Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin
© Lemuth et al; licensee BioMed Central Ltd. 2011
Received: 13 January 2011
Accepted: 26 April 2011
Published: 26 April 2011
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.
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).
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.
Xanthophylls comprise the group of oxygenated carotenoids that are synthesized by many photosynthetic organisms and also by some non-photosynthetic yeasts, fungi, and bacteria via condensation of isoprenoid units and subsequent oxidation reactions. The xanthophyll astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione) has gained considerable attention due to its beneficial effect on human health. It has been shown that astaxanthin bears a strong antioxidant and singlet oxygen-quenching activity  which can modulate biological functions ranging from lipid peroxidation to tissue protection against UV-light damage . Preclinical studies have further shown that astaxanthin exhibits anti-inflammatory properties and reduces rethrombosis after thrombolysis . Even more than the encouraging beneficial health properties, astaxanthin is used as a food colorant. The red-orange color of astaxanthin is closely connected with the quality of salmon or trout, for example. Therefore, the supplementation of astaxanthin or other carotenoids to their diets improves their value. Furthermore, the natural carotenoids in the diet of fish play an important role in reproduction .
At present, no recombinant xanthophyll-producing E. coli host has been reported that is able to accumulate astaxanthin as the sole carotenoid compound. However, significant improvements have been achieved by protein engineering of the β-carotene ketolase. By using random mutagenesis, crtW mutants form Paracoccus sp. and Sphingomonas sp., were generated that showed an up to 81%  and 90%  production of astaxanthin, respectively, when expressed in zeaxanthin-producing E. coli strains.
So far, all studies on the formation of xanthophyll compounds in E. coli use plasmids for the expression of the heterologous genes. To avoid the use of recombinant plasmids and to allow dispensing with selection makers like antibiotics, we present here a strain harboring the biosynthetic genes crtE, crtB, crtI, crtY, and crtZ from P. ananatis and crtW148 (NpF4798) from N. punctiforme PCC 73102 that are required for the formation of astaxanthin in E. coli stably inserted into the chromosome, along with an enhanced expression of the native E. coli genes idi and dxs. This strain is based on a β-carotene-producing basis strain (E. coli BW-CARO) recently developed in our lab . Furthermore, by balancing the expression of crtZ and crtW148, which was determined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), a plasmid-free E. coli strain was engineered that accumulated astaxanthin as the exclusive carotenoid.
Materials and methods
Bacterial strains, media, and chemicals
Bacterial strains and plasmids
Relevant genotype or sequences
E. coli DH5α
F-, ϕ80d, lacZ ΔM15, endA 1, recA 1, hsdR 17(rK-mK-), supE 44, thi-1, gyrA 96, relA 1, Δ(lacZYA-argF)U169
E. coli BW-CARO
E. coli BW25113, malEG::crtE, fucIK::crtB,
E. coli BW-CAROdxs
E. coli BW25113, malEG::crtE, fucIK::crtB,
xylAB::crtI, lacZA::crtY, PT5-dxs
E. coli BW-CAROdxs-idi
E. coli BW25113, malEG::crtE, fucIK::crtB,
xylAB::crtI, lacZYA::crtY, PT5-dxs, galEM::idi
E. coli BW-CANT
E. coli BW25113, malEG::crtE, fucIK::crtB,
xylAB::crtI, lacZA::crtY, PT5-dxs, galEM::idi, melAB::crtW148
E. coli BW-ASTA
E. coli BW25113, malEG:: Ptac-crtE, fucIK:: Ptac-crtB,
xylAB:: Ptac-crtI, lacZA:: Ptac-crtY, PT5-dxs, galEM:: Ptac-idi, melAB:: Ptac-crtW148, rbsDK:: Prha-crtZ
ParaB γ β exo (red recombinase), AmpR
pPQE32, N. punctiforme PCC 73102 crtW148148 gene, AmpR
cloning vector, RBS, Ptac, AmpR
pET11a, P. putida KT2440 hpd gene, AmpR,
pJF119ΔN, E. coli idi gene, AmpR
pJF119ΔN, E. coli idi gene, FRT-sites, AmpR, CmR
cloning vector, RBS, PT5, AmpR
pJF119ΔN, P. ananatis crtE gene, FRT-sites, AmpR, CmR
pJF119ΔN, N. punctiforme crtW148 gene, AmpR,
pJF119ΔN, N. punctiforme crtW148 gene, FRT-sites, AmpR, CmR
pAW223, P. ananatis crtZ gene, AmpR
pAW223, P. ananatis crtZ gene, FRT-sites, AmpR, CmR
Construction of plasmids
The crtW148 reading frame was obtained from plasmid pPQE32-148 . After Nde I/Hind III digestion, the fragment was ligated into Nde I/Hind III digested pJF119ΔN  and transformed into E. coli DH5α. The resulting plasmid pJF119-crtW148 was further treated with Hind III and ligated with a Hind III digested FRT-cat-FRT fragment derived from plasmid pCAS30-FRT-cat-FRT .
