Part:BBa_K4432220
CrtW expression cassette under the control of the pLac promoter
This part is an expression cassette of the Nostoc punctiforme's CrtW gene (BBa_K4432020) under the control of the pLac promoter regulated by LacI (BBa_R0010) and a custom-made RBS (BBa_K4432010).
The CrtW gene encodes a ß-carotene-ketolase that catalyses the conversion of ß-carotene into echinenone and of echinenone into canthaxanthin.
Usage and Biology
Canthaxanthin belongs to the carotenoid class of compounds [1] which unifies antioxidants usually known for their vivid colors. For health, color and stability canthaxanthin has become renowned in a list of human industries from cosmetics to health products. It is likely due to versatility of canthaxanthin applications, that it’s market value in 2017 has reached $75 million [2]. Among other intriguing factors, as a carotenoid, it can be expressed via heterologous engineering of endogenous MEP pathway. The latter, combined with its appealing red-orange color makes canthaxanthin a perfect target for engineering and streamlining biosynthetic pathways.
Design
The canthaxanthin production pathway (Figure 1) can be divided in un upper (E. coli endogenous) module consisting in production of the intermediate farnesyl diphosphate (FPP), the middle module for the production of ß-carotene and the bottom module for the conversion of ß-carotene to canthaxanthin.
From ß-carotene to canthaxanthin
Canthaxanthin is derived from ß-carotene through the action of the enzyme ß-carotene-ketolase (EC 1.14.99.63) which oxidizes the two rings of ß-carotene sequentially and converts it first to echinenone than to canthaxanthin [3].
Two structurally different ß-carotene-ketolases, the CrtW-type and the CrtO-type were identified in various organisms where, depending on their substrate specificity, are able to catalyze only the first reaction (ß-carotene to echinenone), only the second one (echinenone to canthaxanthin) or both. Moreover, some CrtW and CrtO enzymes are promiscuous and able to oxidize other carotenoids like zeaxanthin, adonixanthin.
To achieve the canthaxanthin production, we choose to use the CrtW148 gene from Nostoc punctiforme PCC 73102 (BBa_K4432020) which was shown to efficiently catalyze both steps of the canthaxanthin synthesis from ß-carotene [4].
In addition, we also choose to use the gene responsible for the canthaxanthin production in Dietzia sp. RNV-4 strain [5]. To the best of our knowledge, no biochemical analysis was described in the literature for any CrtW or CrtO enzymes of any Dietzia sp. Moreover, the genome of the Dietzia sp. RNV-4 strain is not available, nor the sequence of a CrtW or a CrtO enzyme. However, based on the 16S rRNA sequence analysis, the Dietzia psychralcaliphila ILA-1 species, whose genome is available, has 99% sequence similarity with the Dietzia sp. RNV-4 strain. Using different CrtW and CrtO protein sequences for the EC 1.14.99.63 enzymes found in the Kegg database, we performed protein homology searches on the D. psychralcaliphila ILA-1 genome (GenBank Acc. N° CP015453) and thus uncovered a potential CrtO-like enzyme (BBa_K4432021) encoded by the A6048_06315 locus (annotated as a FAD-dependent oxidoreductase by automated computational analysis using protein homology gene prediction method). No CrtW-like enzyme was spotted.
From FPP to ß-carotene
One prerequisite for canthaxanthin production is the ß-carotene synthesis. This can be ‘easily’ achieved in E. coli by using part constructed by previous iGEM teams, like for instance the BBa_K274220 in which four genes CrtE, CrtB, CrtI and CrtY from Pantoea ananatis were assembled in an operon under the control of pBad promoter inducible by L-arabinose by the iGEM 2009 Cambridge team.
To FPP in E. coli'
E. coli is not able to naturally produce carotenoids, but is able to synthesize the farnesyl diphosphate (FPP) which is a key intermediate in the isoprenoids biosynthesis. In E. coli, FPP production is achieved via the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway which is a ten steps pathway starting from pyruvate and glyceraldehyde 3-phosphate. An alternative, the mevalonate pathway starting with acetyl, is present in eukaryotic organisms (including examples of fungi and algae) and archaea, using as Acetyl-CoA precursor.
