US007309602B2
`
`(12) United States Patent
`David
`
`(10,) Patent N0.:
`(45) Date of Patent:
`
`US 7,309,602 B2
`*Dec. 18, 2007
`
`(54)
`
`(75)
`
`COMPOSITIONS AND METHODS FOR
`PRODUCING FERMENTATION PRODUCTS
`AND RESIDUALS
`
`Inventor: Peter R. David, Palo Alto, CA (US)
`
`(73)
`
`Assigncc: Ambrolea, Inc.. Palo Alto, CA (US)
`
`(*5)
`
`Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. l54(b) by 0 days.
`
`This patent is subject to a terminal dis-
`claimer.
`
`(21)
`
`Appl. No.: 11/383,748
`
`4/2005 Thompson et al.
`6,878,860 B1
`2/2006 Stewart
`7,001,610 B2
`10/2002 Walker et al.
`2002/0155192 A1
`3/2003 Jocobson et al.
`2003/0049241 A1
`6/2003 Verser et al.
`2003/0104587 A1
`8/2003 Cordero Otero e a],
`2003/0157675 A1
`2004/0248280 A1 * 12/2004 Bolla et al.
`............ .. 435/254.2
`2006/0008546 A1
`1/2006 de Souza et al.
`2006/0039955 A1
`2/2006 Messinan et al.
`2006/0057251 A1
`3/2006 1)awley et a1.
`2006/0286645 A1
`12/2006 11 et al.
`2007/0037267 A1 *
`2/2007 Lewis et al.
`.............. .. 435/161
`
`FOREIGN PATENT DOCUMENTS
`0450430 A2
`10/1991
`0450430 A3
`10/1991
`W0 2002/03812 A2
`1/2002
`W0 2002/03812 A3
`1/2002
`V\’O 2005/118795 A2
`12/2005
`V\’O 2005/118795 A3
`12/2005
`
`EP
`EP
`W0
`W0
`VVO
`VVO
`
`OTI IER PUBLICATIONS
`
`Parekh et a1.. Pilot-scale production of bumnol by Clostridium
`beijerinckii BAl0l using low-cost fermentation medium based on
`com steep water, Appl Microbiol Biotechnol. 1999 512152-l57.*
`Romanos et al., Yeast vol. 8, Issue 6. pp. 423-488. 1992.*
`Zeikus J.G. Chemical and fuel production by anaerobic bacteria.
`Annu Rev Microbiol. 1980;34:423-64.*
`Birkelo, et al. The energy content of wet corn distillers grains for
`lactating dairy cows. .1 Dairy Sci. 2004; 87(6): 1815-9.
`Casey, et al. High Gravity Brewing" Effects of Nutrition on Yeast
`Compostition, Fcrmcntativc Ability, and Alcohol Production Appl
`Environ Microbiol. 1984: 48(3):639-646.
`
`(Continued)
`
`Joseph Woitach
`Primary Examiner
`,4ssistar2t F_mminer—Maria 1.eavit
`(74) Attorney, Agent, or Firm—Wilson Sonsini Goodrich &
`Rosati
`
`U.S. PATENT DOCUMENTS
`
`(57)
`
`ABSTRACT
`
`4,567,145 A
`4,828,846 A
`5,145,695 A
`5,151,354 A *
`5,219,596 A
`5,480,805 A
`5,530,188 A
`5,656,319 A
`5,656,472 A
`5,766,925 A
`6,410,755 B1
`6,538,182 B1
`6,774,284 B1
`6,849,782 B2
`6,855,529 B2
`6,867,237 B1
`
`............. .. 426/11
`
`1/1986 Faber et al.
`5/1989 Rasco et al.
`9/1992 Smith et al.
`9/1992 Strasser et al.
`6/1993 Smith et al.
`1/1996 Wolf et :11.
`6/1996 Ausich et :11.
`8/1997 Barclay
`8/1997 Ausich et al.
`6/1998 Sugirnoto ct al.
`6/2002 Millis et al.
`3/2003 Thompson et 211.
`8/2004 Thompson et 211.
`2/2005 Thompson et al.
`2/2005 Thompson et a1.
`3/2005 Taylor et a1.
`
`The present invention provides compositions and methods
`designed to increase value output of 21 fermentation reaction,
`111 particular,
`the present
`invention provides a business
`method of increasing value output of a fermentation plant.
