Part:BBa_K1172303

Riboflavin synthesis gene cluster from shewanella oneidensis

Figure 1: From left to right: e. coli KRX wild type compared to e. coli KRX with BBa_K1172303 under control of a medium constitutive promoter (BBa_K525998) and e. coli KRX with BBa_K1172303 under control of a strong constitutive promoter (BBa_K608002).

The riboflavin synthesis gene cluster (rib-gene-cluster) consists of four different genes that form a single operon.

Figure 4: Riboflavin synthesis gene cluster, cloned from Shewanella oneidensis MR-1, 3697 bp; with non-coding sequences. * The nomenclature was taken from the [http://www.ncbi.nlm.nih.gov/ NCBI site]. Depending on the source there are slight differences in nomenclature. For example, ribE is often referred to as ribH.

These genes are pivotal in the riboflavin biosythesis pathway of Shewanella oneidensis and are transcribed polycystronic. The original operon in Shewanella oneidensis has additional activator and repressor genes. We came to the conclusion that these are not sufficient for riboflavin overproduction. Therefore these genes were not isolated from genomic DNA.

Below we shortly describe each functional member of this cluster. (All information and data were taken from the [http://www.ncbi.nlm.nih.gov/ NCBI site]).

• Gene: ribD http://www.ncbi.nlm.nih.gov/gene/1171145 Sequence, 1145 bp
• Protein: bifunctional diaminohydroxyphosphoribosylaminopyrimidine deaminase/5-amino-6-(5-phosphoribosylamino) uracil reductase RibD
• Enzyme: (EC: 3.5.4.26)
• Molecular weight: 41,257 kDa

• Function: Catalyzes the deamination of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, which is an intermediate step in the biosynthesis of riboflavin.

• Gene: SO_3468 http://www.ncbi.nlm.nih.gov/gene/1171144 Sequence, 656 bp
• Protein: Riboflavin synthase alpha subunit RibC-like protein
• Enzyme: EC 2.5.1.9
• Molecular weight: 23,483 kDa
  • Function: Catalyzes the reaction: 2 6,7-dimethyl-8-(1-D-ribityl)lumazine + H(+) = 5-amino-6-(D-ribitylamino)uracil + riboflavin.

• Gene: ribBA → ribA & ribB http://www.ncbi.nlm.nih.gov/gene/1171143 Sequence, 1103 bp
• ribA
  • Protein: GTP cyclohydrolase-2
  • Enzyme: EC 3.5.4.25
  • Molekular weight: 22,852 kDa
    • Function: Catalyzes the first committed step in the biosynthesis of riboflavin.
• ribB
  • Protein: 3,4-dihydroxy-2-butanone-4-phosphate synthase
  • Enzyme: EC 3.5.4.25
  • Molecular weight: 22,956 kDa
    • Function: Catalyzes the formation of 3,4-Dihydroxy-2-butanone 4-phosphate which serves as the biosynthetic precursor for the xylene ring of riboflavin.

• Gene: ribE http://www.ncbi.nlm.nih.gov/gene/1171142 Sequence, 476 bp
• Protein: 6,7-dimethyl-8-ribityllumazine synthase (aka: riboflavin synthase beta subunit RibE)
• Enzyme: EC 2.5.1.78
• Molecular Weight: 16,689 kDa
  • Function: Catalyzes the penultimate step in the biosynthesis of riboflavin

Usage and Biology

Riboflavin, or Vitamin B2 is a redox-active substance that plays an essential role in living cells. As precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) it is crucial for diverse energy supplying metabolic processes, e.g. beta-oxidation or oxidative phosphorylation. Riboflavin is water soluble and shows a distinct yellow coloration. For that reason it is also used for food coloration. It is easily detectable through absorbance and fluorescence measurement. Due to its fluorescent properties and non-toxicity it is used to detect leaks or to control cleaning processes.

Figure 2: Riboflavin (Vitamin B2) and flavin-coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide).