The reading frame of crtZ was isolated from plasmid pJF119-crtZ  after Nde I/Bam HI treatment and cloned into pAW223 [Nde I/Bam HI]. The resulting plasmid pAW-crtZ was further treated with Hind III and ligated with a Hind III digested FRT-cat-FRT fragment. The DNA sequence of idi was amplified by polymerase chain reaction (PCR) from chromosomal DNA of E. coli LJ110 using the following oligonucleotides: a) GTGAGAACATATG CAAACGGAACACGTC b) CAAATGTCGGGATCC TTTTATTTAAGCTGGG. The PCR-product was treated with Nde I and Bam HI and was ligated into expression vector pJF119ΔN hydrolyzed with Nde I and Bam HI. The resulting plasmid pJF-idi was further treated with Hind III and ligated with a Hind III digested FRT-cat-FRT fragment to gain plasmid pJF119-idi- FRT-cat-FRT.
The dxs promoter replacement was conducted according to the method used by Yuan et al . Thus, for the construction of pQE31-FRT-cat-FRT, the DNA sequence of FRT-cat-FRT was amplified by PCR from plasmid pCAS30-FRT-cat-FRT by using the following oligonucleotides: a) CACAGACTGAGGATCCTCGAG AGTCGACCTGCAGG and b) CTGTTTTATCAGACCGCTCGAG CGTTCTGATTTA. The PCR product was hydrolyzed with Xho I and ligated into the Xho I hydrolyzed plasmid pQE31.
The individual expression cassettes of idi, crtW148, and crtZ from plasmid pJF-idi- FRT-cat-FRT, pJF-crtW-FRT-cat-FRT, and pAW-crtZ-FRT-cat-FRT, respectively, were amplified by PCR. The oligonucleotide primers for the Ptac-crtW expression cassette were: 1) CGGCGCATATTGCCCTGATGGACATTGACCCCACCCGCCTGGAAGAGTCGCATATTGTTCAAGGCGCACTCCCGTTCTGG and 2) AGCGCAACGATGGCTTTAAGTGTCAGATGGCTTCCTTCAGCAGACGGTTGATTGTCTGCAGGGTTATTGTCTCATGAGCG. Primers for the Prha-crtZ expression cassette were: ACCGTTCTTAATTCTGATATTTCATCGGTGATCTCCCGTCTGGGACATACCGATATGCATGCATCGATCACCACAATT and ATTCACGCTAGCCCATACACCACGACTTCCTAAAGTAATCAGTACAGTACGGATACC CAGGGTTATTGTCTCATGAGCGGATAC. Primers for the Ptac-idi expression cassette were: TGCAGGCATGAAACCGCGTCTTTTTTCAGATAAAAAGCGCAATCAGTCGCTCAA GGCGCACTCCCGTTCTGG and TAACATTACTCAGCAATAAACTGATATTCCGTCAGGCTGGAATAAGGATGGCCT TCTGCTTAATTTGATGCCTG.
