USOO8846352B2
(12) United States Patent (10) Patent No.: US 8,846,352 B2 Chua et al. (45) Date of Patent: Sep. 30, 2014
(54) GENETICALLY ENGINEERED 5,270,175 A 12, 1993 Moll et al. MCROORGANISMIS THAT METABOLIZE 5,304,481. A 4/1994 Davies et al. XYLOSE 5,338,673 A 8, 1994 Thepenier et al. 5,391,724 A 2f1995 Kindlet al. 5,395.455 A 3, 1995 Scott et al. (75) Inventors: Penelope Chua, San Francisco, CA 5,455,167 A 10, 1995 Rt. al. (US); Aravind Somanchi, Redwood 5.492.938 A 2/1996 Kyle et al. City, CA (US) 5,518,918 A 5/1996 Barclay et al. s 5,547,699 A 8, 1996 Lizuka et al. (73) Assignee: Solazyme, Inc., South San Francisco, 5,685,2185,680,812 A 10,1 1/1997 1997 LikE d CA (US) 5,693,507 A 12/1997 Daniellet al. 5,711,983 A 1/1998 Kyle et al. (*) Notice: Subject to any disclaimer, the term of this 5,792,631 A 8/1998 Running patent is extended or adjusted under 35 E. A ck g 3. RE tal 435,158 U.S.C. 154(b) by 39 days. 5,900,370sy s A 5/1999 RunningaO C. a...... 6,166,231 A 12/2000 Hoeksema (21) Appl. No.: 13/464,948 6.255,505 B1 7/2001 Bijl et al. 6,338,866 B1 1/2002 Criggallet al. (22) Filed: May 4, 2012 6,344,231 B1 2/2002 Nakajo et al. 6,372.460 B1 4/2002 Gladue et al. (65) Prior PublicationO O Data 6,441,2086,680,426 B2 8/20021/2004 BijlDaniell et al. et al. US 2012/0329.109 A1 Dec. 27, 2012 6,750,048 B2 6/2004 Ruecker et al. 7,053,267 B2 5, 2006 Knaufetal. 7,063,957 B2 6/2006 Chen Related U.S. Application Data 7,081,567 B2 7/2006 Xue et al. 7,135,620 B2 11/2006 Daniell et al. (60) Provisional application No. 61/483,550, filed on May 7,268,276 B2 9, 2007 Eniy et al. 6, 2011, provisional application No. 61/497,501, filed Continued on Jun. 15, 2011. (Continued) (51) Int. Cl FOREIGN PATENT DOCUMENTS CI2P 7/64 (2006.01) CN 1940.021. A 4, 2007 CI2N L/12 (2006.01) CN 101.037,639 A 9, 2007 CI2P2/06 (2006.01) (Continued) % 3.08: OTHER PUBLICATIONS A23D 9/00 (2006.015 Broun et al., Catalytic plasticity of fatty acid modification enzymes C7H 2L/04 (2006.01) underlying chemical diversity of plant lipids. Science, 1998, vol. 282: C07K I4/00 (2006.01) 1315-1317. CI2N L/22 (2006.01) Chica et al., Semi-rational approaches to engineering enzyme activ (52) U.S. Cl ity: combining the benefits of directed evolution and rational design. CPC. C12P 7/64 (2013.01); C12N 1/22(2013.01); Devos"E.E.E.Y.: et al., Practical limits of function prediction. Proteins:a Struc Y02T 50/678 (2013.01); CI2P 7/6463 ture, Function, and Genetics. 2000, vol. 41: 98-107. (2013.01); Y02E 50/13 (2013.01); C12P Jeffries et al., Genome sequence of the lignocellulose-bioconverting 2203/00 (2013.01); C12N 1/12 (2013.01) and xylose-fermenting yeast Pichia stipitis. Nat. Biotechnol., 2007. USPC ...... 435/134:435/257.2:435/69.1; 435/91.1; Vo. 25(3): 319-326.* 435/320.1:554/227: 536/23.1:536/23.2: Kimchi-Sarfaty et al. A “Silent” polymorphism in the MDR1 gene 530/350 changes substrate specificty. Science, 2007, vol. 315: 525-528.* (58) Field of Classification Search Kisselev L., Polypeptide release factors in prokaryotes and USPC ...... 435/134, 257.2, 69.1, 91.1, 320.1; eukaryotes:10:8-9. same function, different structure. Structure, 2002, vol. 554/227: 536/23.1, 23.2:530/350 See application file for complete search history. (Continued) (56) References Cited Primary Examiner — Ganapathirama Raghu (74) Attorney, Agent, or Firm — Alston & Bird LLP U.S. PATENT DOCUMENTS (57) ABSTRACT 3,280,502 A 10, 1966 Farrow et al. The invention provides methods of cultivating oil-bearing 3,475,274 A 10, 1969 Harned microbes using Xylose as a fixed carbon source. Also pro 3,957,578 A 5, 1976 Narita et al. vided are microorganisms that have been genetically engi 3,983,008 A 9, 1976 Shinozaki et al. 4,341,038 A 7, 1982 Bloch et al. neered to metabolize Xylose as a fixed carbon source allowing 4,373,434 A 2, 1983 Alexander et al. them to convert xylose into oils. Particular advantages of the 4,390,561 A 6, 1983 Blair et al. processes provided herein include production of oils rather 4,901,635 A 2, 1990 Williams than alcohols through the microbial fermentation processes 4,992,605 A 2, 1991 Craig et al. utilizing Xylose. 5,001,059 A 3, 1991 Skatrudet al. 5,130,242 A 7/1992 Barclay 11 Claims, No Drawings US 8,846,352 B2 Page 2
