Biosynthesis of Pinene in Purple Non-Sulfur Photosynthetic Bacteria

Abstract Background: Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. Results: Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L. Conclusions: In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

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Biosynthesis of pinene in purple non-sulfur photosynthetic bacteria

Microbial Cell Factories ◽

10.1186/s12934-021-01591-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Xiaomin Wu ◽

Guang Ma ◽

Chuanyang Liu ◽

Xin-yuan Qiu ◽

Lu Min ◽

...

Keyword(s):

Fusion Protein ◽

Heterologous Expression ◽

Rhodobacter Sphaeroides ◽

Photosynthetic Bacteria ◽

Fine Chemicals ◽

Isopentenyl Diphosphate ◽

Isopentenyl Diphosphate Isomerase ◽

Geranyl Diphosphate ◽

Ribosome Binding ◽

Geranyl Diphosphate Synthase

Abstract Background Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. Results Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-d-xylulose 5-phosphate synthase, 1-deoxy-d-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L. Conclusions In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

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Heteromeric geranyl diphosphate synthase from mint: construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2003.12.003 ◽

2004 ◽

Vol 422 (1) ◽

pp. 52-60 ◽

Cited By ~ 16

Author(s):

Charles Burke ◽

Karin Klettke ◽

Rodney Croteau

Keyword(s):

Fusion Protein ◽

Geranyl Diphosphate ◽

Substrate Analogs ◽

Geranyl Diphosphate Synthase

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Improved Squalene Production via Modulation of the Methylerythritol 4-Phosphate Pathway and Heterologous Expression of Genes from Streptomyces peucetius ATCC 27952 in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01402-09 ◽

2009 ◽

Vol 75 (22) ◽

pp. 7291-7293 ◽

Cited By ~ 44

Author(s):

Gopal Prasad Ghimire ◽

Hei Chan Lee ◽

Jae Kyung Sohng

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Farnesyl Diphosphate ◽

Farnesyl Diphosphate Synthase ◽

Isopentenyl Diphosphate ◽

Isopentenyl Diphosphate Isomerase ◽

Streptomyces Peucetius ◽

E Coli ◽

Expression Of Genes ◽

Genes Encoding

ABSTRACT Putative hopanoid genes from Streptomyces peucetius were introduced into Escherichia coli to improve the production of squalene, an industrially important compound. High expression of hopA and hopB (encoding squalene/phytoene synthases) together with hopD (encoding farnesyl diphosphate synthase) yielded 4.1 mg/liter of squalene. This level was elevated to 11.8 mg/liter when there was also increased expression of dxs and idi, E. coli genes encoding 1-deoxy-d-xylulose 5-phosphate synthase and isopentenyl diphosphate isomerase.

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Cloning and heterologous expression of isopentenyl diphosphate isomerase gene from Lycium chinense

Journal of Plant Biochemistry and Biotechnology ◽

10.1007/s13562-015-0306-x ◽

2015 ◽

Vol 25 (1) ◽

pp. 40-48 ◽

Cited By ~ 4

Author(s):

Zhaodi Li ◽

Jing Ji ◽

Gang Wang ◽

Tchouopou Lontchi Josine ◽

Jiang Wu ◽

...

Keyword(s):

Heterologous Expression ◽

Isopentenyl Diphosphate ◽

Lycium Chinense ◽

Isopentenyl Diphosphate Isomerase

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Purification and characterization of recombinant human farnesyl diphosphate synthase expressed in Escherichia coli

Biochemical Journal ◽

10.1042/bj2750061 ◽

1991 ◽

Vol 275 (1) ◽

pp. 61-65 ◽

Cited By ~ 23

Author(s):

V D H Ding ◽

B T Sheares ◽

J D Bergstrom ◽

M M Ponpipom ◽

L B Perez ◽

...

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Molecular Mass ◽

Fetal Liver ◽

Farnesyl Diphosphate ◽

Blue Staining ◽

Isopentenyl Diphosphate ◽

Native Form ◽

Geranyl Diphosphate ◽

Apparent Homogeneity

We previously reported the isolation of a partial-length human fetal-liver cDNA encoding farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) and the expression of an active FPP synthase fusion protein in Escherichia coli. The expressed human FPP synthase fusion protein has now been purified to apparent homogeneity by using two chromatographic steps. The purification scheme allowed the preparation of 1.8 mg of homogeneous protein from 149 mg of crude extract in a 64% yield with a 52-fold enrichment. A single band with a subunit molecular mass of 39 kDa was observed by Coomassie Blue staining after SDS/PAGE. A molecular mass of 78-80 kDa was calculated for the native form of the fusion protein by h.p.l.c. on a SEC-250 column, suggesting that the active fusion protein is a dimer. The purified fusion protein has FPP synthase condensation activities in the presence of both substrates, isopentenyl diphosphate and geranyl diphosphate. Enzyme activity was inhibited by a bisubstrate analogue of isopentenyl diphosphate and dimethylallyl diphosphate, and a small amount of higher prenyltransferase was observed. Michaelis constants for isopentenyl diphosphate and geranyl diphosphate were 0.55 and 0.43 microM respectively, and Vmax for synthesis of farnesyl diphosphate from these substrates was 1.08 mumol/min per mg. These results suggest that the structure and catalytic properties of the expressed FPP synthase fusion protein are virtually identical with those of the native human liver enzyme.

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