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Genetic Manipulation of Yeasts for Ethanol Production from Xylose

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Genetic Manipulation of Yeasts for Ethanol Production from Xylose GENETIC MANIPULATION OF YEASTS FOR ETHANOL PRODUCTION FROM XYLOSE NANCY J. ALEXANDER o YEASTS are commercially valuable to a variety of indus­ Protoplast Fusion tries. These microorganisms, mainly known i~ the ?aking an~ a The development of the protoplast fusion technique allows brewin industries, play an important role m alllmal nutn­ genetic manipulation of strains that nor~ally do not mate. In tion (single-cell protein) and in production of alcohol fuels, this procedure, the cells are treated wIth an enzyme, often chemical feedstocks, and, more recently, enzymes. Although zymolyase (Miles Laboratories, Elkhart, IN) or gluculase t~e yeas~s, species of Saccharomyce.s are most widely studied (Endo Laboratories, Garder: City, NY), to r~move most. of the both aenetically and bIOchemIcally, they do have certam drawb~cks cell wall (Fig. 1). The resultmg spheroplast IS very fragIle and such as the limited secretion of products, low must be maintained in an osmotically suitable buffer, usually ethanol tolerance and restricted carbohydrate utilization. whi~h sti~u­ 0.8-1.2 M sorbitol, pH 7. After a gentle washing with this Other yeasts, respond to uniqu: environmen.tal buffer, the spheroplasts are then subjected to a ca}cium Ii, may actually be preferred. PrevIOusly, orga?lSmS wIth chloride or lithium acetate treatment. The actual fusIOn of desirable traits have been selected by screelllng culture the spheroplasts occurs when polyethylene glycol is added or collections or by mutagenesis and subseque~ltisolation of. the when the cells are subjected to an electrical field (electrofu­ desired strains. However, mutations are dIfficult to achIeve sion). The fused cells are placed in a suitable medium.to with commercial polyploid yeast strains. The polyploid undergo cell wall regeneration, then placed on selective nature hinders mutagenesis and sele.ctio~ ?f dominan~ ?har­ media to eliminate the parental cells. acteristics, yet it is important for mamtammg the stablhty of A major problem with protoplast fusion is the selecting out the strain. ~echlllques, of the fused cells from the parentals. To aid in selecting fused With the development of molecular biological cells, the two parentals must have some unique, e~sily improvement of polyploid or imperfect yeast stra~ns may .~e assoclate~ \~llh identifiable trait. This may take the form of an ammo aCId or obtained by circumventing the problems vitamin requirement. However, sinc.e sing~e gene t~aits ~re conventional aenetic methods. Fusion of protoplasts wlthm a often difficult to obtain in a polyplOld stram, other Identifi­ sinale speciesb or across the species barrier has been success­ ful~ an~ able characteristics, such as ability to grow on a unique Unfortunately, fusion products are often unstable carbohydrate, may be substituted. T~e choice must be such may express undesirable parental traits. The b.est ?pportulll­ expressl(~n ~vlth that the parentals have a low reverSIOn rate of the marker ty for successful, stable gene may he the use gene, for the successful rate of fusion is often quite low of recombinant DNA technology that mserts desll'able .genes p~asmlds (l/10,000). .. into vectors (plasmids) and then transfel;"s the to wor~mg mdustr~al ~e In spite of the difficulties of Wlth yeast host organisms. Expression. of the.desIred trait can strains, success with protoplast fusIOn has been achIeved by optimized through the selectIOn of sUitable vectors that wIll using nutritional requirers (Perez et ai., 1984; Leg1?-1ann a~d increase either intracellular or extracellular levels of prod­ Margalith, 1983; Johanssen et ai., 1984) and dIfferential ucts. carbohydrates (Spencer et ai., 1985; Figueroa et ai., 1984; Ethanol Production 1985; Taya et ai., 1984; Groves and Oliver, 1984). The hybrids are often unstable, but once a stable isolate is selected, Modification of yeasts to increase ethanol production for analysis of the DNA content may be performed. In general, liquid fuels and chemical feedstock industries is an open area the nuclear contents of a hybrid are often one parental type for research. Improvement of strains could ovel;"come low with a few aenes or an extra chromosome of the other tolerance to ethanol and high substrate concentratIOns, char­ parental; the~efore, there is no significant increase in the acteristics of most commercial yeast strains. If the two amount of DNA present in the fused product (Taya ~t ai., characteristics are related, then an osmotolerant strain could 1984). Occasionally, a doubling of the nuclear contents m the possibly be altered to achieve high ethanol and substrate fused cell occurs (Groves and Oliver, 1984). tolerance more easily than a strain