Riboflavin consists of two functional subunits, a short-chain ribitol and a tricyclic heterosubstituted isoalloxazine ring. The latter, also known as a riboflavin ring, exists in three redox states and is responsible for the diverse chemical activities of riboflavin. A fully oxidized quinone, a one-electron semiquinone and a fully reduced hydroquinone state are the three stages of riboflavin oxidation. In an aqueous solution, the quinone (fully oxidized) form of riboflavin has a typical yellow coloring. It becomes red in a semi-reduced anionic or blue in a neutral form and is colorless when fully reduced.

Secreted into the medium, it can be effectively used by some bacteria for electron transfer. Presence of riboflavin in anaerobic cultures enables a higher current flow in a Microbial Fuel Cell, which makes riboflavin overproduction a suitable target for optimization of our MFC.
We have shown that cloning of the riboflavin cluster from the metal-reducing bacteria Shewanella oneidensis MR-1 in E. coli is sufficient to achieve significant riboflavin overproduction detectable both in supernatant and in cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1114
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Part uses

For overproduction of riboflavin, the BioBrick BBa_K1172303 was combined with promoters of different strenghts.

Device: BBa_K1172303 under control of accordant promoter.	Promoters used	RBS / Activity
BBa_K1172304	BBa_K525998 “T7 induced"	strong / very strong
BBa_K1172305	BBa_K608006 “Anderson 0.33”	medium / medium
BBa_K1172306	BBa_K608002 “Anderson 0.77”	strong / strong

Since the Regional in Lyon, we were able to combine BBa_K1172303 with other BioBricks from our project.

Device: Combination of BBa_K1172303 with accordant parts	Part 1 of the new device	Explanation
BBa_K1172588	BBa_K1172502	Combination of oprF from P. fluorescence under control of T7 promoter and the rib-gene-cluster from S. oneidensis
BBa_K1172599	BBa_K1172501	Combination of the coding sequences oprF from P. fluorescence and the rib-gene-cluster from S. oneidensis

Results

Confirming overexpression of the rib-gene cluster

The overexpression of BBa_K1172303 and its derived devices BBa_K1172306,BBa_K1172305 and BBa_K1172304 is assured by verifying the protein Riboflavin synthase beta subunit RibE. The protein RibE is part of the riboflavin synthesis pathway of Shewanella oneidensis. The corresponding gene is ribE. This gene belongs to the rib-gene cluster, which we managed to isolate, removing all the illegal restriction sites and subsequently cloned into pSB1C3.

SDS-PAGE

The SDS-PAGE shows a distinct band at ~15 kDa. The exact size of the riboflavin synthase beta subunit RibE is 16.7 kDa. The band was cut out and analyzed using MALDI-TOF.

Figure 10: SDS-PAGE with 20% separating gel for the verification of proteins from the rib-cluster. From left to right: Thermo PageRuler 150 kDa prestained ladder; E. coli KRX wild type; E.coli KRX wild type; rib-T7 uninduced; rib-T7 induced; rib-medium-Anderson33; rib-strong-Anderson77

MALDI-TOF

The spot, described above, was picked, washed and digested with trypsine. Afterwards the sample was spotted on the target and analyzed using [http://2013.igem.org/Team:Bielefeld-Germany/Labjournal/ProtocolsPrograms#MALDI-TOF MALDI-TOF] Measurement of the sample produced valid data: RibE was clearly identified by MALDI-TOF MS/MS with a Mascot Score of 906 against the NCBI database concerning bacterial organisms.

Figure 11: Exported MALDI-TOF results.

Figure 12: Screenshot of the BioTools user interface showing the pure results of the MALDI-TOF, precisely the intensity and the mass to charge ratio of the measured peaks (on top) as well as the peptide coverage (bottom).

Analysis of riboflavin in supernatants

Absorbance measurement

Riboflavin has an absorption peak at 446 nm. The absorbance was measured in a TECAN infinite plate reader. The samples consisted of supernatant derived from E. coli KRX with BBa_K1172306 and KRX as the "wild type" (both strains were cultivated over 72 hours). Further intracellular measurements of both strains were obtained. Therefore, the cells were disrupted via a ribolysation step, centrifugated and the yielded supernatant was evaluated.

