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Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin.

Lemuth K, Steuer K, Albermann C - Microb. Cell Fact. (2011)

Bottom Line: Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors.The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products.This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Microbiology, Universität Stuttgart, Stuttgart, Germany.

ABSTRACT

Background: The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.

Results: Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798) and the β-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).

Conclusions: By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this E. coli strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.

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HPLC analysis of the carotenoids accumulated during cultivation of plasmid-free E. coli strains: A BW-CARO-dxs-idi in LB medium (48 h), B BW-ASTA in LB medium (48 h), C BW-CANT in LB medium (48 h), D BW-ASTA in LB medium plus L-rhamnose (48 h), E BW-ASTA in MM medium plus IPTG (24 h), F BW-ASTA in MM medium plus IPTG and L-rhamnose (48 h), G BW-ASTA in MM medium plus IPTG (48 h), H astaxanthin standard. Detection wavelength 470 nm. (1) β-carotene, (3) echinenone, (6) zeaxanthin, (7) canthaxanthin, (8) adonixanthin, (9) adonirubin, (10) astaxanthin, (10') cis-astaxanthin.
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Figure 2: HPLC analysis of the carotenoids accumulated during cultivation of plasmid-free E. coli strains: A BW-CARO-dxs-idi in LB medium (48 h), B BW-ASTA in LB medium (48 h), C BW-CANT in LB medium (48 h), D BW-ASTA in LB medium plus L-rhamnose (48 h), E BW-ASTA in MM medium plus IPTG (24 h), F BW-ASTA in MM medium plus IPTG and L-rhamnose (48 h), G BW-ASTA in MM medium plus IPTG (48 h), H astaxanthin standard. Detection wavelength 470 nm. (1) β-carotene, (3) echinenone, (6) zeaxanthin, (7) canthaxanthin, (8) adonixanthin, (9) adonirubin, (10) astaxanthin, (10') cis-astaxanthin.

Mentions: Recently our group developed a method for the integration of heterologous expression cassettes into the chromosome of E. coli and the subsequent screening [24]. From this work resulted a plasmid-free strain (E. coli BW-CARO) carrying the four genes crtEBIY from P. ananatis in individual expression cassettes under the control of IPTG inducible tac-promoters. To enhance the expression level of the native 1-deoxyxylulose-5-phosphate synthase gene (dxs), a rate-limiting enzyme of the isoprenoid pathway [32], the native dxs promoter was exchanged by the strong bacteriophage promoter T5 [28]. First, a chloramphenicol resistance gene, flanked by FRT sites, was cloned upstream of the T5-promoter into pQE31. The resistance gene and promoter region of the plasmid gained (pQE31-FRT-cat-FRT) was PCR amplified and subsequently inserted upstream of the native dxs in E. coli BW-CARO using the λ-Red recombineering technique. After elimination of the resistance gene, the resulting strain E. coli BW-CARO-dxs was further modified by the insertion of an additional copy of the native isopentenyl diphosphate isomerase gene (idi), a further rate-limiting enzyme of the isoprenoid pathway [33]. The E. coli idi gene was cloned into the expression vector pJF119ΔN and to allow transient selection for chromosomal integration, the cloned gene was hooked up to a downstream chloramphenicol resistance cassette. The expression cassette of the resulting plasmid pJF119-FRT-cat-FRT, including resistance gene, was PCR amplified and transformed into E. coli BW-CARO-dxs harboring the λ-Red recombinase expression plasmid pKD46. The selected PCR primers led to the homologous recombination of the expression cassette into the galactose locus (galETKM). The engineered E. coli strains gained (E. coli BW-CARO, E. coli BW-CARO-dxs, and E. coli BW-CARO-dxs-idi) were cultivated in complex medium and the carotenoid content was analyzed. All strains produce β-carotene as the sole carotenoid (Figure 2A). As expected, the amount of β-carotene increased due to the enhanced expression of dxs and idi. The exchange of the native dxs promoter by the T5-promoter in E. coli BW-CARO increased the amount of β-carotene per cell by a factor of 2.6. After insertion of the additional idi copy into E. coli BW-CARO-dxs the amount of β-carotene per cell increased by the factor of 1.9 (β-carotene content in E. coli BW-CARO 1.24 ± 0.17 mg/g cellular dry weight (cdw); E. coli BW-CARO-dxs 3.254 ± 0.24 mg/g cdw; E. coli BW-CARO-dxs-idi 6.18 ± 0.78 mg/g cdw).


Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin.

