WO2000050610A1 - Genetically engineered microorganisms useful in expression of bioproducts from starch waste - Google Patents

Genetically engineered microorganisms useful in expression of bioproducts from starch waste Download PDF

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WO2000050610A1
WO2000050610A1 PCT/US2000/004658 US0004658W WO0050610A1 WO 2000050610 A1 WO2000050610 A1 WO 2000050610A1 US 0004658 W US0004658 W US 0004658W WO 0050610 A1 WO0050610 A1 WO 0050610A1
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starch
microorganism
foreign
bioproduct
yeast
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PCT/US2000/004658
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French (fr)
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Jianwei Gao
Brian S. Hooker
Rodney S. Skeen
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Battelle Memorial Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor

Definitions

  • the present invention relates generally to a method of using certain microorganisms to express useful industrial foreign bioproducts directly from starch wastes.
  • certain yeasts and/or fungi can be cultured with a carbon source comprising only a starch waste by-product, in order to express desired bioproteins or biocatalysts.
  • the present invention also relates to a genetically engineered starch-degrading microorganism, which is cultured with the sole carbon source being a waste starch stream.
  • the microorganism is preferably either a yeast or mold, and the foreign bioproducts expressed by the microorganism are preferably either proteins or catalysts, or mixtures thereof.
  • potato waste is rich in starch and other nutritional ingredients that can be directly used as a growth medium by bacteria and fungi.
  • the production and sale of proteins have increased over the past twenty years.
  • enzyme biocatalysts active proteins that facilitate chemical and biochemical reactions under mild temperature and pressure conditions, are experiencing increasing industrial application.
  • Biocatalysts such as glucose isomerase and glucoamylase, and protease have been widely used in corn syrup and detergent industries, respectively.
  • Other enzymes such as cellobiohydrolase, xylanase, chitinase, and pectinase are becoming more important in industrial processing (Rotheim, 1994).
  • yeast strains have been used for the production of transgenic proteins, including pharmaceuticals and industrial enzymes.
  • Yeast strains such as Pichia pastoris have been genetically altered for the expression of human tumor necrosis factor (TNF), epidermal growth factor, invertase, lysozyme, ⁇ -galactosidase, xylanase, among others (Sreekrishna et al., 1989; Clare et al., 1991 ; Tschopp et al., 1987; Digan et al., 1989; Li and Ljungdahl, 1994; Zhu et al., 1995).
  • all reported yeast transformants use processed chemical substrates (including glucose, glycerol and methanol) as the primary carbon source for cell growth.
  • yeast in the genus Schwanniomyces can hydrolyze starch to glucose as the result of the production of two extracellular enzymes, namely amylase ( ⁇ -1 ,4-glucan 4-glucanohydrolase E.C.3.2.1.1) and glucoamylase (syn. amyloglucosidase) ( ⁇ -1 ,4-glucanglucohydrolase, E.C.3.1.2.).
  • amylase ⁇ -1 ,4-glucan 4-glucanohydrolase E.C.3.2.1.1
  • glucoamylase syn. amyloglucosidase
  • TNF tumor necrosis factor
  • Pichia pastoris is able to synthesize large amounts of recombinant protein (up to 30% of the total cell protein) and its glycosylation abilities are very similar to those of animal cells (Sreekrishna et al., 1989; Cregg et al., 1993), though the glycosylation in another yeast strain, Saccharomyces cerevisiae is different from that of an animal (Cregg et al., 1993).
  • the doubling time of yeast strain is usually one to three hours, and can be grown in either a batch or a continuous culture as easily as bacteria.
  • Natural yeast strains have been identified that can use starch as a primary growth substrate via complete or partial enzymatic hydrolysis (Sills and Stewart, 1982; Hongpattarakere and H-Kittikun, 1995). These yeast strains include Saccharomycopsis fibuligera, Schwanniomyces castellii, and Saccharomyces diastaticus (Lemmel et al., 1980; Sills and Stewart, 1982; Hongpattarakere and H-Kittikun, 1995). A fusion yeast cell strain of Saccharomyces diastaticus and Saccharomyces cerevisiae could degrade 60% of starch present in culture media within two days (Kim et al., 1988).
  • the present invention comprises a process for the manufacture of useful industrial foreign bioproducts from a microorganism utilizing starch waste byproducts as the predominant carbon source.
  • the process comprises the steps of: (1 ) selecting the microorganism from the group of starch-degrading fungi capable of utilizing starch as the predominant carbon source; (2) genetically engineering the microorganism by insertion of a plasmid expression vector into the microorganism that will selectively express the foreign bioproduct; (3) feeding a waste starch stream to the microorganism as its predominant carbon source; and (4) culturing the microorganism to produce the foreign bioproduct, and thereafter separating the bioproduct therefrom.
  • the invention comprises genetically-modified, starch- biotransforming yeast containing a specific DNA sequence.
  • the specific DNA sequence can comprise any gene, combination of genes, gene fragments, or combination of gene fragments and may code for proteins or peptides.
  • the specific DNA sequence can further code for mRNA segments where the mRNA segment encodes antisense for genes involved in metabolic pathways. Further, the specific DNA sequence can be a naturally derived or synthetic sequence.
  • the present invention is also directed to compositions useful for producing proteins, peptides, metabolites, or metabolic intermediates that comprise genetically-modified, starch-biotransforming yeast comprising and containing a specific DNA sequence.
  • the specific DNA sequence can comprise any gene, combination of genes, gene fragments, or combination of gene fragments and may code for proteins or peptides.
  • the specific DNA sequence can further code for mRNA segments where the mRNA segment encodes antisense for genes involved in metabolic pathways. Further, the specific DNA sequence can be naturally derived, or a synthetic sequence.
  • Primary substrates for the genetically-modified starch-biotransforming yeast can be either starch compounds or refined carbon glucose, sucrose, maltose, methanol, and glycerol.
  • the starch materials include any low-cost waste stream, including but not limited to those derived from potatoes, sweet potatoes, corn, wheat, cassava, rice or any other starch containing waste stream from food processing operations.
  • FIG. 1 is a graphic representation of the degradation and utilization of starch by a batch culture of a starch-utilizing Schwanniomyces castellii;
  • FIG. 2 is a graphic representation of the degradation and utilization of starch by a batch culture of a starch-utilizing Saccharomyces strain
  • FIG. 3 is a schematic representation of the process for construction of vector pGA2026
  • FIG. 4 is a schematic representation of the process for construction of vector pGA2028
  • FIG. 5 is a table illustrating transgenic glucuronidase activity in transformed starch degrading Saccharomyces strain
  • FIG. 6 is a graphic representation of glucuronidase expression under induction conditions in both potato and corn-starch media;
  • FIG. 7 is a schematic representation of the process for construction of vector pGA2033
  • FIG. 8 is a table illustrating the antibiotic aureobasidin resistance of transformed starch degrading yeast strains, Saccharomyces, Schwanniomyces castellii, and
  • FIG. 9 is a schematic representation of the process for construction of vector pGA2035.
