Team:Lund/Design/Theory

Design

Introduction

Vitreoscilla hemoglobin (VHb) is a protein found in the aerobic gram negative bacteria Vitreoscilla spp. It is regulated by the Vitreoscilla hemoglobin promoter, which is oxygen dependent and activates the transcription of the hemoglobin under microaerobic conditions to increase oxygen uptake [1]. It was first discovered in 1966 and was initially believed to be a cytochrome, until its primary structure was disclosed in 1986 and it was recognized as a hemoglobin [3]. The gene coding for VHb (vgb) was cloned for the first time and expressed in Escherichia coli in 1988, and its capability to increase oxygen uptake was quickly noted [4][5].

The discovery led to a new approach in combating the reduced yields in fermentors caused by low oxygen conditions arising from high density cell culture: instead of modifying external parameters in order to increase oxygen availability, hemoglobin is co-expressed in order to enhance the cellular oxygen utilization. In this way, the cells are able to aerobically catabolize the available carbon sources under microaerobic conditions, without generating growth-inhibitory metabolites such as lactate and acetate [5]. Furthermore, it has been found that in addition of being able to increase protein yields under microaerobic conditions, the presence is also correlated with improved cell growth and survivability [5]. This seems to be the case not only for Vitreoscilla and E. coli, but also for other bacterial species [6], yeast [8], plants [9] and even some vertebrates [10].

Structure

VHb is a homodimeric protein, consisting of two subunits of 146 amino acid residues each and with a close resemblance to the globin fold [2]. The globin fold is a three-dimensional motif shared among the members of the globin superfamily, consisting of six to eight alpha-helices packed at an approximate 50 degree angle from each other. Starting from the N-terminus, the helices are denoted as helix A-H. Between each helix and the following one is a loop connecting the segments, denoted by AB, BC, CD etc., corresponding to each pair of helices. The pocket in which heme is located is formed by a hydrophobic cavity, created by the distal E-helix and proximal F-helix, in which the conserved proximal histidine F8 coordinates with the upper portion of the heme group.

The VHb monomer is illustrated in fig. 1 along with the sperm whale myoglobin (Mb) which is commonly considered as the reference monomeric hemoglobin. The globin fold is clearly visible and the similarities are apparent, with the heme residing in the pocket enclosed by the E and F alpha helices. However, while the globin fold of Mb consists of eight alpha helices, the VHb has only seven as there is no apparent structure in the expected D-helix region [19]. This gives rise to a more hydrophobic CE transition in contrast to the usual CD, which along with the initial hydrophobic and disordered E7-E10 segment of the E helix is believed to be one of the key reasons for the high lipid binding propensity of VHb [11][19].

Figure 1: Upper: monomeric sperm whale myoglobin illustrating the globin fold. pdb: 1JP6 Lower: VHb momoner, showing the globin fold and the heme pocket. pdb: 3TM3

Role in oxygen delivery

It seems that the way in which VHb enhances oxygen uptake is by acting as an oxygen transporter, directly binding oxygen and delivering it to the respiratory chain. This claim is supported by several studies covering different mechanisms of VHb. More specifically, there are two key observations that motivate this.

First, it has been shown in vitro that VHb has a high binding affinity to lipid monolayers extracted from E. coli and that once bound, the oxygen affinity is reduced by more than 20-fold [11]. This is believed to be caused by the hydrophobic region between the CE-corner and the E-helix at the distal pocket serving as an anchor to the acyl chains of the phospholipid bilayer of the cell membrane. Due to the interaction between VHb and the membrane, a conformational change would occur around the heme pocket and the oxygen binding affinity would immediately be reduced.

Secondly, it has been shown by immunogold labeling in electron microscopy that VHb is localized in the cytoplasm and to a large extent near the cell membrane in both Vitreoscilla and E. coli [12]. These two observations indicate that VHb indeed transports oxygen to the respiratory chain, since if VHb is not in proximity to the cell membrane, it has high affinity for oxygen and vice versa, if bound the affinity is greatly reduced. Since respiration occurs around cell membranes, it seems highly plausible that VHb transports oxygen to the respiratory chain via direct interaction.

