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Abstract Background Microbial mutagenesis is an important avenue to acquire microbial strains with desirable traits for industry application. However, mutagens either chemical or physical used often leads narrow library pool due to high lethal rate. The T4 DNA ligase is one of the most widely utilized enzymes in modern molecular biology. Its contribution to repair chromosomal DNA damages, therefore cell survival during mutagenesis will be discussed. Results Expression of T4 DNA ligase in vivo could substantially increase cell survival to ionizing radiation in multiple species. A T4 mediated survival-coupled mutagenesis approach was proposed. When polyhydroxybutyrate (PHB)-producing E. coli with T4 DNA ligase expressed in vivo was subjected to ionizing radiation, mutants with improved PHB production were acquired quickly owing to a large viable mutant library generated. Draft genome sequence analysis showed that the mutants obtained possess not only single nucleotide variation (SNV) but also DNA fragment deletion, indicating that T4 DNA ligase in vivo may contribute to the repair of DNA double strand breaks. Conclusions Expression of T4 DNA ligase in vivo could notably enhance microbial survival to excess chromosomal damages caused by various mutagens. Potential application of T4 DNA ligase in microbial mutagenesis was explored by mutating and screening PHB producing E. coli XLPHB strain. When applied to atmospheric and room temperature plasma (ARTP) microbial mutagenesis, large survival pool was obtained. Mutants available for subsequent screening for desirable features. The use of T4 DNA ligase we were able to quickly improve the PHB production by generating a larger viable mutants pool. This method is a universal strategy can be employed in wide range of bacteria. It indicated that traditional random mutagenesis became more powerful in combine with modern genetic molecular biology and has exciting prospect.