  • FIG. 10 is a table illustrating transgenic E1 endoglucanase in transformed starch degrading Saccharomyces strain. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is not limited to specifically identified components or attributes. It is to be understood that the specific embodiments set forth herein are to be considered exemplary only, as specific embodiments of the invention. That is, the invention disclosed herein is not limited to a particular microorganism, although fungi (yeast and mold) have been demonstrated to be effective herein. Likewise, while particular plasmid expression vectors may be disclosed herein, the invention is not to be considered limited to a particular vector, or a specific method of insertion of such vector. In its broadest embodiment, the present invention comprises the insertion of an expression vector for a particular protein product into a starch- degrading microorganism for the purpose of producing commercial quantities of the protein as a foreign bioproduct of the microorganism.
  • the invention includes genetically-modified starch-biotransforming microorganisms having a specific DNA sequence and specifically, compositions useful for producing proteins, peptides, metabolites, or metabolic intermediates which comprise genetically-modified, starch-biotransforming yeast comprising and containing a specific DNA sequence.
  • the DNA sequence in either case can comprise any gene, combination of genes, gene fragments, or combination of gene fragments and may code for proteins or peptides.
  • the specific DNA sequence can further code for mRNA segments where the mRNA segment encodes antisense for genes involved in metabolic pathways. Further, the specific DNA sequence can be a naturally derived or synthetic sequence.
  • GENE a discrete chromosomal region that is responsible for a discrete cellular product.
  • GENETICALLY-MODIFIED a term that denotes the insertion and expression of DNA within microorganisms that did not previously exist in such microorganism.
  • MICROORGANISM a member of one of the following classes: bacteria, fungi, protozoa, and viruses. Without intending to be limited hereby, Applicants have found that, among others, Saccharomycopsis fibuligera, Saccharomyces cerevisiae, Saccharomyces diastaticus, Trichosporon pullulans, Hansenula anomala, Lipomyces starkeyi, Candida albicans, Schwanniomyces alluvius,
  • Pichia burtonii Schwanniomyces castellii
  • Candida oregonensis are useful in the present invention.
  • STARCH a linear or branched polysaccharide composed of glucose subunits lined by ⁇ (1 - 4) and/or ⁇ (1 ->6) bonds.
  • STARCH-BIOTRANSFORMATION the ability of microorganisms to use starch materials as a primary substrate in catabolism and/or other reactions.
  • yeast and bacteria species are known to posses a natural ability to rapidly degrade starch, such as Schwanniomyces castellii, Saccharomycopsis fibuligera, Trichosporon pullulans, Hansenula anomala, and Lipomyces starkeyi. Because yeasts and fungi are eukaryotes, they are more closely related to animals than are bacteria and therefore have many of the same post- translational processes that are necessary for forming functional proteins. However, there has been no indication that these starch degrading eukaryotes have been successfully genetically engineered to express foreign proteins or peptides when the culture utilizes waste starch as the sole, or primary, carbon source.
  • FIG. 1 illustrates a typical batch culture of Schwanniomyces castellii in a 250 ml flask with a 50 ml starch culture medium supplemented with appropriate nitrogen source. The initial concentration of waste potato starch in the culture medium was 16 g/l. The culture was inoculated with 10% of overnight seed culture that was grown in the same starch medium. The culture was incubated at 30°C on an orbital shaker operated at 150 rpm.
  • Results show that Schwanniomyces castellii degraded over 90% of the added starch within 12 hours, during which a starch hydrolysate, glucose, was formed at a concentration of about 0.9 g/l and consumed subsequently by the culture within the next 10 hours. Biomass growth showed as an increase of optical density (OD) from 0.8 to 2.1 in the culture.
  • OD optical density
  • the data shown here indicates that waste potato starch can be completely degraded and utilized by starch biotransforming yeast, as exemplified by Schwanniomyces castellii, within a short period of time. Similar results are obtained when other starch utilizing microorganisms (noted above) are used.
  • Starch used in the test was potato starch waste obtained from a local potato processing plant. Potato starch waste was used as the primary carbon source for cell growth in the culture.
  • Figure 2 shows a typical batch culture of the Saccharomyces strain in a 250-ml flask with 50-ml starch culture medium supplemented with appropriate nitrogen source. The initial concentration of waste potato starch in the culture medium was 15 g/l. The culture was inoculated with 10% of overnight seed culture which was grown in the same starch medium. The culture was incubated at 30°C on an orbital shaker operated at 150 rpm.
  • Results show that the Saccharomyces strain degraded about 40 % of the added starch within 32 hours, during which a starch hydrolysate, glucose, was formed at a concentration of about 0.5 g/l and consumed subsequently by the culture within the next 18 hours. Biomass growth showed as an increase of optical density (OD) from 0.9 to 2.1 in the culture.
  • OD optical density
  • Plasmid vectors were constructed to effectively transform the starch- degrading Saccharomyces strain and select the transformants after transformation.
  • a plasmid vector pYES2 (5,857 bp) was obtained from Invitrogen, Inc, Carlsbad, CA.
  • the plasmid was modified to form plasmid pGA2026 (4,961 bp) by replacing the ampicillin resistance and URA3 genes at Nhe I and BspH I restriction enzyme sites with an antibiotic Zeocin resistance gene (1 ,186 bp) of a plasmid vector pGAPZ ⁇ -A, also obtained from Invitrogen Inc.
  • the plasmid vector pGA2026 enables the selection of transformed Saccharomyces strains without using uracil-deficient selection medium.
  • the plasmid pGA2026 contains an expression cassette for foreign gene expression under the control of an inducible galactokinase promoter (GAL1), a T7 RNA promoter and a Tcyd terminator.
  • GAL1 galactokinase promoter
  • the plasmid also contains a 2 ⁇ m DNA fragment for plasmid replication in Saccharomyces strains, a ColEI origin for plasmid replication during gene manipulation in E. coli strains, a f1 phage origin, and an antibiotic Zeocin resistance gene for both yeast and E. coli selection during gene manipulation after transformation.
  • An EasySelect Expression Kit (invitrogen, Inc.) was used for preparing competent yeast cells, which were subsequently used for the transformation of pGA2028.
  • the transformed yeast cells were plated onto YPD agar medium plate containing 1.0% glucose, 0.5% yeast extract, 1.0% peptone, and 200 mg/l antibiotic Zeocin. After a three-day incubation period at 30°C, transformed yeast colonies were obtained on the selective culture plate.
  • yeast colonies were streak-purified on fresh selective YPD agar plates and single colonies were used in batch cultures for glucuronidase expression.
  • the yeast colonies were first grown aerobically in YPD medium for 16 hours.
  • the propagated biomass was transferred into production medium containing promoter activity inducer, galactose for glucuronidase expression under aerobic condition.
  • yeast biomass was harvested and intracellular protein was extracted using the glass-bead disintegrating method.