Other supporting experiments include showing that VHb can stimulate oxygenase activity [13][14] and even itself serve as a terminal oxidase [15]. The latter was shown by cultivating cells with no terminal oxidases under aerobic conditions, in which only cells containing VHb could maintain an aerobic respiration. Moreover, it has been shown that VHb protects from oxidative stress [16][17] and that increased expression levels are correlated with increased cell growth [18].

The putative mechanism is illustrated in fig. 2. When VHb is free in the cytoplasm, it has a high affinity to oxygen and binds accordingly. Then, anchoring to the cell membrane causes a decrease in affinity and a subsequent oxygen release. This oxygen may then be consumed within the respiratory chain.

Figure 2: VHb binds to membrane and releases oxygen

Previous research

Several studies have investigated the influence of the VHb expression level on the cell growth and the production of various desired products. In 2010, Sanny et al. managed to increase production of ethanol in E. coli by as much as 119% when expressing VHb in their ethanol-producing strain. However, they found that when they increased the VHb expression level by inserting the vgb gene into other plasmids, the ethanol production decreased to levels below that of the strain with no VHb expression at all, indicating that only low levels of VHb expression were beneficial [20].

Yu et al. studied the effect of VHb on the production of the biopolymer poly(β-hydroxybutyrate) (PHB) in E. coli both by integrating the vgb gene into the host chromosome and by keeping it on a plasmid. What they found was that chromosomal integration if vgb increased PHB content in cells grown in flasks to 83.8%, as compared to the non-vgb carrying strain where the content was less than 70% under the same conditions. However, when the vgb gene was instead carried on a plasmid, both PHB production and cell growth were far lower than with no VHb at all [21]. They theorized that the negative effect might be due to the burden of carrying an additional or a larger plasmid to accommodate the vgb gene. However, it has been shown that integrating vgb on the chromosome of the E. coli strain in question lowers the expression level compared to when a multicopy plasmid is used [22], again suggesting that a lower expression level may be more beneficial than a high one.

In both the studies described above, VHb was shown to only be beneficial when expressed at a low level, with higher VHb expression leading to a poorer outcome than with no VHb at all. This is in contrast to a study done in 1996 by Tsai et al., who by modulating the concentration of the inducer Isopropyl β-D-1-thiogalactopyranoside (IPTG) showed that increasing levels of VHb expression was associated with increasing final cell density (up to 2.7-fold compared to the VHb- strain) and a reduction in fermentation byproducts such as acetate and lactate [18]. However, as opposed to the other two groups above, they did not study the formation of any specific industrially relevant product.

The three studies described above all employed different methods to obtain variation in the VHb expression level - changing the IPTG concentration [18], inserting the vgb gene into different plasmids [20] and chromosomal integration [21]. However, we have not found any articles studying the effect on yield or productivity where the only variable in the expression system is the strength of the promoter under which VHb is expressed. In addition, VHb seems underutilized, especially within iGEM. We wanted to investigate its relevance in the synthetic biology community.

Previous iGEM teams

Until now, only two previous iGEM teams have to our knowledge adopted VHb technology in their projects. Imperial college 2014 co-expressed vgb in Gluconacetobacter xylinus to promote the production of cellulose. By doing so, they achieved an almost twofold increase in cell density using the G. xylinus iGEM strain. SCU-China 2016 used the same gene in E. coli to enhance growth in oxygen limited environments. It was shown that by expressing vgb, an improvement of both growth and protein production was obtained in the early stage of cell cultivation. However, no consistent improvements were observed in the stationary cultivation phase.