Microbial biosynthesis of carotenoids is a well-studied and optimized metabolic engineering case thanks to the introduction of a biosynthetic pathway to classical E. coli chassis. The first metabolic engineering approaches for the carotenoid biosynthesis rapidly included optimizing the flux through the FPP to increase production yields. Over-expressing endogenous rate limiting enzymes of the MEP pathway [6,7] or expressing heterologous mevalonate pathway [8] have both been used. The latter gave better results so far, but MEP pathway is nonetheless promising because its theoretical yield is higher [9].
To maximize FPP production through the MEP pathway, understanding its regulation is critical to bypass cellular control, since it is an endogenous pathway [10]. The new tools provided by the systems biology field established that the steps catalyzed by the 1-deoxyxylulose-5-phosphate synthase (dxs) (BBa_K3166061) and isopentenyl diphosphate isomerase (idi) (BBa_K3166068) enzymes are limiting steps of the MEP pathway [11]. Indeed, overexpression of dxs and idi enzymes increased carotenoids production yields in E. coli [12].
Thus, for improving the canthaxanthin yield in E. coli, we decided to overexpress the E. coli dxs and idi genes and thus increase the amount of FPP precursor available.
Figure 1. Biochemical pathway of canthaxanthin synthesis composed of the endogenous E. coli MEP pathway, the heterologous ß-carotene producing pathway (the CrtEBIY genes from Pantoea ananatis) and the canthaxanthin producing step catalyzed either by the CrtW enzyme from Nostoc punctiforme or the CrtO enzyme from Dietzia psychralcaliphila.
Build
To implement the canthaxanthin production pathway in E. coli and enhance its yield we used biobricks assembled by previous iGEM teams, as well as new ones designed by us.
Upper module: enhancing the FPP in E. coli'
To overexpress the E. coli idi and dxs genes we assembled through the Golden Gate technique, an expression vector in the both pSB1A3 and pSB3T5 backbones (BBa_K4432122) in which the idi and dxs gene were placed under the control of a hybrid T5 promoter regulated by LacI. For this, the idi and dxs gene sequences were PCR-amplified from the genome of E. coli equipped by custom-made RBSes that we specifically designed using the online tools provided by Salis’s De Novo DNA company.
Middle module: from FPP to ß-carotene
We used BBa_K274220 comprising the CrtEBIY operon (containing four genes from P. ananatis allowing the conversion of FPP to ß-carotene) placed by the iGEM 2009 Cambridge team under the control of pBad promoter inducible by L-arabinose.
As a control, we also used BBa_K274120 assembled by the same team, in which the CrtY is not present and the carotenoids pathway thus stops at lycopene.
Both these biobricks were recovered from the iGEM distribution kits where they were made available in the pSB2K3 backbone.
Bottom module: from ß-carotene to canthaxanthin The two selected genes, CrtW from N. punctiforme and CrtO from D. psychralcaliphila were placed under the control of either a hybrid T5 promoter regulated by LacI (BBa_K4432120 and BBa_K4432121), or the classical pLac promoter (BBa_K4432220 and BBa_K4432221) and assembled through the Golden Gate technique in the pSB1A3 backbone. For a proper protein expression, custom-made RBSes were specifically designed using the online tools provided by Salis’s De Novo DNA company.
Upper and Bottom modules Moreover, to lower the burden of plasmid maintenance, we decided to combine the Upper and Bottom modules in a single backbone, the pSB1A3. Thus, both CrtW and CrtO were expressed under the control of the hybrid T5 promoter regulated by LacI as operons together with the E. coli idi and dxs genes (BBa_K4432320 and BBa_K4432321).
Test
To demonstrate the whole-cell bioproduction of canthaxanthin, E. coli MG1655 cells were co-transformed with 2 or 3 plasmids encoding the genes for the bottom, middle and upper parts of the pathway described above. Bacterial cultures were fully grown in LB medium containing the appropriate antibiotics (100 µg/mL ampicillin, 25 µg/mL kanamycin, 5 µg/mL tetracycline) and induced with 100 µM IPTG and 1.5 mM L-arabinose. After 24 hours, 15 mL were centrifuged (15 minutes at 4000 g, 4°C) in order to separate the biomass from the culturing medium, and carotenoids were extracted from the pellets with acetone twice followed by methanol. For this, the cell pellet was resuspended in 2 mL acetone in the presence of 0.3 g of glass beads (diameter 1 mm) and homogenized by vortexing for 5 minutes. After centrifuging the sample for 5 minutes at 4000 g, 4°C, the acetone-soluble fraction was recovered and the pellet was subject to a second round of acetone extraction. Finally, as for canthaxanthin it was shown that methanol improves the extraction yield [13], we performed a tried extraction using 2 mL of methanol and the same protocol for acetone.
