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Itaconic acid (IA), an important five-carbon unsaturated dicarboxylic acid, is one of the top 12 renewable chemicals with an urgent need to reduce industrial production costs. Halomonas bluephagenesis, which possesses the potential for cost-effective bioproduction of chemicals and organic acids due to its ability to grow under open nonsterile conditions and high tolerance to organic acid salts, was genetically engineered and used to produce IA from citrate by a cell catalytic strategy. Here, two essential genes ( cis -aconitate decarboxylase encoding gene cadA and aconitase (ACN) encoding gene acn ) were introduced into H. bluephagenesis to construct an IA biosynthesis pathway. Further engineering modifications including coexpression of molecular chaperones GroESL, increasing the copy number of the gene encoding rate-limiting enzyme ACN, and weakening the competing pathway were implemented. Under the optimized condition for the cell catalytic system, the engineered strain TAZI-08 produced 451.45 mM (58.73 g/L) IA from 500 mM citrate, with 93.24% conversion in 36 h and a productivity of 1.63 g/(L h). An intermittent feeding strategy further increased the IA titer to 488.86 mM (63.60 g/L). The IA titer and citrate conversion in H. bluephagenesis are the highest among heterologous hosts reported so far, demonstrating that this strain is a suitable chassis for hyperproduction of IA.

Adaptive laboratory evolution (ALE) is a widely used and highly effective tool for improving microbial phenotypes and investigating the evolutionary roots of biological phenomena. Serving as the raw materials of evolution, mutations have been extensively utilized to increase the chances of engineering molecules or microbes with tailor-made functions. The generation of genetic diversity is therefore a core technology for accelerating ALE, and a high-quality mutant library is crucial to its success. Because of its importance, technologies for generating genetic diversity have undergone rapid development in recent years. Here, we review the existing techniques for the construction of mutant libraries, briefly introduce their mechanisms and applications, discuss ongoing and emerging efforts to apply engineering technologies in the construction of mutant libraries, and suggest future perspectives for library construction.