  • the extraction buffer contains 50 mM sodium phosphate at a pH 7.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM beta- mercaptoethanol, and 0.1 % triton X-100. After cell disruption, the sample was centrifuged at 20,000xg in a centrifuge for 5 minutes. The supernatant was saved for both protein and glucuronidase activity assays.
  • PMSF phenylmethylsulfonyl fluoride
  • the extracted protein samples were assayed for protein concentration using a BioRad protein assay reagent (Bio-Rad Laboratories, Hercules, CA) and glucuronidase activity using an enzymatic reaction in which a substrate 4-methylumbelliferyl-beta-D- glucuronide (MUG) can be hydrolyzed by glucuronidase to a fluorescent compound 4-methylumbelliferone.
  • a substrate 4-methylumbelliferyl-beta-D- glucuronide MUG per minute at 37 °C.
  • the specific activity of glucuronidase is calculated as the units of glucuronidase per milligram of total protein in the sample.
  • Fig. 5 illustrates the results of glucuronidase specific activities of ten different transformed clones.
  • Glucuronidase can be highly expressed in the transgenic yeast host under the control of GAL1 promoter. The highest specific activity obtained in the culture was 10,057 units per mg of extracted intracellular protein.
  • Starch medium was used to cultivate transformed yeast clones for glucuronidase expression.
  • Corn starch and waste potato starches were used.
  • the culture medium is a sugar-free medium and contains 1.0% corn or potato starch and other growth nutrients.
  • 2-day aerobic culture in the starch medium the biomass was collected and glucuronidase was induced under aerobic condition in a production medium primarily containing 2% galactose as inducer.
  • Cells were harvested periodically and intracellular protein was extracted in the extraction buffer.
  • Fig. 6 shows the results of glucuronidase expression during different inducing periods. After 4-hour induction, the glucuronidase activity could reach 13,396 units/mg of extracted intracellular protein. The glucuronidase activity leveled off thereafter, indicating stable glucuronidase expression in the cultures.
  • a chromosomal integrating vector was also used for expression vector construction and transformation for starch degrading yeast strains. Chromosomal integration vectors incorporate the desired gene into the yeast chromosome based on the underlying principle that linearized plasmid DNA fragments are efficiently repaired during yeast transformation by recombination with a homologous DNA restriction fragments.
  • a yeast chromosomal integrating vector pAUR 101 was obtained from PanVera Corporation in Madison, Wl.
  • the expression cassette containing GAL1 promoter, glucuronidase gene and Tcyd terminator was cloned out from pGA2028 by polymerase chain reaction (PCR) technique using the following primers: 5' end forward primers:
  • the glucuronidase expression cassette was adapted with Kpn I restriction enzyme sites in both 5' and 3' ends of the DNA sequence. After digested with Kpn I restriction enzyme, the expression cassette was cloned into pAUR101 vector at Kpn I site to form plasmid pGA2033 as shown in Fig. 7.
  • the vector pGA2033 contains the expression cassette for glucuronidase expression upon transformation to starch-degrading yeast strains, an antibiotic, aureobasidin resistance gene for yeast transformant selection and chromosomal integration, an ampicillin resistance gene for E. coli transformant selection during DNA manipulation, and an E. coli plasmid replication origin ori.
  • the starch-degrading yeast strains Saccharomyces and Schwanniomyces castellii were used for plasmid transformation test. Overnight cultures of Saccharomyces and Schwanniomyces castellii were used for preparing competent cells using an EasySelect Expression Kit (Invitrogen, Inc.). Plasmid DNA of pGA2033 was linearized with Stu I restriction enzyme site, and subsequently used for the transformation of Saccharomyces and Schwanniomyces castellii. Upon transformation, the transformed yeast cells were plated onto YPD agar medium plate containing 1.0% glucose, 0.5% yeast extract, 1.0% peptone, and 0.2 - 0.4 mg/l antibiotic aureobasidin.
  • EXAMPLE 7 Plasmid vector construction for E1 endoglucanase expression
  • the starch-degrading yeast strain Saccharomyces was also used to express a thermostable cellulolytic enzyme gene, endoglucanase from Acidothermus cellulolyticus (Tucker, et al.,1989; Baker et al., 1994; Laymon et al., 1995).
  • the beta-1 ,4-endoglucanase (E1) precursor gene was obtained from Steven R. Thomas of the National Renewable Energy Laboratory in Golden, CO.
  • the mature endoglucanase gene (1 ,562 bp) was cloned out by PCR from the beta-1 ,4-endoglucanase precursor gene and adapted with a Hind III restriction enzyme site and an initiation codon ATG at the 5' end of the gene and a Sac I restriction enzyme site at the 3' end using the following primers:
  • the PCRed mature E1 gene was digested with Hind III and Sac I restriction enzymes and cloned into plasmid vector pGA2026 at Hind III and Sac I restriction enzyme sites to form pGA2035 (6,518 bp) as shown in Fig. 9.
  • the starch-degrading yeast strain Saccharomyces was used for plasmid transformation and E1 endoglucanase expression.
  • An EasySelect Expression Kit (Invitrogen, Inc.) was used for preparing competent yeast cells, which were subsequently used for the transformation of pGA2035.
  • the transformed yeast cells were plated onto YPD agar medium plate containing 1.0% glucose, 0.5% yeast extract, 1.0% peptone, and 200 mg/l antibiotic Zeocin. After a three-day incubation period at 30°C, transformed yeast colonies were obtained on the selective culture plate.
  • yeast colonies were streak-purified on fresh selective YPD agar plates and single colonies were used in batch cultures for E1 endoglucanase expression.
  • the yeast colonies were first grown aerobically in YPD medium for 16 hours.
  • the propagated biomass was transferred into production medium containing promoter activity inducer, galactose for E1 endoglucanase expression under aerobic condition.
  • yeast biomass was harvested and intracellular protein was extracted using the glass-bead disintegrating method.
  • the extraction buffer contains 50 mM sodium phosphate pH 7.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM beta-mercaptoethanol, and 0.1% triton X-100.
  • the sample was centrifuged at 20,000 ⁇ g in a centrifuge for 5 minutes. The supernatant was saved for both protein and E1 endoglucanase activity assays.
  • the extracted protein samples were assayed for protein concentration using the BioRad protein assay reagent and E1 endoglucanase activity using an enzymatic reaction in which a substrate 4-methylumbelliferyl-beta-D-celIobioside (MUC) can be hydrolyzed by E1 endoglucanase to a fluorescent compound 4- methylumbelliferone.
  • MUC 4-methylumbelliferyl-beta-D-celIobioside
  • E1 endoglucanase activity is defined as the amount of E1 endoglucanase that produces one pmole of 4-methylumbelliferone (MU) from MUC per minute at 55°C.
  • the specific activity of E1 endoglucanase is calculated as the units of E1 endoglucanase per milligram of total protein in the sample.
  • Fig. 10 shows the results of E1 endoglucanase specific activities of twelve different transformed clones. The highest specific activity obtained in the culture was 1 ,724 units per mg of extracted intracellular protein.

