Model | TeamFreiburg:chAMBER

Indigo & Indirubin - blue pill or red pill?

Indigo, the colorant that gives our jeans their characteristic appearance, is an industrially relevant compound indispensable in our everyday lives. In a survey of 196 participants, we asked the citizen of Freiburg, how many pairs of jeans they do have in their wardrobe. With an average of six pairs of jeans, we estimate that several 10,000 tons of indigo must be produced each year to cover the needs of the global fashion industry.

Indigo is a deep blue, crystalline organic chemical compound. It is an organic pigment with high color strength, has a rather high melting point of around 390–392°C and is poorly soluble in water, ethanol, and diethyl ether.
Historically, Indigo was extracted from certain plants through a tedious, time-consuming, and inconsistent process [60]. Nowadays, Indigo is produced by chemical synthesis. Although this method is very effective, it is not sustainable, and we think it is necessary to find alternatives for that! The biggest problems that emerge from chemical synthesis are its dependency on fossil fuels, such as oil as a source material, high energy consumption and the production of high quantities toxic wastewater [61]. Considering the environmental crisis, together with the declining availability of fossil fuels, one can quickly see that it is not bearable to continue the production of Indigo by chemical means for a much longer time. A better solution is needed. And where could this solution be harboured? In synthetic Biology of course! Already in the early 1960s, the first Indigo-producing bacteria were discovered and in the 1980s efforts were made to use bacteria to produce Indigo on a large scale [62]. However, until now, microbial production of Indigo hardly reached levels that compete with existing ways of synthesis on a global market.

How to improve indigo production with the use of genetic modified bacteria?
Microbial production of indigo requires four genes (TnaB, TnaA, Fre and XiaI) and the amino acids Tryptophan (Figure 1). L-Tryptophan is imported into bacteria with the help of a membrane protein called low affinity tryptophan permease, emerging from the gene TnaB [63]. Afterwards, L-tryptophan is split up into Indole, NH4+ and Pyruvate by the Tryptophanase that stems from the gene TnaA [64]. The reactions continue by the hydroxylation of Indole through XiaI. To enhance the effectivity of this enzyme, the NAD(P)H-flavin reductase is expressed by the gene Fre, as it provides XiaI with FADH2 by adding Hydrogen to FAD (which in turn emerges from the activity of XiaI) [65]. Finally, after going through two enzymatic reactions, L-Tryptophan is transformed to either 3-Hydroxyindole or 2-Hydroxyindole. These two substances spontaneously react to 3-Oxindole and 2-Oxindole through the secession of a Hydrogen from the OH-group. Through spontaneous dimerization Indigo and Indirubin are formed. Two molecules of 3-Oxindole form one molecule of Indigo, while one molecule of 3-Oxindole combined with one molecule of 2-Oxindole form one molecule of Indirubin (Figure 1) [65].

Figure 1: Schematic representation of the used Indigo/Indirubin pathway. L-Tryptophan is imported by the membrane protein TnaB (low affinity tryptophan permease). L-tryptophan is cleaved into Indole, NH4+ and Pyruvate by a Tryptophanase (TnaA). The reactions continues by the hydroxylation of Indole through XiaI. To enhance the effectivity of this enzyme, the NAD(P)H-flavin reductase provides XiaI with FADH2 by adding Hydrogen to FAD. Finally, Indole is transformed to either 3-Hydroxyindole or 2-Hydroxyindole. These two substances spontaneously react to 3-Oxindole and 2-Oxindole through the secession of a hydrogen from the OH-group. Through spontaneous dimerization Indigo and Indirubin are formed. Graphic was adapted from [65].

