Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system
- Hui-Jeong Jang†1,
- Sang-Hwal Yoon†1,
- Hee-Kyung Ryu2,
- Jung-Hun Kim1,
- Chong-Long Wang1,
- Jae-Yean Kim1,
- Deok-Kun Oh3 and
- Seon-Won Kim1Email author
© Jang et al; licensee BioMed Central Ltd. 2011
Received: 26 May 2011
Accepted: 29 July 2011
Published: 29 July 2011
Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay.
Among the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through in situ extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture.
In this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of E. coli as a promising microbial cell factory for retinoid production.
Retinoids are composed of 3 isopentenyl diphosphate (IPP) units and 1 dimethylallyl diphosphate (DMAPP), which are the common five-carbon building blocks of all isoprenoids. The IPP and DMAPP building blocks are generally synthesized via the 2-C-methyl-D-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways in prokaryotes and eukaryotes, respectively [7–9]. Recombinant E. coli harboring an exogenous MVA pathway has been used for the successful production of isoprenoids, such as amorphadiene, carotenoids, and farnesol [10–14]. In particular, we reported the high-level production of β-carotene (465 mg/L) from E. coli harboring an engineered MVA pathway [13, 15]. The recombinant E. coli can be engineered to produce retinal by introducing β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) as a β-carotene cleavage enzyme (Figure 1B).
The cleavage of β-carotene by BCM(D)O (E.C. 22.214.171.124 or E.C. 126.96.36.199) is the initial key step of synthesis of various retinoids from β-carotene. The cleavage reactions can be classified as central and eccentric. In the central cleavage, BCM(D)O cleaves the central double bond (15, 15') of the polyene chain of β-carotene to yield two molecules of retinal. In the eccentric cleavage, BCM(D)O randomly cleaves any double bond in the polyene chain to produce β-apo-carotenals with different side chain lengths. In the cleavage reactions, BCMO utilizes an oxygen atom derived from molecular oxygen and water via an epoxide intermediate, whereas BCDO employs a molecular oxygen via an unstable dioxetane intermediate [16, 17]. Retinal is converted to retinol and retinoic acid by retinol dehydrogenase and retinal dehydrogenase/oxidase, respectively (Figure 1A) [18, 19]. Retinol is esterified to retinyl esters by retinol acyltransferase .
Retinoids are chemically unstable and readily oxidized and isomerized by heat, oxygen, and light due to their reactive conjugated double bonds [21, 22]. Biologically, retinoids are also easily degraded via retinoic acid. The oxidative degradation begins with the conversion of retinoic acid to more polar metabolites, such as 4-hydroxy- and 4-oxo-retinoic acids [23, 24]. Therefore, successful production of retinoids can be achieved by preventing both chemical and biological degradation. To our knowledge, retinoid production using metabolically engineered microorganisms has never been reported. In this study, we cloned the BCM(D)O genes from several organisms and introduced each gene into recombinant E. coli that produce β-carotene. An exogenous MVA pathway was also utilized to increase retinoid production. A two-phase culture system using a dodecane layer over the culture broth was found to minimize the intracellular degradation of retinoids through in situ extraction from the cells.
Comparison of retinal production from various BCM(D)O genes
Engineering the MEP and MVA pathways to supply building blocks
Effects of E. coli strains, culture conditions and carbon sources on retinoid production
Two-phase culture using dodecane for in situ extraction of retinoids
To prevent intracellular retinoid degradation, a two-phase culture system using the hydrophobic solvent dodecane was performed for in situ extraction of retinoids from the cells. Dodecane was chosen for its low toxicity to E. coli, high hydrophobicity (log PO/W, 6.6) for the extraction of hydrophobic retinoids, and low volatility, which prevents loss due to evaporation. A two-phase culture system using decane has been successfully applied to lycopene production .
The proportions of the retinoids obtained with the various dodecane overlay volumes were determined (Figure 6B). An outstanding difference in the proportions of retinal and retinol obtained with and without the dodecane overlays was found. The proportion of retinal among the retinoids after 48 hours was approximately 51% (w/w) in the dodecane overlaid cultures and 23% in the culture without dodecane overlay, whereas the retinol proportion was 30% to 39% in the dodecane overlaid cultures and 59% in the culture without dodecane overlay. Therefore, the dodecane overlay increased the proportion of retinal but decreased the proportion of retinol. Retinal seemed to be extracted from cells by dodecane before it could be intracellularly converted into retinol, as retinol is formed from retinal in cells. The proportion of retinyl acetate after 48 hours was below 20% in both the dodecane overlaid culture and culture without overlay, which was lower than those of retinal and retinol. In the dodecane-overlaid cultures, the proportion of retinyl acetate decreased with increasing culture time, suggesting that retinyl acetate formation decreased during culture. We concluded that the dodecane overlay prevented the decrease in retinoid production during stationary phase growth and increased retinoid production.
