Establishment of a Visible Reporter System in <i>Zymomonas mobilis</i> through Random Mutagenesis and Rational Design of a Chromoprotein eforRed

The lack of reporter-gene systems that can measure the activities of biological parts quantitatively and qualitatively impedes the development of robust cell factories to meet the needs of fast-growing biomanufacturing. Chromoproteins are homologues of fluorescent proteins, which are good reporter-gene candidates for their special absorption of natural light, making them visible to the naked eye without additional instrumentation. In this study, a fluorescent chromoprotein eforRed was selected to establish the visible reporter system in <i>Zymomonas mobilis</i>, a non-model ethanologenic Gram-negative bacterium with many excellent industrial properties to be developed as a biorefinery chassis for lignocellulosic biochemical production. Coupled with random error-prone PCR and protein rational design, the spectral characteristics of the eforRed chromoprotein were enhanced, particularly the fluorescence intensity. Mechanistic studies revealed that substitutions at the amino acid residues K201 and T24 situated on the surface region of eforRed, especially the double-mutant K201H-T24V, could optimize the protein optical characteristics by concurrently increasing intrinsic fluorescence and elevating protein expression levels. Finally, a visible reporter-gene system was successfully established in <i>Z. mobilis</i> by expressing the mutant eforRed^K201H-T24V under a strong promoter like P<i>gap</i>-4S or P<i>T7</i> that gave colonies a visible red color for phenotypic screening and retained high fluorescence intensity for quantitative analysis. This study thus not only constructed a robust visible reporter-gene system in the non-model bacterium <i>Z. mobilis</i> but also helped expand the applications of chromoproteins in biotechnology and synthetic biology, especially in non-model microorganisms.