Identification, Evolution, and Essentiality of the Mevalonate Pathway for Isopentenyl Diphosphate Biosynthesis in Gram-Positive Cocci

Wilding, E. Imogen; Brown, James R.; Bryant, Alexander P.; Chalker, Alison F.; Holmes, David; Ingraham, Karen; Iordănescu, S; So, Chi Y.; Rosenberg, Martin; Gwynn, Michael N.

doi:10.1128/jb.182.15.4319-4327.2000

Cited by 241 publications

(214 citation statements)

References 51 publications

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“…Plasmid construction processes are listed in Supplementary Materials: Table S2. The genes encoding trans-nerolidol synthase (AcNES1; GenBank-KX064240) from Actinidia chinensis (Green et al, 2012), and 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) synthase (EfmvaS; GenBank-KX064238) and bi-functional acetoacetyl-CoA thiolase /HMG-CoA reductase (EfmvaE; GenBank-KX064239) from E. faecalis (Wilding et al, 2000) were synthesized by GenScript (Piscataway, NJ, USA) with codon optimization according to the codon bias of S. cerevisiae (Supplementary Materials: Table S3). .…”

Section: Plasmid and Strain Constructionmentioning

confidence: 99%

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

Peng

Plan

Chrysanthopoulos

et al. 2017

Metabolic Engineering

101

128

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A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae, Metabolic Engineering, http://dx.doi.org/10. 1016/j.ymben.2016.12.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractSesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1 % biomass; moreover, the specific growth rate declined. In order to redirect carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. 2A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86 % to ~100 mg L -1 . In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes.

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Section: Plasmid and Strain Constructionmentioning

confidence: 99%

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

Peng

Plan

Chrysanthopoulos

et al. 2017

Metabolic Engineering

101

128

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“…In genetic studies that we performed on S. aureus, SA2343 was shown to be upregulated 100-500 fold when any of the genes in the mevalonate pathway, responsible for biosynthesis of the isoprenoid precursor isopentenyl pyrophosphate (IPP), is downregulated (Balibar et al, 2009). Given that IPP is the building block for the cell wall carrier lipid undecaprenyl pyrophosphate (UPP), which is essential in peptidoglycan biosynthesis (Wilding et al, 2000), this massive overexpression is presumed to be a response to cell wall damage. This upregulation of SA2343 was more than 20-fold greater than the upregulation of any other gene in the cell wall stimulon.…”

Section: Introductionmentioning

confidence: 99%

cwrA, a gene that specifically responds to cell wall damage in Staphylococcus aureus

Balibar¹,

Shen²,

McGuire³

et al. 2010

Microbiology

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Transcriptional profiling data accumulated in recent years for the clinically relevant pathogen Staphylococcus aureus have established a cell wall stress stimulon, which comprises a coordinately regulated set of genes that are upregulated in response to blockage of cell wall biogenesis. In particular, the expression of cwrA (SA2343, N315 notation), which encodes a putative 63 amino acid polypeptide of unknown biological function, increases over 100-fold in response to cell wall inhibition. Herein, we seek to understand the biological role that this gene plays in S. aureus. cwrA was found to be robustly induced by all cell wall-targeting antibiotics tested – vancomycin, oxacillin, penicillin G, phosphomycin, imipenem, hymeglusin and bacitracin – but not by antibiotics with other mechanisms of action, including ciprofloxacin, erythromycin, chloramphenicol, triclosan, rifampicin, novobiocin and carbonyl cyanide 3-chlorophenylhydrazone. Although a ΔcwrA S. aureus strain had no appreciable shift in MICs for cell wall-targeting antibiotics, the knockout was shown to have reduced cell wall integrity in a variety of other assays. Additionally, the gene was shown to be important for virulence in a mouse sepsis model of infection.

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“…The MVA pathway is essential for the survival of Streptococcus pneumonia in the lung and serum. 13 It was reported that the product of PMK, diphosphomevalonate (DPM), feed-4 back inhibits MK in S. pneumonia. Therefore, regulation of the MVA pathway would provide novel therapies to intervene in the pathway to inhibit the devastating effect of the organism.…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation Mechanism of Phosphomevalonate Kinase: Implications for Rational Engineering of Isoprenoid Biosynthetic Pathway Enzymes

Huang

Wei

et al. 2016

J. Phys. Chem. B

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Mevalonate pathway is of important clinical, pharmaceutical and biotechnological relevance.However, lack of the understanding of the phosphorylation mechanism of the kinases in this pathway has limited rationally engineering the kinases in industry. Here the phosphorylation reaction mechanism of a representative kinase in the mevalonate pathway, phosphomevalonate kinase, was studied by using molecular dynamics and hybrid QM/MM methods. We find that a conserved residue (Ser106) is reorientated to anchor ATP via a stable H-bond interaction. In addition, Ser213 located on the α-helix at the catalytic site is repositioned to further approach the substrate, facilitating the proton transfer during the phosphorylation. Furthermore, we elucidate that Lys101 functions to neutralize the negative charge developed at the β-, γ-bridging oxygen atom of ATP during phosphoryl transfer. We demonstrate that the dissociative catalytic reaction occurs via a direct phosphorylation pathway. This is the first study on the phosphorylation mechanism of a mevalonate pathway kinase. The elucidation of the catalytic mechanism not only sheds light on the common catalytic mechanism of GHMP kinase superfamily, but also provides the structural basis for engineering the mevalonate pathway kinases to further exploit their applications in the production of a wide range of fine chemicals such as biofuels or pharmaceuticals.3

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Identification, Evolution, and Essentiality of the Mevalonate Pathway for Isopentenyl Diphosphate Biosynthesis in Gram-Positive Cocci

Cited by 241 publications

References 51 publications

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae

cwrA, a gene that specifically responds to cell wall damage in Staphylococcus aureus

Phosphorylation Mechanism of Phosphomevalonate Kinase: Implications for Rational Engineering of Isoprenoid Biosynthetic Pathway Enzymes

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