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Abstract

The production of foreign bioproducts from a starch-utilizing microorganisms is disclosed. By genetically modifiying the starch-utilizing microorganism, it is enabled to rapidly and completely metabolize a carbon source comprised predominantly or exclusively of starch, while producing a commercially valuable bioproduct. Starch-degrading strains of yeast are preferably utilized, and are provided with expression vectors for the expression of foreign proteins, peptides or biocatalysts.

Description

GENETICALLY ENGINEERED MICROORGANISMS USEFUL IN EXPRESSION OF BIOPRODUCTS FROM STARCH WASTE
FIELD OF THE INVENTION
The present invention relates generally to a method of using certain microorganisms to express useful industrial foreign bioproducts directly from starch wastes. For example, certain yeasts and/or fungi can be cultured with a carbon source comprising only a starch waste by-product, in order to express desired bioproteins or biocatalysts.
More specifically, the present invention also relates to a genetically engineered starch-degrading microorganism, which is cultured with the sole carbon source being a waste starch stream. The microorganism is preferably either a yeast or mold, and the foreign bioproducts expressed by the microorganism are preferably either proteins or catalysts, or mixtures thereof.
BACKGROUND OF THE INVENTION
Industrial sources, and particularly the agricultural and food industry, dispose of a significant amount of biomass by-products during the manufacturing of commercial products. U.S. fruit and vegetable processors currently generate approximately 7.4 million dry metric tons of low-value byproduct and treat more than 300 million cubic meters of high BOD waste waters each year. Both of these byproduct streams are usually rich in starch. Such wastes, rich in carbohydrates and other nutritional factors, can be used as a growth medium by bacteria and fungi. For example, a typical potato processing plant (such as a plant for the production of frozen french-fried potatoes) can produce as much as 340,000 pounds of biomass byproduct per day, in the form of wet potato waste. Such biomass byproducts are typically sold as livestock feed or even disposed as waste. In particular, potato waste is rich in starch and other nutritional ingredients that can be directly used as a growth medium by bacteria and fungi. The production and sale of proteins have increased over the past twenty years. For example, enzyme biocatalysts, active proteins that facilitate chemical and biochemical reactions under mild temperature and pressure conditions, are experiencing increasing industrial application. Biocatalysts such as glucose isomerase and glucoamylase, and protease have been widely used in corn syrup and detergent industries, respectively. Other enzymes such as cellobiohydrolase, xylanase, chitinase, and pectinase are becoming more important in industrial processing (Rotheim, 1994). The total world market for industrial enzymes is currently approaching $2.0 billion (Grant, 1994; Hodgson, 1994). However, deployment of these enzymes for practical processing and manufacturing is still limited due to the high production costs. Nutrient requirements for enzyme producing organisms in presently-employed processes may comprise as much as 50% of this cost.
Genetically modified yeast strains have been used for the production of transgenic proteins, including pharmaceuticals and industrial enzymes. Yeast strains such as Pichia pastoris have been genetically altered for the expression of human tumor necrosis factor (TNF), epidermal growth factor, invertase, lysozyme, α-galactosidase, xylanase, among others (Sreekrishna et al., 1989; Clare et al., 1991 ; Tschopp et al., 1987; Digan et al., 1989; Li and Ljungdahl, 1994; Zhu et al., 1995). However, all reported yeast transformants use processed chemical substrates (including glucose, glycerol and methanol) as the primary carbon source for cell growth.
It has been shown that yeast in the genus Schwanniomyces can hydrolyze starch to glucose as the result of the production of two extracellular enzymes, namely amylase (α-1 ,4-glucan 4-glucanohydrolase E.C.3.2.1.1) and glucoamylase (syn. amyloglucosidase) (α-1 ,4-glucanglucohydrolase, E.C.3.1.2.). As disclosed in U.S. Patent No. 5,151,354, recombinant DNA technology is used to clone genes coding for α-amylase and glucoamylase from genus Schwanniomyces into a host Saccharomyces yeast. After transformation, the cloned genes coding for amylolytic enzymes are expressed in the host, and the gene products are secreted into the medium. Similarly, in U.S. Patent No. 5,364,782, using random mutagenesis, α- amylase genes are isolated, with the mutated genes expressed in a host organism using a suitable vector system.
As reported in U.S. Patent No. 5,002,876, the gene coding for human tumor necrosis factor (TNF) has been cloned and expressed in E. coli. Because human TNF has a number of commercially useful biological activities, TNF is produced in yeast by culturing yeast cells transformed with DNA constructs comprising TNF coding regions under the control of yeast regulatory regions. The use of yeast for producing transgenic prokaryotic and eukaryotic heterologous proteins has the added advantage that yeast and filamentous fungus are microbial eukaryotes and they are more closely related to animal cells. Hence, yeasts possess the molecular genetic manipulation and growth characteristics of prokaryotic organisms together with the subcellular machinery for performing eukaryotic post-translational protein modification. For example, Pichia pastoris is able to synthesize large amounts of recombinant protein (up to 30% of the total cell protein) and its glycosylation abilities are very similar to those of animal cells (Sreekrishna et al., 1989; Cregg et al., 1993), though the glycosylation in another yeast strain, Saccharomyces cerevisiae is different from that of an animal (Cregg et al., 1993). In addition, the doubling time of yeast strain is usually one to three hours, and can be grown in either a batch or a continuous culture as easily as bacteria.
Natural yeast strains have been identified that can use starch as a primary growth substrate via complete or partial enzymatic hydrolysis (Sills and Stewart, 1982; Hongpattarakere and H-Kittikun, 1995). These yeast strains include Saccharomycopsis fibuligera, Schwanniomyces castellii, and Saccharomyces diastaticus (Lemmel et al., 1980; Sills and Stewart, 1982; Hongpattarakere and H-Kittikun, 1995). A fusion yeast cell strain of Saccharomyces diastaticus and Saccharomyces cerevisiae could degrade 60% of starch present in culture media within two days (Kim et al., 1988). In addition, other natural Saccharomyces species can ferment starch and dextrin to ethanol (Laluce et al, 1988). For the past two decades, the starch-biotransforming yeast technology was mostly used in cattle feed (single cell protein) production (Lemmel et al., 1980; Hongpattarakere and H-Kittikun, 1995). Others use this technology to improve ethanol production from starch and higher sugars (U.S. Patent Nos. 4,769,324; Pirselova et al., 1993; Ryu et al., 1994).
BACKGROUND REFERENCES
1. Rotheim P. 1994. The enzyme industry: Industrial and chemical applications. Report by Business Communication Company, Inc.
2. Grant R. 1994. Enzyme's future looks bright, as range improves and expands. Pulp & Paper Internal 36:20-21
3. Hodgson J. 1994. The changing bulk biocatalyst market: recombinant DNA techniques have changed bulk enzyme production dramatically. Bio/technology 12:789-790.
4. Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P, Fish W, Fuke M, Holden K, Phelps D, Wood P, Parker K. 1989. High level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry 28:4117-4125.
