Authors' address: Department of Applied Microbiology, Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan. * Correspondent. Fax: +81 774 33 3004.
Summary
The cellular level of methylglyoxal (MG), a highly toxic 2-oxoaldehyde, in Saccharomyces cerevisiae cells transformed with genes for some of the glycolytic enzymes was determined as an index of the safety of genetically engineered yeast and the level was compared with that in non-transformed control cells. The phosphoglucose isomerase (PGI), phosphofructokinase (PFK) and triosephosphate isomerase (TPI) activities significantly increased in the transformants and were approximately five-, three- and sevenfold higher, respectively, than those in the control. When these transformed cells were used for alcohol fermentation from glucose, they accumulated MG in cells at a level sufficient to induce mutagenicity. These results illustrate that careful thought should be given to the potential metabolic products and their safety when a genetically engineered yeast is applied to food-related fermentation processes.
Keywords
Food safety, genetically engineered food, glycolytic bypass, methylglyoxal, recombinant yeast.
Introduction
Recent advances in gene engineering technology have greatly enhanced the potential of various organisms for use in food, chemical and pharmaceutical areas. Recombinant microbes have been used for the production of chemically and pharmaceutically important classes of compounds. Transgenic plants and animals are expected to contribute as novel food and food-stuffs in the near future. However, the safety of gene-altered organisms such as food, food-stuffs and neutraceuticals has not been fully analysed, and a recent report described the appearance of a novel and unknown virus from transgenic plants through RNA recombination (Greene & Allison, 1994). The safety aspects of this virus remain to be determined.
The safety of gene-altered organisms, especially genetically engineered foods, has been viewed either from a standpoint of `process (gene engineering technique) or product (gene-altered organisms)'. The `process' is thought not to contain either essentially or potentially dangerous steps, since the gene engineering technique is apparently more precise and intentional in comparison with current random mutagenic methods. It has therefore been concluded that safety studies should be concentrated on and limited to the `product (i.e. genetically engineered foods)', but not the `process'. This decision is based on the scientific concept recommended by the US Food and Drug Administration (FDA) policy or the International Food Biotechnology Council. This concept centres on a demonstration of substantial equivalency between gene-altered organisms and non-transformed counterparts.
The safety defined by the concept of `substantially equivalent' represents the nutritional, biochemical and morphological equivalencies between genetically engineered and non-engineered organisms. The safety issue addressed by the concept may be: (i) Are there sharp morphological changes in the gene-altered organisms in comparison with traditional organisms? (ii) Are there broad changes in the level of essential nutrients or natural toxins in the gene-altered organisms? and (iii) Are there environmental hazards resulting from the release of the gene-altered organisms? Based on the concept of `substantially equivalent', the safety of the gene-altered tomato, which is the first genetically engineered whole food, was recently guaranteed (Redenbaugh et al., 1994) and the tomato has now been sold in California and Midwestern states, although it has faced new protests by consumers preparing `boycotts' and public `tomato squashes' (Lehrman, 1994).
Contrary to the case of transgenic plants, the food safety of recombinant microbes has not been studied except for a system of monitoring the presence of genetically engineered microbes in the environment (Prosser, 1994). Procaryotic and eucaryotic microbes have the capability to convert dihydroxyacetonephosphate (DHAP) into lactate via methylglyoxal (MG), a highly toxic 2-oxoaldeyde (Fig. 1). The metabolic route called the `glycolytic bypass' was first demonstrated in Escherichia coli by Cooper & Anderson (1970), and the details of the bypass have been studied in yeast cells (Murata et al., 1989). Although the bypass generates no high energy compounds, it contains highly reactive intermediates such as MG, glutathione (GSH) and S-lactoylglutathione (S-LG). However, despite the enzymic and genetic studies, no definite physiological function of the bypass has been assigned so far. The MG is highly toxic and completely represses proliferation of yeast cells at around 2 mm (Murata et al., 1989). In order to assess the safety of recombinant microbes, we determined the change in content of MG as an index of the safety of genetically engineered Saccharomyces cerevisiae cells during alcohol fermentation.
Figure 1. Glycolytic pathway in S. cerevisiae. Abbreviations used are: PGI, phosphoglucose isomerase; PFK, phosphofructokinase; TPI, phosphoglucose isomerase; MGS, MG synthase; Glo I, glyoxalase I; Glo II, glyoxalase II; MGR, MG reductase; LALD H, lactoaldehyde dehydrogenase. The pathway indicated by thick and thin lines represent generally accepted glycolysis and glycolytic bypass, respectively.
