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Powerful Duet

Fly enzyme produced in yeast fights harmful bacteria in the manufacture of fuel alcohol

LUCIANO JOSÉ SILVEIRARecombining a strain of yeast that produces lysozymeLUCIANO JOSÉ SILVEIRA

A small protein called lysozyme, found in saliva and tears, showed in the laboratory that it can increase the efficiency of the ethanol production process, with considerable savings for refineries, which have already stated their interest in this new biotechnological breakthrough. To obtain these results researchers from the University of São Paulo (USP) produced a line of Saccharomyces cerevisiae yeast, a microorganism responsible for transforming sugar into fuel alcohol and that is capable of producing this protein. The yeast gains the capacity to fight the bacteria that contaminate the vats where the sugar-cane juice, also known as the wash, is fermented. “The wash is a great substrate not only for yeast but for various other microorganisms to grow in because it has high levels of nutrients, in addition to a favorable pH and temperature”, says Professor Ana Clara Guerrini Schenberg, from the Institute of Biomedical Sciences of the University São Paulo (USP), the research coordinator. In addition to competing for sucrose and other nutrients in the wash, the bacteria introduce undesirable metabolism products to the process, mainly organic acids.

Refineries normally add antibiotics to the wash to guard against bacterial contamination. With the passage of time, however, new antibiotics or combinations of medication are needed to fight resistant strains of bacteria. It is estimated that refineries, for every cubic meter of ethanol produced, spend an average of US$ 3 to 5 on antibiotics. “Besides the economic problem there is the environmental pollution aspect, because everything ends up going into the wild in the form of effluents that run off into the rivers”, says Ana Clara, a specialist in the molecular genetics of micro-organisms, who is coordinating various pieces of work with yeasts and bacteria in her laboratory in the Department of Microbiology.

Antibacterial substance
The search for an alternative method to antibiotics for controlling the microorganisms found in the fermentation of ethanol was the subject of postgraduate work by Elza Grael Marasca and Luciano José Silveira, under the guidance of Ana Clara. “We thought that if the yeast itself contained a gene codifying an antibacterial substance it could solve the problem itself without the need to add antibiotics to the wash”, says the research coordinator, the daughter of renowned Brazilian theoretical physicist, Mário Schenberg (1916-1990). Lysozyme, an enzyme that is not exactly an antibiotic, but that is highly effective against bacteria because it breaks down cell walls, was chosen for this purpose. “This is a very interesting protein that is produced by practically all living beings but not by yeast”, she says. In some industrial processes, such as food conservation and the production of wine and medication, an imported commercial lysozyme is used that is extracted from egg white, using a process created almost 40 years ago.

Before modifying the yeast’s genetic information, the researchers looked at the literature to check whether some lysozyme had been described that might tolerate the acidification that occurs at the end of alcoholic fermentation. At the same time, in 1996, Professor Sirlei Daffre, a researcher from the Department of Parasitology, which is in the same building as the Department of Microbiology, had just arrived from Sweden where she had worked on the lysozymes of the Drosophila melanogaster fly. “Because it acts on the digestive tract of the fly this lysozyme works extremely well when there is an acidic pH, which is similar to that found in the alcoholic fermentation process”, says Ana Clara. The object of Sirlei’s study was insects and how they fight from infection, research that eventually contributed to the preparation of a technological process. “She had cloned the cDNA of the lysozyme, the part of the gene that is interesting and that is transformed into a messenger within the cells”, says Ana Clara.

Ceding the cloned gene helped shorten the route, but there was still a lot of work ahead. The next step was to find a promoter that would make the gene express itself in the yeast, a process called the DNA recombining technique, and to ensure that the lysozyme would be secreted into the growing environment. It was Elza Marasca’s responsibility to place the cDNA of the fruit fly under the control of the promoter of alcohol-dehydrogenase 1, an enzyme that comes from the very yeast used during the fermentation process. Everything fitted together perfectly. “Both the promoter and the yeast function in perfect harmony during the fermentation process, while the production of lysozyme takes place”, declares Ana Clara.

Alcoholic fermentation
Having done this it was necessary to check whether the use of the strain of recombining yeast that produces lysozyme in alcoholic fermentation processes would help reduce the use of antibiotics. It is a known fact that most of the microorganisms found in vats are sensitive to the action of lysozyme, which acts primarily on the wall of gram-positive bacteria, such as Bacillus coagulans and Lactobacillus fermentum. “Since in alcohol refineries in the state of São Paulo 98.5% of the contaminants are gram-positive bacteria, in theory this is a great choice”, says Gabriela Ribeiro dos Santos, a post-doctoral student who is taking part in the project. “Now we have to carry out a study that mimics the conditions found in the refinery to evaluate, in practice, the effect of the modified yeast.” To do so it will be necessary to adjust the amount of enzyme secreted relative to the amount of contaminants that exist in the industrial process.