E. coli harboring the synthetic BCDO (SR) gene, which was the codon-optimized blh gene from the uncultured marine bacterium 66A03, successfully produced retinal from β-carotene. Interestingly, the E. coli also produced retinol and retinyl acetate. We hypothesize that promiscuous enzymes in E. coli are able to metabolize retinal to produce retinol and retinyl acetate. Retinal is metabolized to retinol and retinyl acetate in a sequential manner by retinol dehydrogenase and retinol acyltransferase, respectively. Therefore, we investigated a presence of a potential retinol dehydrogenase in E. coli. The ybbO gene in E. coli 83972 (Accession No. ZP_04002297) was identified as a possible retinol dehydrogenase in the NCBI protein database, although we did not expect to find specific enzymes that metabolize foreign compounds, such as retinoids. The ybbO gene is annotated in E. coli strain MG1655 as a predicted oxidoreductase and has the highest identity and similarity (31% and 52%, respectively) in the E. coli genome to H. sapiens retinol dehydrogenase (Accession No. AAC72923) based on the BLASTP analysis of NCBI http://www.ncbi.nlm.nih.gov/blast/. It has been reported through sequence comparisons and phylogenetic analysis that the ybbO gene may be an example of horizontal gene transfer from a eukaryotic retinol dehydrogenase ancestor . To identify putative homologues of retinol acyltransferase in E. coli, BLASTP analysis was performed with retinol acyltransferases of H. sapiens (Accession No. NP_004735) and two bacteria, Shewanella putrefaciens CN-32 (YP_001185024) and Trichodesmium erythraeum IMS 101 (YP_723688), because the protein sequence of bacterial retinol acyltransferase was available in only these two bacteria. No homologue of retinol acyltransferase was identified from the BLASTP analysis. An alternative approach for the identification of a homologous gene would be to delete the genes of all acyltransferases present in E. coli. Biological degradation of retinoids is initiated from retinoic acid. We observed significant intracellular degradation of retinoids during stationary phase growth. If retinoic acid is quickly degraded in E. coli, it would not be detected in the culture during retinoid production. We hypothesize that retinoic acid is formed in our E. coli strain engineered to produce retinoids. Retinal is converted to retinoic acid by retinal dehydrogenase. Salmonella enterica is known to have a retinal dehydrogenase (Accession No. CBY96723). BLASTP analysis was performed on the E. coli genome with the retinal dehydrogenase of S. enterica, and eutE (predicted aldehyde dehydrogenase/ethanolamine utilization protein) was identified as a homologue (with 94% identity and 97% similarity). The retinal dehydrogenase of H. sapiens (Accession No. NP_733798) was also used for the same BLASTP analysis, and puuC (gamma-Glu-gamma-aminobutyraldehyde dehydrogenase) was found to have the highest homology (42% identity and 63% similarity) to the retinal dehydrogenase. If eutE or puuC are involved in the formation of retinoic acid, deletion of these genes will prevent the biological degradation of retinoids via retinoic acid, resulting in an enhancement in total retinoid production.