For the integration of the expression cassettes, the purified PCR products were each transformed into E. coli BW-CARO and its variant strain carrying plasmid pKD46. Expression of the λ-Red enzymes and the preparation of competent cells were carried out as described previously . Competent cells were electroporated with 0.2-0.4 μg of PCR product. After electroporation, the cells were resuspended in 1 ml LB-medium and incubated at 30°C for 12 h with shaking. Subsequently, the cell suspension was spread onto MacConkey-agar plates containing chloramphenicol and 1% of the respective sugar (D-galactose, D-ribose, or melibiose), corresponding to the targeted genes responsible for sugar degradation. MacConkey agar plates were incubated over-night at 37°C. 5-10 pale colonies per transformation were picked  and checked regarding correct recombination by control PCR. The chloramphenicol resistance cassette was eliminated using the plasmid pCP20 as previously described .
Extraction and analysis of carotenoids
E. coli strains carrying carotenoid biosynthetic genes were cultivated in shake flasks in 50 ml LB medium or minimal medium. Cultivations were carried out at 30°C. At an optical density (OD600) of 0.3 - 0.4 the cultures were induced, if required, by addition of IPTG (0.5 mM, final conc.) and/or L-rhamnose (0.2% w/v, final conc.). Samples (1 ml) were withdrawn from cultures after different time points within a period of 48 h. The cells were harvested by centrifugation, washed with cold water, and subsequently extracted by vigorous shaking with acetone (500 μl) for 15 min at 50°C. Insoluble components of the extract were removed by centrifugation (20,000 × g). HPLC analysis was performed on Dionex HPLC Instrument (Idstein, Germany), installed with a Chromeleon Software, Gina autosampler, P580 pumps, and a diode array detector. Products were analyzed by loading 50 μl of the supernatant onto a C30-reverse-phase HPLC column (250 mm × 4.6 mm, 5 μm, YMC-Europa GmbH, Dinslaken, Germany) attached with a guard column containing matrix of the same material as the column. A solvent flow rate of 1.0 ml/min was used. The solvents used were Solvent A, consisting of MTBE (methyl-tert-butylether)/methanol/water (19/80/1) and Solvent B, consisting of MTBE/methanol/water (90/9/1). Gradient conditions: equilibration conditions at 2% B; 0 to 22 min linear gradient from 2% B to 100% B. The spectra of the eluted carotenoids were recorded online with a Dionex UVD340 diode array detector (200 - 600 nm).
Carotenoid compounds were identified by co-chromatography using authentic standard compounds and by analysis of its UV-Vis spectra.
For the quantification of the carotenoid compounds the integrated peak areas were compared to HPLC standard curves of authentic standards.
Detection and quantification of crtW148 and crtZ
Although isoprenoid products of CrtW148 and CrtZ were detected, neither enzyme activity tests nor SDS-PAGE were able to verify the presence of these proteins. Therefore, mRNA from crtW148 and crtZ was detected by means of absolute RT-qPCR using an internal standard.
(I) Generation of the internal standard
Internal hpd (hydroxyphenyl-pyruvate dioxygenase gene from Pseudomonas putida) RNA standard was generated. This was similar to a protocol published by Schuhmacher et al.  with the following exceptions: as template for the internal standard, pCAS1 was used .
(II) Sampling, total RNA isolation, and cDNA synthesis
E. coli BW-ASTA was cultivated in shaking flasks at 37°C (140 rpm) either in LB; LB plus L-rhamnose [12 mM]; LB plus L-rhamnose [12 mM] and IPTG [0.5 mM] or in minimal medium (MM); MM plus IPTG [0.5 mM]; or MM plus IPTG [0.5 mM] and L-rhamnose [12 mM]. For transcript level quantification, samples from the late exponential phase were taken. In total, two biological replicates were processed.
