Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in Thiocapsa roseopersicina

被引:24
作者
Maroti, Gergely [1 ]
Tong, Yingkai [1 ]
Yooseph, Shibu [1 ]
Baden-Tillson, Holly [1 ]
Smith, Hamilton O. [1 ]
Kovacs, Kornel L. [2 ]
Frazier, Marvin [1 ]
Venter, J. Craig [1 ]
Xu, Qing [1 ]
机构
[1] J Craig Venter Inst, Rockville, MD 20850 USA
[2] Univ Szeged, Dept Biotechnol, Szeged, Hungary
关键词
MOLECULAR-BIOLOGY; CRYSTAL-STRUCTURE; LARGE SUBUNIT; MATURATION; BIOSYNTHESIS; NICKEL; PURIFICATION; ALCALIGENES; COMPLEX; PROTEIN;
D O I
10.1128/AEM.00580-09
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Using a metagenomics approach, we have cloned a piece of environmental DNA from the Sargasso Sea that encodes an [NiFe] hydrogenase showing 60% identity to the large subunit and 64% to the small subunit of a Thiocapsa roseopersicina O-2-tolerant [NiFe] hydrogenase. The DNA sequence of the hydrogenase identified by the metagenomic approach was subsequently found to be 99% identical to the hyaA and hyaB genes of an Alteromonas macleodii hydrogenase, indicating that it belongs to the Alteromonas clade. We were able to express our new Alteromonas hydrogenase in T. roseopersicina. Expression was accomplished by coexpressing only two accessory genes, hyaD and hupH, without the need to express any of the hyp accessory genes (hypABCDEF). These results suggest that the native accessory proteins in T. roseopersicina could substitute for the Alteromonas counterparts that are absent in the host to facilitate the assembly of a functional Alteromonas hydrogenase. To further compare the complex assembly machineries of these two [NiFe] hydrogenases, we performed complementation experiments by introducing the new Alteromonas hyaD gene into the T. roseopersicina hynD mutant. Interestingly, Alteromonas endopeptidase HyaD could complement T. roseopersicina HynD to cleave endoproteolytically the C-terminal end of the T. roseopersicina HynL hydrogenase large subunit and activate the enzyme. This study refines our knowledge on the selectivity and pleiotropy of the elements of the [NiFe] hydrogenase assembly machineries. It also provides a model for functionally analyzing novel enzymes from environmental microbes in a culture-independent manner.
引用
收藏
页码:5821 / 5830
页数:10
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