`The present invention also provides an modified fermentation
`residual of higher commercial value. Also provided in the
`present invention are complete animal feeds, nutritional
`supplements comprising the subject ferment residuals. Fur-
`ther provided by the present
`invention is a method of
`performing fermentation, a modified fermentative 1nicroor-
`ganism and a genetic vehicle for modifying such microo-
`ganism.
`
`24 Claims, 2 Drawing Sheets
`
`(22)
`
`Filed:
`
`May 16, 2006
`Prior Publication Data
`
`US 2007/0243592 A1
`
`Oct. 18, 2007
`
`Related U.S. Application Data
`Provisional application No. 60/797,431, filed on May
`3, 2006, provisional application No. 60/744,833, filed
`on Apr. 13, 2006.
`Int. Cl.
`(2006.01)
`CI2N 1/19
`(2006.01)
`A23B 7/10
`(2006.01)
`/1233 7/154
`(2006.01)
`CI2P 7/06
`U.S. Cl.
`................. .. 435/254.2; 435/161; 435/256;
`426/53; 426/56
`Field of Classification Search ................... .. None
`See application file for complete search history.
`References Cited
`
`(65)
`
`(60)
`
`(51)
`
`(52)
`
`(58)
`
`(56)
`
`(cid:0)H(cid:0)Y(cid:0)D(cid:0)R(cid:0)I(cid:0)T(cid:0)E(cid:0) (cid:0)E(cid:0)X(cid:0)H(cid:0)I(cid:0)B(cid:0)I(cid:0)T(cid:0) (cid:0)1(cid:0)0(cid:0)1(cid:0)3
`

`
`US 7,309,602 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Coon, C The present and future utilization of biotechnology in the
`feed industry. a poultry nutritionist’s perspective. University Of
`Arkansas. Available at http://www.asa-europe.orgpdf’present.pdf.
`Accessed Oct. 16, 2006.
`Feed-Use Amino Acids Business. Ajinornoto Co., Inc. Oct. 2006. 6
`pages.
`Gasent-Ramirez, et al. Lysine-overproducing mutants of San-
`charonzy/ces cerevisiae baker’s yeast isolated in continous culture.
`Appl Environ Microbiol 1997; 63(12):4800-6.
`Hao. C. T. J. Yeast dilution. Bachelor of engineering thesis pre-
`sented at The University of Queensland on May 19. 2004. 39 pages.
`Ingledew. W. M. Yeast—could you base business 011 this bug? In
`T.P.
`Lyons
`and
`K.A.
`Jacques,
`editors.
`Under
`the
`miccroscope—l-'ocal Points for the New Millennium-Biotechnology
`in the Feed Industry. Proceedings of Al1tech’s 15th Annual Sy1n-
`posiurn. Nottingham University Press, Nottingham, UK. 1999. pp.
`27-47.
`technology through
`Lngledew. V\’. M. Improvements in alcohol
`advancements in fermentation technology. Getreidetechnologie
`(Getreidetechnologie).2005; 59(5):308-311.
`Larkins. B. A. Boosting lysine improves nutritional value of corn.
`US Department of Agriculture. Cooperative State Research. Edu-
`cation, and Extension Service. 2001 No. 4. 2 pages.
`Lyons, et al. (Ed) Biotechnology in the Feed Lndustry. Proceedings
`o1‘Alltech’s 10”‘ Annual Symposium. Nottingham University Press.
`I oughtorough, Leicestershire, UK. 1994. (Table of Contents only).
`Mason, S. Rumen-protected amino acids. Available at http://www.
`westerndairyscienee conflhtml/CALRT%20articles/html/Aa1 .html.
`Accessed Oct. 11, 2006.
`Meyers. et al. trans-Recessive mutation in the first structural gene of
`histidine operon that results in a constitutive expression of the
`operon. J. Bacteriology 1975. 124 (3) 1227-1235.
`Moller, K. Glucose metabolism in the petite-negative yeast Sac-
`charomyces kluyveri. Ph. D. thesis presented technical University
`of Denmark. 2001. 132 pages.
`Nagai, et al. Transcriptional regulation of the heat shock regulatory
`gene rpoH in Escherichia Cali: involvement of a novel catabolite-
`sensitive promoter J. Bacteriol. 1990; 172(_5):2710-2715.
`National Renewable Energy Laboratory. The DOE bioethanol pilot
`plant—A tool for commercialization. DOE/GO-102000-1114. Sep.
`2000. 4 pages.
`Nutrient Requirements of Beef Cattle. '/"h Revised Edition. National
`Academy Press. Washington, D. C. 1996 (Table of Contents only).
`2 pages.
