Comparisons between the principle of “Mutagenesis followed-by Selection” and the principle of “Mutagenesis coupled-with Selection”. (A) Traditional “Mutagenesis followed-by Selection” principle was usually performed by iterative rounds of mutagenesis and selection. Exogenous mutagens or genetic manipulations were required for mutagenesis and the following selection manipulations isolated cells with improved phenotypes, which could be used in next rounds of “mutagenesis-selection”. (B) As for “Mutagenesis coupled-with Selection” principle, the two steps are synchronized, so that iterative and lengthy manual interventions are greatly simplified, leading to a continuous and efficient strain improvement process. “S” represents for “selection”, “M” represents for “mutagenesis”, and red triangles represent for manual interventions such as mutagen treatments or genetic manipulations or selections for improvement phenotypes.