Metabolic engineering of Escherichia coli for the production of riboflavin
© Lin et al.; licensee BioMed Central 2014
Received: 14 May 2014
Accepted: 9 July 2014
Published: 16 July 2014
Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions.
The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose.
The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.
Riboflavin (vitamin B2) is the universal precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), both of which act as hydrogen carriers and are essential for the activity of a wide variety of metabolic enzymes in higher eukaryotes [1,2]. Riboflavin is synthesized by all plants, fungi and most bacteria, but not by higher animals including humans. So humans and animals must obtain riboflavin through dietary sources . Traditionally, riboflavin was synthesized and produced by chemical procedures or microbial fermentation. Since chemical synthesis of riboflavin has many disadvantages such as high cost and energy wasting, the microbial fermentation method has been widely applied in industrial production.
In a series of previous reports, a numbers of conceivable strategies were carried out to develop B. subtilis as a riboflavin-producing strain after selection for resistance to different antimetabolites. In B. subtilis, the riboflavin production was increased by amplifying copies of rib operon or ribA gene [6,7], or by enhancing the energy generation and reducing the maintenance metabolism [8–10]. Other efforts focused on modulating precursor metabolites supply by increasing the carbon flow through the pentose phosphate pathway (PP pathway) [11–14], by deregulating the gluconeogenesis flux via inactivating the ccpN , or by enhancing the flux of purine synthesis pathway [16,17].
E. coli has been employed for efficient production of biofuels, amino acids, and bulk chemicals [18–20]. Although wild type E. coli does not accumulate riboflavin under natural conditions, it may be an efficient host for the production of riboflavin due to its clear genetic background, fast-growing, low maintenance metabolism, and the presence of convenient metabolic engineering tools. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the production of riboflavin by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions.
In this study, the riboflavin synthetic pathway from E. coli and B. subtilis was constructed and compared in E. coli MG1655. Then, we examined the impact of overexpressing mutation type of zwf and gnd genes from C. glutamicum and pgl from E. coli on riboflavin production. Third, the glycolysis and ED pathway were modified to reroute carbon flux to the PP pathway for improving the riboflavin synthesis. Fourth, the acetate accumulation was reduced by overexpressing the acs gene. Finally, riboflavin production was significantly increased by modulating the expression of ribF. We also optimized the fermentation conditions for the riboflavin production. The final recombinant strain RF05S-M40 produced 2702.8 mg/L riboflavin with a yield of 137.5 mg riboflavin/g glucose under the optimized batch fermentation condition.
Results and discussion
Construction of riboflavin synthetic pathway
Previous researches indicated that overexpression of the genes of the riboflavin synthetic pathway contributed to the efficient production of riboflavin. In B. subtilis, introduction of multiple copies of rib operon into the chromosome resulted in improving the riboflavin production . In Pichia pastoris, overexpression of the riboflavin biosynthetic pathway caused riboflavin production increased to 175 mg/L riboflavin although the wild type P. pastoris did not accumulate riboflavin . In C. famata, overexpressing the gene related to the riboflavin production significantly increased riboflavin production .
Activities of GTP cyclohydrolase II and Riboflavin synthase
Enzyme activities (nmol/min/mg protein)
GTP cyclohydrolase II a
Riboflavin synthase b
E. coli MG1655/p20C-EC10
E. coli MG1655/p20C-BSrib
E. coli MG1655/p20C-BS15
Effects of zwf, gnd, and pgl overexpression on riboflavin production
Effect of overexpressing pentose phosphate pathway genes on riboflavin production and enzyme activities
Riboflavin yield (mg/g) b
Enzyme activity (nmol/min/mg protein) c
Disruption of pgi for improving riboflavin production
Riboflavin production of the various strains constructed in the study
Biomass (g/L) b
Riboflavin yield (mg/g) c
Specific productivity (mg-RF/g-DCW/h)
Disruption of edd and eda for improving riboflavin production
The ED pathway has been shown to be inactive with glucose as the carbon source in wild-type E. coli , but it was activated in the pgi mutant strain . Previous studies indicated that glucose was mainly metabolized through PP pathway and partially through ED pathway after pgi was disrupted .
The edd and eda transcription levels in the Δpgi strain RF02 were compared with parent strain MG1655 through RT-qPCR analysis. Consistent with previously research , the transcript abundance of edd and eda increased 2.21-fold and 2.36-fold in RF02, respectively, which indicated that ED pathway was up-regulated in Δpgi strain. Thus, edd and eda were disrupted in RF02 for further increasing the flux from 6-phosphogluconate to Ru-5-P, resulting in strain RF03. RF03 harboring p20C-EC10 (strain RF03S) produced 559.88±8.99 mg/L riboflavin, which increased by 41.8% compared to that of RF02S (Table 3). These results suggested that further increasing riboflavin precursors can be achieved via disrupting ED pathway in the pgi mutation strain.