In the cultures without a dodecane overlay, there was a significant decrease in retinoid production during stationary phase growth. This might be due to increased oxidative degradation of retinoids by reactive oxygen species, such as hydrogen peroxide and superoxide, which are generated at high levels during stationary phase. Retinoids are easily oxidized as antioxidants by reactive oxygen species. Oxidative retinoid degradation could be decreased by overexpression of catalases (Kat E/G) and superoxide dismutases (Sod A/B/C), which scavenge reactive oxygen species. To prevent biological degradation of retinoids inside of the cells, in situ extraction of retinoids was performed with a two-phase culture system using dodecane. Oxidizable compounds are easily oxidized and degraded by molecular oxygen dissolved in the aqueous phase, whereas compounds in hydrophobic solvents, including dodecane, are sequestered and more stable [30, 31]. Thus, dodecane was found to efficiently extract hydrophobic retinoids from cells and preserve the products during two-phase culture. In a previous report, a two-phase culture system using decane was successfully applied for lycopene production ; however, lycopene was inefficiently extracted from recombinant E. coli without partial digestion of the cell wall by lysozyme. In this study, the use of lysozyme for cell wall digestion was not required for the in situ extraction of retinoids. Retinoids are efficiently released from cells without removing the cell walls because retinoids (C20, isoprenoid molecule) are half the size of lycopene (C40). In the two-phase culture for retinoid production, β-carotene should be retained inside of the cells because it is the immediate precursor of retinoids. If it is extracted in the dodecane phase, it would not be available for the cleavage reaction by BCM(D)O located in the cytosol. Even though extraction of β-carotene by dodecane would not be expected because it is a C40 carotenoid like lycopene, two-phase culture for β-carotene production was performed to confirm that β-carotene is retained in the cells (Additional file 5). A negligible amount of β-carotene was detected in the dodecane phase and almost all of the β-carotene was retained in the cells. There was no significant difference in both β-carotene production and cell growth between cultures with and without a dodecane overlay. The two-phase culture system prevents intracellular degradation of retinoids and provides a driving force for further retinoid production. Physical sequestration of the product from a reaction system drives the reaction to high efficiency without the effects of product inhibition or reaction equilibrium [32, 33]. In the two-phase culture system, the retinoids were sequestered from the cells in the dodecane phase, and production was enhanced. A total retinoid production of 122 mg/L was obtained after 48 hours in a culture with a 5 mL dodecane overlay, whereas half of this amount (60 mg/L) was produced after 48 hours without a dodecane overlay. Thus, the dodecane-overlaid two-phase culture system could be employed for other engineered systems that produce lipophilic small molecules.
Our results represent the first report on retinoid biosynthesis using metabolically engineered E. coli. In this study, we successfully produced 136 mg/L retinoids, which were composed of retinal (67 mg/L), retinol (54 mg/L), and retinyl acetate (15 mg/L), using a two-phase culture system with dodecane, which was a 68-fold improvement from the initial level of retinoid production (2.2 mg/L). This improvement was achieved with use of (1) an efficient marine bacterial BCDO gene that was codon-optimized for expression in E. coli, (2) introduction of an exogenous MVA pathway that successfully provided the building blocks IPP and DMAPP for retinoid synthesis, and (3) the use of a two-phase culture system with dodecane that prevented intracellular degradation of the retinoids and provided a driving force for retinoid production. Retinal, retinol and retinyl acetate were contained in the retinoids produced from the recombinant E. coli, which suggests that E. coli has the potential to synthesize multiple retinoids, which can be used for different commercial applications. Based on this potential, the retinoid synthesis pathway of E. coli can be reengineered to produce a specific retinoid through elaborative genetic manipulations, such as gene deletions and overexpression of genes involved in the modification of retinoids. Therefore, E. coli is a genetically tractable host that is a promising microbial cell factory for the engineered production of retinoids.
Bacterial strains and culture conditions