To improve production, we target TnaA and XiaI inside microcompartments, by adding the SnoopCatcher and SpyCatcher, respectively to the genes. By adding L-Cysteine to the mix, we can shift the production to Indirubin, an isomer of Indigo [65]. This is especially interesting as this compound is currently being tested as a supportive drug to treat leukaemia: current trials show the beneficial effects of indirubin while being scarce in side effects. A reason why indirubin might be of significant importance in the medical sector in the near future [66]. However, as of now, Indirubin is very costly even in small quantities. In case of being used as a drug, its production is likely to be the threshold limiting how many people will have the financial means to be treated with the help of Indirubin. Improvement of synthesis to reduce cost and enhance accessibility to this product are of high priority. Therefore, our compartmentalisation toolbox could be one way to achieve a higher yield of Indirubin, providing a solution to this scarceness of indirubin and possibly improving the life of many leukaemia patients by allowing for a fair distribution of this upcoming drug.

Trehalose

Trehalose is a non-reducing disaccharide consisting of two glucose molecules with an α (1-1) linkage. It has a wide range of applications, for example, as a sweetener, preservative, or as additive in pharmaceutical products [67] [68] [69] [70].

Trehalose occurs naturally in various plants, fungi and in the haemolymph of many insects. At first it was believed to be a rare sugar, but later it was found that many bacteria, fungi, plants, and invertebrates produce it. It is difficult to detect in most organisms because it is usually rapidly metabolized by an enzyme called trehalase [71]. In bacteria, it is expressed under osmotic stress [72]. It acts as an osmoprotectant in bacteria. One possible way in which trehalose preserves and protects macromolecules is by replacing the binding of water molecules with the binding of the sugar, thereby maintaining the structural integrity of these biological structures [73]. Two enzymes are involved in trehalose synthesis. The trehalose-6-phosphate synthetase (TPS) is encoded by the gene otsA. This enzyme converts glucose-6-phosphate (G-6-P) and UDP-glucose to trehalose-6-phosphate (T-6-P). The gene otsB expresses trehalose-6-phosphatase (TPP), which converts T-6-P to trehalose (Figure 2) [68].

Due to the wide range of applications and the need for only two enzymes, we decided to use trehalose as another pathway to encapsulate in our microcompartments.

Figure 2: Schematic representation of the used Trehalose pathway. The trehalose-6-phosphate synthetase (TPS) is encoded by the gene otsA. This enzyme converts glucose-6-phosphate (G-6-P) and UDP-glucose to trehalose-6-phosphate (T-6-P). The gene otsB expresses trehalose-6-phosphatase (TPP), which converts T-6-P to trehalose.

References

[1] Y. G. Zhao and H. Zhang, “Phase Separation in Membrane Biology: The Interplay between Membrane-Bound Organelles and Membraneless Condensates,” Dev. Cell, vol. 55, no. 1, pp. 30–44, 2020, doi: 10.1016/j.devcel.2020.06.033.

[2] E. Gomes and J. Shorter, “The molecular language of membraneless organelles,” J. Biol. Chem., vol. 294, no. 18, pp. 7115–7127, 2019, doi: 10.1074/jbc.TM118.001192.

[3] J. M. Volland et al., “A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles,” Science (80-. )., vol. 376, no. 6600, pp. 1453–1458, 2022, doi: https://doi.org/10.1101/2022.02.16.480423.

[4] C. A. Kerfeld, C. Aussignargues, J. Zarzycki, F. Cai, and M. Sutter, “Bacterial microcompartments,” Nat. Rev. Microbiol., vol. 16, no. 5, pp. 277–290, 2018, doi: 10.1038/nrmicro.2018.10.

[5] A. H. Chen and P. A. Silver, “Designing biological compartmentalization,” Trends Cell Biol., vol. 22, no. 12, pp. 662–670, 2012, doi: 10.1016/j.tcb.2012.07.002.

[6] A. M. Ladouceur et al., “Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation,” Proc. Natl. Acad. Sci. U. S. A., vol. 117, no. 31, pp. 18540–18549, 2020, doi: 10.1073/pnas.2005019117.

[7] B. Guilhas et al., “ATP-Driven Separation of Liquid Phase Condensates in Bacteria,” Mol. Cell, vol. 79, no. 2, pp. 293-303.e4, 2020, doi: 10.1016/j.molcel.2020.06.034.

