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Latest revision as of 04:34, 17 December 2021

Riboflavin synthesis gene cluster

The riboflavin synthesis gene cluster consists of the genes ribd (BBa_K3724013) and the SO_3468+ribBA+ribE cluster (BBa_K3724014). This gene cluster consists of genes involved in the biosynthesis pathway of riboflavin in Shewanella oneidensis MR-1.

Usage and Biology

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]. Two extracellular electron transfer pathways have been identified in the reduction of GO by Shewanella oneidensis MR-1. These are indirect electron transfer, mediated by secreted electron shuttles, and direct extracellular electron transfer (DET) which involves direct contact with the extracellular material [3].

The riboflavin gene synthesis cluster comprises genes that are integral in the riboflavin biosynthesis pathway in S. oneidensis MR-1 without the regulatory elements. These include the riboflavin biosynthesis protein (ribD) which converts 2,5-diamino-6-(ribosylamino)-4(3h)-pyrimidinone 5'-phosphate into 5-amino-6-(ribosylamino)-2,4(1h,3h)-pyrimidinedione 5'-phosphate, the riboflavin synthase alpha subunit (SO_3468) which catalyzes the formation of riboflavin and 5-amino-6-(D-ribitylamino)uracil from two molecules of 6,7-dimethyl-8-ribityllumazine, the GTP cyclohydrolase-2 (ribA), 3,4-dihydroxy-2-butanone-4-phosphate synthase (ribB) and riboflavin synthase beta subunit (RibE). The latter three proteins catalyzes the conversion of GTP to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate , formate and pyrophosphate, catalyzes the conversion of D-ribulose 5-phosphate to formate and 3,4-dihydroxy-2-butanone 4-phosphate and catalyzes the formation of 6,7-dimethyl-8-ribityllumazine by condensation of 5-amino-6-(D-ribitylamino)uracil with 3,4-dihydroxy-2- butanone 4-phosphate, respectively.

It has been proposed that flavins may act as electron shuttles in the reduction of extracellular material by S. oneidensis MR-1 [4]. These flavins initially begin as riboflavin and are then converted to flavin adenine dinucleotide (FAD) in the cytoplasm. FAD exits the cytoplasm where it is then reconverted to riboflavin in the periplasm or in the extracellular space through the intermediate flavin, flavin mononucleotide (FMN). The overexpression of the genes in the riboflavin synthesis gene cluster in S. oneidensis MR-1 has previously been shown to increase the concentration of extracellular flavins [3]. This increase in flavins has been shown to increase the rate of extracellular electron transfer in microbial fuel cells for bioelectricity generation [3].

It is thought that the overexpression of these genes in S. oneidensis MR-1 would lead to an increase in extracellular flavins that can then reduce GO, ultimately leading to an increase in the rate of reduction.

Bielefeld iGEM 2013 team expressed this riboflavin gene synthesis cluster in Escherichia coli (BBa_K1172303). They discovered that expression of the gene cluster in E. coli resulted in a significant increase in extracellular flavins. We utilized the sequences from this biobrick which were initially obtained from the genome of S. oneidensis MR-1 and improved on this part by inserting a ribosome binding site (RBS) - BBa_0032 - before the SO_2468 sequence and the ribe sequence for expression of each of the genes in the cluster and transformed this gene cluster into S. oneidensis MR-1. The gene cluster was successfully transformed into S. oneidensis MR-1 and was used for microbial reduction of GO.

Results

Optical density measurements (O.D.₆₀₀)

Previous studies have shown that the magnitude of reduction of graphene oxide can be measured using optical density measurements at a wavelength of 600 nm. It has also been shown that the presence of graphene oxide has no significant effect on the growth rate of S. oneidensis MR-1 [5]. So, we utilized this technique to measure and compare the magnitude of reduction with our different transformed strains and the wildtype.

Expression of our genes 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: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to riboflavin cluster (light blue) for the reduction period. Here, time zero reflects the start of induction with 1.5mM IPTG.

Figure 1 shows the microbial reduction of graphene oxide with the transformed strain, riboflavin cluster. It shows that transformation of S. oneidensis MR-1 with riboflavin cluster still allows for reduction at a rate comparable to that of the wildtype. This figure represents the combined O.D.₆₀₀ measurements for the reduced graphene oxide absorbance and bacterial absorbance. Without adjusting for bacterial growth, this figure shows that riboflavin cluster had a faster rate of reduction than pcD8.

Figure 2: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to riboflavin cluster (turquoise) over a 48-hour period. Here, time zero reflects the start of induction with 1.5mM IPTG.

Figure 3: 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.

The bacterial growth over the 48-hour period was measured by means of O.D.₆₀₀ to determine the impact of riboflavin cluster expression on bacterial growth. Figure 2 shows that bacteria expressing the riboflavin cluster gene (turquoise) had a similar growth curve to the wildtype (dark gray) and the empty vector, pcD8. This indicated that the expression of riboflavin cluster in our transformed strain was likely not toxic to the cells.

To get the absolute O.D.₆₀₀, representative of the reduced graphene oxide content in the microbial reduction, the measured O.D.₆₀₀ was corrected for the O.D.₆₀₀ for bacterial growth in TSB only. Figure 3 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 than both after the 5 hour point.