5. Clare JJ, Romanos MA, Rayment FB, Rowedder JE, Smith MA, Payne MM, Sreekrisna K, Henwood CA. 1991. Production of mouse epidermal growth factor in yeast: high-level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105:205-212.
6. Tschopp JF, Sverlow G, Kosson R, Craig W, Grinna L. 1987. High-level secretion of glycosylated invertase in the methylotrophic yeast Pichia pastoris. Bio/Technology 5:1305-1308.
7. Digan ME, Lair SV, Brierley RA, Siegel RS, Williams ME, Ellis SB, Kellaris PA, Provow SA, Craig WS, Velicelebi G, Harpold MM, Thill GP. 1989. Continuous production of a novel lysozyme via secretion from the yeast, Pichia pastoris. Bio/Technology 7: 160-164. 8. Li XL, Ljungdahl LG. 1994. Cloning, sequencing, and regulation of a xylanase gene from the fungus Aureobasidium pullulans Y2311-1. Appl Environ Microbiol. 60:3160-3166. 9. Zhu A, Monahan C, Zhang Z, Hurst R, Leng L, Goldstein J. 1995. High-level expression and purification of coffee bean alpha-galactosidase produced in the yeast Pichia pastoris. Arch. Biochem. Biophys. 324:65-70.
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12. U.S. Patent No. 5,002,876, 1991.
13. Cregg JM, Vedvick TS, Raschke WC. 1993. Recent advances in the expression of foreign genes in Pichia pastoris
14. Lemmel SA, Heimsch RC, Korus RA. 1980. Kinetics of growth and amylase production of Saccharomycopsis fibuligera on potato processing. Appl
Environ Microbiol. 39:387-393.
15. Sills AM, Stewart GG. 1982. Production of amylolytic enzymes by several yeast species. J. Inst. Brew. 88:313-316.
16. Hongpattarakere T, H-Kittikun A. 1995. Optimization of single-cell-protein production from cassava starch using Schwanniomyces castellii. J.
Microbiol. Biotech nol. 11 :607-609.
17. Kim K, Park CS, Mattoon JR. 1988. High-efficiency one-step starch utilization by transformed Saccharomyces cells which secrete both yeast glucoamylase and mouse alpha amylase. Appl Environ Microbiol. 54:966-971.
18. Laluce C, Bertolini MC, Ernandes JR, Martini AV, Martini A. 1988. New amylolytic yeast strains for starch and dextrin fermentation. Appl. Environ. Microbiol. 54:2447-2451.
19. U.S. Patent No.4,769,324, 1988. 20. Pirselova K, Smogrovicova D. Balaz S. 1993. Fermentation of starch to ethanol by a co-culture of Saccharomycopsis fibuligera and Saccharomyces cerevisiae. World J. Microbiol. Biotechnol. 9:338-341. 21. Ryu YW, Ko SH, Byun SY, Kim C. 1994. Direct alcohol fermentation of starch by a derepressed mutant of Schwanniomyces castellii. Biotechnol. Lett. 16:107-112. SUMMARY OF THE INVENTION
The present invention comprises a process for the manufacture of useful industrial foreign bioproducts from a microorganism utilizing starch waste byproducts as the predominant carbon source. The process comprises the steps of: (1 ) selecting the microorganism from the group of starch-degrading fungi capable of utilizing starch as the predominant carbon source; (2) genetically engineering the microorganism by insertion of a plasmid expression vector into the microorganism that will selectively express the foreign bioproduct; (3) feeding a waste starch stream to the microorganism as its predominant carbon source; and (4) culturing the microorganism to produce the foreign bioproduct, and thereafter separating the bioproduct therefrom.
More specifically, the invention comprises genetically-modified, starch- biotransforming yeast containing a specific DNA sequence. The specific DNA sequence can comprise any gene, combination of genes, gene fragments, or combination of gene fragments and may code for proteins or peptides. The specific DNA sequence can further code for mRNA segments where the mRNA segment encodes antisense for genes involved in metabolic pathways. Further, the specific DNA sequence can be a naturally derived or synthetic sequence.
The present invention is also directed to compositions useful for producing proteins, peptides, metabolites, or metabolic intermediates that comprise genetically-modified, starch-biotransforming yeast comprising and containing a specific DNA sequence. The specific DNA sequence can comprise any gene, combination of genes, gene fragments, or combination of gene fragments and may code for proteins or peptides. The specific DNA sequence can further code for mRNA segments where the mRNA segment encodes antisense for genes involved in metabolic pathways. Further, the specific DNA sequence can be naturally derived, or a synthetic sequence. Primary substrates for the genetically-modified starch-biotransforming yeast can be either starch compounds or refined carbon glucose, sucrose, maltose, methanol, and glycerol. The starch materials include any low-cost waste stream, including but not limited to those derived from potatoes, sweet potatoes, corn, wheat, cassava, rice or any other starch containing waste stream from food processing operations.
Accordingly, it is an object of the present invention to produce foreign proteins using genetically engineered starch-utilizing microorganisms directly from starch materials, which comprise the primary carbon source.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphic representation of the degradation and utilization of starch by a batch culture of a starch-utilizing Schwanniomyces castellii;
FIG. 2 is a graphic representation of the degradation and utilization of starch by a batch culture of a starch-utilizing Saccharomyces strain;
FIG. 3 is a schematic representation of the process for construction of vector pGA2026;
FIG. 4 is a schematic representation of the process for construction of vector pGA2028;
FIG. 5 is a table illustrating transgenic glucuronidase activity in transformed starch degrading Saccharomyces strain; FIG. 6 is a graphic representation of glucuronidase expression under induction conditions in both potato and corn-starch media;
FIG. 7 is a schematic representation of the process for construction of vector pGA2033;
FIG. 8 is a table illustrating the antibiotic aureobasidin resistance of transformed starch degrading yeast strains, Saccharomyces, Schwanniomyces castellii, and
Saccharomycopsis fibuligera.
FIG. 9 is a schematic representation of the process for construction of vector pGA2035;
FIG. 10 is a table illustrating transgenic E1 endoglucanase in transformed starch degrading Saccharomyces strain. DETAILED DESCRIPTION OF THE INVENTION
In its broadest embodiment, the present invention is not limited to specifically identified components or attributes. It is to be understood that the specific embodiments set forth herein are to be considered exemplary only, as specific embodiments of the invention. That is, the invention disclosed herein is not limited to a particular microorganism, although fungi (yeast and mold) have been demonstrated to be effective herein. Likewise, while particular plasmid expression vectors may be disclosed herein, the invention is not to be considered limited to a particular vector, or a specific method of insertion of such vector. In its broadest embodiment, the present invention comprises the insertion of an expression vector for a particular protein product into a starch- degrading microorganism for the purpose of producing commercial quantities of the protein as a foreign bioproduct of the microorganism.