that is not tolerant to high Efforts to improve ethanol production by fusion have been osmotic conditions. Characteristics such as flocculence and successful between S. cerevisiae and S. diastaticus, the latter relative stability to fluctuations in temperature and pH may beina a yeast which produces enzymes that degrade starch. also be transferable. How~ver most reports thus far describe the success of the Another area for strain improvement, one that could fusion l~adina to the conclusion that the achievement of substantially reduce ethanol production costs, is t.hat of better'fermentations is apparently limited. However, one S. increasing the variety of carbohydrates that yeast wIll eco­ cerevisiaelS. diastaticus fusion product did produce more nomically ferment. Starch, cellulose, .and xylose are a.bundant ethanol than either parent, presumably by utilizing both the carbohydrates found in plant matenals. Other fungI, as \,:,ell starch and the glucose (Figueroa et ai., 1985). It is unknown as bacteria have been identified as being capable of breakmg thes~ ~he ge~etic what actually caused the greater increase in ethanoi. down complex carbohydrates. Combining Increases in ethanol production rates for fused products material of these various organisms could result m a ulllque over parentals have been noted in other strains. A fusion of individual capable of coding for the enzymes necessary to an osmotolerant strain (S. mellis) with S. cerevisiae pro­ attack a number of carbohydrates simultaneously. duced a better fermentor (as measured by CO 2 evolution) In the immediate future, the primary goals for .increased than either parent (Legmann and ~argalith, 1983). Brewing alcohol production are the development of orgalllsms that strains have also been used to obtam better ethanol produc­ have high osmotolerance; degrade cellulose, starch,.and ers (Johansson and Sjostrom, 1984; Seki et al., 1983; Panchal xylose; and have high tolerance to ethanoi. Such 0l;"f5.alllsms et ai., 1982). .. could make ethanol production much more competitIve. In many studies involving protoplas~ fusIOn, res~lrator.y­ deficient (RD) strains are used. RespIratory deficIency IS The author is IVlicrobiologist, Northern Regional Rese~rch Ce~te~, characterized by a loss of mitochondrial function so that U.S. Dept. of Agriculture, Agricultural Research ServIce, 1810 N. oxidative processes no longer occur and. the cell ~ust, University St., Peoria, IL 61604 therefore, function fermentatively. RD strams can easIly be detected by their lack of growth on glycerol, a carbohydrate Reprinted from Food Technology 40(10) 99-103 OCTOBER 1986-FOOD TECHNOLOGY 99 Purchased by USDA for Official Use Only Genetic Manipulation of Yeasts (continued) ======================== most yeasts use oxidatively. The genetic lesion for respiratory deficiency can be located within the mitochondrial genome (forming rho- or mit- mutants) or in the nuclear genome (forming pet- mutants). Unless the lesion for respiratory deficiency is clearly defined (which is often not the case), fusion of an RD cell with a respiratory-competent cell may C) produce a recombinant that is due to either nuclear or mitochondrial complementation, a fact that may be impor­ a-b+ tant in the cell's oxidative processes. However, respiratory deficiency has proven to be a suitable marker for interspecific 1Enzyme Trealmenl crosses involving S. cerevisiae and Candida uti/is (Perez et aI., 1984; Richard and van Broock, 1984) and S. cerevisiae and S. diastaticus (Figueroa et aI., 1984; 1985; Spencer et aI., 1980). If the end result is an increase in ethanol production, the source of the lesion, at least initially, may not be important. G Protoplasl 0 Transformation by Recombinant DNA Techniques Although a good deal of work has been done using proto­ \ plast fusion methods, it is obvious that successful production / of improved yeast strains has been limited. Transformation of yeast by recombinant DNA techniques is more likely to Fuse with PEG, produce stable isolates carrying a desired characteristic. However, to increase the chance of successful transformation. Electrofusion one needs to know the background of both the donor and recipient cells. Increasing the knowledge of the basic genetic background by knowing the biochemical pathways, cofactor requirements, genetic controls, and genes involved will allow for more successful genetic manipulation. The basic techniques for yeast transformation are present­ ed in Figure 2. Nuclear DNA of the donor cell is isolated, restricted with an endonuclease (such as by partial digestion with Sau3A to give a range in the sizes of pieces), and mixed with a plasmid that has been cut once by a comparable recognition-site enzyme. The mixture of DNA fragments undergoes ligation (to fuse the cut ends) and the plasmids are then inserted into an Escherichia coli host.
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