Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.
The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, including Escherichia coli. Although the ionic strength of the H. volcanii cytosol differs over 15-fold from that of E. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like the E. coli purified enzyme, ZmPDC from H. volcanii catalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected as H. volcanii transitioned from log phase to late stationary phase that was inversely proportional to the amount of pdc-specific mRNA. Based on these results, proteins from non-halophilic organisms can be actively synthesized in haloarchaea; however, post-transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.
Kaczowka
Steven J
SJ
Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611-0700, USA.
Reuter
Christopher J
CJ
Talarico
Lee A
LA
Maupin-Furlow
Julie A
JA
D001709:Biotechnology / Q000379:methods; D015153:Blotting, Western; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D000431:Ethanol / Q000378:metabolism; D019622:Haloferax volcanii / Q000201:enzymology* / Q000235:genetics*; D008970:Molecular Weight; D011767:Pyruvate Decarboxylase / Q000096:biosynthesis* / Q000235:genetics*; D019642:RNA, Archaeal / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic; D017355:Zymomonas / Q000201:enzymology* / Q000235:genetics*
D004269:DNA, Bacterial; D019642:RNA, Archaeal; D011994:Recombinant Proteins; D000431:Ethanol; D011767:Pyruvate Decarboxylase