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The in vitro biosynthesis of high-value compounds has become popular and attractive. The convenient and simple strategy of enzyme immobilization has been significant for continuous and efficient in vitro biosynthesis. On the basis of that, this work established a one-step self-assembly-based immobilization strategy to efficiently biosynthesize isobutyraldehyde in vitro. Isobutyraldehyde is a crucial precursor for the synthesis of foods and spices. The established CipA scaffold-based strategy can express and immobilize enzymes at the same time, and purification requires only one centrifugation step. Structural simulations indicated that this scaffold-dependent self-assembly did not influence the structure or catalytic mechanisms of the isobutyraldehyde production-related enzymes leucine dehydrogenase (LeuDH) and ketoisovalerate decarboxylase (Kivd). Immobilized LeuDH and Kivd displayed a higher conversion capacity and thermal stability than the free enzymes. Batch conversion experiments demonstrated that the recovered immobilized LeuDH and Kivd have similar conversion capacities to the enzymes used in the first round of reaction. The continuous production of isobutyraldehyde was achieved by filling the immobilized enzymes into the column of a constructed device. This study not only expands the application range of self-assembly systems but also provides guidance for the in vitro production of value-added compounds.

Background Branched chain amino acids (BCAAs) are widely applied in the food, pharmaceutical, and animal feed industries. Traditional chemical synthetic and enzymatic BCAAs production in vitro has been hampered by expensive raw materials, harsh reaction conditions, and environmental pollution. Microbial metabolic engineering has attracted considerable attention as an alternative method for BCAAs biosynthesis because it is environmentally friendly and delivers high yield. Main text Corynebacterium glutamicum (C. glutamicum) possesses clear genetic background and mature gene manipulation toolbox, and has been utilized as industrial host for producing BCAAs. Acetohydroxy acid synthase (AHAS) is a crucial enzyme in the BCAAs biosynthetic pathway of C. glutamicum, but feedback inhibition is a disadvantage. We therefore reviewed AHAS modifications that relieve feedback inhibition and then investigated the importance of AHAS modifications in regulating production ratios of three BCAAs. We have comprehensively summarized and discussed metabolic engineering strategies to promote BCAAs synthesis in C. glutamicum and offer solutions to the barriers associated with BCAAs biosynthesis. We also considered the future applications of strains that could produce abundant amounts of BCAAs. Conclusions Branched chain amino acids have been synthesized by engineering the metabolism of C. glutamicum. Future investigations should focus on the feedback inhibition and/or transcription attenuation mechanisms of crucial enzymes. Enzymes with substrate specificity should be developed and applied to the production of individual BCAAs. The strategies used to construct strains producing BCAAs provide guidance for the biosynthesis of other high value-added compounds.