`Nutritional Requirements of Dairy Cattle. 7”‘ Revised bdition.
`National Acedemy Press. Washington, DC. 2001. (Table of Con-
`tents only). 2 pages.
`Nutritional Requirements of Swine. 10th Revised Edition. National
`Academy Press. Washington, D.C. 1998 (Table of Contents only).
`9 pages.
`O’Connor-Cox, et al. Wort nitrogenous sources—Their use by
`brewing yeasts: Areview. J. Am. Soc. Brew. Chem. 1989, 120-108.
`Potera, C. Progress with biofuels will depend on. d.rive nricrobology
`resea.rch—As interest in biofuels surges. finding co st—e1Tective ways
`to converting biomass to fuels and feedstocks poses challenges to
`microbiologists. Vlicrobe. 2006;
`l(7):317-322.
`Program and Abstracts. 27”“ Symposium on Biotechnology for fuels
`and chemicals hosted by the National Renewable Energy Labora-
`tory in Denver Marriott City Center Hotel. Denver, Colorado May
`1-4. 2005.
`
`In Riso
`
`Sambrook. et al. Molecular cloning: A laboratory manual. Cold
`Spring IIarbor Labs Press. Planview. NY. 1989. Table of Contents
`only. 30 pages.
`Shimazu, et al. A Family of Basic Amino Acid Transporters of the
`Vacuolar Membrane from Saccharomyces' cerevisiae. J. Biol. Chem.
`2005; 280(_6)4851—4857.
`Shurson, et al. Corn By-Product Diversity and Feeding Value to
`Non-Ruminants. Minnesota Nutrition Conference Proceedings.
`2005; 19 pages.
`Shurson, et al. Nutritional and value added benefits of maize DDGS
`and other dry-mill co-products to swine. University of Minnesota.
`Eastern Nutrition Conf., Ottawa. Ontario, Canada. May 10-11.
`2004. 20 pages.
`Soto, et al. Estimation of ethanol yield in corn mash fermentations
`using mass of ash as a marker. Journal of the Institute of Brewing,
`2005; 111(2):137—143.
`Thomas, et al. Fllecls of particulate materials and osmoprotectants
`on very-high-gravity ethanolic fermentation by Saccharomyces
`cerevisiae. Appl Environ Microbiol. 1994; 60(5):1519-24
`Thomas, et al. Fuel alcohol production: effects of free amino
`nitrogen on ferementation on very-high-gravity wheat mashes. Appl
`Environ Microbiol. 1990;56(7):2046-50.
`Thomas, et al. Production of fuel alcohol from hull-less barley by
`very high gravity technology. Cereal Chemistry. 1995; 72(4):360-
`364.
`'1'homsen, et al. Biotechnology in ethanol production.
`Energy Report 2. 2003; 40-44.
`Widyaratne, G. P. Characterization and improvement of the nutri-
`tional value of ethanol by-products for swine. Master of science
`degree thesis presented at University of Saskatchenwan. Dec. 2005.
`140 pages.
`the
`al. Heterologous protein expression ir1
`et
`Cereghino.
`methylotropic yeast Pichia pastoris. FEMS Microbiology Reviews.
`2000; 24:45-66.
`Dansen, et al. Regulation of sterol carrier protein gene expression by
`the horkhead transcription factor FOXO3a. J. Lipid Research. 2004;
`45:81-88.
`He, et al. Overexpression of a sterol C-240.8) reductase increases
`ergosterol production in Saccharomyces cerevisiae Biotechnology
`Letters. 2003; 25(10):773-8.
`Kim. et al. A role in Vacuolar arginine transport for yeast Btnlp and
`for htunan CLN3, the protein defective ir1 Batten disease. PNAS.
`2003; 100:15458-15462.
`Rippert, et al. Engineering Plant Shikinrate Pathway for Production
`of'1'ocotrienol and Improving Herbicide Resistance. Plant Physiol.
`2004; 134292-100.
`Stepanova, et al. Lysine Overproduction Mutations in the Yeast
`Sacz*}z(1rorriyces cerevisiae Are Introduced into Industrial Yeast
`Strains. Russian J. Genetics. 2001; 37:460-463.
`Sychrova, et al. Kinetic properties of yeast lysine permeases coded
`by genes on multi-copy vectors. FEMS Microbiol Lett. 1993;
`113(1):57-61.
`Szczebara, et al. Total biosynthesis of hydrocortisone from a simple
`carbon source in yeast. Nat Biotechnol. 2003;21(_2):143-9.
