Industrial biotechnology has become a key technology for the sustainable economics, playing important roles for the production of fuels, pharmaceuticals, and materials. Engineering strains with high yield, productivity and robustness is essential for the success of modem industrial biotechnology. The complexity of cellular metabolic network and various regulations from atomic level to systems level make strain development a highly complicated work. Metabolic engineering has shown some success in optimizing strain performance by the ways of gene deletion, overexpression or substitution. However, it is not efficient in removing the inherent limitations of strain performance which are based on molecular level, such as the substrate/product inhibition, enzyme specificity or stereoselectivity.In this work, we have developed new tools of protein design and apply these tools in metabolic engineering to overcome the depicted limitations for the development of amino acid producer. More specifically, this work provided efficient solutions to overcome the key obstacles towards lysine production with regard to: (i) feedback inhibition of enzymes in terminal pathways; (ii) metabolic control of anaplerotic pathways for precursor supply, (iii) NADPH generation, and (iv) byproduct synthesis. All of these obstacles can be solved by structure-based rational protein design, providing a fast and efficient way to develop new strains.Based on minimal point mutations, the procedure demonstrated in this work avoided the drawbacks of traditional ways of strain development. The promising results demonstrated the high potential of protein design for the construction of minimally mutated industrial strain.