Effect of the acs promoter insertion on riboflavin production
Acetate, which led to growth retardation, was the main byproduct of these recombinant strains. Thus, the strategy of overexpressing the acs gene encoding acetyl-CoA synthetase was selected to reduce acetate secretion. The trc promoter was inserted in the upstream of acs gene in RF03, resulting in strain RF05. RT-qPCR analysis showed that the expression of acs gene in RF05S (RF05 harboring p20C-EC10) increased 4.80-fold compared with RF03S, indicating successful overexpression of acs gene. RF05S produced 585.2±13.6 mg/L riboflavin with a yield of 59.30 mg/g glucose, which did not change significantly compared to RF03S (Table 3). However, the acetate production decreased from 4.0 g/l to 1.5 g/L in strain RF05S, which was in accordance with the results presented by Lin et al. .
Modulating the expression ribF for enhanced riboflavin production
According to previous studies, introducing a mutation in ribC gene (encoding flavokinase/flavin adenine dinucleotide synthetase) in B. subtilis could reduce enzymatic activity and resulted in riboflavin overproduction in the engineering strains . The ribC mutation of B. subtilis also reduced the synthesis of FMN, which is a negative regulatory factor of rib operon transcription . In E. coli, these enzymes were encoded by ribF, which is an essential gene for strain growth . Therefore, we speculate that reducing the expression level ribF might reduce the conversion of riboflavin to FMN/FAD and increase riboflavin production.
Optimization of fermentation conditions for riboflavin production
To optimize fermentation condition, we investigated the effect of different culture temperatures on riboflavin production. Changing the fermentation temperature from 37°C to 31°C increased riboflavin titer to 1507.0±56.8 mg/L from 1036.1±54.6 mg/L (Figure 4B). Many factors may have contributed to the enhanced production. For example, growth temperature was reported to influence not only recombinant gene expression and protein folding, but also the global metabolism of the strains . In addition, low growth temperature was reported to stabilize the recombinant proteins and improve the plasmid stability .
Riboflavin production of RF05S-M40 in different cultural conditions
Yeast extract (g/L)
Biomass b (g/L)
Riboflavin yield (mg/g) c
Specific productivity (mg-RF/g-DCW/h)
For the first time, we constructed a genetically defined riboflavin producing E. coli strain. Riboflavin synthetic pathway, PP pathway, central metabolic pathways, and riboflavin consumption pathway were systematically engineered for direct and efficient conversion of glucose to riboflavin. The E. coli strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work also demonstrates that E. coli has a potential to be a host for riboflavin bioproduction.
Strain, medium, and cultivation condition
Strains used in this study
Escherichia coli DH5α
Coli Genetic Stock Center strain (CGSC) No. 12384
E. coli MG1655
Coli Genetic Stock Center strain (CGSC) No. 7740
Bacillus subtilis 168
E. coli MG1655 containing p20C-EC10
E. coli MG1655, Δpgi
E. coli MG1655, Δpgi, Δedd, Δeda
RF03 containing p20C-EC10
E. coli MG1655, Δpgi, Δedd, Δeda, Ptrc-acs
RF05 containing p20C-EC10
Plasmids used in this study
pUC18 PvegI-gnd361, Ampr, Spcr
pUC18 PvegI-zwf243, Ampr, Spcr
Expression vector, pSC101 replication, Ptrc, Ampr
Expression vector, p15A replication, Ptrc, Ampr
Expression vector, pBR322 replication, Ptrc, Ampr
pSC101 replication, Ampr, Ptrc-synRBS-ribA-synRBS-ribB- synRBS-ribD-synRBS-ribE-synRBS-ribC
p15A replication, Ampr, Ptrc-synRBS-ribA-synRBS-ribB-synRBS- ribD-synRBS-ribE-synRBS-ribC
pBR322 replication, Ampr, Ptrc-synRBS-ribA-synRBS-ribB- synRBS-ribD-synRBS-ribE-synRBS-ribC
pBR322 replication, Ampr, Ptrc-synRBS-ribB/A-synRBS-ribD- synRBS-ribE-synRBS-ribC
pBR322 replication, Ampr, Ptrc-BSrib operon
Expression vector, p15A replication, Cmr
p15A replication, Cmr, Ptrc-synRBS-zwf-synRBS-gnd, zwf and gnd genes from C. glutamicum
p15A replication, Cmr, Ptrc-synRBS-zwf-synRBS-gnd-synRBS-pgl, zwf and gnd genes from C. glutamicum, pgl gene from E. coli
p15A replication, Cmr, Tetr, I-SceI restriction sites
pSC101 replication, temperature sensitive replication origin, Spcr, ParaBAD-driven I-SceI gene, Red recombinase expression plasmid, lac-inducible expression
For the construction of p15Trc-zg, zwf (A243T) and gnd (S361F) from C. glutamicum were firstly cloned in a p15A origin vector under the control of Ptrc individually by CPEC  with primer sets p15Trc-1BF, p15Trc-1BR, p15Trc-zwfF, p15Trc-zwfR, p15Trc-gndF, p15Trc-gndR respectively. Then, the zwf (A243T) and gnd (S361F) genes from C. glutamicum and pgl gene from E. coli were assembled into a p15A origin vector under the control of Ptrc by CPEC, obtaining plasmid p15Trc-zgp. All primers were listed in Additional file 1: Table S1, and plasmids constructed were listed in Table 6.