[8] N. Al-Husini, D. T. Tomares, O. Bitar, W. S. Childers, and J. M. Schrader, “α-Proteobacterial RNA Degradosomes Assemble Liquid-Liquid Phase-Separated RNP Bodies,” Mol. Cell, vol. 71, no. 6, pp. 1027-1039.e14, 2018, doi: 10.1016/j.molcel.2018.08.003.

[9] X. Jin et al., “Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness,” Sci. Adv., vol. 7, no. 43, 2021, doi: 10.1126/sciadv.abh2929.

[10] W. Bonacci et al., “Modularity of a carbon-fixing protein organelle,” Proc. Natl. Acad. Sci. U. S. A., vol. 109, no. 2, pp. 478–483, 2012, doi: 10.1073/pnas.1108557109.

[11] B. B. Menon, Z. Dou, S. Heinhorst, J. M. Shively, and G. C. Cannon, “Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species,” PLoS One, vol. 3, no. 10, 2008, doi: 10.1371/journal.pone.0003570.

[12] D. F. Savage, B. Afonso, A. H. Chen, and P. A. Silver, “Bacterial Carbon Fixation Machinery,” Science (80-. )., vol. 327, no. March, pp. 1258–1261, 2010.

[13] M. Fischbach and C. A. Voigt, “Prokaryotic gene clusters: A rich toolbox for synthetic biology,” Biotechnol. J., vol. 5, no. 12, pp. 1277–1296, 2010, doi: 10.1002/biot.201000181.

[14] H. Chen, J. Wilson, S. Ottinger, Q. Gan, and C. Fan, “Introducing noncanonical amino acids for studying and engineering bacterial microcompartments,” Curr. Opin. Microbiol., vol. 61, pp. 67–72, 2021, doi: 10.1016/j.mib.2021.03.004.

[15] J. C. Jackson, S. P. Duffy, K. R. Hess, and R. A. Mehl, “Improving nature’s enzyme active site with genetically encoded unnatural amino acids,” J. Am. Chem. Soc., vol. 128, no. 34, pp. 11124–11127, 2006, doi: 10.1021/ja061099y.

[16] I. Drienovská and G. Roelfes, “Expanding the enzyme universe with genetically encoded unnatural amino acids,” Nat. Catal., vol. 3, no. 3, pp. 193–202, 2020, doi: 10.1038/s41929-019-0410-8.

[17] D. Choe, S. Cho, S. C. Kim, and B. K. Cho, “Minimal genome: Worthwhile or worthless efforts toward being smaller?,” Biotechnol. J., vol. 11, no. 2, pp. 199–211, 2016, doi: 10.1002/biot.201400838.

[18] M. McNutt, “Climate change impacts,” Science (80-. )., vol. 341, no. 6145, p. 435, 2013, doi: 10.1126/science.1243256.

[19] F. Johnsson, J. Kjärstad, and J. Rootzén, “The threat to climate change mitigation posed by the abundance of fossil fuels,” Clim. Policy, vol. 19, no. 2, pp. 258–274, 2019, doi: 10.1080/14693062.2018.1483885.

[20] M. Höök and X. Tang, “Depletion of fossil fuels and anthropogenic climate change-A review,” Energy Policy, vol. 52, pp. 797–809, 2013, doi: 10.1016/j.enpol.2012.10.046.

[21] J. Du, Z. Shao, and H. Zhao, “Engineering microbial factories for synthesis of value-added products,” J. Ind. Microbiol. Biotechnol., vol. 38, no. 8, pp. 873–890, 2011, doi: 10.1007/s10295-011-0970-3.

[22] N. Ferrer-Miralles, J. Domingo-Espín, J. Corchero, E. Vázquez, and A. Villaverde, “Microbial factories for recombinant pharmaceuticals,” Microb. Cell Fact., vol. 8, pp. 1–8, 2009, doi: 10.1186/1475-2859-8-17.

[23] L. Sanchez-Garcia, L. Martín, R. Mangues, N. Ferrer-Miralles, E. Vázquez, and A. Villaverde, “Recombinant pharmaceuticals from microbial cells: A 2015 update,” Microb. Cell Fact., vol. 15, no. 1, pp. 1–7, 2016, doi: 10.1186/s12934-016-0437-3.

