Journal of Undergraduate Research
Volume 1, Issue 4 - January 2000

Cloning and Characterization of the Pyruvate Decarboxylase Gene from Sarcina ventriculi

Jason S. Cesario

ABSTRACT

In the Gram positive eubacterium, Sarcina ventriculi, following glycolysis a branch point occurs in which pyruvate can either follow one of two pathways for fermentation. The first pathway occurs as the oxidative decarboxylation of pyruvate under the direction of the enzyme, pyruvate dehydrogenase, yielding acetyl CoA and ultimately acetate via phosphotransacetylase and acetate kinase. The second possibility, which is the focus of this study, is non-oxidative and yields ethanol under direction of the enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Central metabolism is redirected from acidic fermentation products to ethanol in the presence of ADH and PDC. Although ADH is abundant in bacteria, PDC is quite rare. PDC has been detected and even purified in several yeast and a few bacteria, S. ventriculi being one of those. To date, the only bacterial PDC gene, which has been isolated and sequenced, is from the Gram negative eubacterium Zymomonas mobilis. This has somewhat restricted the host flexibility of the current portable ethanol-production operons and the kinetics of metabolism. So, it is the aim of this project to isolate the gene of S. ventriculi encoding PDC in expectation of developing a portable ethanol-production operon to be expressed in thermotolerant gram-positive bacteria. At the present time, a partial fragment of the S. ventriculi PDC gene has been isolated and characterized.

INTRODUCTION

In glycolytic pathways, glucose is first broken down forming two molecules of pyruvate, supplying the cell with energy in the form of ATP and NADH. Upon formation, pyruvate has several metabolic fates. First is pyruvate oxidation in which the presence of pyruvate dehydrogenase catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA. This reaction is very important to the cell since it acts as one of the major cellular sources of acetyl-CoA, which is often oxidized to CO2 under aerobic conditions in the citric acid cycle. As an alternative to pyruvate oxidation, the anaerobic process of alocoholic fermentation occurs, where under the control of the enzyme, pyruvate decarboxylase (PDC), the nonoxidative decarboxylation of pyruvate takes place, producing acetaldehyde and giving off CO2. This reaction is followed by the reduction of acetaldehye to ethanol, catalyzed by alcohol dehydrogenase (ADH).

The production of ethanol from pyruvate during fermentation requires the presence of ADH and PDC. In order to successfully redirect the metabolic pathway of bacteria into an ethanol-production machine it is necessary to have both PDC and ADH present. ADH is widely distributed in several organisms including bacteria, yeast, plants, and higher eukaryotes (Cannio et al., 1994). However, PDC is scarce in prokaryotes, having been detected only in Sarcina ventriculi (Stephenson & Dawes, 1971), Zymomonas mobilis (Hoppner & Doelle, 1983), Acetobacter peroxydans (DeLey & Schell, 1959), Acetobacter suboxydans (King & Cheldelin, 1954), and Erwinia amylovora (Haq & Dawes, 1971) so finding both PDC and ADH together in nature is extremely uncommon explaining the low ethanol production of natural bacteria. To date, the gene that encodes the PDC enzyme has been isolated from Z. mobilis (Lowe & Zeikus et al., 1992).

The pdc genes are present in many yeast, upon which much research has already taken place. Currently the pdc gene sequences from Saccharomyces cerevisiae, Kluyveromyces marxianus, and Hanseniaspora uvarum are known, and has enabled the characterization of several PDC primary structures (Lu et al., 1998).

Although the sequence of the Z. mobilis pyruvate decarboxlase gene is known, finding a suitable host for the production of ethanol is still a problem. In eukaryotes and prokaryotes, such as Z. mobilis, exposure to elevated levels of ethanol is a microbial stress and stimulates a stress response leading to a declining level of growth and thus a lower overall yield of ethanol (Barbosa et al.., 1994). Extensive research has been performed testing the expression of Z. mobilis pdc gene in many other species of bacteria in an attempt to produce ethanol while maintaining high growth rates. For instance in Bacillus it was found that ethanol production was increased using the Z. mobilis pdc gene although the efficiency for ethanol production was rather low, and improvements in the level of expression are still needed (Barbosa & Ingram., 1994). Recombinant strains of Eschericia coli and Klebsiella spp. containing the Z. mobilis PDC gene have proven to be efficient ethanol-producers, but these strains relative sensitivity to ethanol limited bacterial growth and therefore they would not prove useful as a source for ethanol production (Gold et al., 1996). The Z. mobilis pdc genes has also been transformed into Lactobacillus casei. The recombinant L. casei produced more than twice the amount of ethanol that was made by the original strain (Gold et al., 1996). Expression of the pdc genes did divert the carbon flow away from the production of lactic acid and toward the production of ethanol but not completely since most of the glucose being metabolized still resulted in lactate rather than ethanol formation (Gold et al., 1996) Therefore, the search continues for an ethanol-tolerant, and ultimately thermo-tolerant bacteria in which a pdc gene can be cloned producing high levels of ethanol while not limiting growth.

Ongoing research to deal with these problems involves finding a gene that can be cloned in vectors, which will optimize ethanol production. S. ventriculi will be utilized as the gene source, since PDC activity has been previously observed (Lowe & Zeikus et al., 1992). Unlike Z.mobilis, S. ventriculi is a Gram positive bacteria, and although the bacteria itself is an obligate anaerobe, the pyruvate decarboxylase enzyme is oxygen stable so as to allow for easier manipulation in the laboratory and purification. Thus, the implications and expectations of this research include a portable pdc operon which will be available for cloning in vectors and will optimize both growth and ethanol production in an overall attempt to find a renewable source of energy in the form of ethanol.

MATERIALS AND METHODS

A partial fragment of the S. ventriculi gene has been successfully cloned and partly sequenced. At this time however, due to proprietary reasons, the methods used can not be disclosed.

RESULTS AND DISCUSSION

Since a partial fragment of the gene has been cloned and isolated, the genes presence on the S. ventriculi genome is evident. For the duration of the experiment attempts will be made to clone and sequence the entire gene.

Photos by John Elderkin and Jason Cesario.

REFERENCES

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