LUCIANO JOSÉ SILVEIRAAlpha-amylase enzyme secreted by the yeast breaks down the starch (in blue) and forms a clear haloLUCIANO JOSÉ SILVEIRA

Confirmed action
The first step in this direction has already been taken. One of the studies recently carried out by the research group consisted of inoculating, for comparison purposes, the original strain of yeast and the strain that produces lysozyme in a non-sterile wash, as is used in the refinery, but in USP’s own laboratory. An interesting aspect of this work, which reflects an advance for arriving at the industrial process and resulted in filing for a patent, under the management of the USP Innovation Agency, is that the genetic information of the lysozyme was stabilized by integrating it into one of the chromosomes of a strain of industrial yeast used by the refineries. “We saw a very significant difference between the quantity of contaminants under the original conditions with and without lysozyme”, says Gabriela. This means that the action of the recombining yeast on the bacteria was confirmed in the tests. Nevertheless, the researchers want to increase production of the enzyme so that it responds satisfactorily to the conditions faced on an industrial scale.

The modified yeast has just one copy of the lysozyme gene integrated into its chromosome. “Now a strategy is being designed to extend the number of copies of the gene inserted in the yeast”, says Ana Clara. “As some of the chromosomal sites of the Saccharomyces cerevisiae yeast that can be used for inserting foreign genes without disturbing the life of the strain are known, we can increase the production capacity of lysozyme without interfering with the efficiency of ethanol production.”

All these modifications are being thought of because of the interest being shown by businessmen from the sugar and alcohol sector in genetically modified yeast. The first step that is required for this interest to turn into reality has already been taken. Researchers submitted the modified yeast to concept proof. In other words, various tests were carried out to prove that it works not only under laboratory conditions but also has the potential for use in industrial processes. “We concluded this concept proof and we passed in all the requirements”, says Gabriela. The businessmen now want to know whether in the future there will be any problems regarding authorization for the use of genetically modified organism (GMOs) to produce ethanol. “We’re going to consult the National Technical Bio-safety Committee (CTNBio) before we can provide a reply”, says Ana Clara.

The ethanol production studies coordinated by the researcher started when Proálcool, the Brazilian program set up in late 1975, was being implemented. The idea was to produce fuel alcohol from cassava starch rather than from the simple sugars such as sucrose and glucose found in cane juice. It just so happens that the Saccharomyces cerevisiae yeast, whose comes from sacaro, sugar, and myces, fungus, can metabolize glucose and sucrose, but not starch, which is a complex molecule with various glucose units in linear and ramified chains. This means that in all processes involving amylase substrates, like barley, cassava or corn, prior enzymatic treatment is needed in order to produce ethanol.

To abbreviate this prior substrate treatment stage and reduce the fermentation process of starches, the research group started a program of genetic improvement of S. cerevisiae almost 25 years ago. “A lot of work was done to provide the yeast strain with the capacity of breaking down starch and processing the cassava sugar in the fermentation stage to transform it into alcohol”, says Gabriela, whose master’s degree dissertation was devoted to this subject. As starch is a ramified molecule it needs a complex of amylolytic enzymes to be completely broken down.

Complex molecule
The group’s first triumph, published in Nature Biotechnology in 1986, was to make the yeast produce and secrete into the growing medium a gene that codifies the alpha-amylase produced in the pancreas of guinea pigs. Alpha-amylase works on the linear starch chains, breaking this complex molecule down into smaller molecules consisting of two or more units of glucose, so that the yeast still wastes a substantial portion of the sugar within the starch. At the next improvement stage Gabriela placed the gene of another enzyme in the yeast, that of glycoamylase from another yeast species. Glycoamylase helps the alpha-amylase break down the starch and take it to the glucose molecule. “One helps the other in this process, but we still couldn’t take advantage of 100% of it”, says Gabriela. “In fact, experiments on a pilot scale carried out in USP’s Department of Chemical Engineering showed that the factor limiting fermentation efficiency was precisely the activity of the glycoamylase”, she adds.

New constructions then followed so that the yeast could express more powerful gycoamylases, and above all in a stable form and in an industrial yeast. Today, the laboratory has some industrial recombining strains, one of which stands out thanks to having the glycoamylase gene in five copies. The new strains must still be evaluated on a pilot scale, using the starch from cassava, a cheap and abundant raw material in Brazil, and an alternative source to the sugar from sugarcane.

Micro-organisms programmed to remove metals
Bacteria and yeast capable of removing heavy metals from mining effluents are being produced with genetic engineering tools by the research group coordinated by Professor Ana Clara Guerrini Schenberg. The project was commissioned two years ago by Companhia Vale do Rio Doce, the world’s leading iron ore producer and the second largest global producer of manganese and iron alloys.

“The important thing is to find a suitable microorganism for carrying out a particular function”, says Ana Clara. “When they don’t work exactly as we want them to we can improve the characteristics of these microorganisms.” Since the bio-remediation of materials is a new area, the project is divided into several sub-projects. One of the participants, Ronaldo Biondo, is building bacteria that can link heavy metals together in order to make it easier to remove them from mining effluent. Meanwhile, researcher Gabriela Ribeiro dos Santos is building a suicide system for these bacteria so that when their task is finished they are not a risk to other organisms found in nature. “I’m working to develop a genetic system that causes the bacteria’s death, but like a time-bomb – it only goes into action after the bioremediation stage has been accomplished by the micro-organism”, says Gabriela.