`Misawa, et al. Production of [5-carotene in Aymomonas mobilis and
`Agrobacterium tumefaciens by intoduction of the biosynthesis
`genes
`from Erwinia uredovora. Applied and Environmental
`Microbiology. 1991; 57(6): 1847-9.
`Foreign Search Report of Jul 2. 2007 Reguarding Application No.
`GB07067788.
`
`* cited by examiner
`
`(cid:0)H(cid:0)Y(cid:0)D(cid:0)R(cid:0)I(cid:0)T(cid:0)E(cid:0) (cid:0)E(cid:0)X(cid:0)H(cid:0)I(cid:0)B(cid:0)I(cid:0)T(cid:0) (cid:0)1(cid:0)0(cid:0)1(cid:0)3
`

`
`U.S. Patent
`
`Dec. 18,2007
`
`Sheet 1 of2
`
`Us 7,309,602 B2
`
`Biofuelsz Ethanol
`
`
`
`
`
`Figure 1
`
`
`
`_Saccharification
`
`
`Distillation
`I Centrifugation
`
`(cid:0)H(cid:0)Y(cid:0)D(cid:0)R(cid:0)I(cid:0)T(cid:0)E(cid:0) (cid:0)E(cid:0)X(cid:0)H(cid:0)I(cid:0)B(cid:0)I(cid:0)T(cid:0) (cid:0)1(cid:0)0(cid:0)1(cid:0)3
`

`
`U.S. Patent
`
`Dec. 18,2007
`
`Sheet 2 of 2
`
`Us 7,309,602 B2
`
`Vector for expressing exogenous sequence in yeast
`
`replication
`
`Pl’0m0teT
`
`origin
`
`regulatory
`sequence
`
`multiple cloning
`site
`
`
`
`’
`selection
`marker
`
`EXOQEFIOLIS
`sequence
`
`terminator
`
`multiple cloning
`site
`
`Exemplary Selection Marker: Zeocin
`
`Exemplary Regulatory Sequence:
`
`Glucose suppressor operon
`Regulatory sequence of a heat shock gene
`Regulatory sequence of a toxicity gene
`Regulatory sequence of a spore formation gene
`
`Figure 2
`
`(cid:0)H(cid:0)Y(cid:0)D(cid:0)R(cid:0)I(cid:0)T(cid:0)E(cid:0) (cid:0)E(cid:0)X(cid:0)H(cid:0)I(cid:0)B(cid:0)I(cid:0)T(cid:0) (cid:0)1(cid:0)0(cid:0)1(cid:0)3
`

`
`US 7,309,602 B2
`
`1
`CONIPOSITIONS AND METHODS FOR
`PRODUCING FERl\/IENTATION PRODUCTS
`AND RESIDUALS
`
`CROSS REFERENCE
`
`This application claims the benefit of US. Provisional
`Application No. 60/744,833 filed Apr. 13, 2006, U.S. Pro-
`visional Application No. 60/797,431 filed May 3, 2006, all
`of which are incorporated herein by reference in their
`entirety.
`
`BACKGROUND OF THE INVENTION
`
`industry is growing at a rapid pace.
`The ethanol fuel
`Numerous federal and state incentives, such as clean burn-
`i11g fuel programs, have fostered the exponential growth of
`n1ore than five times over the past two decades. In 2004,
`high oil prices, a bumper corn crop, and limited processing
`capacity created new market opportunities and resulted in
`record production of more than 3.4 billion gallons of fuel
`ethanol. Today, ethanol represents the third largest market
`for US. corn. At
`this pace,
`fuel ethanol production is
`positioning itself as an integral part of rural economic
`development and environmental improvement.
`Ethanol can be made through fermentation and distillation
`of starch found in crops such as con1, sorgl1un1, potatoes,
`sugar cane, as well as in cornstalks. Ethanol
`is usually
`produced in either dry grind or wet mill facilities. The
`primary co-products generated from the wet mills or “corn
`refineries” include high fructose corn syrup. corn oil, gluten
`feed, and gluten meal. Co—products from the dry grind
`process include distillers grains and carbon dioxide. While
`both types of facilities have similar operating costs, the dry
`grind facilities are usually smaller and require a lower initial
`investment, making their capital costs two to four times less
`per gallon. The dry mill types of ethanol production process
`the starch portion of corn, which is about 60% of the kernel.