Table 1: Pipetting scheme and measurement results of riboflavin standards and cell samples for absorbance measurement at 446 nm in the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]. WT = wild type, And77 = E. coli equipped with BBa_K1172306, sn = supernatant, cd = cell disruption.

Riboflavin in known concentrations (5.31 * 10^-5 M) and dilutions was measured to generate a calibration curve. The subsequently computed riboflavin concentrations were 5773.3 µg / L for the supernatant of E. coli KRX with BBa_K1172306 and 6112.63 µg /L for the cell disruption samples of E. coli KRX with BBa_K1172306. The concentration of putative riboflavin in the wild type strain was not detectable.

• Absorbance measurement is the least sensitive method used for riboflavin detection. Therefore the slightly higher yields (compared to fluorescence and HPLC measurement) should be taken with a grain of salt.

Fluorescence measurement

Riboflavin absorbs light at 440 nm with a corresponding emission at 535 nm. The fluorescence was measured in a TECAN infinite plate reader. The samples consisted of supernatant samples from E. coli KRX with BBa_K1172306 (grown for 72 hours) , E. coli KRX with BBa_K1172306 (grown for 12 hours) and E. coli KRX wild type bacteria (grown for 72 hours)

Table 2: Pipetting scheme and measurement results of riboflavin standards and cell samples for fluorescence measurement, emission at 535 nm. Measured in the [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]. WT = wild type, And77 = E. coli equipped with BBa_K1172306, sn = supernatant, cd = cell disruption.

Riboflavin in known concentrations and dilutions was measured to generate a calibration line. The subsequently computed riboflavin concentrations were 308.1 µg / L for the supernatant sample after 12 hours and 3821.5 µg /L for the supernatant sample after 72 hours. The concentration of putative riboflavin in the wild type strain was not detectable.

HPLC measurement

Supernatant and cell disruption samples of E. coli KRX with BBa_K1172306 (grown for 72 hours) , E. coli KRX with BBa_K1172306 (grown for 12 hours) and E. coli KRX wild type bacteria (grown for 72 hours) were measured in a HPLC detector.

Table 3: HPLC measurement results for riboflavin concentrations in supernatant (sn) and cell disruption (cd) samples after 72 hours and 12 hours of cultivation respectively.

Figure 13: Results of the HPLC measurement shown as graph. Figure 13 was centered on the riboflavin peak for a better view.

In summary, the obtained data showed, that after 72 hours of cultivation the concentration of riboflavin in the supernatant and cell disruption samples of E. coli KRX with BBa_K1172306 was 60fold higher than in the E. coli KRX wild type: Approx. 4400 µg/L, yielded with KRX carrying BBa_K1172306 compared to 67.05 µg/L for the KRX wild type. Even after 12 hours, the riboflavin producing strain had generated ten times as much riboflavin as the wild type: Approx. 700 µg/L compared to 76.64 µg/L in the wild type.

• HPLC yielded the only results for riboflavin concentration in KRX wild type supernatants due to its sensitivity.

LC/MS measurement

Supernatant samples of a wild type E. coli KRX (grown for 72 hours) and E. coli KRX with BBa_K1172306 (grown for 72 hours and 36 hours) were measured. One sample of E. coli KRX with BBa_K1172306, which had been grown in a biased M9 medium for 72 hours was measured additionally.

Figure 14: LC/MS results presented as list depiction.

Figure 15: LC/MS results presented as overlay depiction.

The BBa_K1172306 carrying strains produced a much higher amount of riboflavin compared to the wild type E. coli KRX strain. The LC/MS results do not allow for a statement on how much more riboflavin E. coli KRX with BBa_K1172306 produces. Nevertheless, it is obvious that riboflavin was overproduced in a remarkable quantity.

Evaluation of the measurements

The quantitative data obtained using absorbance, fluorescence and HPLC measurements shows a distinct trend. All samples generated from E. coli KRX with BBa_K1172306 (grown for 72 h) showed similar values of approx. 4000 µg/ml. This is a considerable increase in riboflavin production compared to the wild type KRX strains.