A cell number - OD correlation was generated by counting E. coli BW-ASTA using a Neubauer counting chamber. According to this correlation, 5 × 108 cells were pipetted into twice the amount RNA protect bacteria reagent (Qiagen) and processed according to manufacturer's protocol to prevent RNA degradation. Prior to RNA isolation, cell pellets were spiked with 10 μL internal hpd standard RNA [6.3 × 109 copies/μL]. RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany). On-column DNAse digestion was performed using RNase-free DNase set from Qiagen. RNA concentration and quality were assessed photometrically (NanoDrop ND 1000) and analyzed using the Agilent Bioanalyzer 2100. In total, RNA concentrations ranged from 396 ng/μL to 710 ng/μL. Only RNA with 260/280 nm ratios of 1.8 to 2.0 and 260/230 nm ratios greater than 1.8 were used for reverse transcription. Reverse transcription (RT) of 1 μg total RNA was performed using QuantiTect Rev. Transcription Kit (Qiagen, Hilden, Germany) in a total volume of 20 μL according to manufacturer's protocol. cDNA was stored at -80°C until further use.
(III) Quantitative real-time PCR
Primer and hydrolysis probe sequences for qPCR, amplicon length and PCR efficiencies
Primer 1 (5' - 3')
Primer 2 (5' - 3')
with N: copies × μL-1; NA: copies/mol (Avogadro constant with 6.022 × 1023 × mol-1), Conc.hP: concentration hydrolyzed plasmid (g × μL-1); MW: molecular weight (g × mol-1) and MWhP: molecular weight hydrolyzed plasmid.
(IV) Calculation of internal standard recovery and absolute copy numbers
with NmRNA, cell being the absolute copy number per cell and NmRNAα being the copy number of the mRNA of interest in total volume used for RNA isolation (50 μL).
with NmRNA,quant: copy number estimated by qPCR.
Construction of a β-carotene-producing strain
Chromosomal integration of crtW and crtZ
The reading frame of the β-carotene ketolase gene (crtW148) from N. punctiforme PCC73102 was amplified from plasmid pQE32-148 and cloned into the expression vector pJF119ΔN, resulting in plasmid pJF-crtW148. In transformants of β-carotene-producing E. coli BW-CARO-dxs-idi with pJF-crtW148 the di-keto carotenoid canthaxanthin represented 85% (0.896 ± 0.12 mg/g cdw) of the total carotenoid content with about 10% β-carotene and small amounts of echinenone (Figure 2C). The ratio of the three carotenoids did not vary during the 48 h cultivation. The addition of the inducer IPTG had neither influence on the carotenoid formation nor on growth (data not shown).
After cloning of a FRT-cat-FRT cassette into pJF-crtW148, chromosomal integration of the Ptac-crtW148 expression cassette into the melibiose locus (melAB) of E. coli BW-CARO-dxs-idi, and elimination of the cat resistance cassette, the engineered strain E. coli BW-CANT showed an about 20% higher formation of canthaxanthin (1.085 ± 0.15 mg/g cdw) but the percentage of canthaxanthin of all carotenoids was 85% as in the plasmid carrying strain E. coli BW-CARO-dxs-idi pJF-crtW148. The addition of IPTG up to 1 mM into LB-medium cultures of E. coli BW-CARO-dxs-idi pJF-crtW148 and E. coli BW-CANT, respectively, did not change the ratio of canthaxanthin, β-carotene, and echinenone as well as the total carotenoid yield, which we take as evidence of the leakiness of the tac-promoter in LB-medium.