`All
`the remaining nutrients—protein,
`fat, minerals, and
`vitamins—are concentrated into distillers grain which is a
`valuable feed for livestock. A bushel ofcorn weighing nearly
`56 pounds may produce approximately 2.8 gallons of etha-
`nol and 18 pounds of distillers grain.
`Distillers grain ca11 provide a high quality feedstuff ration
`for dairy cattle, beef cattle, swine, poultry, pets, a11d aquac-
`ulture. The feed is an economical partial replacement for
`corn, soybean meal, and dicalcium phosphate in livestock
`a11d poultry feeds. Distillers grain continues to be an excel-
`lent, economical feed ingredient for use in ruminant diets.
`DDGS (distillers dried grains with solubles) production has
`been expected to double from 3.5 million metric tons in
`2002 to over 7 million metric tons by 2006. The sale of
`distillers grain is an important part of the total profitability
`a11d growth of the ethanol industry. If dried distillers grain
`sales lag behind the increasing production of ethanol, the
`current ethanol industry could be significantly affected. An
`eifective marketing of distillers grain as animal feed will
`undoubtedly contribute to the efficiency and overall profit-
`ability of am ethanol facility.
`Current ethzmol production schemes by fermentation are
`far from being optimized. While eiforts have been directed
`to improve ethanol production,
`little research has been
`focused on enhancing the value output of the fermentation
`residuals including the distillers grai11 that contributes to a
`significant portion of the animal feed market.
`Thus, there remains a considerable need for compositions
`and methods that are designed to increase the value output
`
`15
`
`20
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`of a fermentation facility. An ideal fermentation scheme
`would maintain the high ethanol production, and at the same
`time yield fermentation residuals of higher commercial
`value. The present invention satisfies this need and provides
`related advantages as well.
`SUMMARY OF IH A INVWNTION
`
`The present invention provides compositions and meth-
`ods designed to increase value output of a fermentation
`reaction. In one embodiment, the present invention provides
`a business method of increasing value output of a ferrner1—
`tation plant. The method comprises the steps of (a) perform-
`ing a fermentation reaction with the use of a modified
`microorganism; and (b) marketing or selling one or r11ore of
`the products of the fermentation reaction comprising said
`modified microorganism.
`In a related embodiment,
`the
`method of increasing value output of a fermentation plant
`comprises perfonning a fermentation reaction using carbon-
`containing material in the presence of a modified microor-
`ganism to yield fermentation residual
`that has a higher
`commercial value than if the femientation reaction were
`performed in the absence of the modified microorganism. In
`one aspect,
`the fermentation reaction car1 be performed
`under either aerobic or anaerobic conditions. The fennen-
`tation reaction typically produces products such as alcohol,
`including but not limited to methanol, ethanol, propanol, and
`butanol, as well as gaseous co-products such as carbon
`dioxide. In addition, the fermentation reaction also yields
`residuals that are of higher commercial value than conver1—
`tional fermentation residuals. In another aspect, the fennen-
`tation reaction may utilize any carbon-containing starting
`material, eg, carbohydrates that are present
`in a wide
`variety of substances, including but not limited to cellulose,
`wood chips, vegetables, biomass, excreta, animal wastes,
`oat, wheat, com, barley, milo, millet, rice, rye, sorghum,
`potato, sugar beets, taro, cassaya, fruits, fruit juices, and
`sugar cane. The modified microorganism employed iii the
`subject methods can be eukaryotic (e.g., yeast) or prokary—
`otic (e.g., bacteria or archaebacteria). In a preferred embodi-
`ment, the fermentation reaction yields fermentation residu-
`als that have an enhanced nutritional content. In one aspect
`of this embodiment, the femientation residuals are enriched
`in one or more types of cofactors, hormones, proteins,
`preservatives, stabilization agents, nutraceuticals, vitamins,
`essential amino acids, a11d/or lipids. In so111e aspects, the
`reaction is performed with the subject microorganisms to
`increase the value output of the entire fermentation reaction
`by enhancing the process to yield more valuable products
`and/or fermentation residuals. In some other aspects, the
`reaction is performed with the subject microorganisms to
`increase the value output without substantially decreasing
`the amount of fermentation products produced by the fer-
`mentation reaction, and/or without substantially decreasing
`the total values of fermentation products produced by the
`fermentation reaction.
`The present
`invention also provides a fermentation
`residual comprising a genetically modified microorganism,
`wherein the fermentation residual has a commercial value
`(eg. due to increase in nutritional content) higher than that
`of a fermentation residual that is deficient in said modified
`microorganism.
