Pyruvate decarboxylase (PDC), an enzyme central to homoethanol fermentation, catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde with release of carbon dioxide. PDC enzymes from diverse organisms have different kinetic properties, thermal stability and codon usage that are likely to offer unique advantages for the development of desirable Gram-positive biocatalysts for use in the ethanol industry. To examine this further, pdc genes from bacteria to yeast were expressed in the Gram-positive host Bacillus megaterium. The PDC activity and protein levels were determined for each strain. In addition, the levels of pdc-specific mRNA transcripts and stability of recombinant proteins were assessed. From this analysis, the pdc gene of Gram-positive Sarcina ventriculi was found to be the most advantageous for engineering high-level synthesis of PDC in a Gram-positive host. This gene was thus selected for transcriptional coupling to the alcohol dehydrogenase gene (adh) of Geobacillus stearothermophilus. The resulting Gram-positive ethanol production operon was expressed at high levels in B. megaterium. Extracts from this recombinant were shown to catalyse the production of ethanol from pyruvate.
BarbosaM. F. S.,
IngramL. O.
1994; Expression of the Zymomonas mobilis alcohol dehydrogenase II ( adhB ) and pyruvate decarboxylase ( pdc ) genes in Bacillus
. Curr Microbiol 28:279–282[CrossRef]
BeallD. K.,
OhtaK.,
IngramL. O.
1991; Parametric studies of ethanol-production from xylose and other sugars by recombinant Escherichia coli
. Biotechnol Bioeng 38:296–303[CrossRef]
BraüB., SahmH.
1986; Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli
. Arch Microbiol 144:296–301[CrossRef]
ConwayT.,
OsmanY. A.,
KonnanJ. I.,
HoffmannE. M.,
IngramL. O.
1987; Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. J Bacteriol 169:949–954
DienB. S.,
BothastR. J.,
NicholsN. N.,
CottaM. A.
2002; The US corn ethanol industry: an overview of current technology and future prospects. Int Sugar J 104:204–211
DienB. S.,
CottaM. A.,
JeffriesT. W.
2003; Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258–266[CrossRef]
GoldR. S.,
MeagherM. M.,
TongS.,
HutkinsR. W.,
ConwayT.
1996; Cloning and expression of the Zymomonas mobilis “production of ethanol” genes in Lactobacillus casei
. Curr Microbiol 33:256–260[CrossRef]
GurkanC.,
EllarD. J.
2003; Expression of the Bacillus thuringiensis Cyt2Aa I toxin in Pichia pastoris using a synthetic gene construct. Biotechnol Appl Biochem 38:25–33[CrossRef]
HillmanJ. D.,
ChenA.,
SnoepJ. L.
1996; Genetic and physiological analysis of the lethal effect of L-(+)-lactate dehydrogenase deficiency in Streptococcus mutans : complementation by alcohol dehydrogenase from Zymomonas mobilis
. Infect Immun 64:4319–4323
HoppnerT. C.,
DoelleH. W.
1983; Purification and kinetic characteristics of pyruvate decarboxylase and ethanol dehydrogenase from Zymomonas mobilis in relation to ethanol production. Eur J Appl Microbiol Biotechnol 17:152–157[CrossRef]
IngramL. O.,
ConwayT.,
ClarkD. P.,
SewellG. W.,
PrestonJ. F.
1987; Genetic engineering of ethanol production in Escherichia coli
. Appl Environ Microbiol 53:2420–2425
JacksonR. M.,
GelpiJ. L.,
CortesA.
& 7 other authors; 1992; Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase. Biochemistry 31:8307–8314[CrossRef]
KönigS.
1998; Subunit structure, function and organisation of pyruvate decarboxylases from various organisms. Biochim Biophys Acta1385271–286[CrossRef]
LakeyD. L.,
VoladriR. K. R.,
EdwardsK. M.,
HagerC.,
SamtenB.,
WallisR. S.,
BarnesP. F.,
KernodleD. S.
2000; Enhanced production of recombinant Mycobacterium tuberculosis antigens in Escherichia coli by replacement of low-usage codons. Infect Immun 68:233–238[CrossRef]
LoweS. E.,
ZeikusJ. G.
1992; Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi
. J Gen Microbiol 138:803–807[CrossRef]
NealeA. D.,
ScopesR. K.,
WettenhallR. E.,
HoogenraadN. J.
1987; Pyruvate decarboxylase of Zymomonas mobilis : isolation, properties, and genetic expression in Escherichia coli
. J Bacteriol 169:1024–1028
NicholsN. N.,
DienB. S.,
BothastR. J.
2003; Engineering lactic acid bacteria with pyruvate decarboxylase and alcohol dehydrogenase genes for ethanol production from Zymomonas mobilis
. J Ind Microbiol Biotechnol 30:315–321[CrossRef]
OhtaK.,
BeallD. S.,
MejiaJ. P.,
ShanmugamK. T.,
IngramL. O.
1991a; Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II. Appl Environ Microbiol 57:893–900
OhtaK.,
BeallD. S.,
MejiaJ. P.,
ShanmugamK. T.,
IngramL. O.
1991b; Metabolic engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and glucose. Appl Environ Microbiol 57:2810–2815
PuyetA.,
SandovalH.,
LópezP.,
AguilarA.,
MartinJ. F.,
EspinosaM.
1987; A simple medium for rapid regeneration of Bacillus subtilis protoplasts transformed with plasmid DNA. FEMS Microbiol Lett 40:1–5[CrossRef]
RajK. C.,
IngramL. O.,
Maupin-FurlowJ. A.
2001; Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus
. Arch Microbiol 176:443–451[CrossRef]
RajK. C.,
TalaricoL. A.,
IngramL. O.,
Maupin-FurlowJ. A.
2002; Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase ( pdc ): comparison to bacterial homologues. Appl Environ Microbiol 68:2869–2876[CrossRef]
RygusT.,
HillenW.
1991; Inducible high-level expression of heterologous genes in Bacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl Microbiol Biotechnol 35:594–599
TalaricoL. A.,
IngramL. O.,
Maupin-FurlowJ. A.
2001; Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli
. Microbiology 147:2425–2435
WeiW.,
LiuM.,
JordanF.
2002; Solvent kinetic isotope effects monitor changes in hydrogen bonding at the active center of yeast pyruvate decarboxylase concomitant with substrate activation: the substituent at position 221 can control the state of activation. Biochemistry 41:451–461[CrossRef]
WongS. L.
1995; Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr Opin Biotechnol 6:517–522[CrossRef]
WongS. L.,
YeR.,
NathooS.
1994; Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system. Appl Environ Microbiol 60:517–523