Genome engineering: gene deletion, replacement, and insertion
As mentioned above, the strategy of fragment construction and genome manipulation was used to modulate the expression of ribF. The sequence of primer ribF L_F is TACAAGGTATACTCGGACGATTTTCACTGTHKTGWRCCAGMCATG. The RBS library of ribF, design with RBS Calculator , was placed between the DR sequence (italicized) and the start codon of ribF (bold). The primers used during construction were listed in Additional file 1: Table S1.
Quantitative real-time reverse transcription (RT)-PCR analysis
The recombinant strains were cultured in LB medium with 1% glucose. Cells were harvested when OD600 reached 1. Total mRNA were extracted using the RNAprep pure Cell/Bacteria Kit (Tiangen, Beijing, China) as described by the manufacturer. The cDNA was amplified using FastQuant RT Kit (Tiangen, Beijing, China) with the total mRNA as the templates. Samples were then analyzed using a Light Cycler®480 II (Roche, Basel, Switzerland) with RealMasterMix (SYBR Green I) (Tiangen, Beijing, China). Quantity real-time PCR amplification primers were listed in Additional file 1: Table S1. The rrsA gene was selected as internal standard for normalization and three biological replicates were performed. The obtained data were analyzed by using the 2-ΔΔCt method described previously .
Enzyme assay and preparation of cell extracts
The engineering strains were cultivated in LB medium containing 1% glucose. They were cultured to mid-exponential phase and the cells were harvested by centrifugation for 10 min at 12000×g and 4°C, washed twice with 100 mM potassium phosphate (pH 7.5)-1 mM dithiothreitol-0.1 mM EDTA. The cells were resuspended and sonicated for 5 min in an ice bath (130 W, 20 kHz, pulse: 5 s on; 5 s off). After centrifugation (13,000×g, 10 min, 4°C), the supernatants were used as cell extracts. When necessary, IPTG was used at a concentration of 2 mM for induction. The activities of GTP cyclohydrolase II, riboflavin synthase and flavokinase activity were measured as previously described [54–56]. The activities of glucose 6-phosphate-1-dehydrogenase and 6-phosphogluconate dehydrogenase were determined as previously described . Total protein concentrations were determined by the Bradford method using bovine serum albumin as standard .
Production of riboflavin from glucose
For riboflavin production, LBG medium (LB medium with 1% (W/V) glucose), modified M9 medium, or modified minimal salt (MS) medium was selected. An amount of 20 g/liter glucose or 10 g/liter glucose was added as the carbon source except as indicated. The modified M9 medium contained (per liter) 6 g Na2HPO4, 3 g KH2PO4, 1 g NH4Cl, 0.5 g NaCl, 2 mM MgSO4, 0.1 mM CaCl2, and yeast extract as indicated. The MS medium contained (per liter) 3.8 g Na2HPO4, 1.5 g KH2PO4, 1.0 g (NH4)2SO4, 0.2 g MgSO4, yeast extract as indicated, and 2% (v/v) trace element solution as described in previous studies .
For fermentation, transformed strains were cultured overnight in 5 ml LB media at 37°C, then transferred into 50 ml LB media with 1% (v/v) of the seed culture. This was cultured to mid-exponential phase at 240 rpm, 37°C, and frozen at −80°C in 15% glycerol (v/v). The inoculum was prepared from frozen seed stocks in LB medium supplemented with 100 mg/L ampicillin, grown to mid-exponential phase, and then used to inoculate batch cultures with 1% (v/v) of the seed culture, at 37°C or 31°C as indicated. When appropriate, IPTG was used at a concentration of 2 mM for induction. For each strain, three parallel fermentations were performed.
Cell concentration was calculated from OD 600 nm measurements. Glucose concentration was measured enzymatically using a glucose analyzer (Model-SBA40, Shandong, China). To determine the concentration of acetate, culture samples were centrifuged at 12,000×g for 5 min and the aqueous supernatant used for HPLC analysis on an Agilent 1100 Series HPLC system equipped with an Aminex HPX-87H anion exchange column (Bio-Rad Laboratories, Richmond, CA, USA) and refractive index detector. The column was eluted with 5 mM sulfuric acid at a flow rate of 0.4 ml/min. Standards were prepared for acetate for both the refractive index detector, and calibration curves were created. For riboflavin measurements, culture samples were diluted with 0.05 M NaOH to the linear range of the spectrophotometer and the A444 was immediately measured. The results represented the means±S.D. of three independent experiments. Dry cell weight (DCW) was calculated from the optical density at 600 nm (1 OD600=0.38 g DCW/L).
The authors thank Dr. Thomas E. Kuhlman for kindly providing requested plasmids. This work was supported by National 973 Project [2011CBA00804, 2012CB725203]; National Natural Science Foundation of China [NSFC-21176182, NSFC-21206112, NSFC-21390201]; and National High-tech R&D Program of China [2012AA02A702, 2012AA022103].
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