`In one aspect,
`the subject fermentation
`residual has a shelf life that
`is longer than that of a
`fermentation residual
`that
`is deficient
`in said modified
`microorganism. In another aspect, the residual is enriched in
`at least one essential amino acid, a significant faction of
`which (e.g. the majority of which) is encapsulated in a cell
`
`(cid:0)H(cid:0)Y(cid:0)D(cid:0)R(cid:0)I(cid:0)T(cid:0)E(cid:0) (cid:0)E(cid:0)X(cid:0)H(cid:0)I(cid:0)B(cid:0)I(cid:0)T(cid:0) (cid:0)1(cid:0)0(cid:0)1(cid:0)3
`

`
`US 7,309,602 B2
`
`3
`eg, a prokaryotic or eukaryotic cell used in the fermenta-
`tion reaction). Where desired, at least about 25%, or pref-
`erably 50%, preferably at least about 60%. or even more
`wreferably at least about 80% of the essential amino acids
`measured by dry weight are encapsulated in a cell or spore.
`In addition, the essential amino acids may be embodied in a
`iomologous polypeptide with enhanced concentration, or a
`ieterologous polypeptide produced by a microorganism
`used in the fermentation reaction. The heterologous
`mlypeptide car1 be secretory or preferably non—secretory
`e.g., in a vacuole when the polypeptide is in an inclusion
`aody within the fermentation microorganism). The heterolo-
`gous polypeptide enriched ir1 essential amino acid sequences
`can adopt a variety of structural conformations such as a
`aeta-sheet conformation, an alpha-helix conformation, a
`random—coil conformation, and/or a coiled—coil conforma-
`tion, or an aggregate, or a combination thereof.
`Depending on the intended use, the essential amino acid
`may exclude histidi r1e and include any one of the exemplary
`essential amino acids. Non-limiting exemplary essential
`amino acids include lysi11e, methionine, threonine. methion-
`ine, phenylalanine, and arginine. The quantity of essential
`amino acid present in the residuals may vary fron1 at least
`about 0.25%, 1%, at least about 2%. at least about 3% to
`about 95% by dry weight.
`The subject fermentation residuals can be supplemented
`with a desirable flavor tailored for one or more types of
`animals. The residuals can also be packaged With instruc-
`tions for use as animal feed or food supplement for lnunans.
`The present invention further provides a modified micro-
`organism Whose nutritional content increases by a greater
`extent than that of an unmodified corresponding microor-
`ganism when used in a fermentation reaction.
`I11 some
`instances,
`if the nutritional cor1ter1t
`increases due to ar1
`increase i11 at least one essential amino acid, then the at least
`one essential amino acid is not histidine. In a related but
`separate embodiment, the present invention also provides a
`modified microorganism whose nutritional
`content
`is
`enhanced as compared to an unmodified corresponding
`microorganism when used in a fermentation reaction and
`when the fermentation reaction has achieved at least about
`50% completion. In another embodiment. the present inven-
`tion provides a modified microorganism producing an alco-
`hol product in a fermentation reaction that utilizes a carbon-
`containing starting material, wherein said microorganism
`also produces a nutrient subsequent to the initiation of the
`alcohol production
`In another embodiment, the present invention provides a
`modified microorganism that comprises
`an exogenous
`sequence encoding a polypeptide, wherein the polypeptide
`comprises at least one essential amino acid, and wherein
`expression of the exogenous sequence is induced when the
`fermentation reaction has achieved at
`least about 50%
`completion. In yet another embodiment, the present inven-
`tion provides a modified microorganism comprising an
`exogenous sequence encoding a polypeptide that comprises
`at least one essential amino acid, and wherein expression of
`the exogenous sequence is under the control of a glucose
`suppressor operon.
`The progression of fermentation can be monitored by a
`variety of ways. For example, at least 50% completion of a
`fermentation reaction can be evidenced by the consumption
`of at least 50% of the total glucose in the desired fermen-
`tation, when compared to similar fermentations, or when
`50% of the total glucose has been added, or when the total
`amotmt of carbon dioxide emitted, and dissolved is 50% of
`the total amount emitted in similar fermentations. More
`
`l0
`
`I5
`
`20
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`least 50% completion of a fermentation
`specifically, at
`reaction can be evidenced by a decrease in glucose content
`to less than about 50% of the initial content of glucose
`present in a fermentation reaction mixture (i.e., the glucose
`level present prior to the beginning of the fermentation
`reaction), or less than a desired threshold level (e.g., about
`100 grams per liter fermentation reaction). Alternatively, the
`degree of completion can be determined by the amount of
`time during which the fermentation has taken place, typi-
`cally, at least about halfthe time taken by a similar ferrner1—
`tation. The duration of fermentation time may range from
`about 1 hour to several days, depending on the relevant
`amounts of microorganisms and fermentation starting n1ate—
`rial provided. One skilled in the art can readily ascertain the
`normal duration of a fermentation reaction without undue
`experimentation when given the amount of microorganisms
`and starting materials.