Conclusion

Riboflavin possesses the ability to be a potent redoxmediator. By turning the rib-gene cluster from Shewanella oneidensis into a BioBrick and subsequently cloning it into the desired chassi Escherichia coli, the iGEM Team Bielefeld was able to raise the amount of riboflavin produced by E. coli significantly. The results indicate that the transformation of E. coli with BBa_K1172303, respectively BBa_K1172306, represents a viable option when considering genetical optimization of microorganisms intended for usage in microbial fuel cells (MFC).

Methods for Riboflavin analysis

HPLC-Method

• Procedure:
  • Riboflavin was separated from disturbing substances on a C18 column and detected with a fluorescence detector.
• Elution:
  • The sample was injected onto a C18-reversed phase column and eluted with a buffer consisting of 99 % 0.01 M sodium acetat pH 7.4 and 1 % tetrahydrofurane. Total flow is set to 1 ml/min
• Detection:
  • Excitation wavelength of 436 nm and emission wavelength of 535 nm

• C18 reverse phase column
• Isocratic method: 99 % 0.01 M sodium acetate pH 7.4 + 1 % tetrahydrofurane
• Flow = 1 mL min-1
• UV-detection at 535 nm
• Internal standard: 5.31 x 10^-5 mg L^-1 Riboflavin
• Column:
  • Eurospher II 100-5 C18p by Knauer
  • Dimensions: 150 x 4.6 mm with precolumn
  • Particle size: 5 µm
  • Pore size: 100 Å
  • Material: silica gel
• Software:
  • Clarity (Version 3.0.5.505) by Data Apex
• Autosampler:
  • Midas by Spark Holland
  • Tray cooling: 10 °C
• Pump:
  • L-6200A Intelligent Pump by Hitachi
• UV-Detector:
  • Series 1050 by Hewlett Packard

LC-ESI-qTOF-MS(-MS)

• Column: C18 reversed phase (Cogent [http://www.mtc-usa.com/diamond_hydride_specs.asp Diamond Hydride])
  • dimension: 150 x 2,1 mm
  • Pore size: 100 Å
  • Particle size: 4 µm
• Flow: 0.4 mL min^-1
• Column temperature: 40 °C
• Injection: 5 µl
• Prerun: 5 min
• Eluent:
  • A = 50 % acetonitrile + 50 % H2O + 0,1 % formic acid
  • B = 90 % acetonitrile + 10 % H2O + 0,1 % formic acid
• Step gradient:
  • 0:00 min: 0 % eluent A
  • 8:00 min: 100 % eluent A
  • 13:00 min: 100 % eluent A
  • 15:30 min: 0 % eluent A
  • 18:00 min: 0 % eluent A
• VWR Hitachi LaChrom ULTRA HPLC equipment
• Software: HyStar 3.2, mircrOTOF Control, DataAnalysis 4.0

Ionization method

• Using Bruker Daltonics ESI-QTOF micrOTOF_Q
• ESI-QTOF-MS in positive mode
• Mass range: 50 - 1500 m/z
• Source:
  • End plate offset: - 500 V, 107 nA
  • Capillary: - 3500 V, 4 nA
  • Nebulizer: 2 bar
  • Dry gas: 8 L min^-1
  • Dry Temp.: 180 °C
• Transfer:
  • Funnel 1 RF: 200 Vpp
  • Funnel 2 RF: 300 Vpp
  • ISCID Energy: 0 eV
  • Heyapole RF: 80 Vpp
• Quadrupole
  • Ion energy: 4 eV
  • Low mass: 50 m/z
• Collision Cell:
  • Collision energy: 8 eV
  • Collision RF: 130 Vpp
  • Transfer time: 75 µs
  • Pre puls storage: 4 µs

MS-MS

• Isolated mass: 243.1 +/- 0.1
• Collision energy: 30 eV

Direct fluorescens measurement of riboflavins

• This method has been used for measurement of intracellular and extracellular riboflavin concentration.

