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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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Detection of the T. roseopersicina hydrogenase large subunit HynL in S. elongatus.Western blotting was performed on protein extracts from S. elongatus strains (wild-type PCC7942, Hyn4, Hyn5, Hyn5/Hup, and Hyn5/Hoxhup) by using anti-HynL antisera. Hyn4/Hup was not included since the expression level in this strain was similar to that in Hyn4. Each lane contains 25 µg total proteins from cells treated with or without IPTG. Strain Hyn4 carries the T. roseopersicina genes hynSL, hynD, hupK, and hypC1C2DEF while strain Hyn5 carries the genes from Hyn4 as well as isp1 and isp2. Strain Hyn5/Hup contains the T. roseopersicina genes hupCDHIorf and the genes from Hyn5. Strain Hyn5/Hoxhup contains the T. roseopersicina genes hoxEFUYH in addition to the genes from Hyn5/Hup.
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pone-0020126-g004: Detection of the T. roseopersicina hydrogenase large subunit HynL in S. elongatus.Western blotting was performed on protein extracts from S. elongatus strains (wild-type PCC7942, Hyn4, Hyn5, Hyn5/Hup, and Hyn5/Hoxhup) by using anti-HynL antisera. Hyn4/Hup was not included since the expression level in this strain was similar to that in Hyn4. Each lane contains 25 µg total proteins from cells treated with or without IPTG. Strain Hyn4 carries the T. roseopersicina genes hynSL, hynD, hupK, and hypC1C2DEF while strain Hyn5 carries the genes from Hyn4 as well as isp1 and isp2. Strain Hyn5/Hup contains the T. roseopersicina genes hupCDHIorf and the genes from Hyn5. Strain Hyn5/Hoxhup contains the T. roseopersicina genes hoxEFUYH in addition to the genes from Hyn5/Hup.

Mentions: S. elongatus cell cultures expressing different combinations of T. roseopersicina structural and accessory genes were induced with IPTG, and protein extracts prepared from lysed cells were analyzed by immunoblot after SDS-PAGE electrophoresis using anti-HynL antisera. A band corresponding to the correct size of the mature form of HynL was detected (62 kDa), and increased expression was observed in the presence of IPTG (Fig. 4). Similar levels of HynL expression were observed for all strains that contained HynSL (Fig. 4). In vitro hydrogen evolution assays were performed to determine whether the expressed hydrogenase possessed activity. No activity was detected in strains Hyn4, Hyn5, Hyn4/Hup or Hyn5/Hup (data not shown).


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)

Detection of the T. roseopersicina hydrogenase large subunit HynL in S. elongatus.Western blotting was performed on protein extracts from S. elongatus strains (wild-type PCC7942, Hyn4, Hyn5, Hyn5/Hup, and Hyn5/Hoxhup) by using anti-HynL antisera. Hyn4/Hup was not included since the expression level in this strain was similar to that in Hyn4. Each lane contains 25 µg total proteins from cells treated with or without IPTG. Strain Hyn4 carries the T. roseopersicina genes hynSL, hynD, hupK, and hypC1C2DEF while strain Hyn5 carries the genes from Hyn4 as well as isp1 and isp2. Strain Hyn5/Hup contains the T. roseopersicina genes hupCDHIorf and the genes from Hyn5. Strain Hyn5/Hoxhup contains the T. roseopersicina genes hoxEFUYH in addition to the genes from Hyn5/Hup.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102683&req=5

pone-0020126-g004: Detection of the T. roseopersicina hydrogenase large subunit HynL in S. elongatus.Western blotting was performed on protein extracts from S. elongatus strains (wild-type PCC7942, Hyn4, Hyn5, Hyn5/Hup, and Hyn5/Hoxhup) by using anti-HynL antisera. Hyn4/Hup was not included since the expression level in this strain was similar to that in Hyn4. Each lane contains 25 µg total proteins from cells treated with or without IPTG. Strain Hyn4 carries the T. roseopersicina genes hynSL, hynD, hupK, and hypC1C2DEF while strain Hyn5 carries the genes from Hyn4 as well as isp1 and isp2. Strain Hyn5/Hup contains the T. roseopersicina genes hupCDHIorf and the genes from Hyn5. Strain Hyn5/Hoxhup contains the T. roseopersicina genes hoxEFUYH in addition to the genes from Hyn5/Hup.
Mentions: S. elongatus cell cultures expressing different combinations of T. roseopersicina structural and accessory genes were induced with IPTG, and protein extracts prepared from lysed cells were analyzed by immunoblot after SDS-PAGE electrophoresis using anti-HynL antisera. A band corresponding to the correct size of the mature form of HynL was detected (62 kDa), and increased expression was observed in the presence of IPTG (Fig. 4). Similar levels of HynL expression were observed for all strains that contained HynSL (Fig. 4). In vitro hydrogen evolution assays were performed to determine whether the expressed hydrogenase possessed activity. No activity was detected in strains Hyn4, Hyn5, Hyn4/Hup or Hyn5/Hup (data not shown).

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
Related in: MedlinePlus