[24] S. C. Wenzel and R. Müller, “Myxobacteria - ‘microbial factories’ for the production of bioactive secondary metabolites,” Mol. Biosyst., vol. 5, no. 6, pp. 567–574, 2009, doi: 10.1039/b901287g.

[25] N. S. Lau, M. Matsui, and A. A. A. Abdullah, “Cyanobacteria: Photoautotrophic Microbial Factories for the Sustainable Synthesis of Industrial Products,” Biomed Res. Int., vol. 2015, 2015, doi: 10.1155/2015/754934.

[26] E. Cornejo, N. Abreu, and A. Komeili, “Compartmentalization and organelle formation in bacteria,” Curr. Opin. Cell Biol., vol. 26, no. 1, pp. 132–138, Feb. 2014, doi: 10.1016/j.ceb.2013.12.007.

[27] T. W. Giessen and P. A. Silver, “Encapsulation as a Strategy for the Design of Biological Compartmentalization,” J. Mol. Biol., vol. 428, no. 5, pp. 916–927, 2016, doi: 10.1016/j.jmb.2015.09.009.

[28] W. Martin, “Evolutionary origins of metabolic compartmentalization in eukaryotes,” Philos. Trans. R. Soc. B Biol. Sci., vol. 365, no. 1541, pp. 847–855, 2010, doi: 10.1098/rstb.2009.0252.

[29] H. Kirst and C. A. Kerfeld, “Bacterial microcompartments: Catalysis-enhancing metabolic modules for next generation metabolic and biomedical engineering,” BMC Biol., vol. 17, no. 1, pp. 1–11, 2019, doi: 10.1186/s12915-019-0691-z.

[30] C. M. Agapakis, P. M. Boyle, and P. A. Silver, “Natural strategies for the spatial optimization of metabolism in synthetic biology,” Nat. Chem. Biol., vol. 8, no. 6, pp. 527–535, 2012, doi: 10.1038/nchembio.975.

[31] C. Liu et al., “Metabolic Engineering Mevalonate Pathway Mediated by RNA Scaffolds for Mevalonate and Isoprene Production in,” 2022, doi: 10.1021/acssynbio.2c00226.

[32] H. Kirst, B. H. Ferlez, S. N. Lindner, C. A. R. Cotton, A. Bar-Even, and C. A. Kerfeld, “Toward a glycyl radical enzyme containing synthetic bacterial microcompartment to produce pyruvate from formate and acetate,” Proc. Natl. Acad. Sci. U. S. A., vol. 119, no. 8, pp. 1–10, 2022, doi: 10.1073/pnas.2116871119.

[33] M. G. Klein et al., “Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport,” J. Mol. Biol., vol. 392, no. 2, pp. 319–333, 2009, doi: 10.1016/j.jmb.2009.03.056.

[34] M. J. Lee, D. J. Palmer, and M. J. Warren, “Biotechnological Advances in Bacterial Microcompartment Technology,” Trends Biotechnol., vol. 37, no. 3, pp. 325–336, 2019, doi: 10.1016/j.tibtech.2018.08.006.

[35] J. K. Lassila, S. L. Bernstein, J. N. Kinney, S. D. Axen, and C. A. Kerfeld, “Assembly of robust bacterial microcompartment shells using building blocks from an organelle of unknown function,” J. Mol. Biol., vol. 426, no. 11, pp. 2217–2228, 2014, doi: 10.1016/j.jmb.2014.02.025.

[36] A. Hagen, M. Sutter, N. Sloan, and C. A. Kerfeld, “Programmed loading and rapid purification of engineered bacterial microcompartment shells,” Nat. Commun., vol. 9, no. 1, pp. 1–10, 2018, doi: 10.1038/s41467-018-05162-z.

[37] B. Zakeri et al., “Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin,” Proc. Natl. Acad. Sci. U. S. A., vol. 109, no. 12, 2012, doi: 10.1073/pnas.1115485109.

[38] T. W. Giessen, “Encapsulins: Microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science,” Curr. Opin. Chem. Biol., vol. 34, pp. 1–10, 2016, doi: 10.1016/j.cbpa.2016.05.013.