• Protocol:
  • Inoculate an overnight culture (30 mL with 1 mL of pre-culture)
  • Centrifugate (5 min at 5000 g) of 6-10 mL overnight culture (OD 4-6), adjust volume between different samples for the approximation of the number of cells
  • Transfer supernatant in fresh reagent tube until measurement. Wash pellet 3 times with 1 mL of PBS buffer
  • Resuspend pellet in 1 mL PBS buffer
  • Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm)
  • Centrifugation for 10 min at maximum speed
  • Store extra- and intracellular fractions at - 20 ° C or direct measurement with [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader]

• [http://www.tecan.com/platform/apps/product/index.asp?MenuID=1812&ID=1916&Menu=1&Item=21.2.10.1 Tecan Infinite® M200 platereader] parameters:
  • Sample volume = 150 μL supernatant
  • Excitation = 440 nm
  • Emission = 535 nm
  • Concentration calculation by riboflavin calibration curve

Improvement by iGEM Rochester 2021

We improved on the 'Riboflavin synthesis gene cluster from shewanella oneidensis' (BBa_K1172303) to 'riboflavin synthesis gene cluster' (BBa_K3724015) by introducing the ribosome binding site (RBS) - BBa_0032 - before the SO_2468 sequence and the ribe sequence for expression of each of the genes in the riboflavin synthesis gene cluster. We further improved on this part by re-optimizing sequences for S. oneidensis MR-1 and transforming into S. oneidensis MR-1 for microbial reduction of graphene oxide.

Figure 1: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to riboflavin cluster (light blue)adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, time zero reflects the start of induction with 1.5mM IPTG.

Figure 2: Maximum rates of the bacterial reduction curves adjusted for bacterial growth at the IPTG induction conditions of 1.5mM and 1.0 mM IPTG with 0hr induction and 1.5mM and 0.75mM IPTG with 5 hour induction.

Figure 3: D/G ratio for rGO produced under microbial conditions and chemical conditions.

Figure 4: D/G ratio for rGO produced under microbial conditions after 12 hours

Shewanella oneidensis MR-1 are gram-negative bacteria at the center of studies of microbial reduction due to their ability to transfer electrons extracellularly to reduce materials such as graphene oxide (GO)[1]. Such characteristics have made S. oneidensis MR-1 an organism of interest in microbial fuel cells for bioelectricity generation and potential applications in bioremediation[2]. We aimed to use this improved part to increase the rate of graphene oxide reduction by S. oneidensis MR-1.

Optical density measurements (O.D.₆₀₀)

Our part was successfully transformed into S. oneidensis MR-1 and expression of riboflavin cluster in our IPTG-inducible vector, pcD8, was initially induced with 1.5mM IPTG. Immediately following induction, reduction of graphene oxide was carried out under constant shaking at 200 rpm, and the O.D.₆₀₀ was measured every hour for 48 hours for TSB only, graphene oxide only, graphene oxide and TSB only, and graphene oxide and TSB each with riboflavin cluster and the wildtype. Here, the TSB only , graphene oxide only, and graphene oxide and TSB only are our negative controls, and wild type MR-1 is the positive control to which all our transformed strains are compared. The O.D.₆₀₀ measurements obtained from these experiments reflect both bacterial absorbance as well as reduced graphene oxide absorbance. So, the absolute O.D.₆₀₀ due to reduced graphene oxide is obtained by correcting for O.D.₆₀₀ of bacteria and TSB only.

Figure 1 shows that after correction for bacterial growth riboflavin cluster had a comparable rate of reduction with the wildtype and pcD8 where it appears to be slightly faster after the 5 hour point.

The negative controls (TSB only, GO only, and GO and TSB only) show an insignificant change in O.D.₆₀₀ over time indicating that the bacteria are responsible for the increase in O.D.₆₀₀ which is the measure of reduction of graphene oxide in these experiments.

