distribution and gene expression data. The novel insights in FMM architecture reported here will contribute to further explore the biological significance of FMMs and the means by which FMMs can be exploited as heterologous production platforms. Moreover, our lipidomics method enables comparative FMM lipid profiling between different bacteria.

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“…Recently, a synthetic metabolite channel was engineered in Bacillus subtilus to enhance N-acetylglucosamine synthesis ( Lv et al, 2020 ), which provides concrete empirical evidence that metabolite channeling can significantly alter reaction efficiency. The creation of metabolite channels is a promising metabolic engineering strategy because it is orthogonal to many common metabolic engineering strategies such as enzyme overexpressions or knockdowns, allowing for a more complex or fine-tuned approach.…”

Section: Discussionmentioning

confidence: 99%

Probing Light-Dependent Regulation of the Calvin Cycle Using a Multi-Omics Approach

2021

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Photoautotrophic microorganisms are increasingly explored for the conversion of atmospheric carbon dioxide into biomass and valuable products. The Calvin-Benson-Bassham (CBB) cycle is the primary metabolic pathway for net CO2 fixation within oxygenic photosynthetic organisms. The cyanobacteria, Synechocystis sp. PCC 6803, is a model organism for the study of photosynthesis and a platform for many metabolic engineering efforts. The CBB cycle is regulated by complex mechanisms including enzymatic abundance, intracellular metabolite concentrations, energetic cofactors and post-translational enzymatic modifications that depend on the external conditions such as the intensity and quality of light. However, the extent to which each of these mechanisms play a role under different light intensities remains unclear. In this work, we conducted non-targeted proteomics in tandem with isotopically non-stationary metabolic flux analysis (INST-MFA) at four different light intensities to determine the extent to which fluxes within the CBB cycle are controlled by enzymatic abundance. The correlation between specific enzyme abundances and their corresponding reaction fluxes is examined, revealing several enzymes with uncorrelated enzyme abundance and their corresponding flux, suggesting flux regulation by mechanisms other than enzyme abundance. Additionally, the kinetics of 13C labeling of CBB cycle intermediates and estimated inactive pool sizes varied significantly as a function of light intensity suggesting the presence of metabolite channeling, an additional method of flux regulation. These results highlight the importance of the diverse methods of regulation of CBB enzyme activity as a function of light intensity, and highlights the importance of considering these effects in future kinetic models.

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Synthetic metabolic channel by functional membrane microdomains for compartmentalized flux control

Cited by 22 publications

References 56 publications

De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts

De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts

Membrane composition and organization of Bacillus subtilis 168 and its genome‐reduced derivative miniBacillus PG10

Probing Light-Dependent Regulation of the Calvin Cycle Using a Multi-Omics Approach

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