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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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Production of zeaxanthin (A) and astaxanthin (B) by E. coli BW-ASTA (in mg per liter of culture (rhomboid)) and in mg per gram cellular dry weight (cdw; (square)) after cultivation for 48 h under different conditions. Values represent the mean (n = 4) ± standard deviation. * indicates significant changes between un-induced (LB or MM) and induced state (LB Rha, LB IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, unpaired t-test assuming unequal variances.
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Figure 3: Production of zeaxanthin (A) and astaxanthin (B) by E. coli BW-ASTA (in mg per liter of culture (rhomboid)) and in mg per gram cellular dry weight (cdw; (square)) after cultivation for 48 h under different conditions. Values represent the mean (n = 4) ± standard deviation. * indicates significant changes between un-induced (LB or MM) and induced state (LB Rha, LB IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, unpaired t-test assuming unequal variances.

Mentions: Cloning of a FRT-cat-FRT cassette into pAW-crtZ and chromosomal integration of the PrhaBAD-crtZ expression cassette into the ribose locus (rbsDABCK) of E. coli BW-CANT and elimination of the resistance cassette resulted in the strain E. coli BW-ASTA, containing all required biosynthetic genes for the formation of astaxanthin as single expression units, respectively, on the chromosome. Cultivation of E. coli BW-ASTA in LB-medium showed that astaxanthin is the predominant carotenoid product (71%). Besides astaxanthin, zeaxanthin (25%) was the only other detectable carotenoid (Figure 2B, Figure 3). The addition of the inducer L-rhamnose to E. coli BW-ASTA cultures led to an increase of zeaxanthin (69%) compared to astaxanthin (20%) (Figure 2D, Figure 3). The enhanced expression of crtZ by L-rhamnose induction therefore led to a significant increase of zeaxanthin, obviously due to a low activity of CrtW148 for the substrate zeaxanthin. The induction of E. coli BW-ASTA LB medium cultures with IPTG in the presence of 0.2% L-rhamnose or without had neither effect on the amount of carotenoid nor on the ratio of astaxanthin and zeaxanthin.


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)

Production of zeaxanthin (A) and astaxanthin (B) by E. coli BW-ASTA (in mg per liter of culture (rhomboid)) and in mg per gram cellular dry weight (cdw; (square)) after cultivation for 48 h under different conditions. Values represent the mean (n = 4) ± standard deviation. * indicates significant changes between un-induced (LB or MM) and induced state (LB Rha, LB IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, unpaired t-test assuming unequal variances.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111352&req=5

Figure 3: Production of zeaxanthin (A) and astaxanthin (B) by E. coli BW-ASTA (in mg per liter of culture (rhomboid)) and in mg per gram cellular dry weight (cdw; (square)) after cultivation for 48 h under different conditions. Values represent the mean (n = 4) ± standard deviation. * indicates significant changes between un-induced (LB or MM) and induced state (LB Rha, LB IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, unpaired t-test assuming unequal variances.
Mentions: Cloning of a FRT-cat-FRT cassette into pAW-crtZ and chromosomal integration of the PrhaBAD-crtZ expression cassette into the ribose locus (rbsDABCK) of E. coli BW-CANT and elimination of the resistance cassette resulted in the strain E. coli BW-ASTA, containing all required biosynthetic genes for the formation of astaxanthin as single expression units, respectively, on the chromosome. Cultivation of E. coli BW-ASTA in LB-medium showed that astaxanthin is the predominant carotenoid product (71%). Besides astaxanthin, zeaxanthin (25%) was the only other detectable carotenoid (Figure 2B, Figure 3). The addition of the inducer L-rhamnose to E. coli BW-ASTA cultures led to an increase of zeaxanthin (69%) compared to astaxanthin (20%) (Figure 2D, Figure 3). The enhanced expression of crtZ by L-rhamnose induction therefore led to a significant increase of zeaxanthin, obviously due to a low activity of CrtW148 for the substrate zeaxanthin. The induction of E. coli BW-ASTA LB medium cultures with IPTG in the presence of 0.2% L-rhamnose or without had neither effect on the amount of carotenoid nor on the ratio of astaxanthin and zeaxanthin.

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