In order to engineer an astaxanthin-producing strain, the β-carotene hydroxylase gene (crtZ) was inserted into the chromosome of E. coli BW-CANT. To allow a variable expression of crtZ compared to the tac-promoter controlled biosynthetic genes, crtZ was expressed under control of the rhamnose-promoter (PrhaBAD). For this purpose crtZ was cloned from plasmid pJF119-crtZ into pAW223. To verify the functional expression of this construct, pAW-crtZ was introduced into β-carotene-producing E. coli. Cultivation of E. coli BW-CARO-dxs-idi pAW-crtZ and subsequent carotenoid analysis showed that zeaxanthin was produced as the only carotenoid product (1.48 mg/g cdw).
Controlled gene expression in E. coli BW-ASTA using glucose-containing medium
Transcriptional analysis of crtW148 and crtZ
In order to ascertain whether the assumed expression levels of CrtW148 and CrtZ based on carotenoid formation by E. coli BW-ASTA were in accordance with the mRNA levels of crtW148 and crtZ, we performed absolute RT-qPCR studies. A method previously described by Schuhmacher et al.  that uses an external standard for absolute transcript number quantification, was adapted to the Roche UPL system allowing for absolute transcript number determination per cell.
From the cultivation of E. coli BW-ASTA in LB medium and in minimal medium, with or without induction of the tac- and rha-promoter, respectively, cells were withdrawn in the late exponential phase and were analyzed for the crtZ and crtW148 transcript level by RT-qPCR. The number of transcripts of crtW148 and crtZ under the different conditions are shown in Figure 4. In LB medium, comparable transcript numbers per cell could be detected for crtW148 with or without IPTG induction (between 22 ± 4 and 25 ± 1 copies/cell). A 15% higher copy number was detected for crtZ (41 ± 3 copies/cell compared to 48 ± 5 to 53 ± 3 copies/cell) after induction with L-rhamnose. The transcript level of crtZ reflects the formation of zeaxanthin when cultivated in complex medium. An increased expression of crtZ correlated with an increased formation of zeaxanthin by E. coli BW-ASTA. Under all cultivation conditions in LB medium, zeaxanthin and astaxanthin had been detected. By using glucose-containing minimal medium the transcription of the heterologous genes was more tightly regulated than in LB medium. Transcript numbers per cell of crtW148 without the addition of IPTG were 12 ± 1. For crtZ in average, less than one copy number per cell was detected in minimal medium without induction. This reflects the tight regulation of the rhamnose-promoter under these conditions. These results are in good accordance with the product formation; in minimal medium without induction, only small amounts of astaxanthin as the sole carotenoid were detected (Figure 3). The induction by IPTG led to a 2.3 to 2.6 fold increase in mRNA level of crtW148, and resulted in a 20-fold increase of the carotenoid concentration. Here, astaxanthin was the only carotenoid that was accumulated by E. coli BW-ASTA after 48 h (Figure 2). This shows that the low mRNA level of crtZ yielded in a sufficient amount of hydroxylase activity converting all the produced precursors into astaxanthin. The addition of L-rhamnose led to a significant increase in transcript levels of crtZ (8.6 fold). In these cases, both astaxanthin (90%) and zeaxanthin (5%) had been detected as products after 48 h.
The in vivo biosynthesis of a complex natural product in a heterologous host like E. coli first requires the introduction and the functional expression of biosynthetic genes that enable the conversion of available cell intermediates towards the desired product. The heterologous biosynthesis of astaxanthin by E. coli has been achieved in numerous studies by the expression of respective carotenoid biosynthetic genes using recombinant plasmids [20, 23, 33–36]. In this study, we constructed a plasmid-free E. coli strain that carries each of the astaxanthin biosynthetic genes (crtE,B,I,Y,Z,W) as individual expression units on the chromosome. For the integration of the expression cassettes we used a method, recently developed in our lab, that allows fast and reliable integration and screening . It is based on the λ-Red mediated recombination technique developed for the directed knock-out . This method utilizes the replacement of E. coli s' rare sugar degradation genes which are dispensable for most biotechnological applications. The replacement of these genes can easily be visualized by the use of MacConkey differential agar medium carrying the corresponding sugar compound.