The invention includes genetically-modified starch-biotransforming microorganisms having a specific DNA sequence and specifically, compositions useful for producing proteins, peptides, metabolites, or metabolic intermediates which comprise genetically-modified, starch-biotransforming yeast comprising and containing a specific DNA sequence. The DNA sequence in either case can comprise any gene, combination of genes, gene fragments, or combination of gene fragments and may code for proteins or peptides. The specific DNA sequence can further code for mRNA segments where the mRNA segment encodes antisense for genes involved in metabolic pathways. Further, the specific DNA sequence can be a naturally derived or synthetic sequence.
In order to provide a clear and concise understanding of the specification and claims, including the scope given to such terms, the following definitions are provided:
GENE: a discrete chromosomal region that is responsible for a discrete cellular product.
GENETICALLY-MODIFIED: a term that denotes the insertion and expression of DNA within microorganisms that did not previously exist in such microorganism. MICROORGANISM: a member of one of the following classes: bacteria, fungi, protozoa, and viruses. Without intending to be limited hereby, Applicants have found that, among others, Saccharomycopsis fibuligera, Saccharomyces cerevisiae, Saccharomyces diastaticus, Trichosporon pullulans, Hansenula anomala, Lipomyces starkeyi, Candida albicans, Schwanniomyces alluvius,
Pichia burtonii, Schwanniomyces castellii, and Candida oregonensis are useful in the present invention.
STARCH: a linear or branched polysaccharide composed of glucose subunits lined by α (1 - 4) and/or α (1 ->6) bonds. STARCH-BIOTRANSFORMATION: the ability of microorganisms to use starch materials as a primary substrate in catabolism and/or other reactions.
Certain yeast and bacteria species are known to posses a natural ability to rapidly degrade starch, such as Schwanniomyces castellii, Saccharomycopsis fibuligera, Trichosporon pullulans, Hansenula anomala, and Lipomyces starkeyi. Because yeasts and fungi are eukaryotes, they are more closely related to animals than are bacteria and therefore have many of the same post- translational processes that are necessary for forming functional proteins. However, there has been no indication that these starch degrading eukaryotes have been successfully genetically engineered to express foreign proteins or peptides when the culture utilizes waste starch as the sole, or primary, carbon source.
The use of natural strains of starch-biotransforming yeast to convert agricultural wastes into transgenic proteins and peptides has several advantages over current production systems. For example, because these yeast strains can utilize waste starch as their primary carbon source, refined substrates such as glucose, sucrose and other sugars are unnecessary. Based on standard economic analyses, a process producing 7,000-10,000 kg (calculated as pure protein) of industrial enzymes can save over $600,000 per year on substrate costs alone — representing up to a 30% savings in production costs. In addition, the processed yeast biomass can be used as an industrial feedstock for pharmaceutical nucleic acid production and as a single cell protein product, thus eliminating the need to dispose of waste biomass from the process. EXAMPLE 1 Starch utilization by Schwanniomyces castellii
Batch cultures of Schwanniomyces castellii were conducted in shaker flasks. Starch used in the test was potato starch waste obtained from a local potato processing plant. Potato starch waste was used as the primary carbon source for cell growth in the culture. Figure 1 illustrates a typical batch culture of Schwanniomyces castellii in a 250 ml flask with a 50 ml starch culture medium supplemented with appropriate nitrogen source. The initial concentration of waste potato starch in the culture medium was 16 g/l. The culture was inoculated with 10% of overnight seed culture that was grown in the same starch medium. The culture was incubated at 30°C on an orbital shaker operated at 150 rpm. Results show that Schwanniomyces castellii degraded over 90% of the added starch within 12 hours, during which a starch hydrolysate, glucose, was formed at a concentration of about 0.9 g/l and consumed subsequently by the culture within the next 10 hours. Biomass growth showed as an increase of optical density (OD) from 0.8 to 2.1 in the culture. The data shown here indicates that waste potato starch can be completely degraded and utilized by starch biotransforming yeast, as exemplified by Schwanniomyces castellii, within a short period of time. Similar results are obtained when other starch utilizing microorganisms (noted above) are used.
EXAMPLE 2 Starch utilization by Saccharomyces strain
To characterize starch hydrolysis and utilization, batch cultures of starch- degrading Saccharomyces strain were conducted in shaker flasks. Starch used in the test was potato starch waste obtained from a local potato processing plant. Potato starch waste was used as the primary carbon source for cell growth in the culture. Figure 2 shows a typical batch culture of the Saccharomyces strain in a 250-ml flask with 50-ml starch culture medium supplemented with appropriate nitrogen source. The initial concentration of waste potato starch in the culture medium was 15 g/l. The culture was inoculated with 10% of overnight seed culture which was grown in the same starch medium. The culture was incubated at 30°C on an orbital shaker operated at 150 rpm. Results show that the Saccharomyces strain degraded about 40 % of the added starch within 32 hours, during which a starch hydrolysate, glucose, was formed at a concentration of about 0.5 g/l and consumed subsequently by the culture within the next 18 hours. Biomass growth showed as an increase of optical density (OD) from 0.9 to 2.1 in the culture. The data shown here indicates that waste potato starch can be utilized by starch utilizing Saccharomyces strain, though the starch degrading rate of the Saccharomyces strain is slower than that of Schwanniomyces castellii.
EXAMPLE 3 Plasmid vector construction for starch-degrading Saccharomyces strain
Plasmid vectors were constructed to effectively transform the starch- degrading Saccharomyces strain and select the transformants after transformation. A plasmid vector pYES2 (5,857 bp) was obtained from Invitrogen, Inc, Carlsbad, CA. As illustrated in Fig. 3, the plasmid was modified to form plasmid pGA2026 (4,961 bp) by replacing the ampicillin resistance and URA3 genes at Nhe I and BspH I restriction enzyme sites with an antibiotic Zeocin resistance gene (1 ,186 bp) of a plasmid vector pGAPZα-A, also obtained from Invitrogen Inc. The plasmid vector pGA2026 enables the selection of transformed Saccharomyces strains without using uracil-deficient selection medium. The plasmid pGA2026 contains an expression cassette for foreign gene expression under the control of an inducible galactokinase promoter (GAL1), a T7 RNA promoter and a Tcyd terminator. In addition, the plasmid also contains a 2 μm DNA fragment for plasmid replication in Saccharomyces strains, a ColEI origin for plasmid replication during gene manipulation in E. coli strains, a f1 phage origin, and an antibiotic Zeocin resistance gene for both yeast and E. coli selection during gene manipulation after transformation. A bacterial glucuronidase gene (1 ,923 bp) (Jefferson et al., 1987) was subsequently cloned into pGA2026 at Hind III and Sac I restriction enzyme sites to form pGA2028 (6,884 bp) as shown in Fig. 4. EXAMPLE 4 Glucuronidase expression in starch-degrading Saccharomyces strain
The selected starch-degrading yeast strain Saccharomyces obtained from James R. Mattoon of University of Colorado, Colorado Springs, CO, was used for plasmid transformation and glucuronidase expression. An EasySelect Expression Kit (invitrogen, Inc.) was used for preparing competent yeast cells, which were subsequently used for the transformation of pGA2028. Upon transformation, the transformed yeast cells were plated onto YPD agar medium plate containing 1.0% glucose, 0.5% yeast extract, 1.0% peptone, and 200 mg/l antibiotic Zeocin. After a three-day incubation period at 30°C, transformed yeast colonies were obtained on the selective culture plate.