Materials and methods
Yeast strains
Strains of Saccharomyces cerevisiae DF406pTPI and DF409pPGI were obtained from D.G. Fraenkel. DF406pTPI and DF409pPGI contain plasmids harbouring triosephosphate isomerase (TPI) and phosphoglucose isomerase (PGI) genes, respectively, on a multi-copy plasmid YEp 13 (Kawasaki & Fraenkel., 1982). These genes were extracted and introduced into S. cerevisiae DKD-5D-H (MATa leu2 leu2-1 trpl his3 ), a gift from Y. Oshima, and resulting transformants containing pPGI and pTPI were designated DKD-5D-H/pPGI and DKD-5D-H/pTPI. A strain DKD-5D-H/pPFK with a plasmid containing phosphofructokinase (PFK) gene was constructed by us (unpublished results). These plasmids pPGI, pPFK and pPTI were also introduced into MG-resistant mutant MGRI derived from DKD-5D-H (Murata & Kimura, 1986) and resulting transformants were designated MGR1/pPGI, MGR1/pPFK and MGR1/pTPI, respectively. The MGRI contains GSH at the lowest level among MG-resistant mutants isolated (Murata & Kimura, 1986).
Culture conditions
All the yeast strains were pre-cultured in a SD:minimal medium (50 ml) consisting of 2% glucose, 0.67% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA) (pH 5.0). Amino acids were added to SD at 20 µg ml-1 if required. After overnight culture at 30°C with reciprocation (120 r.p.m.), the pre-culture was added to fresh medium (2 l in Sakaguchi flask) with the same composition and incubation was continued for a further 40 h under the same conditions. The cells were collected, washed once in chilled water, and then used for the fermentation experiments and assays for enzymes and metabolites as follows.
Fermentation
For fermentation experiments, to yeast cells (10 g as wet weight) placed in a 300-ml Ehrlenmeyer flask, was added 50 ml of reaction mixture [0.5 m glucose, 10 mm MgCl2, and 0.5 mm potassium phosphate buffer (pH 7.0)] and the mixture was incubated at 30°C with reciprocal shaking until the end of fermentation, determined by the disappearance of glucose.
Enzyme assay
The collected cells were suspended in 5.0 mmTris-HCl buffer (pH 7.0) containing 0.05 mm phenylmethanesulphonyl fluoride and disrupted by Braun Homogenizer. The cell debris was removed by centrifugation and the supernatant was dialysed against the same Tris buffer at 4°C overnight before enzyme assays. PGI, PFK, TPI and methylglyoxal synthase (MGS) activities were determined enzymically (Shigematsu et al., 1993; Murata et al., 1985). Protein was determined by the method of Lowry et al. (1951).
Metabolite assay
After fermentation, the cells were collected by centrifugation, washed once in chilled water, suspended in 30 ml of water, placed in boiling water for 5 min, chilled quickly in iced water, centrifuged and the clear supernatant (10 ml) was lyophilized; dry sample was then dissolved in 2.0 ml of water and used for the assay of GSH and MG. GSH was determined by the method of Tietze (1969) using GSH reductase. MG was assayed according to the method of Klotzsch & Bergmeyer (1965) using glyoxalase I. All the chemicals and enzymes used for the assay were purchased from Sigma Chemical Co., St Louis, MO, USA.
Mutagenicity
Lyophilized samples were dissolved in water (3.0 ml) and mutagenicity of the solution was determined according to the method of Levin et al. (1982) using Salmonella tester strain TA97. Mutagenicity of MG (Sigma Chemical Co., St. Louis, MO, USA) was also determined as a control.
Results and discussion
Activity of glycolytic enzymes
The activities of PGI, PFK and TPI in transformed cells with the relevant genes, which were ligated into a multi-copy plasmid YEp13 (Kawasaki & Fraenkel, 1982), were determined and compared with those in cells transformed with YEp13 (control) (Table 1). The activities of PGI, PFK and PTI in DKD-5D-H or MGR1 cells significantly increased (fivefold for PGI, threefold for PFK and sevenfold for TPI) by the introduction of these enzyme genes. Although the data are not shown here, the increases in these enzyme activities are due to the over-expression of the relevant genes (Kawasaki & Fraenkel, 1982). The activity of MGS responsible for the synthesis of MG from DHAP was not affected by the glycolytic enzyme genes introduced into the yeast cells.