Strains, plasmids and primers used in this study
Strains, plasmids and primers
Reference or source
E. coli strains
K12, Wild type
F-, ϕ 80dlac ZΔ M15, Δ(lacZYA-argF)U169, deoR, recA 1 endA 1, hsdR 17(rK- mK+), phoA, supE 44, λ-, thi-1, g yrA96, rel A1
hsdR 17, supE 44, recA 1, endA 1, gyrA 46, thi, relA 1, lac/F ' [proAB + , lacIq, lacZΔ M15::Tn10(tetr)]
recA pro hsdR RP4-2-Tc::Mu-Km::Tn7
F-, ompT, hsdSB(r B - m B - ), gal (λc I 857, ind 1, Sam 7, nin 5, lac UV5-T7gene 1), dcm (DE3)
Plac cloning vector, ColE1 origin, lacZ, Ampr
Plac expression vector, pACYC184 origin, lacZ, Cmr
Ptrc expression vector, pBR322 origin, lacIq, Cmr
pTrc99A containing crtE, crtB, and crtI from P. agglomerans, crtY from P. ananatis, and ipiHP1 from H. pluvialis
pT-HB containing dxs from E. coli
pT-HB containing blh from Halobacterium sp. NRC-1
pT-HB containing brp from Halobacterium sp. NRC-1
pT-HB containing brp2 from N. pharaonis
pT-HB containing Bcmo1 from M. musculus
pT-HB containing the codon-optimized blh gene (SR) from uncultured marine bacterium 66A03
pT-DHB containing the codon-optimized blh gene (SR) from uncultured marine bacterium 66A03
pSTV28 containing mvaE and mvaS from E. faecalis; mvaK1, mvaK2, and mvaD from S. pneumoniae; and idi from E. coli
5'-GGAATTC AGGAGGTGTTCGGC ATG CCACACGG-3'
5'-GACTAG TTA GAGGACGCCCTGCACGCGGTC-3'
5'-GGAATTC AGGAGGTATTCAT ATG AGCAATAGGTC-3'
5'-GACTAG TTA TGGGACGTACCAGATGCCG-3'
5'-GGAATTC AGGAGGCCGAGT ATG AGTAACGCGTC-3'
5'-GACTAGTT A TGCTCCGGGTCGCCAGAG-3'
5'-GGAATTC AGGAGCGGTTCC ATG GAGATAATATTTG-3'
5'-GACTAG TTA AAGACTTGAGCCACCATG-3'
5'-GACTAGTGAATTC AGGAGGTAATAAAT ATG G-3'
5'-CACTAG TTA GTTTTTGATTTTG-3'
Gene cloning and plasmid construction
The plasmids and PCR primers used in this study are listed in Table 1. Common procedures, including genomic DNA preparation, restriction digests, transformations, and other standard molecular biological techniques, were carried out as described in the literature (Sambrook and Russell 2001). PCR was performed using pfu DNA polymerase (Solgent Co., Korea) with a standard protocol. The pBluescript, pTrc99A, and pSTV28 plasmids were used for gene cloning and gene expression (Table 1). The pS-NA plasmid containing the operon for the MVA pathway was used as described previously (Yoon et al., 2009). The BCM(D)O genes blh and brp, brp2, and bcmo1 were amplified using PCR from Halobacterium sp. NRC-1, Natronomonas pharaonis and Mus musculus, respectively. The PCR products were cloned into the EcoR I and Spe I sites of pT-HB, resulting in the retinal plasmids pT-HBblh, pT-HBbrp, pT-HBbrp2 and pT-HBBcmo1 (Table 1) . The blh gene (Genbank accession number AAY68319) of the uncultured marine bacterium 66A03 was synthesized by Genofocus (Daejeon, Korea) according to the codon-optimization function of the company in-house software for expression in E. coli. The synthetic gene named SR (s ynthetic r etinoid gene) was amplified using the PCR primers SR-F and SR-R, and cloned into the Eco RI and Spe I sites of pT-HB, resulting in pT-HBSR. The SR gene cleaved from pT-HBSR with Spe I was cloned into the corresponding site of pT-DHB, which resulted in pT-DHBSR.
Analysis of β-carotene and retinoids
β-Carotene and retinoids were extracted from bacterial cell pellets with acetone . In the two-phase culture system with a dodecane overlay, the upper dodecane phase containing the retinoids was collected and centrifuged for 10 min at 14,000 rpm to remove all cellular particles. The acetone extracts and dodecane phases were analyzed with HPLC (LC-20A, Shimadzu, Kyoto, Japan) at detection wavelengths of 370 nm (retinal), 340 nm (retinol and retinyl acetate), and 454 nm (β-carotene) and using the Symmetry C18 (250 mm × 4.6 mm, 5 μm) with Sentry Guard C18 (15 mm × 4.6 mm, 5 μm) HPLC columns (Waters, Milford, USA). The mobile phases were 95:5 and 70:30 methanol and acetonitrile for the retinoid and β-carotene analyses, respectively. A flow rate of 1.5 ml/min and column temperature of 40°C were applied for the HPLC analysis. Retinal (Cat. No. R2500), retinol (Cat. No. R7632), retinyl acetate (Cat. No. R4632) and β-carotene (Cat. No. C4582) were purchased from Sigma (USA), dissolved in acetone, and used as standard compounds. The results are presented in the means ± SD from three independent experiments.
This work was supported by a grant (2009-0084490) from the Basic Research Program, a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, MEST, and a grant from the Next-Generation BioGreen 21 Program (No. 2010-0000), Rural Development Administration, Korea. HJ Jang and JH Kim are supported by scholarships from the BK21 Program, MEST, Korea.
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