In this research, metabolic engineering was employed to synthesize the artificial major ampullate spidroin 2 (MaSp2) in the engineered Escherichia coli. An iterative seamless splicing strategy was used to assemble the MaSp2 gene, which could reach 10000 base pairs, and more than 100 kDa protein was expected. However, only 55 kDa recombinant MaSp2 was obtained. Because MaSp2 is rich in alanine and glycine residues, Glycyl/alanyl-tRNA pool and extra amino acids adding were adopted in order to supplement alanine and glycine in the protein translation process. With the supplementary alanine and glycine (0.05 wt%) in the medium, MaSp2 constructed in pET28a(+) and Gly/Ala-tRNA constructed in pET22b(+) were co-expressed in Escherichia coli BL21 (DE3). As results, the artificial MaSp2 with 110 kDa molecular weight was obtained in the present work. This work demonstrates a successful example of applying metabolic engineering approaches and provided a potential way with the enhanced Glycyl/alanyl-tRNA pool to achieve the expression of high molecular weight protein with the repeated motifs in the engineered Escherichia coli.