A typical expression plasmid, pRK415, was widely used to introduce cloned DNA fragment into a broad range of Gram-negative bacteria, including Rhodobacter sphaeroides. Tetracy-cline (Tc) is required for stable inheritance of the plasmid, but it is subject to photooxidation, inhibitory to the growth of the host strain. However, in this study, the introduction of pRK415 into R. sphaeroides HY01 under tetracycline stress showed clear enhancement on H-2 production performance of the recombinant strain pRK415/HY01. The maximum H-2 production rate of pRK415/HY01 was enhanced by 10%-70% compared with the control group in the absent of Tc. Supplementing with Tc, the wild type strain HY01 showed repressed cell growth and reduced H-2 production performance. The tetAR genes knockout test demonstrated that the expression of tetAR genes on pRK415 promoted the H-2 production performance. And within tolerance concentration (Tc < 2.5 mg/L), higher Tc concentration led to higher H-2 production performance of pRK415/HY01. Expression of tetAR genes in the genome of R.sphaeroides HY01 by substituting partial of hupSL genes enhanced the H-2 production performance as well. The mechanism of the H-2 production performance enhancement was discussed. (C) 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.