In order to test glucuronidase gene expression, ten transformed yeast colonies were streak-purified on fresh selective YPD agar plates and single colonies were used in batch cultures for glucuronidase expression. The yeast colonies were first grown aerobically in YPD medium for 16 hours. The propagated biomass was transferred into production medium containing promoter activity inducer, galactose for glucuronidase expression under aerobic condition. After a 5-hour inducing period, yeast biomass was harvested and intracellular protein was extracted using the glass-bead disintegrating method. The extraction buffer contains 50 mM sodium phosphate at a pH 7.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM beta- mercaptoethanol, and 0.1 % triton X-100. After cell disruption, the sample was centrifuged at 20,000xg in a centrifuge for 5 minutes. The supernatant was saved for both protein and glucuronidase activity assays. The extracted protein samples were assayed for protein concentration using a BioRad protein assay reagent (Bio-Rad Laboratories, Hercules, CA) and glucuronidase activity using an enzymatic reaction in which a substrate 4-methylumbelliferyl-beta-D- glucuronide (MUG) can be hydrolyzed by glucuronidase to a fluorescent compound 4-methylumbelliferone. One unit of glucuronidase activity is defined as the amount of glucuronidase that produces one pmole of 4- methylumbelliferone (MU) from MUG per minute at 37 °C. The specific activity of glucuronidase is calculated as the units of glucuronidase per milligram of total protein in the sample. Fig. 5 illustrates the results of glucuronidase specific activities of ten different transformed clones. Glucuronidase can be highly expressed in the transgenic yeast host under the control of GAL1 promoter. The highest specific activity obtained in the culture was 10,057 units per mg of extracted intracellular protein.
Starch medium was used to cultivate transformed yeast clones for glucuronidase expression. Corn starch and waste potato starches were used. The culture medium is a sugar-free medium and contains 1.0% corn or potato starch and other growth nutrients. After 2-day aerobic culture in the starch medium, the biomass was collected and glucuronidase was induced under aerobic condition in a production medium primarily containing 2% galactose as inducer. Cells were harvested periodically and intracellular protein was extracted in the extraction buffer. Fig. 6 shows the results of glucuronidase expression during different inducing periods. After 4-hour induction, the glucuronidase activity could reach 13,396 units/mg of extracted intracellular protein. The glucuronidase activity leveled off thereafter, indicating stable glucuronidase expression in the cultures.
EXAMPLE 5 Chromosomal expression vector construction for the transformation and glucuronidase expression of starch-degrading yeast strains
A chromosomal integrating vector was also used for expression vector construction and transformation for starch degrading yeast strains. Chromosomal integration vectors incorporate the desired gene into the yeast chromosome based on the underlying principle that linearized plasmid DNA fragments are efficiently repaired during yeast transformation by recombination with a homologous DNA restriction fragments. A yeast chromosomal integrating vector pAUR 101 was obtained from PanVera Corporation in Madison, Wl. The expression cassette containing GAL1 promoter, glucuronidase gene and Tcyd terminator was cloned out from pGA2028 by polymerase chain reaction (PCR) technique using the following primers: 5' end forward primer
AGG CCT GGT ACC ATG ATC CAC TAG TAC GGA TTA GAA GCC 3' end reverse primer
AGA TCT GGT ACC GGC CGC AAA TTA AAG CCT TCG AGC GTC
The glucuronidase expression cassette was adapted with Kpn I restriction enzyme sites in both 5' and 3' ends of the DNA sequence. After digested with Kpn I restriction enzyme, the expression cassette was cloned into pAUR101 vector at Kpn I site to form plasmid pGA2033 as shown in Fig. 7. The vector pGA2033 contains the expression cassette for glucuronidase expression upon transformation to starch-degrading yeast strains, an antibiotic, aureobasidin resistance gene for yeast transformant selection and chromosomal integration, an ampicillin resistance gene for E. coli transformant selection during DNA manipulation, and an E. coli plasmid replication origin ori.
EXAMPLE 6 Transformation of starch-degrading yeast strains, Schwanniomyces, Saccharomyces, and Saccharomycopsis with chromosomal expression vector
The starch-degrading yeast strains Saccharomyces and Schwanniomyces castellii were used for plasmid transformation test. Overnight cultures of Saccharomyces and Schwanniomyces castellii were used for preparing competent cells using an EasySelect Expression Kit (Invitrogen, Inc.). Plasmid DNA of pGA2033 was linearized with Stu I restriction enzyme site, and subsequently used for the transformation of Saccharomyces and Schwanniomyces castellii. Upon transformation, the transformed yeast cells were plated onto YPD agar medium plate containing 1.0% glucose, 0.5% yeast extract, 1.0% peptone, and 0.2 - 0.4 mg/l antibiotic aureobasidin. After a three- day incubation period at 30°C, transformed yeast colonies from both Saccharomyces and Schwanniomyces castellii were obtained on the selective culture plate. The results in Fig. 8 show that the transformed colonies of Saccharomyces and Schwanniomyces castellii exhibited the antibiotic aureobasidin resistance while the none-transformed cells did not have the antibiotic resistance. Similar results were also shown in Fig. 8 for the transformation of another starch-degrading strain Saccharomycopsis fibuligera using a similar vector of pGA2033.
EXAMPLE 7 Plasmid vector construction for E1 endoglucanase expression The starch-degrading yeast strain Saccharomyces was also used to express a thermostable cellulolytic enzyme gene, endoglucanase from Acidothermus cellulolyticus (Tucker, et al.,1989; Baker et al., 1994; Laymon et al., 1995). The beta-1 ,4-endoglucanase (E1) precursor gene was obtained from Steven R. Thomas of the National Renewable Energy Laboratory in Golden, CO. The mature endoglucanase gene (1 ,562 bp) was cloned out by PCR from the beta-1 ,4-endoglucanase precursor gene and adapted with a Hind III restriction enzyme site and an initiation codon ATG at the 5' end of the gene and a Sac I restriction enzyme site at the 3' end using the following primers:
5' end forward primer
AGG CCT AAG CTT ATG GCG GGC GGC GGC TAT TGG CAC ACG 3' end reverse primer
GTC GAC GAG CTC TTA ACT TGC TGC GCA GGC GAC TGT CGG
The PCRed mature E1 gene was digested with Hind III and Sac I restriction enzymes and cloned into plasmid vector pGA2026 at Hind III and Sac I restriction enzyme sites to form pGA2035 (6,518 bp) as shown in Fig. 9.