The chromosomal insertion of heterologous biosynthetic genes has for obvious reasons some advantages compared to the use of heterologous plasmids. Thus, plasmids may be the best choice for the cloning and short-term expression of recombinant genes, in particular for the maximum overproduction of a given protein. Especially in metabolic engineering applications, however, a too strong gene expression may be unfavorable for long-term productivity . Yoon et al.  observed that a high expression of lycopene biosynthetic genes in E. coli leads to a decrease in growth and lycopene production. Therefore, the chromosomal integration of heterologous expression cassettes can be favorably compared to multi-copy plasmids in terms of metabolic burden effects, structural instability, and most important segregational instability . Furthermore, a stable chromosomal insertion obviates the use of selection-markers (e.g. antibiotics) that are commonly used for the maintenance of plasmids during cultivation. Especially antibiotics are both costly and can hamper the product purification in food and pharmaceutical production processes. On the other hand, a low enzyme activity of a heterologous downstream pathway can result in a reduced product yield or in an accumulation of pathway intermediates . Thus, the in vivo biosynthesis of carotenoids in E. coli requires an appropriate heterologous gene expression, adapted to the supply of isoprenoid precursors to avoid the effect of metabolic burden and to avoid the accumulation of pathway intermediates. The increased biosynthesis of β-carotene (up to 6.2 mg/g cdw) by the enhanced expression of idi and dxs in E. coli BW-CARO demonstrates that the expression of the heterologous biosynthetic genes and accordingly the enzyme activity of the corresponding proteins in this strain do not limit the formation of β-carotene. Similar observations were made by Chiang et al.  for a lycopene-producing strain that carries a single copy of a lycopene biosynthetic gene cluster on the chromosome. Surprisingly, the additional expression of crtW148 and crtZ in the β-carotene-producing strain reduced the overall formation of carotenoids about three times compared to E. coli BW-CARO-dxs-idi. This leads to the suggestion that the recombinant proteins (CrtW148 or CrtZ) or a product of their enzymatic reaction effect the formation of the carotenoid precursors upstream of phytoene, because no other carotenoid accumulated in the cell. This stands in contrast to the study by Scaife et al.  who found that expression of a β-carotene-ketolase and -hydroxylase within a β-carotene-producing strain significantly increases the total carotenoid yield. The maximum astaxanthin yield of almost 2 mg/g in this study and ours is in the same range and represents the highest astaxanthin content in E. coli that has been achieved so far.
Besides the chromosomal integration, another aim of our work was to find conditions by which E. coli synthesizes astaxanthin as the sole carotenoid. For the conversion of β-carotene to astaxanthin, the β-carotene ketolase gene from Nostoc punctiforme (crtW148) and the β-carotene hydroxylase gene from Pantoea ananatis (crtZ) were chosen, because the corresponding proteins (CrtW148, CrtZ; see Figure 1) are known to be bifunctional and therefore accept β-carotene as well as hydroxylated or ketolated products, respectively, as substrate [12, 20]. In order to vary the expression level of crtW148 and crtZ, the hydroxylase gene was expressed under control of the rha-promoter in contrast to the other heterologous genes that were controlled by a tac-promoter, respectively. The cultivation of E. coli BW-ASTA in LB medium showed the formation of astaxanthin as the predominant product but also a significant amount of zeaxanthin that increased upon the additional induction of the rha-promoter. In contrast, the additional IPTG induction of the crtW148 controlling tac-promoter had no significant influence on the product formation. The carotenoid formation is in concordance with the results of mRNA quantification. The qPCR measurement showed that both, tac-promoter (crtW148) and rha-promoter (crtZ), are only marginally repressed under the given conditions (Figure 4).