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In some cases, the microorganism is capable of accu 61/497.501, filed Jun. 15, 2011. Each of these applications is 10 mulating at least 10, 20,30,40, 50, 60, 70, or 80% triglyceride incorporated herein by reference in its entirety. by dry cell weight when cultured on pure Xylose. In some cases, the microorganism is capable of accumulating at least GOVERNMENT INTERESTS 50% triglyceride by dry cell weight when cultured on glucose This invention was made with State of California support 15 as a carbon source and is capable of accumulating at least under California Energy Commission Grant Number PIR-08 20% triglyceride by dry cell weight when cultured on pure 018. The Energy Commission has certain rights to this inven Xylose. In some cases, the microorganism is cultured on a tion. carbon source that is 60% glucose and 40% xylose under nitrogen limiting conditions, the cells produce lipid in which REFERENCE TO ASEQUENCE LISTING 2O the carbonatoms are at least 5, 10, 20, 30, 40, or 50% derived from carbonatoms of xylose, as measured by isotopic tracing. This application includes a sequence listing as shown in In some cases, the microorganism is capable of consuming at pages 1-49, appended hereto. least 90% of the xylose in a culture media having 2.5%xylose as a sole carbon source in less than or equal to 21, 14 or 7 days. FIELD OF THE INVENTION 25 In some cases, the recombinant nucleic acid is operable to The present invention relates to the expression of xylose encode one or more of an active Xylose transporter, a Xylu metabolizing pathways in oleaginous microorganisms to lose-5-phosphate transporter, a xylose isomerase, a xyluloki allow them to metabolize xylose so as to produce lipid or nase, a xylitol dehydrogenase, or a Xylose reductase. In some other useful biomass, and to oleochemical, food, fuel, and cases, the recombinant nucleic acid encodes an active Xylu other products produced from the biomass. 30 lose-5-phosphate transporter in operable linkage with a plas tid-targeting signal sequence and wherein the Xylulose-5- BACKGROUND OF THE INVENTION phosphate transporter is operable to transport xylulose-5- phosphate into a plastid. In some cases, the recombinant An important and difficult challenge in biotechnology is nucleic acid is operable to encode (a) a xylulose-5-phosphate unleashing the vast potential of carbon capture in cellulosic 35 transporter protein with a plastid-targeting signal sequence, materials for conversion into valuable Substances such as So as to cause transport of xylulose-5-phosphate into a plastid, liquid fuel, chemicals, food, and other products. One specific (b) a xylulokinase, and (c) either a Xylose isomerase, or both challenge relates to using Xylose, commonly found in depo of a Xylitol dehydrogenase and a Xylose reductase. lymerized hemicellulose, as a carbon source for the het In some cases, the microorganism is of the Subkingdom erotrophic cultivation of microorganisms. Although much a Viridiplantae. In some cases, the microorganism is of the work has been done in using Xylose in the production of infrakingdom or phyla Chlorophytae, of the subphylum Tet ethanol using yeast, most yeast strains either cannot utilize xylose or utilize xylose only very inefficiently (see Jeffries, raphytina, the class Trebouxiophyceae, or the order Chlorel 2006, Curr. Op. Biotech. 17: 320-326; and Wang et al., 2004, lales. In some cases, the microorganism is of the genus Chlo Biotechnol. Lett. 26(11): 885-890). rella or Prototheca. In some cases, the microorganism is of Certain oleaginous microorganisms (e.g. oleaginous yeast 45 the species Prototheca moriformis or Chlorella prototh and oleaginous microalgae) are capable of converting fixed ecoides. carbon energy sources into higher value products such as In some cases, the microorganism further comprising a triglycerides, fatty acids, carbohydrates, and proteins. In recombinant modification operable to alter the fatty acid pro addition, the microalgae themselves can be valuable as a food file of lipid produced by the microorganism. In some cases, source. For example, certain species of oleaginous microal- 50 the recombinant modification comprises an exogenous gene gae have been genetically engineered to produce “tailored that encodes an active acyl-ACP thioesterase, ketoacyl-ACP oils', which means that their triglyceride content shows synthase or a fatty acid desaturase, or is operable to reduce or altered distributions offatty acid chain lengths and Saturation ablate an acyl-ACP thioesterase, ketoacyl-ACP synthase or relative to the strains from which they were derived. See PCT fatty acid desaturase. Pub. Nos. 2008/151149, 2009/126843, 2010/045358, 