Colored Proteins Act as Biocolorants in Escherichia coli

Colored proteins play an important role in synthetic biology research, providing a systematic labeling tool for visualizing microscopic biological activities in vivo. They can exhibit visible colors to the naked eye under natural light, and some of them are well-known fluorescent proteins. Here, several colored proteins were taken into consideration for acting as biocolorants in Escherichia coli, including green fluorescent proteins (eGFP and sfGFP), a red fluorescent protein (mKate2), and three chromoproteins (GfasPurple, AmilCP, and AeBlue). All of them can significantly change the colors of their bacterial colonies. The color of GfasPurple was much more stable after the heat treatments at 65 °C with 75% or 95% ethanol. In addition, several factors commonly occurring under natural conditions that lead to color dissolution, such as heat, ethanol, H₂O₂, vitamin C, acid, and alkali treatments, were further tested on GfasPurple. Visual observation and absorption spectroscopy analysis results showed an excellent tolerance of GfasPurple against these unfriendly conditions. GfasPurple could withstand temperatures of 65 °C for 2 h or 70 °C for 1 h in aqueous solutions, but it fades rapidly in 50% ethanol. The color of GfasPurple is more stable in 80% ethanol than in 50% ethanol, which could be attributed to its poor solubility in high-concentration ethanol. The visible light absorption curves of GfasPurple were basically not affected by physiological concentrations of vitamin C or H₂O₂, but reversible effects of high-concentration H₂O₂ were found. GfasPurple maintains its color within the pH range of 7-11; the chromophore of GfasPurple will suffer irreversible damage when pH is up to thirteen or as low as three. These results suggest that GfasPurple is an excellent biocolorant far beyond its application in prokaryotes. Furthermore, GfasPurple variants created via mutagenesis expanded the color library of chromoproteins, which have a potential value in the color manipulation of living organisms.