Integration of heterologous 4-hydroxybenzoic acid transport proteins in Rhodobacter sphaeroides for enhancement of coenzyme Q<sub>10</sub>production

Qi, Feng; Limei, Zou; Jiang, Xianzhang; Cai, Shaoli; Zhang, Mingliang; Zhao, Xuebing; Huang, Jianzhong

doi:10.1039/c7ra02346d

Cited by 9 publications

(5 citation statements)

References 27 publications

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“…Although other work has focused on increasing yields of endogenous MK, such as in E. coli and Bacillus subtilis , this is the first metabolic engineering of the synthesis of the entire MK biosynthetic pathway from chorismate and polyprenyl diphosphate in a heterologous host, in this instance using nine genes from E. coli . The synthesis of MK in R. sphaeroides , coupled with the versatile ability to heterologously express integral membrane proteins, , may provide a platform enabling further studies of proteins from recalcitrant or pathogenic organisms (especially those lacking genetic tools) that interact with or incorporate MK into integral membrane proteins, such as the TtRC from T. tepidum , and MenA from M. tuberculosis . This approach could also be used to engineer RCs from green filamentous photosynthetic bacteria such as the thermophile C. aurantiacus for thermostable biophotovoltaic applications, , and possibly RCs and other MK-requiring enzymes encoded in metagenomes from environmental DNA.…”

Section: Discussionmentioning

confidence: 99%

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins

et al. 2020

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Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chlorof lexus aurantiacus. Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides, nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA, and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the Q A pocket of this heterologously expressed reaction center.

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Section: Discussionmentioning

confidence: 99%

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins

et al. 2020

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“…The exogenous expression of MFS transporters can improve metabolite production by microbes (41,42). For instance, a system for coenzyme Q 10 production from 4-hydroxybenzoate and glucose using a Rhodobacter strain expressing Corynebacterium or Klebsiella pcaK demonstrated 1.18-to 1.21-fold greater yield than the wild-type Rhodobacter strain (43). Similarly, our laboratory found that pcaK overexpression in a SYK-6-derived strain enhanced PDC production 1.24-fold from protocatechuate (22).…”

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confidence: 70%

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6

Mori

Niinuma

Fujita

et al. 2018

Appl Environ Microbiol

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The microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. In sp. strain SYK-6, most of the enzyme genes involved in 5,5'-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 () and SLG_07780 (), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA-demethylase oxygenase component gene (), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled transcriptional regulatory system. A UT26S transformant expressing acquired DDVA uptake capacity, indicating that encodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects of overexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene () cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a -expression plasmid into a mutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives. The bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5'-dehydrodivanillate, a lignin-derived biphenyl compound, in sp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5'-dehydrodivanillate.

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“…In 2017, Qi and co-workers successfully engineered Rhodobacter sphaeroides , a natural producer of CoQ10 by expressing three membrane transport proteins (AcPcaK from Acinetobacter calcoaceticus , KpPcaK from Klebsiella pneumoniae , and CgPcaK from Corynebacterium glutamicum ) involved in the transport of 4-hydroxybenzoic acid, which is a key intermediate in the biosynthesis of CoQ10, therefore improving the production of CoQ10. 297 The same group built on this existing work, 298 and in 2020 they identified a bottle neck in the production of farnesyl pyrophosphate (FPP) 64 (Fig. 13), which is generated from the MEP pathway.…”

Section: Anti-ageing Ingredientsmentioning

confidence: 99%

The beauty of biocatalysis: sustainable synthesis of ingredients in cosmetics

2022

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Integration of heterologous 4-hydroxybenzoic acid transport proteins in Rhodobacter sphaeroides for enhancement of coenzyme Q₁₀production

Abstract: This work provides a novel genetic engineering strategy that improves uptake of extracellular 4-hydroxybenzoic acid by heterologously expressing the membrane transport protein PcaK in R. sphaeroides for enhancement of CoQ10 production.

Cited by 9 publications

References 27 publications

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6

The beauty of biocatalysis: sustainable synthesis of ingredients in cosmetics

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Integration of heterologous 4-hydroxybenzoic acid transport proteins in Rhodobacter sphaeroides for enhancement of coenzyme Q10production

Abstract: This work provides a novel genetic engineering strategy that improves uptake of extracellular 4-hydroxybenzoic acid by heterologously expressing the membrane transport protein PcaK in R. sphaeroides for enhancement of CoQ10 production.

Cited by 9 publications

References 27 publications

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins

DdvK, a Novel Major Facilitator Superfamily Transporter Essential for 5,5′-Dehydrodivanillate Uptake by Sphingobium sp. Strain SYK-6

The beauty of biocatalysis: sustainable synthesis of ingredients in cosmetics

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Integration of heterologous 4-hydroxybenzoic acid transport proteins in Rhodobacter sphaeroides for enhancement of coenzyme Q₁₀production