Highly toxic and flammable H2S gas has become an environmental threat. Because of its ability to efficiently remove H2S by oxidation, Thioalkalivibrio versutus is gaining more attention. Haloalkaliphilic autotrophs, like the bio-desulfurizing T. versutus, grow weakly. Weak growth makes any trial for developing potent genetic tools required for genetic engineering far from achieved. In this study, the fed-batch strategy improved T. versutus growth by 1.6 fold in maximal growth rate, 9-fold in 0.D-600 values and about 3-fold in biomass and protein productions. The strategy also increased the favorable desulfurization product, sulfur, by 2.7 fold in percent yield and 1.5-fold in diameter. A tight iron-inducible expression system for T. versutus was successfully developed. The system was derived from fed-batch cultivation coupled with new design, build, test and validate (DPTV) approach. The inducible system was validated by toxin expression. Fed-batch cultivation coupled with DPTV approach could be applied to other autotrophs.