[39] J. A. Jones and T. W. Giessen, “Advances in encapsulin nanocompartment biology and engineering,” Biotechnol. Bioeng., vol. 118, no. 1, pp. 491–505, 2021, doi: 10.1002/bit.27564.

[40] J. Fontana et al., “Phage capsid-like structure of Myxococcus xanthus encapsulin, a protein shell that stores iron,” Microsc. Microanal., vol. 20, no. 3, pp. 1244–1245, 2014, doi: 10.1017/S1431927614007958.

[41] M. Sutter et al., “Structural basis of enzyme encapsulation into a bacterial nanocompartment,” Nat. Struct. Mol. Biol., vol. 15, no. 9, pp. 939–947, 2008, doi: 10.1038/nsmb.1473.

[42] W. J. Altenburg, N. Rollins, P. A. Silver, and T. W. Giessen, “Exploring targeting peptide-shell interactions in encapsulin nanocompartments,” Sci. Rep., vol. 11, no. 1, pp. 1–9, 2021, doi: 10.1038/s41598-021-84329-z.

[43] A. Van de Steen et al., “Bioengineering bacterial encapsulin nanocompartments as targeted drug delivery system,” Synth. Syst. Biotechnol., vol. 6, no. 3, pp. 231–241, 2021, doi: 10.1016/j.synbio.2021.09.001.

[44] I. Boyton, S. C. Goodchild, D. Diaz, A. Elbourne, L. Collins-Praino, and A. Care, “Exploring the Self-Assembly of Encapsulin Protein Nanocages from Different Structural Classes,” bioRxiv, 2021, doi: 10.1101/2021.06.06.447285.

[45] C. A. McHugh et al., “A virus capsid‐like nanocompartment that stores iron and protects bacteria from oxidative stress,” EMBO J., vol. 33, no. 17, pp. 1896–1911, 2014, doi: 10.15252/embj.201488566.

[46] Y. H. Lau, T. W. Giessen, W. J. Altenburg, and P. A. Silver, “Prokaryotic nanocompartments form synthetic organelles in a eukaryote,” Nat. Commun., vol. 9, no. 1, 2018, doi: 10.1038/s41467-018-03768-x.

[47] D. R. Hamill, A. F. Severson, J. C. Carter, and B. Bowerman, “Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains,” Dev. Cell, vol. 3, no. 5, pp. 673–684, 2002, doi: 10.1016/S1534-5807(02)00327-1.

[48] J. B. Woodruff, B. Ferreira Gomes, P. O. Widlund, J. Mahamid, A. Honigmann, and A. A. Hyman, “The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin,” Cell, vol. 169, no. 6, pp. 1066-1077.e10, 2017, doi: 10.1016/j.cell.2017.05.028.

[49] A. K. Tiwary and Y. Zheng, “Protein phase separation in mitosis,” Curr. Opin. Cell Biol., vol. 60, no. 1, pp. 92–98, Oct. 2019, doi: 10.1016/j.ceb.2019.04.011.

[50] C. D. Reinkemeier, G. E. Girona, and E. A. Lemke, “Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes,” Science (80-. )., vol. 363, no. 6434, 2019, doi: 10.1126/science.aaw2644.

[51] L. Wang, A. Brock, B. Herberich, and P. G. Schultz, “Expanding the Genetic Code of Escherichia coli,” Science (80-. )., vol. 292, no. 5516, pp. 498–500, Apr. 2001, doi: 10.1126/science.1060077.

[52] M. Amiram et al., “Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids,” Nat. Biotechnol., vol. 33, no. 12, pp. 1272–1279, 2015, doi: 10.1038/nbt.3372.

[53] L. Wang and P. G. Schultz, “A general approach for the generation of orthogonal tRNAs,” Chem. Biol., vol. 8, no. 9, pp. 883–890, 2001, doi: 10.1016/S1074-5521(01)00063-1.

[54] E. C. Fischer et al., “New codons for efficient production of unnatural proteins in a semisynthetic organism,” Nat. Chem. Biol., vol. 16, no. 5, pp. 570–576, 2020, doi: 10.1038/s41589-020-0507-z.