`The modified microorganism can be a eukaryote (e.g.,
`yeast) or a prokaryote (e.g., bacteria or archaebateria). It can
`be modified to overproduce a nutritional component includ-
`ing but not limited to amino acid, vitamin. and/or lipid. This
`is typically achieved by genetically modifying the metabolic
`pathway of the microorganism for producing such nutri-
`tional component, and/or directly introducing a11 exogenous
`sequence that encodes the nutritional component (e.g,, a
`particular type of amino acid contained in a polypeptide).
`Where desired, genetic modification can be carried out with
`the use of genetic Vehicles that carry one or more of the
`metabolic pathway gene sequences, or the sequences that
`code for the exogenous polypeptides carrying the nutritional
`component such as essential amino acids. A Wide variety of
`genetic vehicles are applicable for such use. They include an
`array of expression vectors including both viral and non-
`viral vectors. Ir1 a preferred embodiment, expression of the
`exogenous sequence is under the control of a regulatory
`sequence selected from the group consisting of a regulatory
`sequence of a heat shock gene, regulatory sequence of a
`oxicity gene, regulatory sequence of a spore fonnation
`gene, and glucose suppressor operon. When regulated under
`hese sequences, the increase in production of the nutritional
`component by the microorganisms can be induced at a time
`when the fermentation has substantially been completed,
`oreferably at least about 50% completed, n1ore preferably at
`east about 70% completed, more preferably about 90%
`completed. Such regulation allows production of fermenta-
`ion products of enhanced nutritional value and maximizing
`he profit from the fermentation reaction.
`The present invention further provides a method of fer-
`mentation using carbon-containing material. The method
`ypically comprises the steps of (a) mixing a carbon-con-
`aining material with a modified microorganism of the
`oresent invention, ar1d (b) subjecting the mixture of (a) to
`conditions suitable for production of a fermentation product.
`Where desired, the method can further comprise the step of
`iarvesling one or more fermentation products. Exemplary
`ermentation products include alcohol such as methanol,
`ethanol, propanol, butanol and the like, as well as gaseous
`oroducts such as carbon dioxide. The fermentation method
`can be performed under aerobic or anaerobic conditions. A
`wide variety of carbon-containing raw materials can be used
`ir1 the fermentation reaction Exemplary materials include
`jut are not limited to cellulose, oat, wheat, corn, milo, millet,
`aarley, rice, rye, sorghum, potato, sugar beets, taro, cassaya,
`ruils, fruit juices, and sugar cane,
`Further embodied in the present invention is an expres-
`sion vector suitable for modifying the subject microorgan-
`ism. The expression vector typically comprises an exog-
`
`(cid:0)H(cid:0)Y(cid:0)D(cid:0)R(cid:0)I(cid:0)T(cid:0)E(cid:0) (cid:0)E(cid:0)X(cid:0)H(cid:0)I(cid:0)B(cid:0)I(cid:0)T(cid:0) (cid:0)1(cid:0)0(cid:0)1(cid:0)3
`

`
`US 7,309,602 B2
`
`5
`enous sequence encoding a polypeptide that comprises at
`least one essential amino acid, wherein expression of the
`exogenous sequence is induced when the fermentation reac-
`tion has achieved at least about 50% completion. Where
`desired, the expression vector comprises one or more of the
`following regulatory sequences so as to control the expres-
`sion of the exogenous polypeptide. F.xemplary regulatory
`sequences include glucose suppressor operon, a regulatory
`sequence of a heat shock ge11e, regulatory sequence of a
`toxicity gene, or regulatory sequence of a spore formation
`gene.
`The present invention also embodies variations and all
`combination of the composition and methods described
`herein.
`
`INCORPORATION BY R A F A RENCE
`
`All publications and patent applications mentioned in this
`specification are herein incorporated by reference to the
`same extent as if each individual publication or patent
`application was specifically and individually indicated to be
`incorporated by reference.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`specification
`included within this
`illustrations
`The
`describe many of the advantages and features of the inven-
`tion. It shall be understood that similar reference numerals
`and characters noted within the illustrations herein may
`designate the same or like features of the invention. The
`illustrations and features depicted herein are not necessarily
`drawn to scale.