Accumulation of MG
The transformed cells of DKD-5D-H with PGI, PFK and TPI genes were incubated in the mixture containing a large amount of glucose (9%) and the changes in GSH and MG contents were analysed (Table 1). MG content in transformed DKD-5D-H cells having GSH at a normal level was about 30-fold higher than that in control cells similarly incubated. Large amounts of MG were accumulated when the MGR1 mutant containing GSH at a decreased level was used, and the MG content reached about 1 mm (Table 1). This was presumably due to the repression of glyoxalase I (GLO-I) activity that requires GSH for conversion of MG to S-LG (Fig. 1). The cellular level of MG attained in both DKD-5D-H and MGR1 cells was sufficient to induce a mutation in Salmonella typhilirum tester strain TA97 (Table 2). The authentic MG also showed mutagenic activity at a concentration more than 10 µg ml-1 (Table 2). However, the mutagenic activity of MG and the extracts prepared from recombinant cells was eliminated by treating MG and the extracts with glyoxalase I and glyoxalase II in the presence of trace amount of GSH.
Table 1. Glycolytic enzyme activity and content of glutathione (GSH) and methylglyoxal (MG)
Activity (a) Content (b) Strain PGI PFK TPI MGS GSH MG DKD-5D-H/YEp13 2.85 113 18.3 0.177 53.2 0.33 DKD-5D-H/pPGI 13.2 104 16.4 0.201 38.4 5.44 DKD-5D-H/pPFK 2.01 345 20.3 0.193 33.3 13.2 DKD-5D-H/pTPI 2.11 116 122 0.235 27.2 11.1 MGR1/YEp13 1.77 102 16.4 0.211 5.31 2.33 MGR1/pPGI 11.2 99.5 13.2 0.181 4.22 65.4 MGR1/pPFK 2.23 293 10.1 0.233 4.55 72.3 MGR1/pTPI 1.43 97.4 105 0.153 4.37 58.4
(a) Activity was expressed as nanomoles of product formed per min per mg of protein. PGI-phosphoglucose isomerase; PFK-phosphofructokinase; MGS-methylglyoxal synthase. (b) Contents of MG and GSH were expressed as µg per g of wet weight cells.
Table 2. Mutagenicity of extract prepared from transformed yeast cells with phosphofructokinase (PFK) gene
Revertants of TA97 per plate
Yeast extract DKD-5D-H/ DKD-5D-H/
used (ml) YEpI3 pPFK MGR1/YEp13 MGR1/pPFK
0 34 43 29 35
0.3 36 121 31 244
1.0 42 341 38 538
1.0* 33 72 40 83
Authentic MG
(ml) Revertants of TA97 per plate
0 44
1.0 672
1.0* 86
Lyophilized yeast extracts were prepared from DKD-5D-H and MGR 1 cells transformed with YEp13 or PFK gene (pPFK) as described in Materials and methods. The samples were dissolved in 3 ml of water and used for the mutagenicity assay. Authentic MG solution (10 µg ml-1) was prepared and 1.0 ml of the solution was also used for assays. Assay plates were incubated for 2 days at 37°C and the number of His+ revertants was scored.
* To eliminate MG, the extracts (1.0 ml) prepared from DKD-5D-H and MGR1 cells transformed with YEp13 or PFK gene was mixed with a solution (20 pl) containing 2.0 pg glyoxalase I, 2.0 pg glyoxalase II and 0.05 µmole GSH and incubated at 37°C for 2 h. One milliliter of authentic MG solution (10 pg ml-') was also treated similarly. After MG elimination reaction, whole reaction mixture (1.02 ml) was subjected to the mutagenicity assay as described above.
This was presumably due to the conversion of MG in the samples of D-lactate via S-lactoylGSH (Fig. 1) by using and regenerating GSH in the samples. The results indicated that the observed mutagenicity of the extracts prepared from recombinant yeast cells was due to the presence of MG formed in the course of glucose assimilation probably through glycolytic bypass (Fig. 1).
Although, except for the case of microbes, we have no information as to the toxic effect of MG in foods on human beings, the results presented here indicate that, in genetically engineered yeast cells, the metabolism is significantly disturbed by the introduced genes or their gene products and the disturbance brings about the accumulation of the unwanted toxic compound MG in cells. Such accumulation of highly reactive MG may cause a damage in DNA, thus suggesting that the scientific concept of `substantially equivalent' for the safety assessment of genetically engineered food is not always applied to genetically engineered microbes, at least in the case of recombinant yeast cells. In order to apply recombinant yeast cells to practical fermentation processes, the safety level of MG in cells should be established.
Thus, the results presented may raise some questions regarding the safety and acceptability of genetically engineered food, and give some credence to the many consumers who are not yet prepared to accept food produced using gene engineering techniques.
Acknowledgments
We thank Y. Oshima and D.G. Fraenkel for yeast strain (DKD-5D-H) and plasmids (pPGI and pTPI), respectively.
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source: http://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2621.1995.tb01365.x
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