References

  1. [1] Dikshit, K., Spaulding, D., Braun, A., and Webster, D. (1989) Oxygen Inhibition of Globin Gene Transcription and Bacterial Haemoglobin Synthesis in Vitreoscilla. Microbiology 135, 2601-2609.
  2. [2] Stark, B., Dikshit, K., and Pagilla, K. (2012) The Biochemistry of Vitreoscilla hemoglobin. Computational and Structural Biotechnology Journal 3, e201210002.
  3. [3] Wakabayashi, S., Matsubara, H., and Webster, D. (1986) Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature 322, 481-483.
  4. [4] Dikshit, K., and Webster, D. (1988) Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene 70, 377-386.
  5. [5] Khosla, C., and Bailey, J. (1988) Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli. Nature 331, 633-635.
  6. [6] Wang S, Liu F, Hou Z, Zong G, Zhu X, Ling P. Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb) World J Microb Biot. 2014;30:1369–1376. doi: 10.1007/s11274-013-1561-4
  7. [8] Wu, JM. and Fu, WC. (2012) Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris. J Biosci Bioeng 113(3), 332–337.
  8. [9] Holmberg, N., Lilius, G., Bailey, J., and Bülow, L. (1997) Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production. Nature Biotechnology 15, 244-247.
  9. [10] Guan, B., Ma, H., Wang, Y., Hu, Y., Lin, Z., Zhu, Z., and Hu, W. (2010) Vitreoscilla Hemoglobin (VHb) Overexpression Increases Hypoxia Tolerance in Zebrafish (Danio rerio). Marine Biotechnology 13, 336-344.
  10. [11] Rinaldi A. C., Bonamore A., Macone A., Boffi A., Bozzi A., Di Giulio A. (2006) Interaction of Vitreoscilla hemoglobin with membrane lipids. Biochemistry 45:4069–4076
  11. [12] Ramandeep D., Hwang K. W., Raje M., Kim K.J., Stark B.C., Dikshit K.L., Webster D. A. (2001) Vitreoscilla hemoglobin: intracellular localization and binding to membranes. J. Biol. Chem. 277:24781–24789.
  12. [13] Fish P. A., Webster D. A., Stark B. C. (2001) Vitreoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo. J. Mol. Catal. B. Enzym. 9:75–82.
  13. [14] Lin J. M., Stark B. C., Webster D. A. (2003) Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (DNT) dioxygenase activity of Burkholderia and on DNT degradation in two-phase bioreactors. J. Ind. Microbiol. Biotechnol. 30:362–368
  14. [15] Dikshit R. P., Dikshit K. L., Liu Y., Webster D. A. (1992) The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of E. coli lacking terminal oxidases. Arch. Biochem. Biophys. 293:241–245.
  15. [16] Geckil H., Gencer S., Kahraman H., Erenler S. O.(2003) Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress. Res. Microbiol. 154:425–431.
  16. [17] Kvist M., Ryabova E. S., Nordlander E., Bulow L. (2007) An investigation of the peroxidase activity of Vitreoscilla hemoglobin. J. Biol. Inorg. Chem. 12:324–334.
  17. [18] Tsai PS, Hatzimanikatis V, Bailey JE. (1996). Effect of Vitreoscilla Hemoglobin Dosage on Microaerobic Escherichia coli Carbon and Energy Metabolism. Biotechnology and Bioengineering, 49, 139-150.
  18. [19] Tarricone, C., Galizzi, A., Coda, A., Ascenzi, P., and Bolognesi, M. (1997) Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp. Structure 5, 497-507.
  19. [20] Sanny, T., Arnaldos, M., Kunkel, S.A. et al (2010). Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Appl Microbiol Biotechnol 88, 1103-1112. https://doi.org/10.1007/s00253-010-2817-7
  20. [21] Yu, H., Yin, J., Li, H., Yang, S. and Shen, Z. (2000). Construction and Selection of the Novel Recombinant Escherichia coli Strain for Poly(β-Hydroxybutyrate) Production. J Biosci Bioeng 89(4), 307-311.
  21. [22] Wu, Y., and Yang, S. (1996) Construction and Study of an Integrated Expression Vector of Vitreoscilla Hemoglobin Gene (vgb) by Double Homologous Recombination Between Plasmid and Chromosome. Chinese Journal of Biotechnology 12, 276-283.
Gant Logo LMK Logo Lund Logo Kunskappsartner Logo