It is supposed that hydroxylase and ketolase compete for their substrate and that only a balanced expression of these two enzymes might lead to a complete conversion of β-carotene to astaxanthin [12, 42–44]. This hypothesis is supported by our results. We suppose that in the astaxanthin biosynthesis by E. coli BW-ASTA the hydroxylation reaction occurs fast with β-carotene as well as with the ketolated intermediates as substrates. No intermediates were detected under these conditions which are mostly ketolated (Figure 2). The CrtW148 ketolase does utilize β-carotene and to a minor extent hydroxylated intermediates. But during the course of cultivation the ketolase was not able to convert the accumulated zeaxanthin into adonixanthin or astaxanthin, completely. Although the bifunctionality of CrtW148 from N. punctiforme was proven , the conversion of zeaxanthin to astaxanthin by CrtW148 is obviously the most limiting step towards the efficient biosynthesis of astaxanthin in our system. To improve the conversion of zeaxanthin to astaxanthin, protein engineering of a CrtW-type ketolase had been used successfully, but a complete transformation to astaxanthin was not achieved [22, 23]. In contrast to this research, our working hypothesis was to avoid the accumulation of zeaxanthin by increasing the activity ratio of CrtW148 to CrtZ not by enhanced crtW148 expression or enzyme activity/specificity but instead by lowering the crtZ expression level. This was achieved by the use of D-glucose containing minimal medium that led to a better balanced regulation of both heterologous promoters. Especially the rhamnose-promoter, that regulates the expression of crtZ, is more tightly controlled by the catabolite repression [45, 46]. Cultivation of E. coli BW-ASTA under this condition with induction of the IPTG-controlled heterologous promoters led in the early phase of the cultivation to the formation of astaxanthin and the intermediates adonirubin and canthaxanthin (Figure 2) that vanished, presumably due to the transformation into astaxanthin during the course of cultivation. We take this as evidence that the adjustment of the expression level can direct the pathway towards the desired product, astaxanthin. The low expression of crtZ and the, in contrast, high expression of crtW148 (Figure 4) make it obvious that the synthesized β-carotene is due to the kinetics of the reactions, preferentially converted into canthaxanthin, which is secondly completely transformed by the slower hydroxylation reaction via adonirubin into astaxanthin. Similar observations concerning the competition between hydroxylase and ketolase have been made for transgenic maize where plants harboring only an endogenous hydroxylase but an exogenous ketolase gene under an endospecific promoter accumulated astaxanthin. Astaxanthin, however, was not accumulated by plants harboring both exogenous hydroxylase and ketolase genes . The authors indicated that the avoidance of the adonixanthin accumulation is crucial for astaxanthin production in transgenic maize endosperm . In contrast, we find the avoidance of zeaxanthin accumulation to be the crucial step for sole astaxanthin synthesis in the bacterium E. coli BW-ASTA.
In this study, we engineered a plasmid-free E. coli strain that carries biosynthetic genes for the in vivo biosynthesis of astaxanthin. The stable chromosomal insertion of the heterologous genes enables dispensing with selection makers that are required for the maintenance of recombinant plasmid. The biosynthetic genes were each integrated as single expression units into the chromosome of E. coli. This approach allows the control of individual gene expression levels, which is complicated to achieve if the astaxanthin biosynthetic genes are organized within one operon. This E. coli engineering strategy might also be useful as a general approach for the construction of stable production strains for the heterologous biosynthesis of natural products for which excessive protein overexpression is a hindrance.
The adjustment of the crtZ expression level in the astaxanthin-producing strain was applied successfully for the complete in vivo conversion of β-carotene into astaxanthin by recombinant E. coli cells.
The authors are grateful to Prof. Gerhard Sandmann (Universität Frankfurt) for providing us with plasmid pPQE32-148, to Holger Beuttler (Institute of Technical Biochemistry, Universität Stuttgart) for providing carotenoid standards, to Cristina Prada for her help with the construction of E. coli BW-CARO-dxs-idi, and particularly to Prof. Georg Sprenger for helpful discussions, continuous support, and critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 706/TP B3).
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