Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli

Abstract

HynSL from Alteromonas macleodii 'deep ecotype' (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (hynSL), encoding small and large hydrogenase subunits, are surrounded by eight genes (hynD, hupH and hypCABDFE ) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (orf2, cyt and orf1) was cloned into an IPTG-inducible expression vector and transferred into an Escherichia coli mutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed in E. coli and was active, as determined by an in vitro hydrogen evolution assay. Subsequent genetic analysis revealed that orf2, cyt, orf1 and hupH are not essential for assembling an active hydrogenase. However, hupH and orf2 can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and its Thiocapsa roseopersicina homologue. When the structural genes for the T. roseopersicina hydrogenase, hynSL, were expressed along with known T. roseopersicina accessory genes (hynD, hupK, hypC1C2 and hypDEF ), no active hydrogenase was produced. Further, co-expression of AltDE accessory genes hypA and hypB with the entire set of the T. roseopersicina genes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with the T. roseopersicina structural genes generated an active T. roseopersicina hydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts from T. roseopersicina and that the two hydrogenases share similar maturation mechanisms.

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

“…Heterologous expression of the A. macleodii hydrogenase has been previously reported in both E. coli [15] and the cyanobacterium S. elongatus [16]. These reports address general issues of aerobic expression of the A. macleodii hydrogenase, as in both these reports, aerobic handling was used throughout, established as safe in the original report on the enzyme [14].…”

“…Finally, because A. macleodii hydrogenase can be expressed as active enzyme in both E. coli [15] and in the photoautotrophic cyanobacterium S. elongatus [16], our strategy moving forward is to use expression in E. coli as a preliminary model system to rapidly generate and test promising modifications of the enzyme prior to experimentation in S. elongatus , which has a longer turnaround time for genetic manipulations. The H230C/P285C enzyme demonstrated the same qualitative performance over the wild type enzyme in extracts prepared from both S. elongatus and E. coli , validating this strategy.…”

“…A closely-related sequence was later identified in the Alteromonas macleodii ‘ Deep ecotype’ genome (hereafter referred to as the A. macleodii hydrogenase), and was characterized as active in the presence of 2% oxygen [14]. The DNA encoding the enzyme was then cloned and used to express active enzyme in both Escherichia coli [15] and S. elongatus [16]. Expressing active A. macleodii hydrogenase in both E. coli and cyanobacteria requires the co-expression of at least nine additional maturation factors to properly assembly an active enzyme [17].…”

Cyanobacterial Hydrogenases and Hydrogen Metabolism Revisited: Recent Progress and Future Prospects

“…In accordance, the authors cloned a 13 kb fragment including the A. macleodii structural genes ( hynSL ) and 11 adjacent hypothetical accessory genes in an IPTG-inducible expression vector. The vector was transformed into an Escherichia coli ( E. coli ) mutant strain lacking its native hydrogenases [100]. Upon induction, HynSL from A. macleodii expressed in E. coli and was active, as determined by the in vitro hydrogen evolution assays.…”

“…Upon induction, HynSL from A. macleodii expressed in E. coli and was active, as determined by the in vitro hydrogen evolution assays. The HynSL from A. macleodii shares about 60% identity to the HynSL from T. roseopersicina [100]. The authors also showed successful complementation of the T. roseopersicina structural gene with the accessory gene cluster from A. macleodii .…”