Microbial cells have been widely used in the industry to obtain various biochemical products, and evolutionary engineering is a common method in biological research to improve their traits, such as high environmental tolerance and improvement of product yield. To obtain better integrate functions of microbial cells, evolutionary engineering combined with other biotechnologies have attracted more attention in recent years. Classical laboratory evolution has been proven effective to letting more beneficial mutations occur in different genes but also has some inherent limitations such as a long evolutionary period and uncontrolled mutation frequencies. However, recent studies showed that some new strategies may gradually overcome these limitations. In this review, we summarize the evolutionary strategies commonly used in industrial microorganisms and discuss the combination of evolutionary engineering with other biotechnologies such as systems biology and inverse metabolic engineering. Finally, we prospect the importance and application prospect of evolutionary engineering as a powerful tool especially in optimization of industrial microbial cell factories.

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.

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Keratinase is an important industrial enzyme, but its application performance is limited by its low activity. A rational design of 5′-UTRs that increases translation efficiency is an important approach to enhance protein expression. Herein, we optimized the 5′-UTR of the recombinant keratinase KerZ1 expression element to enhance its secretory activity in Bacillus subtilis WB600 through Spacer design, RBS screening, and sequence simplification. First, the A/U content in Spacer was increased by the site-directed saturation mutation of G/C bases, and the activity of keratinase secreted by mutant strain B. subtilis WB600-SP was 7.94 times higher than that of KerZ1. Subsequently, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R was further increased to 13.45 times that of KerZ1 based on the prediction of RBS translation efficiency and the multi-site saturation mutation screening. Finally, the keratinase activity secreted by the mutant strain B. subtilis WB600-SP-R-D reached 204.44 KU mL−1 by reducing the length of the 5′ end of the 5′-UTR, which was 19.70 times that of KerZ1. In a 5 L fermenter, the keratinase activity secreted by B. subtilis WB600-SP-R-D after 25 h fermentation was 797.05 KU mL−1, which indicated its high production intensity. Overall, the strategy of this study and the obtained keratinase mutants will provide a good reference for the expression regulation of keratinase and other industrial enzymes.

The unique role of keratinases in keratin hydrolysis has garnered huge interest in the recovery of feather waste. However, owing to the high hydrophobicity of feather keratins, the catalytic capacity of keratinases for hydrolyzing feathers is typically low. In this study, we aimed to improve the keratinase feather hydrolysis efficiency by fusing a substrate-binding domain into the enzyme. We screened several carbohydrate-binding modules (CBMs) and linking peptides. We selected the most promising candidates to construct, clone, and express a fusion keratinase enzyme KerZ1/CBM-L8 with a feather hydrolysis efficiency of 7.8 × 10–8 g/U. Compared with those of KerZ1, KerZ1/CBM-L8 has a feather hydrolysis efficiency that is 2.71 times higher, a k cat value that is 179% higher, which translates to higher catalytic efficiency, and K m and binding constant (K) values that are lower, which indicate a higher KerZ1/CBM-L8–keratin binding affinity. Moreover, the number of binding sites to the substrate (N), determined using isothermal titration calorimetry, was 24.1 times higher than that of KerZ1. Thus, the fusion of the substrate-binding domain improved the binding ability of the keratinase enzyme to the hydrophobic substrate, which improved its feather hydrolysis efficiency. Therefore, using the fusion keratinase would significantly improve the recovery of feather waste.