Acetone and methanol extracts were analyzed by high pressure liquid chromatography (HPLC) using a Shimadzu Prominence LC20/SIL-20AC equipped with a Kinetex XB-C18 reversed phase column (250 mm × 4.5 mm, 5 μm) and an UV–Vis detector. Mobile phases A (methanol) and B (acetonitrile) were set at a flow rate of 1 mL/min and the separation was performed isocratically with an A:B ratio of 90:10. The sample injection volume was 10 μL, the column was thermostated at 40°C, and the metabolites were monitored at 452 nm and 472 nm. The product quantification was done by interpolation of the integrated peak areas using calibration curves prepared with standard samples for lycopene, ß-carotene and canthaxanthin as detailed on the Measurement page on this wiki. Data was normalized to grams of dry cell weight (gDW) estimated by converting first the cell density (OD600nm) to number of cells (based on the approximate conversion of OD600nm of 1.0 = 8 x 108 cells/mL) and finally to gDW knowing that the dry weight of one E. coli cell is 3 x 10-13 g according to the E. coli Metabolome Database (ECMDB) [14].
Learn
Carotenoids production was readily visible by naked eye. Indeed, bacterial pellets were colored in different shades from pale yellow to dark red, while the negative control performed with E. coli cells not expressing any Crt gene had the characteristic beige color (Figure 2).
When only the CrtEBI genes were present, the cells were slightly but visibly red, indicative of lycopene expression, as expected. The color intensity is enhanced in the presence of the idi and dxs expression device. This same trend was observed when the ß-carotene production operon composed of the 4 genes CrtEBIY was expressed in the E. coli cells: strains exhibited higher orange coloration with the induced metabolic boost plasmid (idi and dxs) being it in the high copy plasmid pSB1A3 or the low copy one pSB3T5.
The presence of either CrtW or CrtO genes led to the appearance of a coral color, strongly suggesting the production of canthaxanthin.
Figure 2. E. coli MG1655 cell pellets expressing the lycopene (CrtEBI), ß-carotene (CrtEBIY) or canthaxanthin (CrtEBIYW/O) devices along or not with with the idi and dxs synthetic operon.
To assess each carotenoid production in each strain, both qualitatively and quantitatively, the extracts were subject to reverse phase chromatography and spectrophotometric analysis. Comparaisons with standard commercial lycopene, ß-carotene and canthaxanthin confirmed their production in E. coli cells expressing the lycopene (CrtEBI), ß-carotene (CrtEBIY) and canthaxanthin (CrtEBIYW/O) devices respectively.
Indeed, the reverse phase chromatography allowed us to clearly and effectively separate the 3 carotenoids and thus unambiguously identify them (Figure 3). For instance, on the HPLC chromatogram of the acetone extraction from E. coli cells expressing the lycopene (CrtEBI) operon a single peak with the same retention time as commercial lycopene is observed, while on the HPLC chromatogram of the acetone extraction from E. coli cells expressing the ß-carotene (CrtEBIY) operon, one can observe a single peak with the same retention time as commercial ß-carotene. As expected, no peak is visible on the HPLC chromatogram of the acetone extraction from E. coli cells not expressing any Crt gene.
Moreover, when either CrtW or CrtO genes are present, the characteristic peak of canthaxanthin appears indicating that both enzymes are capable of synthesizing canthaxanthin. It should be noted that residual amounts of ß-carotene are visible on the HPLC chromatogram in the case of CrtO suggesting a less efficient conversion compared to CrtW.
Spectroscopic analysis further supports the chromatography results (Figure 4). Indeed, the acetone extractions from E. coli MG1655 cells expressing the lycopene (CrtEBI), ß-carotene (CrtEBIY) and canthaxanthin (CrtEBIYW) devices have the same spectral properties as commercial lycopene, ß-carotene and canthaxanthin respectively.
