Synthetic small regulatory RNA (sRNA) can efficiently downregulate target gene expression at translational level in metabolic engineering, but cannot be used in engineered strain already having incompatible plasmid(s). To address this problem and make the sRNA gene expression modulation platform universally applicable, we report the development and applications of expanded synthetic sRNA expression platforms for rapid, multiplexed and genome-scale target gene knockdown in engineered Escherichia coll. As proof-of-concept, high performance strains capable of producing L-proline (54.1 g l(-1)) and L-threonine (22.9 g l(-1)) are rapidly developed by combinatorial knockdown of up to three genes via one-step co-transformation of sRNA expression vectors. Furthermore, a genome-scale sRNA library targeting 1,858 E. coli genes is employed to construct crude violacein (5.19 g l(-1)) and indigo (135 mg l(-1)) producers by high-throughput colorimetric screening. These examples demonstrate that the expanded sRNA expression vectors developed here enables rapid development of chemical overproducers regardless of plasmid compatibility.