[55] K. C. Schultz, L. Supekova, Y. Ryu, J. Xie, R. Perera, and P. G. Schultz, “A genetically encoded infrared probe,” J. Am. Chem. Soc., vol. 128, no. 43, pp. 13984–13985, 2006, doi: 10.1021/ja0636690.

[56] Y. Won, A. D. Pagar, M. D. Patil, P. E. Dawson, and H. Yun, “Recent Advances in Enzyme Engineering through Incorporation of Unnatural Amino Acids,” Biotechnol. Bioprocess Eng., vol. 24, no. 4, pp. 592–604, 2019, doi: 10.1007/s12257-019-0163-x.

[57] Q. Gan, B. P. Lehman, T. A. Bobik, and C. Fan, “Expanding the genetic code of Salmonella with non-canonical amino acids,” Sci. Rep., vol. 6, no. November, pp. 1–7, 2016, doi: 10.1038/srep39920.

[58] K. J. Lee, D. Kang, and H. S. Park, “Site-Specific Labeling of Proteins Using Unnatural Amino Acids,” Mol. Cells, vol. 42, no. 5, pp. 386–396, 2019, doi: 10.14348/molcells.2019.0078.

[59] N. D. Pham, R. B. Parker, and J. J. Kohler, “Photocrosslinking approaches to interactome mapping,” Curr. Opin. Chem. Biol., vol. 17, no. 1, pp. 90–101, 2013, doi: 10.1016/j.cbpa.2012.10.034.

[60] N. Wenner, “The Production of Indigo Dye from Plants,” Fibershed, no. December, pp. 1–13, 2017, [Online]. Available: https://bit.ly/3ymh4Xp.

[61] T. M. Hsu et al., “Sustainable Indigo Dyeing Strategy,” Nat. Publ. Gr., vol. 14, no. january, pp. 256–261, 2018, doi: 10.1038/nchembio.2552.Employing.

[62] K. Aino, K. Hirota, T. Okamoto, Z. Tu, H. Matsuyama, and I. Yumoto, “Microbial communities associated with indigo fermentation that thrive in anaerobic alkaline environments,” Front. Microbiol., vol. 9, no. SEP, pp. 1–16, 2018, doi: 10.3389/fmicb.2018.02196.

[63] S. Bhatt, A. Anyanful, and D. Kalman, “CsrA and TnaB coregulate tryptophanase activity to promote exotoxin-induced killing of Caenorhabditis elegans by enteropathogenic Escherichia coli,” J. Bacteriol., vol. 193, no. 17, pp. 4516–4522, 2011, doi: 10.1128/JB.05197-11.

[64] G. Li and K. D. Young, “Indole production by the tryptophanase TnaA in escherichia coli is determined by the amount of exogenous tryptophan,” Microbiol. (United Kingdom), vol. 159, no. 2, pp. 402–410, 2013, doi: 10.1099/mic.0.064139-0.

[65] H. Yin et al., “Efficient Bioproduction of Indigo and Indirubin by Optimizing a Novel Terpenoid Cyclase XiaI in Escherichia coli,” ACS Omega, vol. 6, no. 31, pp. 20569–20576, 2021, doi: 10.1021/acsomega.1c02679.

[66] S. Nam et al., “Indirubin derivatives induce apoptosis of chronic myelogenous leukemia cells involving inhibition of Stat5 signaling,” Mol. Oncol., vol. 6, no. 3, pp. 276–283, 2012, doi: 10.1016/j.molonc.2012.02.002.

[67] A. B. Richards et al., “Trehalose: A review of properties, history of use and human tolerance, and results of multiple safety studies,” Food Chem. Toxicol., vol. 40, no. 7, pp. 871–898, 2002, doi: 10.1016/S0278-6915(02)00011-X.

[68] H. Li et al., “Enhanced production of trehalose in Escherichia coli by homologous expression of otsBA in the presence of the trehalase inhibitor, validamycin A, at high osmolarity,” J. Biosci. Bioeng., vol. 113, no. 2, pp. 224–232, 2012, doi: 10.1016/j.jbiosc.2011.09.018.