Since ydeh (BBa_K3724008), ribf (BBa_K3724010) and oprf (BBa_K3724011) showed slower growth than wild-type MR-1 (Figure 2), we hypothesized that the induced genes may have been slightly toxic to the cells. We then observed the growth curve when the concentration of IPTG was decreased to 1.0mM to reduce the amount of gene expression for all of our transformed strains including riboflavin cluster.

Figure 4: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to riboflavin cluster (light blue). Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 5: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (dark gray) compared to riboflavin cluster (turquoise) over a 48-hour period. Here, time zero reflects the start of induction with 1.0mM IPTG.

Figure 6: 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 1.0mM IPTG.

Figure 4 shows that with 1.0mM IPTG induction, microbial reduction of graphene oxide with the transformed strain riboflavin cluster had comparable reduction rates to wild-type MR-1 and the empty vector, pcD8. From the figure it is seen that the rate of reduction of riboflavin cluster without corrections for bacterial growth is slightly slower than that of the wildtype.

Figure 5 shows that there was no significant alteration in the growth for riboflavin cluster for induction with 1.0mM IPTG as compared with 1.5mM IPTG (Figure 2) as was expected. The curve showed similar bacterial growth as compared to the wildtype and the empty vector, pcD8.

Figure 6 shows that with the correction for bacterial growth, microbial reduction with riboflavin cluster with 1.0mM IPTG induction had a similar rate of reduction to the wildtype and a faster rate of reduction than pcD8 after the first 5 hours.

We carried out an induction time of 5 hours prior to reduction to allow riboflavin cluster to already have the riboflavin gene synthesis protein production fully active before we began the reduction reactions. This was carried out with 1.5mM IPTG as IPTG concentration had no effect on riboflavin cluster growth curves (Figure 2,5). The reductions were carried out under the same conditions of shaking at 200 rpm.

Figure 7: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to riboflavin cluster (green) for the reduction period. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

From Figure 7, the microbial reduction of graphene oxide with the transformed strain riboflavin cluster shows comparable rates of reduction to that of the wildtype and pcD8 without any corrections to the O.D.₆₀₀ measurements for bacterial growth.

Figure 8: O.D.₆₀₀ of bacteria and TSB only with wild-type MR-1 (red) compared to riboflavin cluster (light blue) for the 48-hour period. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

The bacterial growth measured by O.D.₆₀₀ in Figure 8 shows that bacteria expressing the riboflavin cluster (light blue) is still similar to the wildtype and the empty vector, pcD8, over the 48-hour period, with the 5 hour induction. This indicates that expression of riboflavin cluster over time is not likely to be toxic to the cells.

Figure 9: O.D.₆₀₀ of microbial reduction with wild-type MR-1 (deep purple) compared to riboflavin cluster (green) adjusting for the O.D.₆₀₀ values due to bacterial growth. Here, reduction was initiated following a 5 hour induction with 1.5mM IPTG (5hr induction)

After corrections for bacterial growth, Figure 9 shows that riboflavin cluster had a rate of reduction comparable to that of wild type and the empty vector, pcD8 where the reduction rate was slightly slower.

The growth curves of riboflavin cluster show similar bacterial growth compared to the wildtype and pcD8 for each of the IPTG conditions (Figure 2,5,8). The rate of reduction of our transformed strain with riboflavin cluster were also similar to the wildtype and pcD8, only lagging slightly behind.

The slopes at the steepest point on the curves were obtained for each induction condition to give the maximum reduction rate measured during the reduction period, and this was compared to that of wild-type MR-1.

Figure 13: 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.

The results show that the rate of reduction for riboflavin cluster varied across the different IPTG induction conditions. Figure 13 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 max rate of 8.936 hr^-1. There are also comparable reduction rates with wildtype following 5 hr induction with 1.5mM IPTG for reduction.

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.

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.

Figure 14: D/G ratio for rGO produced under microbial conditions and chemical conditions.

As Figure 14 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.

Figure 15: D/G ratio for rGO produced under microbial conditions after 12 hours

As figure 15 shows, not all of the bacteria reduced the GO at the same rate. 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.

Conclusion

We were able to successfully transform the riboflavin gene cluster into S. oneidensis MR-1. This transformed strain reduced the graphene oxide to levels expected with microbial reduction for S. oneidensis MR-1. It also had a greater degree of reduction at 12 hours than the wildtype. This indicates that expression of riboflavin cluster in S. oneidensis MR-1 ,can decrease the time it takes for graphene oxide to be reduced to expected reduction levels for microbial reduction.

Sequence and Features

References

[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] Schwalb, C.; Chapman, S. K.; Reid, G. A. The Tetraheme Cytochrome Cyma Is Required for Anaerobic Respiration with Dimethyl Sulfoxide and Nitrite in Shewanella Oneidensis. Biochemistry 2003, 42, 9491–9497.
[3] 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.

[4] Kouzuma, A.; Kasai, T.; Hirose, A.; Watanabe, K. Catabolic and Regulatory Systems in Shewanella Oneidensis MR-1 Involved in Electricity Generation in Microbial Fuel Cells. Frontiers in Microbiology 2015, 6.
[5] Lehner, B. A.; Janssen, V. A.; Spiesz, E. M.; Benz, D.; Brouns, S. J.; Meyer, A. S.; van der Zant, H. S. Creation of Conductive Graphene Materials by Bacterial Reduction Using Shewanella Oneidensis. ChemistryOpen 2019, 8, 888–895.