Enhanced production of bio-indigo in engineered Escherichia coli, reinforced by cyclopropane-fatty acid-acyl-phospholipid synthase from psychrophilic Pseudomonas sp. B14-6

Indigo has been one of the most widely used dyes in the food and textile industry for a long time and it is naturally extracted from the leaves of few plants, such as Persicaria tinctoria and Indigofera sp. (Berry et al., 2002, Erkan et al., 2014). By the end of the 19th century, it changed from traditionally obtaining indigo from plants to chemically synthesizing indigo (Bechtold et al., 2002, Namgung et al., 2019). Compared to growing plants, which take a long time and are heavily affected by climate, chemical synthesis of indigo is economically advantageous (Kim et al., 2017). However, chemical synthesis can cause environmental pollution and adversely affect health owing to the use of toxic substrates and catalysts (Pathak and Madamwar, 2010, Qu et al., 2012b).

Therefore, microbial production of indigo (bio-indigo) has been studied as a method for effectively producing indigo as an eco-friendly alternative. Various studies have reported the production of indigo in microorganisms, such as Pseudomonas, Acinetobacter, Comamonas, and recombinant Escherichia coli (Table 1). Researches have shown that single- and multi-component oxygenases including naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, styrene monooxygenase, P450 monooxygenase, and flavin-containing monooxygenase (FMO) are required to produce bio-indigo (Berry et al., 2002, Bhushan et al., 2000, Cheng et al., 2016, Mercadal et al., 2010, Murdock et al., 1993, Qu et al., 2012a, Qu et al., 2010, Qu et al., 2013, Woo et al., 2000).

FMOs catalyze NADPH-dependent N- or S-oxygenation of heteroatom-containing compounds (Chen et al., 2011, Ziegler, 1993). FMOs are distributed in all mammals, yeast, plants, and bacteria and perform various functions (Chen et al., 2011). Some bacterial and plant FMOs produce indigo by converting indole to indoxyl (Ameria et al., 2015, Choi et al., 2003, Inoue et al., 2021). Among them, a FMO originated from Methylophaga aminisulfidivorans MPT (maFMO) is well-known, and E. coli, which contains maFMO, produces 920 mg/L of bio-indigo from 2 g/L L-tryptophan (Han et al., 2008).

Numerous studies have been conducted to produce bio-indigo using indole, the precursor of indigo, as the substrate. However, indole is cytotoxic and cannot be used as a substrate in large amounts (Chant and Summers, 2007). Therefore, to produce a large amount of indigo, L-tryptophan must be used as a substrate (Han et al., 2011, Han et al., 2008). Tryptophan is converted to indole by an enzyme called tryptophanase (TnaA). As explained earlier, indole is converted to indoxyl by oxygenase, and indoxyl is oxidized and dimerized to form indigo without any enzyme reaction.

Microorganisms establish their own defense systems to survive in harsh environments such as pH, salinity, and temperature. The cell membrane is the first line directly attached to the external environment. Phospholipid fatty acids (PLFAs) are major components of the cell membrane. PLFA analysis is used to determine the composition of microorganisms in microbial communities in soil or water environments because it provides accurate phospholipid information (Bligh and Dyer, 1959). An enzyme called cyclopropane-fatty acid-acyl-phospholipid synthase (Cfa) regulates the levels of cyclopropane fatty acids (CFAs) in the microbial cell membrane (Choi et al., 2020b, Cronan et al., 1979, Garba et al., 2016). Overexpression of cfa in microorganisms increases the proportion of CFA in the cell membrane and decreases the proportion of unsaturated fatty acids, resulting in changes that increase the fluidity and permeability of the cell membrane (Loffhagen et al., 2007, Smittle et al., 1974). This increases resistance to temperature change, osmotic pressure, high pressure, and growth inhibitors.

In this study, Cfa, which originated from Pseudomonas sp. B14-6 was introduced into an indigo-producing system expressing FMO derived from Methylophaga aminisulfidivorans and TnaA derived from Escherichia coli K12. We determined the cytotoxicity of indole as a substrate or intermediate product in indigo production and how the expression of Cfa affects indigo production. Finally, several culture conditions that affect indigo production were optimized to increase indigo production. These experiments not only increased indigo production by using engineered E. coli as a host, but also revealed the correlation between indigo production and phospholipid composition in cell membranes.

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