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Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus.

Weyman PD, Vargas WA, Tong Y, Yu J, Maness PC, Smith HO, Xu Q - PLoS ONE (2011)

Bottom Line: Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus.A protein of the correct size was expressed but had no activity.This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, Maryland, United States of America.

ABSTRACT
Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.

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Heterologous expression of AltDE hydrogenase HynSL in S. elongatus.A. Western blotting of protein extracts from S. elongatus cell cultures. Each lane contains 25 µg total proteins from cells treated with or without IPTG. The arrow indicates the position of the HynL band. B. Coomassie blue staining of a duplicate protein gel as in Panel A showing equal loading in each lane. C. Determination of the hydrogenase activity of the heterologously expressed HynSL. In vitro hydrogen evolution assays were performed on protein samples described in Panel A. Two controls, 7942-IPTG and PW416-IPTG, were not included since their activity levels are same as those from 7942+IPTG and PW416+IPTG, respectively. Error bars indicate standard deviations of the mean from at least three samples. The experiment was repeated at least three times with similar results.
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pone-0020126-g002: Heterologous expression of AltDE hydrogenase HynSL in S. elongatus.A. Western blotting of protein extracts from S. elongatus cell cultures. Each lane contains 25 µg total proteins from cells treated with or without IPTG. The arrow indicates the position of the HynL band. B. Coomassie blue staining of a duplicate protein gel as in Panel A showing equal loading in each lane. C. Determination of the hydrogenase activity of the heterologously expressed HynSL. In vitro hydrogen evolution assays were performed on protein samples described in Panel A. Two controls, 7942-IPTG and PW416-IPTG, were not included since their activity levels are same as those from 7942+IPTG and PW416+IPTG, respectively. Error bars indicate standard deviations of the mean from at least three samples. The experiment was repeated at least three times with similar results.

Mentions: To examine the expression of HynSL, RC41 cultures expressing the AltDE hydrogenase gene operon were grown and induced with IPTG, and protein extracts from lysed cells were separated on an SDS-PAGE gel for immunoblotting. Western blotting was performed with antiserum raised against the T. roseopersicina [NiFe] hydrogenase large subunit, HynL. When induced with IPTG, a single band corresponding to the mature form of the AltDE HynL (67 kDa) was detected (Fig. 2A). Without IPTG induction, no HynL band was detected. As a control, a duplicate gel was stained with Coomassie blue to confirm equal loading in each lane (Fig. 2B). IPTG was added to a final concentration of 5, 20, 100, or 200 µM to RC41 cultures to determine the optimal concentration of IPTG for protein expression. Western blotting analysis indicated that 100 µM IPTG yielded maximal expression of HynL (data not shown) and this concentration of IPTG was used for all future experiments.


Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus.

Weyman PD, Vargas WA, Tong Y, Yu J, Maness PC, Smith HO, Xu Q - PLoS ONE (2011)

Heterologous expression of AltDE hydrogenase HynSL in S. elongatus.A. Western blotting of protein extracts from S. elongatus cell cultures. Each lane contains 25 µg total proteins from cells treated with or without IPTG. The arrow indicates the position of the HynL band. B. Coomassie blue staining of a duplicate protein gel as in Panel A showing equal loading in each lane. C. Determination of the hydrogenase activity of the heterologously expressed HynSL. In vitro hydrogen evolution assays were performed on protein samples described in Panel A. Two controls, 7942-IPTG and PW416-IPTG, were not included since their activity levels are same as those from 7942+IPTG and PW416+IPTG, respectively. Error bars indicate standard deviations of the mean from at least three samples. The experiment was repeated at least three times with similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102683&req=5

pone-0020126-g002: Heterologous expression of AltDE hydrogenase HynSL in S. elongatus.A. Western blotting of protein extracts from S. elongatus cell cultures. Each lane contains 25 µg total proteins from cells treated with or without IPTG. The arrow indicates the position of the HynL band. B. Coomassie blue staining of a duplicate protein gel as in Panel A showing equal loading in each lane. C. Determination of the hydrogenase activity of the heterologously expressed HynSL. In vitro hydrogen evolution assays were performed on protein samples described in Panel A. Two controls, 7942-IPTG and PW416-IPTG, were not included since their activity levels are same as those from 7942+IPTG and PW416+IPTG, respectively. Error bars indicate standard deviations of the mean from at least three samples. The experiment was repeated at least three times with similar results.
Mentions: To examine the expression of HynSL, RC41 cultures expressing the AltDE hydrogenase gene operon were grown and induced with IPTG, and protein extracts from lysed cells were separated on an SDS-PAGE gel for immunoblotting. Western blotting was performed with antiserum raised against the T. roseopersicina [NiFe] hydrogenase large subunit, HynL. When induced with IPTG, a single band corresponding to the mature form of the AltDE HynL (67 kDa) was detected (Fig. 2A). Without IPTG induction, no HynL band was detected. As a control, a duplicate gel was stained with Coomassie blue to confirm equal loading in each lane (Fig. 2B). IPTG was added to a final concentration of 5, 20, 100, or 200 µM to RC41 cultures to determine the optimal concentration of IPTG for protein expression. Western blotting analysis indicated that 100 µM IPTG yielded maximal expression of HynL (data not shown) and this concentration of IPTG was used for all future experiments.

Bottom Line: Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus.A protein of the correct size was expressed but had no activity.This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Synthetic Biology and Bioenergy, The J. Craig Venter Institute, Rockville, Maryland, United States of America.

ABSTRACT
Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.

Show MeSH