Genetics of isoprenoid biosynthesis in Paracoccus zeaxanthinifaciens

Hümbelin, Markus; Thomas, Ann; Lin, Junyu; Li, Juan; Jore, J.p.m.; Berry, Alan

doi:10.1016/s0378-1119(02)00877-6

Cited by 36 publications

(16 citation statements)

References 25 publications

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“…Most relevantly, when concentrations of CnVS and CitrusVS proteins were tested by Western blot, we observed far more soluble and insoluble synthase in CnVS cells than in CitrusVS cells (Figure S3). To further improve yields, plasmids were constructed in which the mevalonate operon from Paracoccus zeaxanthinifaciens is co‐expressed with the valencene synthases to increase the availability of FPP substrate (Hümbelin et al ., ). Expression of CnVS in this engineered R. sphaeroides strain in shaker flasks resulted again in a strongly (six‐fold) improved valencene production (Table ).…”

Section: Resultsmentioning

confidence: 97%

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene

Beekwilder

Houwelingen²,

Cankar³

et al. 2013

Plant Biotechnology Journal

118

136

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Summary Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large‐scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm‐specific TPS‐d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14‐fold to 352 mg/L, bringing production to levels with industrial potential.

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Section: Resultsmentioning

confidence: 97%

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene

Beekwilder

Houwelingen²,

Cankar³

et al. 2013

Plant Biotechnology Journal

118

136

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“…The numbers below the genes represent their position in the pathway depicted in panel A), while Sp/Sm R refers to spectinomycin/streptomycin resistance. The image is adapted from(Hümbelin et al , 2002). Other abbreviations: GAP (glyceraldehyde‐3‐phosphate), PYR (pyruvate), CDP‐ME (4‐(cytidine 5'‐diphospho)‐2‐C‐methyl‐D‐erythritol), CDP‐MEP (2‐phospho‐4‐(cytidine 5'‐diphospho)‐2‐C‐methyl‐D‐erythritol), MEcPP (2‐C‐methyl‐D‐erythritol 2,4‐cyclodiphosphate), pyrF (uridylate kinase, gene), frr (ribosome recycling factor, gene), uppS (undecaprenyl‐diphosphate synthase, gene), cdsA (phosphatidate cytidylyltransferase, gene), rseP (Regulator of sigma E protease, gene) Ac‐CoA (acetyl‐CoA), AA‐CoA (acetoacetyl‐CoA), HMG‐CoA (S)‐3‐hydroxy‐3‐methylglutaryl‐CoA), MVA‐P ((R)‐5‐phosphomevalonate), MVA‐PP ((R)‐5‐diphosphomevalonate), mvaA (HMG‐CoA reductase, gene), idi (IPP isomerase, gene), hcs (HMG‐CoA synthase, gene), mvk (mevalonate kinase, gene), pmk (phosphomevalonate kinase, gene), mvd (MVA‐PP decarboxylase, gene).…”

Section: Resultsmentioning

confidence: 99%

“…Phylogenetically, the traditional distinction that couples MEP pathway to the prokaryotic domain and MVA to eukaryotes and archaea has been revised (Lombard and Moreira, 2011). The α‐proteobacterium Paracoccus zeaxanthinifaciens (Hümbelin et al , 2002) harbours a complete MVA pathway, with all required genes organized in an operon. This operon already proved to be functional when expressed on a replicative plasmid in R. sphaeroides (Beekwilder et al , 2014).…”

Section: Resultsmentioning

confidence: 99%

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Orsi

Beekwilder²,

Gelder

et al. 2020

Microbial Biotechnology

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Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-13 C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.

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“…The E. coli harboring pha genes (phaA, phaB and phaC) of R. eutropha was proved to be highly active to produce more than 80% of PHB in dry cell weight (Resch et al, 1998). Also, PhaA rather than AtoB was found to be responsible for most of the acetyl-CoA acetyltransferase activity in Paracoccus zeaxanthinifaciens (Humbelin et al, 2002).…”

Section: Comparison Of Various Top Portions Of Mva Pathway On Mevalonmentioning

confidence: 99%

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

Yoon

Lee

Das

et al. 2009

Journal of Biotechnology

197

132

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Genetics of isoprenoid biosynthesis in Paracoccus zeaxanthinifaciens

Cited by 36 publications

References 25 publications

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene

Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

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