`FIG. 1 is a flow chart describing an exemplary ethanol
`production process that results in formation of ethanol,
`carbon dioxide, and fem1er1tatio11 residuals such as distillers
`dried grain with solubles or solids (DDGS).
`FIG. 2 is a schematic representation of an exemplary
`genetic vehicle useful for modifying a microorganism used
`i11 the subject fermentation reaction.
`T)F.TAII.F.T) DESCRIPTION OF THE
`INVENTION
`
`VVhile preferred embodiments of the invention have been
`shown and described herein,
`it will be obvious to those
`skilled in the art that such embodiments are provided by way
`of example only. Numerous variations, changes. and stIbsti—
`tutions will now occur to those skilled in the art without
`departing from the invention. It should be understood that
`various alternatives to the embodiments of the invention
`described herein may be employed in practicing the inven-
`tion.
`The term “animal” means any organism belonging to the
`kingdom Animalia and includes, without limitation, poultry,
`cattle, swine, goat, sheep, cat, dog, mouse, aquaculture,
`horse, etc.
`The term “fermentation residuals” as used herein means
`any residual substances directly resulting from a fermenta-
`tion reaction. In some instances, a fennentation residual
`contains modified microorganisms such that it has a nutri-
`tional content enhanced as compared to a fermentation
`residual that is deficient in such modified microorganism.
`The
`fermentation
`residuals may
`contain
`suitable
`constituent(s) from a fermentation broth. For example, the
`fermentation residuals may include dissolved and/or sus-
`pended constituents from a fermentation broth. The sus-
`pended constituents may include undissolved soluble con-
`
`I0
`
`15
`
`20
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6
`stituents (e.g., where the solution is supersaturated with one
`or more components) and/or insoluble materials present in
`the fermentation broth. The fermentation residuals may
`include substantially all of the dry solids present at the end
`of a fermentation (e.g., by spray drying a fermentation broth
`and the biomass produced by the fermentation) or may
`include a portion thereof. The fermentation residuals may
`include crude fermentation product
`from fermentation
`where a 1nodified—microorganism may be fractionated a11d/or
`partially purified to increase the nutrient content of the
`material.
`"he term “fatty acid” as used herein means an aliphatic or
`aromatic monocarboxylic acid.
`"he term “lipids” as used herein means fats or oils
`including without limitation the glyceride esters of fatty
`acids along with associated phosphatides, sterols, alcohols,
`hydrocarbons, ketones, and related compounds.
`'
`'he term “nutrient” as used herein means any substances
`with nutritional value. It can be part of an animal feed or
`food supplement for humans. Exemplary nutrients include
`but are not
`limited to fats,
`fatty acids,
`lipids such as
`phospholipid, vitamins, essential amino acids, peptides, pro-
`teins, carbohydrates, sterols, enzymes, and trace minerals
`such as, iron, copper, zinc. manganese, cobalt, iodine, sele-
`nium, molybdenum, nickel, fluorine, vanadiimr,
`ti11, and
`silicon. The nutrient may be secreted by a modified micro-
`organism in a fermentation broth or contained within the
`microorganism. (e.g. in inclusion bodies in the microorgan-
`ism.) The nutrient may also be added to the feed containing
`the fermentation residuals.
`“Heterologous polypeptide” or “heterologous protein”
`means derived from (i.e., obtained from) a genotypically
`distinct entity from the rest of the entity to which it is being
`compared, or that it is genetically indistinct but produced at
`an abnormally high or low concentration as compared to a
`native Lurmodified enviromnent or microorganism.
`The term “unsaturated fatty acid” as used herein means a
`faty acid with I to 3 double bonds and a “highly unsaturated
`faty acid” means a fatty acid with 4 or more double bonds.
`Fermentation Process
`Fennentation as used herein can be anaerobic (deficient in
`oxygen) as well as aerobic (oxygenated). Under aerobic
`conditions, microorganisms such as yeast cells can break
`down sugars to end products such as CO: and H20. Under
`anaerobic conditions, yeast cells utilize an alternative path-
`way to produce CO2 a11d ethanol. The fennentation reaction
`of the present invention is preferably anaerobic, i.e., par-
`tially or completely deficient in oxygen. Fermentation czm
`also be used to refer to the bulk growth of microorganisms
`on a growth medium where no distinction is made between
`aerobic and anaerobic metabo