2010/ 55 In another aspect, the present invention provides a method 063031 and 2010/063032 and PCT App. Nos. U.S. Ser. No. for producing microbial biomass or a product produced from 11/038,463 and U.S. Ser. No. 11/038,464. the biomass, in which the method comprises heterotrophi The ability to convert xylose to lipid and other products in cally cultivating a recombinant microorganism, as discussed meaningful amounts using oleaginous microorganisms above or herein, in a culture medium comprising Xylose so as would be of major environmental significance because it 60 to convert Xylose into the microbial biomass. In some cases, would reduce our dependence on fossil fuels and reduce the the microbial biomass comprises microbial lipid. In some cost of microbial oils. cases, the lipid is used to make a product selected from the group consisting of a chemical, a lubricant, a detergent, a SUMMARY OF THE INVENTION fuel, a food oil, and a cosmetic ingredient. In some cases, the 65 product is a fuel selected from biodiesel or renewable diesel. In one aspect, the present invention provides a microorgan In some cases, the culture medium comprises a carbon ism of the kingdom Plantae, the microorganism comprising a Source that is greater than 50% Xylose. In some cases, the US 8,846,352 B2 3 4 carbon source is greater than 55%, 60%. 65%, 70%, 75%, selected from XylA (SEQID NO: 15) or XYL3 (SEQID NO: 80%, 85%, 90%, or 95% xylose. 51), and a fourth exogenous gene that encodes a xylose In another aspect, the present invention provides a natural isomerase pathway protein selected from XYL1 (SEQ ID oil produced by any of the microorganisms, as discussed NO:35),XYL2(SEQID NO:50) orXYL3 (SEQIDNO:51). above or herein, or produced by any of the methods discussed 5 In one embodiment, the recombinant oleaginous microbe above or herein. comprises exogenous genes that encode XylA,XYL3, SUT1, In another aspect, the present invention provides an ole and GPT-F-XPT. In one embodiment, the recombinant ole ochemical, food oil, fuel, or other product produced from the aginous microbe comprises exogenous genes that encode natural oil discussed above. XylA, XYL2, XYL3, XLT1, and S106SAD-XPT. In one In another aspect, the present invention provides recombi 10 nant oleaginous microbes that utilize Xylose as a carbon embodiment, the recombinant oleaginous microbe comprises Source. In one embodiment, the oleaginous microbe is a exogenous genes that encode XylA, XYL1, XYL3, XLT1, microalgae, oleaginous yeast, oleaginous fungi or oleaginous and S106SAD-XPT. In one embodiment, the recombinant bacteria. In another embodiment, the recombinant microbes oleaginous microbe comprises exogenous genes that encode are microalgae cells, including but not limited to cells of the 15 XylA, XYL1, XYL3, GXS1, and GPT-A-XPT. In one genus Prototheca, comprising one or more exogenous genes embodiment, the recombinant oleaginous microbe comprises that allow the cells to utilize, or utilize more efficiently, xylose exogenous genes that encode XYL1, XYL2, XYL3, sym as a carbon Source. In some embodiments, the cell is a strain porter, and GPT-F-XPT. In one embodiment, the recombinant of the species Prototheca moriformis, Prototheca krugani, oleaginous microbe of comprises exogenous genes that Prototheca stagnora or Prototheca Zopfii, and in other encode XYL1,XYL2, XYL3, GXS1, and GPT-F-XPT. In one embodiments, the cell has a 23S rRNA sequence with at least embodiment, the recombinant oleaginous microbe comprises 70, 75, 80, 85 or 95% nucleotide identity to one or more of exogenous genes that encode XYL2, XYL3, Symporter, and SEQ ID NOs: 1-9. The exogenous gene comprises coding S106SAD-XPT. In one embodiment, the recombinant oleagi sequence in operable linkage with a promoter, and in some nous microbe comprises exogenous genes that encode XYL2. embodiments the promoter is from a gene endogenous to a 25 XYL3, XLT1, and S106SAD-XPT. In one embodiment, the species of the genus Prototheca. In further embodiments the recombinant oleaginous comprises exogenous genes that coding sequence encodes a protein selected from the group encode XYL2, XYL3, GXS1, and GPT-A-XPT. consisting of oxidoreductase pathway proteins, Xylose In some cases, the recombinant oleaginous microbes are isomerase pathway proteins, Xylose translocator proteins and propagated or cultivated in a culture medium comprising a Xylose transporter proteins. 