Quantitative analysis of the HPLC data (Figure 5) confirms the conclusions derived from the visual observation of the color of the cells pellets. Indeed, the production of all three carotenoids is strongly enhanced when the MEP pathway is deregulated by the overexpression of the idi and dxs genes. Our best canthaxanthin yield is obtained upon co-expression of the CrtEBIY (BBa_K274220) and CrtW+idi+dxs (BBa_K4432320) operons and it reaches 17.9 µmol (10.1 mg) per gram of E. coli dried weight (gDW) which is in the same range of previous studies that reported 16.1 mg/g of 90% pure canthaxanthin [15].
Figure 3. HPLC chromatograms of commercial lycopene, ß-carotene and canthaxanthin and of acetone extractions from E. coli MG1655 cells expressing the lycopene (CrtEBI), ß-carotene (CrtEBIY) and canthaxanthin (CrtEBIYW/O) devices along with the idi and dxs synthetic operon. The negative control was performed using E. coli cells not expressing any Crt gene. Images were produced using Shimadzu's LabSolutions Postrun Analysis software.
Figure 4. Spectroscopic analysis of commercial lycopene, ß-carotene and canthaxanthin and of acetone extractions from E. coli MG1655 cells expressing the lycopene (CrtEBI), ß-carotene (CrtEBIY) and canthaxanthin (CrtEBIYW/O) devices along with the idi and dxs synthetic operon. The negative control was performed using E. coli cells not expressing any Crt gene.
Figure 5. Canthaxanthin, ß-carotene or lycopene production yields by E. coli MG1655 cells expressing the lycopene (CrtEBI), ß-carotene (CrtEBIY) or canthaxanthin (CrtEBIYW/O) devices along or not with with the idi and dxs synthetic operon.
References
[1] Rebelo BA, Farrona S, Ventura MR, Abranches R. Canthaxanthin, a red-hot carotenoid: applications, synthesis, and biosynthetic evolution. Plants (2020) 9: 1039.
[2] Ahuja K, Rawat A. Global Canthaxanthin Market to exceed $85 mn by 2024. Global Market Insights Inc. https://www.gminsights.com/pressrelease/canthaxanthin-market.
[3] Sandmann G. Diversity and origin of carotenoid biosynthesis: its history of coevolution towards plant photosynthesis. The New Phytologist (2021) 232: 479–493.
[4] Steiger S, Sandmann G. Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin. Biotechnology Letters (2004) 26: 813–817.
[5] Sacco NJ, Bonetto MC, Cortón E. Isolation and characterization of a novel electrogenic bacterium, Dietzia sp. RNV-4. PloS One (2017) 12: e0169955.
[6] Kim SW, Keasling JD. Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnology and Bioengineering (2001) 72: 408–415.
[7] Alper H, Fischer C, Nevoigt E, Stephanopoulos G. Tuning genetic control through promoter engineering. Proceedings of the National Academy of Sciences of the United States of America (2005) 102: 12678–12683.
[8] Yoon S-H, Lee S-H, Das A, Ryu H-K, Jang H-J, Kim J-Y, Oh D-K, Keasling JD, Kim S-W. Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. Journal of Biotechnology (2009) 140: 218–226.
[9] Ajikumar PK, Xiao W-H, Tyo KEJ, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science (New York, N.Y.) (2010) 330: 70–74.
[10] Banerjee A, Sharkey TD. Methylerythritol 4-phosphate (MEP) pathway metabolic regulation. Natural Product Reports (2014) 31: 1043–1055.
[11] Volke DC, Rohwer J, Fischer R, Jennewein S. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microbial Cell Factories (2019) 18: 192.
[12] Albrecht M, Misawa N, Sandmann G. Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin. Biotechnology Letters (1999) 21: 791–795.
[13] Scaife MA, Ma CA, Armenta RE. Efficient extraction of canthaxanthin from Escherichia coli by a 2-step process with organic solvents. Bioresource Technology (2012) 111: 276–281.
[14] Sajed T, Marcu A, Ramirez M, Pon A, Guo AC, Knox C, Wilson M, Grant JR, Djoumbou Y, Wishart DS. ECMDB 2.0: A richer resource for understanding the biochemistry of E. coli. Nucleic Acids Research (2016) 44: D495-501.
[15] Scaife MA, Prince CA, Norman A, Armenta RE. Progress toward an Escherichia coli canthaxanthin bioprocess. Process Biochemistry (2012) 47: 2500–2509.
Sequence and Features
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- 1000COMPATIBLE WITH RFC[1000]
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