Proteomic Analysis and Promoter Modification of Bacillus thuringiensis to Improve Insecticidal Vip3A Protein Production

Abstract

Insecticidal protein Vip3A secreted from B. thuringiensis is a potential biocontrol agent for control of lepidopteran pests. Under laboratory conditions, high albeit variable Vip3A production from the local isolate Bt294 was only obtained from a much enriched TB culture medium. Proteomic analysis and strain improvement were therefore performed to improve Vip3A production. Studies indicated that the buffer capacity, carbon source, and nitrogen source are critical to efficiently produce Vip3A. Medium with lower amounts of peptone and yeast extract (compared to TB), with an additional carbon source and phosphate buffer (LB*G medium) was found to give reasonable yields of Vip3A. Proteomic analysis revealed higher expression of proteins involved in glutamate and histidine biosynthesis in cells cultured in TB compared to LB about 58 and 33 times, respectively. Experiments confirmed that glutamate supplementation could increase Vip3A production. In addition, promoter substitution with that of cry3A increased Vip3A yields by about 20-30%. Overall, very high yields of Vip3A could be obtained by culturing Bt294 (P_cry3A-vip3Aa64) in LB*G medium with glutamate supplementation.

Keywords: Bacillus thuringiensis; Biocontrol agent; Protein production; Proteomic analysis; Vip3A.