[69] C. Schiraldi, I. Di Lernia, and M. De Rosa, “Trehalose production: Exploiting novel approaches,” Trends Biotechnol., vol. 20, no. 10, pp. 420–425, 2002, doi: 10.1016/S0167-7799(02)02041-3.

[70] R. Ruhal, R. Kataria, and B. Choudhury, “Trends in bacterial trehalose metabolism and significant nodes of metabolic pathway in the direction of trehalose accumulation,” Microb. Biotechnol., vol. 6, no. 5, pp. 493–502, 2013, doi: 10.1111/1751-7915.12029.

[71] G. Iturriaga, R. Suárez, and B. Nova-Franco, “Trehalose metabolism: From osmoprotection to signaling,” Int. J. Mol. Sci., vol. 10, no. 9, pp. 3793–3810, 2009, doi: 10.3390/ijms10093793.

[72] A. R. Strom and I. Kaasen, “Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression,” Mol. Microbiol., vol. 8, no. 2, pp. 205–210, 1993, doi: 10.1111/j.1365-2958.1993.tb01564.x.

[73] A. Patist and H. Zoerb, “Preservation mechanisms of trehalose in food and biosystems,” Colloids Surfaces B Biointerfaces, vol. 40, no. 2, pp. 107–113, 2005, doi: 10.1016/j.colsurfb.2004.05.003.

[74] R. J. Dutton, D. Boyd, M. Berkmen, and J. Beckwith, “Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation,” Proc. Natl. Acad. Sci. U. S. A., vol. 105, no. 33, pp. 11933–11938, 2008, doi: 10.1073/pnas.0804621105.

[75] D. Hatlem, T. Trunk, D. Linke, and J. C. Leo, “Catching a SPY: Using the SpyCatcher-SpyTag and related systems for labeling and localizing bacterial proteins,” Int. J. Mol. Sci., vol. 20, no. 9, 2019, doi: 10.3390/ijms20092129.

[76] H. J. Kang and E. N. Baker, “Intramolecular isopeptide bonds: Protein crosslinks built for stress?,” Trends Biochem. Sci., vol. 36, no. 4, pp. 229–237, 2011, doi: 10.1016/j.tibs.2010.09.007.

[77] G. Veggiani et al., “Programmable polyproteams built using twin peptide superglues,” Proc. Natl. Acad. Sci. U. S. A., vol. 113, no. 5, pp. 1202–1207, 2016, doi: 10.1073/pnas.1519214113.

[78] S. A. H. Heyde and M. H. H. Nørholm, “Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity,” Commun. Biol., vol. 4, no. 1, pp. 1–9, 2021, doi: 10.1038/s42003-021-02493-4.

[79] H. Jeong et al., “Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3),” J. Mol. Biol., vol. 394, no. 4, pp. 644–652, 2009, doi: 10.1016/j.jmb.2009.09.052.

[80] G. L. Rosano, E. S. Morales, and E. A. Ceccarelli, “New tools for recombinant protein production in Escherichia coli: A 5-year update,” Protein Sci., vol. 28, no. 8, pp. 1412–1422, 2019, doi: 10.1002/pro.3668.

[81] L. A. Burdette, S. A. Leach, H. T. Wong, and D. Tullman-Ercek, “Developing Gram-negative bacteria for the secretion of heterologous proteins,” Microb. Cell Fact., vol. 17, no. 1, pp. 1–16, 2018, doi: 10.1186/s12934-018-1041-5.

[82] G. Lozano Terol, J. Gallego-Jara, R. A. Sola Martínez, A. Martínez Vivancos, M. Cánovas Díaz, and T. de Diego Puente, “Impact of the Expression System on Recombinant Protein Production in Escherichia coli BL21,” Front. Microbiol., vol. 12, no. June, pp. 1–12, 2021, doi: 10.3389/fmicb.2021.682001.

