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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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Transcript copy numbers/cell quantified by absolute RT-qPCR. Copy numbers ± STABW of crtW148 (grey) and crtZ (black) from E. coli BW-ASTA cultivated in LB or minimal medium (MM) and harvested in late exponential phase are shown. Rha: Induction with L-rhamnose [12 mM]. IPTG: Induction with IPTG [0.5 mM]. * indicates significant transcript number changes between un-induced (LB or MM) and induced state (LB Rha, LB Rha IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, n = 4, unpaired t-test assuming unequal variances.
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Figure 4: Transcript copy numbers/cell quantified by absolute RT-qPCR. Copy numbers ± STABW of crtW148 (grey) and crtZ (black) from E. coli BW-ASTA cultivated in LB or minimal medium (MM) and harvested in late exponential phase are shown. Rha: Induction with L-rhamnose [12 mM]. IPTG: Induction with IPTG [0.5 mM]. * indicates significant transcript number changes between un-induced (LB or MM) and induced state (LB Rha, LB Rha IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, n = 4, unpaired t-test assuming unequal variances.

Mentions: In order to avoid the formation of zeaxanthin by E. coli BW-ASTA, an increase of the ketolase activity or a decrease of the hydroxylase activity would be necessary. To better control the expression by the tac- and, in particular, by the rha-promoter that are both not tightly controlled in LB-medium (see Figure 4), minimal medium with glucose as C- and energy source was used. The cultivation of E. coli BW-ASTA in minimal medium without the addition of inducer molecules resulted in the formation of only small amounts (0.11 mg/g cdw) of astaxanthin, which we interpret as the result of the higher repression of the tac-promoter in minimal medium in contrast to LB-medium. On the other hand, the addition of IPTG to the cultures, which induces the Ptac controlled gene expression of dxs, idi, crtE, crtB, crtI, crtY, and crtW, resulted after 48 h in a 12-fold increase in the formation of carotenoids. After 24 h of incubation in minimal medium, E. coli BW-ASTA synthesized 0.96 ± 0.14 mg/g cdw astaxanthin and the by-products adonirubin (13%), canthaxanthin (12%), and β-carotene (1%) (Figure 2E). In contrast to the cultivation in LB medium no zeaxanthin formation was observed. After an incubation time of 48 h astaxanthin was produced as exclusive carotenoid (>95%) in a concentration of (2.07 ± 0.15 mg/l; 1.41 ± 0.11 mg/g cdw) (Figure 3). This result shows that during the cultivation of E. coli BW-ASTA in minimal medium with glucose and IPTG the β-carotene produced is predominantly converted by the ketolase (CrtW148) into cantaxanthin, which is subsequently slowly hydroxylated by CrtZ. The concurrent addition of both inducers, IPTG and L-rhamnose, also led after 24 h to the formation of astaxanthin (68%), adonirubin (14%), cantaxanthin (12%), and traces of β-carotene (1%). In the late stationary phase (48 h) of the culture astaxanthin was still the predominant carotenoid with >90%, however, the cells also contained about 5% of zeaxanthin as a by-product (Figure 2F).


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)

Transcript copy numbers/cell quantified by absolute RT-qPCR. Copy numbers ± STABW of crtW148 (grey) and crtZ (black) from E. coli BW-ASTA cultivated in LB or minimal medium (MM) and harvested in late exponential phase are shown. Rha: Induction with L-rhamnose [12 mM]. IPTG: Induction with IPTG [0.5 mM]. * indicates significant transcript number changes between un-induced (LB or MM) and induced state (LB Rha, LB Rha IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, n = 4, unpaired t-test assuming unequal variances.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111352&req=5

Figure 4: Transcript copy numbers/cell quantified by absolute RT-qPCR. Copy numbers ± STABW of crtW148 (grey) and crtZ (black) from E. coli BW-ASTA cultivated in LB or minimal medium (MM) and harvested in late exponential phase are shown. Rha: Induction with L-rhamnose [12 mM]. IPTG: Induction with IPTG [0.5 mM]. * indicates significant transcript number changes between un-induced (LB or MM) and induced state (LB Rha, LB Rha IPTG or MM IPTG, MM Rha IPTG, respectively). p ≤ 0.05, n = 4, unpaired t-test assuming unequal variances.
Mentions: In order to avoid the formation of zeaxanthin by E. coli BW-ASTA, an increase of the ketolase activity or a decrease of the hydroxylase activity would be necessary. To better control the expression by the tac- and, in particular, by the rha-promoter that are both not tightly controlled in LB-medium (see Figure 4), minimal medium with glucose as C- and energy source was used. The cultivation of E. coli BW-ASTA in minimal medium without the addition of inducer molecules resulted in the formation of only small amounts (0.11 mg/g cdw) of astaxanthin, which we interpret as the result of the higher repression of the tac-promoter in minimal medium in contrast to LB-medium. On the other hand, the addition of IPTG to the cultures, which induces the Ptac controlled gene expression of dxs, idi, crtE, crtB, crtI, crtY, and crtW, resulted after 48 h in a 12-fold increase in the formation of carotenoids. After 24 h of incubation in minimal medium, E. coli BW-ASTA synthesized 0.96 ± 0.14 mg/g cdw astaxanthin and the by-products adonirubin (13%), canthaxanthin (12%), and β-carotene (1%) (Figure 2E). In contrast to the cultivation in LB medium no zeaxanthin formation was observed. After an incubation time of 48 h astaxanthin was produced as exclusive carotenoid (>95%) in a concentration of (2.07 ± 0.15 mg/l; 1.41 ± 0.11 mg/g cdw) (Figure 3). This result shows that during the cultivation of E. coli BW-ASTA in minimal medium with glucose and IPTG the β-carotene produced is predominantly converted by the ketolase (CrtW148) into cantaxanthin, which is subsequently slowly hydroxylated by CrtZ. The concurrent addition of both inducers, IPTG and L-rhamnose, also led after 24 h to the formation of astaxanthin (68%), adonirubin (14%), cantaxanthin (12%), and traces of β-carotene (1%). In the late stationary phase (48 h) of the culture astaxanthin was still the predominant carotenoid with >90%, however, the cells also contained about 5% of zeaxanthin as a by-product (Figure 2F).

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