EXAMPLE 8 Thermostable E1 endoglucanase expression in starch-degrading Saccharomyces strain
The starch-degrading yeast strain Saccharomyces was used for plasmid transformation and E1 endoglucanase expression. An EasySelect Expression Kit (Invitrogen, Inc.) was used for preparing competent yeast cells, which were subsequently used for the transformation of pGA2035. Upon transformation, the transformed yeast cells were plated onto YPD agar medium plate containing 1.0% glucose, 0.5% yeast extract, 1.0% peptone, and 200 mg/l antibiotic Zeocin. After a three-day incubation period at 30°C, transformed yeast colonies were obtained on the selective culture plate.
Twelve transformed yeast colonies were streak-purified on fresh selective YPD agar plates and single colonies were used in batch cultures for E1 endoglucanase expression. The yeast colonies were first grown aerobically in YPD medium for 16 hours. The propagated biomass was transferred into production medium containing promoter activity inducer, galactose for E1 endoglucanase expression under aerobic condition. After a 5-hour inducing period, yeast biomass was harvested and intracellular protein was extracted using the glass-bead disintegrating method. The extraction buffer contains 50 mM sodium phosphate pH 7.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM beta-mercaptoethanol, and 0.1% triton X-100. After cell disruption, the sample was centrifuged at 20,000χg in a centrifuge for 5 minutes. The supernatant was saved for both protein and E1 endoglucanase activity assays. The extracted protein samples were assayed for protein concentration using the BioRad protein assay reagent and E1 endoglucanase activity using an enzymatic reaction in which a substrate 4-methylumbelliferyl-beta-D-celIobioside (MUC) can be hydrolyzed by E1 endoglucanase to a fluorescent compound 4- methylumbelliferone. One unit of E1 endoglucanase activity is defined as the amount of E1 endoglucanase that produces one pmole of 4-methylumbelliferone (MU) from MUC per minute at 55°C. The specific activity of E1 endoglucanase is calculated as the units of E1 endoglucanase per milligram of total protein in the sample. Fig. 10 shows the results of E1 endoglucanase specific activities of twelve different transformed clones. The highest specific activity obtained in the culture was 1 ,724 units per mg of extracted intracellular protein.
While a preferred embodiment of the present invention has been shown and described, it will be apparent to those skilled in this art that many modifications may be made to the invention as shown without departing from the broader aspects of the invention. Accordingly, the scope of this invention should not be limited by the description above, but should only be considered in light of the claims appended hereto. The claims affixed hereto are intended to cover all modifications to the invention that fall within the spirit and scope of the invention. EXAMPLE REFERENCES
1. Jefferson RA, Kavanagh TA, Bevan MW. 1987. GUS fusions: beta- glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 6 (13), 3901-3907.
2. Tucker MP, Mohagheghi A, Grohmann K, Himmel ME. 1989. Ultra- thermostable cellulases from Acidothermus cellulolyticus: comparison of temperature optima with previously reported cellulases. Bio/Technology, 7:817-820.
3. Baker JO, Adney WS, Nieves RA, Thomas SR, Wilson DB, Himmel ME. 1994. A new thermostable endoglucanase Acidothermus cellulolyticus E1. Appl. Biochem. Biotechnol. 45/46:245-256.
4. Laymon RA, Himmel ME, Thomas, SR. 1995. Acidothermus cellulolyticus E 1 beta-1 ,4-endoglucanase precursor gene, complete cds. GeneBank database,
NID 988299.

Claims

We claim:
1. A process for the manufacture of useful industrial foreign bioproducts from a microorganism utilizing starch waste by-products as the predominant carbon source, comprising the steps of: a. selecting the microorganism from the group consisting essentially of starch-degrading fungi capable of utilizing starch as the predominant carbon source; b. genetically engineering the microorganism by insertion of a plasmid expression vector into the microorganism for selective expression of the foreign bioproduct of interest; c. providing starch as the predominant carbon source for culturing of the microorganism; d. culturing the genetically engineered microorganism to produce the foreign bioproduct, and thereafter separating the foreign bioproduct therefrom.
2. The process of Claim 1 , further comprising selecting the microorganism from the group consisting of Saccharomycopsis fibuligera, Saccharomyces cerevisiae, Saccharomyces diastaticus, Trichosporon pullulans, Hansenula anomala, Lipomyces starkeyi, Candida albicans, Schwanniomyces alluvius, Pichia burtonii, Schwanniomyces castellii, and Candida oregonensis.
3. The process of Claim 1 , wherein the foreign bioproduct is selected from the group comprising proteins, peptides, metabolites and metabolic intermediates.
4. The process of Claim 1 , wherein the foreign bioproduct comprises a peptide.
5. The process of Claim 1 , wherein the foreign bioproduct comprises a protein.
6. The process of Claim 1 , wherein the waste starch stream comprises process wastes selected from the group consisting of potatoes, sweet potatoes, wheat, rice, corn, and cassava.
7. The process of Claim 1 , wherein a promoter is isolated from the starch-utilizing microorganism using α-amylase gene as a DNA probe.
8. The process of Claim 7, wherein an expression vector using the isolated α-amylase gene is constructed for foreign bioproduct production.
9. The process of Claim 1 , wherein a promoter is isolated from the starch-utilizing microorganism using glucoamylase gene as a DNA probe.
10. The process of Claim 7, wherein an expression vector using the isolated glucoamylase gene is constructed for foreign bioproduct production.
11. The process of Claim 1 wherein the starch comprises a stream of starch-based waste products.
12. A process for the manufacture of foreign bioproducts from a starch- degrading microorganism comprising the steps of: a. selecting the microorganism from the group consisting of starch-degrading yeast and molds; b. genetically engineering the microorganism for selective expression of the bioproduct; c. providing waste starch as the sole carbon source for culturing of the microorganism; and d. culturing the genetically engineered microorganism to produce the bioproduct, and thereafter separating the bioproduct.
13. The process of Claim 12, further comprising selecting the microorganism from the group consisting of Saccharomycopsis fibuligera, Saccharomyces cerevisiae, Saccharomyces diastaticus, Trichosporon pullulans, Hansenula anomala, Lipomyces starkeyi, Candida albicans, Schwanniomyces alluvius, Pichia burtonii, Schwanniomyces castellii, and Candida oregonensis.
14. The process of Claim 12, further comprising genetically engineering the microorganism by inserting therein a plasmid expression vector selected for selective expression of the foreign bioproduct.
15. A genetically engineered microorganism for the manufacture of foreign bioproducts, comprising: a. a microorganism selected from the group consisting of yeast and molds; b. a plasmid expression vector for insertion into the microorganism, said vector selectively expressing the foreign bioproduct; c. a carbon source feedstock for microorganism growth comprising a waste starch stream; and d. a foreign bioproduct comprising a protein, peptide, or mixture thereof.
16. A non-naturally occurring bioproduct produced from a transgenic starch-utilizing microorganism, wherein the microorganism is cultured with a carbon source comprising a waste starch.
17. A non-naturally occurring protein produced from a transgenic starch-utilizing microorganism, wherein the microorganism is cultured with a carbon source comprised primarily of starch.
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