[83] F. R. Blattner et al., “The complete genome sequence of Escherichia coli K-12,” Science (80-. )., vol. 277, no. 5331, pp. 1453–1462, 1997, doi: 10.1126/science.277.5331.1453.

[84] M. J. Lajoie et al., “Genomically Recoded Organisms Expand Biological Functions,” Science (80-. )., vol. 342, no. 6156, pp. 357–360, Oct. 2013, doi: 10.1126/science.1241459.

[85] K. Dryden, D. Lina, and N. Fabris, “Minicells from Highly Genome Reduced,” 2021.

[86] M. Kurokawa and B. W. Ying, “Experimental challenges for reduced genomes: The cell model escherichia coli,” Microorganisms, vol. 8, no. 1, 2020, doi: 10.3390/microorganisms8010003.

[87] C. Pál, B. Papp, and G. Pósfai, “The dawn of evolutionary genome engineering,” Nat. Rev. Genet., vol. 15, no. 7, pp. 504–512, 2014, doi: 10.1038/nrg3746.

[88] S. M. Chiang and H. E. Schellhorn, “Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria,” Arch. Biochem. Biophys., vol. 525, no. 2, pp. 161–169, 2012, doi: 10.1016/j.abb.2012.02.007.

[89] S. Babakhani and M. Oloomi, “Transposons: the agents of antibiotic resistance in bacteria,” J. Basic Microbiol., vol. 58, no. 11, pp. 905–917, 2018, doi: 10.1002/jobm.201800204.

[90] S. Müller, H. Harms, and T. Bley, “Origin and analysis of microbial population heterogeneity in bioprocesses,” Curr. Opin. Biotechnol., vol. 21, no. 1, pp. 100–113, 2010, doi: 10.1016/j.copbio.2010.01.002.

[91] P. Rugbjerg, N. Myling-Petersen, A. Porse, K. Sarup-Lytzen, and M. O. A. Sommer, “Diverse genetic error modes constrain large-scale bio-based production,” Nat. Commun., vol. 9, no. 1, 2018, doi: 10.1038/s41467-018-03232-w.

[92] S. J. Giovannoni et al., “Genetics: Genome streamlining in a cosmopolitan oceanic bacterium,” Science (80-. )., vol. 309, no. 5738, pp. 1242–1245, 2005, doi: 10.1126/science.1114057.

[93] D. J. Martínez-Cano et al., “Evolution of small prokaryotic genomes,” Front. Microbiol., vol. 6, no. JAN, pp. 1–23, 2015, doi: 10.3389/fmicb.2014.00742.

[94] M. Kafri, E. Metzl-Raz, G. Jona, and N. Barkai, “The Cost of Protein Production,” Cell Rep., vol. 14, no. 1, pp. 22–31, 2016, doi: 10.1016/j.celrep.2015.12.015.

[95] M. Juhas, D. R. Reuß, B. Zhu, and F. M. Commichau, “Bacillus subtilis and Escherichia coli essential genes and minimal cell factories after one decade of genome engineering,” Microbiol. (United Kingdom), vol. 160, pp. 2341–2351, 2014, doi: 10.1099/mic.0.079376-0.

[96] J. H. Lee et al., “Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production,” Microb. Cell Fact., vol. 8, no. 1, pp. 1–12, 2009, doi: 10.1186/1475-2859-8-2.

[97] M. Hashimoto et al., “Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome,” Mol. Microbiol., vol. 55, no. 1, pp. 137–149, 2005, doi: 10.1111/j.1365-2958.2004.04386.x.

[98] H. Yu et al., “Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells,” ACS Synth. Biol., vol. 10, no. 10, pp. 2465–2477, 2021, doi: 10.1021/acssynbio.1c00375.

[99] J. I. Kato and M. Hashimoto, “Construction of consecutive deletions of the Escherichia coli chromosome,” Mol. Syst. Biol., vol. 3, no. 132, pp. 1–7, 2007, doi: 10.1038/msb4100174.

[100] G. Pósfai, K. Umenhoffer, V. Kolisnychenko, B. Stahl, and S. S. Sharma, “Emergent Properties of,” vol. 1044, no. 2006, pp. 1044–1047, 2013.