The results show that the rate of reduction for riboflavin cluster varied across the different IPTG induction conditions. Figure 2 shows that the maximum rate obtained for riboflavin cluster at 0hr induction with 1.5mM IPTG as well as 1.0mM IPTG were 10.728 hr^-1 and 9.816 hr^-1, respectively, whereas the wildtype had a rate of 8.936 hr^-1. There are also comparable reduction rates with the wildtype for reduction with 1.5mM IPTG following 5 hr induction.

Raman spectroscopy

Raman spectroscopy was carried out to investigate the amount of carbon-carbon single bonds of the reduced graphene oxide produced by riboflavin cluster after the 48 hour period. We primarily relied on the D/G ratio, which is the intensity of the D peak divided by the intensity of the G peak. The D peak is associated with double-bonded carbon, and the G peak is associated with single-bonded carbon.

We used chemically reduced GO as our positive control as chemical reduction with ascorbic acid results in a much greater degree of reduction than seen in microbial reduction. We also had two negative controls: GO only and GO and TSB media only.

After reduction, we compared the D/G ratios to determine how well our transformed strain had done in reducing the number of Sp² carbons.

As Figure 3 shows, the chemically reduced GO was by far the most reduced, with an average D/G ratio of around 1.2. The microbial reduction with riboflavin cluster resulted in a D/G ratio of 0.99 which is similar to the D/G ratio of wildtype. This demonstrates that our part worked as intended and was able to reduce the GO to expected levels of reduction during the reduction period.

To investigate the degree of reduction at an earlier time point, we took Raman spectra for riboflavin cluster at the 12 hour time point.

As figure 4 shows, after 12 hours, riboflavin cluster was far more reduced than the pcD8 and wildtype strains. This shows that this modified bacteria strain is faster at reducing the GO than either the wild type strain or the strain with the empty expression vector, pcD8.

References iGEM Rochester 2021

[1] Wang, G.; Qian, F.; Saltikov, C. W.; Jiao, Y.; Li, Y. Microbial Reduction of Graphene Oxide by Shewanella. Nano Research 2011, 4, 563–570.
[2] Lin, T.; Ding, W.; Sun, L.; Wang, L.; Liu, C.-G.; Song, H. Engineered Shewanella Oneidensis-Reduced Graphene Oxide Biohybrid with Enhanced Biosynthesis and Transport of Flavins Enabled a Highest Bioelectricity Output in Microbial Fuel Cells. Nano Energy 2018, 50, 639–648.

References

• C. A. Abbas and A. S. Sibirny. (2011) Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers. [http://mmbr.asm.org/content/75/2/321.full#ref-292| Microbiology and Molecular Biology Reviews 75(2): 321-360].

• Hohmann H. P., Stahmann K. P. (2010). Biotechnology of riboflavin production, p. 115–139. In Mander L., Liu H. W. (ed.), Comprehensive natural products. II. Chemistry and biology, vol. 7. Cofactors. Elsevier, Philadelphia, PA.

• von Canstein H., Ogawa J., Shimizu S., Lloyd J. R. (2008). Secretion of flavins by Shewanella species and their role in extracellular electron transfer. [http://aem.asm.org/content/74/3/615.full Appl. Environ. Microbiol. 74:615–623].

• Bacher A., et al. (2001). Biosynthesis of riboflavin. [http://www.ncbi.nlm.nih.gov/pubmed/11153262 Vitam. Horm. 61:1–49.]

• Tesliar G. E., Shavlovskii G. M. (1983). Localization of the genes coding for GTP cyclohydrolase II and riboflavin synthase on the chromosome of Escherichia coli K-12. Tsitol. Genet. 17:54–56. (In Russian.)

• Seong Han Lim, Jong Soo Choi and Enoch Y. (2001). Park Microbial Production of Riboflavin Using Riboflavin Overproducers, Ashbya gossypii, Bacillus subtilis, and Candida famate: An Overview.[http://www.bbe.or.kr/storage/journal/BBE/6_2/6657/articlefile/article.pdf Biotechnol. Bioprocess Eng., 6: 75-88]