Lemuth K, Steuer K, Albermann C - Microb. Cell Fact. (2011)

HPLC analysis of the carotenoids accumulated during cultivation of plasmid-free E. coli strains: A BW-CARO-dxs-idi in LB medium (48 h), B BW-ASTA in LB medium (48 h), C BW-CANT in LB medium (48 h), D BW-ASTA in LB medium plus L-rhamnose (48 h), E BW-ASTA in MM medium plus IPTG (24 h), F BW-ASTA in MM medium plus IPTG and L-rhamnose (48 h), G BW-ASTA in MM medium plus IPTG (48 h), H astaxanthin standard. Detection wavelength 470 nm. (1) β-carotene, (3) echinenone, (6) zeaxanthin, (7) canthaxanthin, (8) adonixanthin, (9) adonirubin, (10) astaxanthin, (10') cis-astaxanthin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: HPLC analysis of the carotenoids accumulated during cultivation of plasmid-free E. coli strains: A BW-CARO-dxs-idi in LB medium (48 h), B BW-ASTA in LB medium (48 h), C BW-CANT in LB medium (48 h), D BW-ASTA in LB medium plus L-rhamnose (48 h), E BW-ASTA in MM medium plus IPTG (24 h), F BW-ASTA in MM medium plus IPTG and L-rhamnose (48 h), G BW-ASTA in MM medium plus IPTG (48 h), H astaxanthin standard. Detection wavelength 470 nm. (1) β-carotene, (3) echinenone, (6) zeaxanthin, (7) canthaxanthin, (8) adonixanthin, (9) adonirubin, (10) astaxanthin, (10') cis-astaxanthin.
Mentions: Recently our group developed a method for the integration of heterologous expression cassettes into the chromosome of E. coli and the subsequent screening [24]. From this work resulted a plasmid-free strain (E. coli BW-CARO) carrying the four genes crtEBIY from P. ananatis in individual expression cassettes under the control of IPTG inducible tac-promoters. To enhance the expression level of the native 1-deoxyxylulose-5-phosphate synthase gene (dxs), a rate-limiting enzyme of the isoprenoid pathway [32], the native dxs promoter was exchanged by the strong bacteriophage promoter T5 [28]. First, a chloramphenicol resistance gene, flanked by FRT sites, was cloned upstream of the T5-promoter into pQE31. The resistance gene and promoter region of the plasmid gained (pQE31-FRT-cat-FRT) was PCR amplified and subsequently inserted upstream of the native dxs in E. coli BW-CARO using the λ-Red recombineering technique. After elimination of the resistance gene, the resulting strain E. coli BW-CARO-dxs was further modified by the insertion of an additional copy of the native isopentenyl diphosphate isomerase gene (idi), a further rate-limiting enzyme of the isoprenoid pathway [33]. The E. coli idi gene was cloned into the expression vector pJF119ΔN and to allow transient selection for chromosomal integration, the cloned gene was hooked up to a downstream chloramphenicol resistance cassette. The expression cassette of the resulting plasmid pJF119-FRT-cat-FRT, including resistance gene, was PCR amplified and transformed into E. coli BW-CARO-dxs harboring the λ-Red recombinase expression plasmid pKD46. The selected PCR primers led to the homologous recombination of the expression cassette into the galactose locus (galETKM). The engineered E. coli strains gained (E. coli BW-CARO, E. coli BW-CARO-dxs, and E. coli BW-CARO-dxs-idi) were cultivated in complex medium and the carotenoid content was analyzed. All strains produce β-carotene as the sole carotenoid (Figure 2A). As expected, the amount of β-carotene increased due to the enhanced expression of dxs and idi. The exchange of the native dxs promoter by the T5-promoter in E. coli BW-CARO increased the amount of β-carotene per cell by a factor of 2.6. After insertion of the additional idi copy into E. coli BW-CARO-dxs the amount of β-carotene per cell increased by the factor of 1.9 (β-carotene content in E. coli BW-CARO 1.24 ± 0.17 mg/g cellular dry weight (cdw); E. coli BW-CARO-dxs 3.254 ± 0.24 mg/g cdw; E. coli BW-CARO-dxs-idi 6.18 ± 0.78 mg/g cdw).

Bottom Line: Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors.The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products.This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Microbiology, Universität Stuttgart, Stuttgart, Germany.

ABSTRACT

Background: The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.

Results: Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798) and the β-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).

Conclusions: By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this E. coli strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.

Show MeSH
Related in: MedlinePlus