30 carbon source that is greater than 50% Xylose. In some cases, In various embodiments, the microalgae cell includes one the microbes are propagated or cultivated in a culture medium or more exogenous genes that allow the cells to utilize, or comprising a carbon source that is greater than 55%, greater utilize more efficiently, xylose as a carbon source. For than 60%, greater than 65%, greater than 70%, greater than example, microalgae cells of the invention include those that 75%, greater than 80%, greater than 85%, greater than 90%, have been engineered to comprise: (i) one or more genes that 35 or greater than 95% Xylose. In some cases, the microbes are encode a Xylose isomerase pathway protein; or (ii) one or propagated or cultivated in a culture medium comprising more genes that encode an oxidoreductase pathway protein; Xylose as the sole carbon Source. In some cases, the microbes, or (iii) one or more genes that encode a Xylose isomerase when propagated or cultivated in a culture medium compris pathway protein; (iv) one or more genese that encode a Xylose ing a carbon Source that is greater than 50% Xylose, or a transporter protein; or (V) one or more genes that encode a 40 culture medium comprising Xylose as the sole carbon Source, Xylose translocator protein; or combinations of (i), (ii), (iii). produce at least 25%, at least 30%, at least 35%, at least 40%, (iv), and (V). In an embodiment, the microbial cell is engi at least 45%, at least 50%, at least 55% or at least 60% of the neered to express a Xylose transporter protein and a Xylose lipid produced by a comparable microbial cell propagated or isomerase pathway protein. In another embodiment, the cultivated in a culture medium comprising glucose as the sole microbial cell is engineered to express a Xylose transporter 45 carbon Source and lacking the one or more exogenous genes. protein and an oxidoreductase pathway protein. In a further Unless otherwise indicated, reference above and through embodiment, the microbial cell is engineered to express a out this specification to an exogenous gene that encodes a Xylose transporter protein, an oxidoreductase pathway pro specified protein refers to a gene that is operable to encode the tein and a Xylose isomerase protein. In yet another embodi specified protein. ment, the microbial cell is engineered to express a Xylose 50 Another embodiment of the invention is microbial lipid transporter protein, an oxidoreductase pathway protein, and a composition produced by propagating or cultivating a recom Xylose translocator protein. In another embodiment, the binant oleaginous microbe in the presence of Xylose. In one microbial cell is engineered to express a Xylose transporter embodiment, the recombinant oleaginous microbes dis protein, a xylose isomerase pathway protein, and a Xylose cussed herein are propagated and/or cultivated in the presence translocator protein. In another embodiment, the microbial 55 of cellulosic material that comprises xylose. Optionally, the cell is engineered to express a xylose transporter protein, a cellulosic material can further comprise glucose and Sucrose. Xylose isomerase pathway protein, a xylose isomerase path The microbial lipid composition is produced by lysing the way protein, and a Xylose translocator protein. microbial cell and isolating the oil. In some embodiments of the present invention, the recom binant oleaginous microbe comprises a first exogenous gene 60 DETAILED DESCRIPTION OF THE INVENTION that encodes a xylose translocator protein selected from GPT A-XPT (SEQID NO: 45), GPT-F-XPT (SEQID NO: 47) or I. Definitions S106SAD-XPT (SEQID NO: 49), a second exogenous gene that encodes a xylose transporter protein selected from SUT1 “Active' refers to a nucleic acid that is functional in a cell. (SEQ ID NO:37), GXS1 (SEQID NO:39), XLT1 (SEQ ID 65 For example, a promoter that has been used to drive an anti NO: 43) or symporter (SEQ ID NO: 41), a third exogenous biotic resistance gene to impart antibiotic resistance to a gene that encodes an oxidoreductase pathway protein microalgae is active in microalgae. US 8,846,352 B2 5 6 Area Percent” refers to the determination of the area per perature and pressure in Solid or liquid formina culture media cent of chromatographic, spectroscopic, and other peaks gen that can be utilized by a microorganism cultured therein. erated during experimentation. The determination of the area “Heterotrophic cultivation' and variants thereof such as under the curve of a peak and the area percent of a particular "heterotrophic culture' and "heterotrophic fermentation' peak is routinely accomplished by one of skill in the art. For 5 refer to the intentional fostering of growth (increases in cell example, in FAME GC/FID detection methods in which fatty size, cellular contents, and/or cellular activity) in the presence acid molecules in the sample are converted into a fatty acid of a fixed carbon source. Typically, heterotrophic cultivation methyl ester (FAME), a separate peak is observed for a fatty is performed in the absence of light. However, the absence of acid of 14 carbon atoms with no unsaturation (C14:0) com light is not a requirement for heterotrophic cultivation. Cul pared to any other fatty acid such as C14:1. The peak area for 10 each class of FAME is directly proportional to its percent tivation in the absence of light means cultivation of microbial composition in the mixture and is calculated based on the Sum cells in the complete absence or near complete absence of of all peaks present in the sample (i.e. area under specific light. peak/total area of all measured peaks)x100). When referring “Increase lipid yield” refers to an increase in the produc to lipid profiles of oils and cells of the invention, “at least 4% 15 tivity of a microbial culture by, for example, increasing dry C8-C14” means that at least 4% of the total fatty acids in the weight of cells per liter of culture, increasing the percentage cell or in the extracted glycerolipid composition have a chain of cells that constitute lipid, or increasing the overall amount length that includes 8, 10, 12 or 14 carbon atoms. of lipid per liter of culture volume per unit time. “Biodiesel’ is a biologically produced fatty acid alkyl ester “In operable linkage' is a functional linkage between two Suitable for use as a fuel in a diesel engine. nucleic acid sequences, such a control sequence (typically a “Biomass” is material produced by growth and/or propa promoter) and the linked sequence (typically a sequence that gation of cells. Biomass may contain cells and/or intracellular encodes a protein, also called a coding sequence). A promoter contents as well as extracellular material, which includes, but is in operable linkage with an exogenous gene if it can medi is not limited to, compounds secreted by a cell. ate transcription of the gene. “Bioreactor is an enclosure or partial enclosure in which 25 “Microalgae is a eukarytotic microbial organism that con cells are cultured, optionally in Suspension. tains a chloroplast or plastid, and optionally that is capable of “Cellulosic material' is the product of digestion of fibrous performing photosynthesis, or a prokaryotic microbial organ material, typically including glucose and Xylose (e.g., from ism capable of performing photosynthesis. Microalgae hemicelullose), and optionally additional compounds such as include obligate photoautotrophs, which cannot metabolize a disaccharides, oligosaccharides, lignin, furfurals and other 30 fixed carbon Source as energy, as well as heterotrophs, which compounds. Nonlimiting examples of Sources of cellulosic can live solely off of a fixed carbon source. Microalgae material include Sugar cane bagasses, Sugar beet pulp, corn include unicellular organisms that separate from sister cells stover, wood chips, sawdust and Switchgrass. shortly after cell division, such as Chlamydomonas, as well as “Expression vector” or “expression construct” or “plas microbes Such as, for example, Volvox, which is a simple mid' or “recombinant DNA construct” refer to a nucleic acid 35 multicellular photosynthetic microbe of two distinct cell that has been generated via human intervention, including by types. Microalgae include cells such as Chlorella, Dunaliella, recombinant means or direct chemical synthesis, with a series and Prototheca. Microalgae also include other microbial pho of specified nucleic acid elements that permit transcription tosynthetic organisms that exhibit cell-cell adhesion, such as and/or translation of a particular nucleic acid in a host cell. Agmenellum, Anabaena, and Pyrobotrys. Microalgae also The expression vector can be part of a plasmid, virus, or 40 include obligate heterotrophic microorganisms that have lost nucleic acid fragment. Typically, the expression vector the ability to perform photosynthesis, such as certain includes a nucleic acid to be transcribed operably linked to a dinoflagellate algae species and species of the genus Prototh promoter. aCC "Exogenous gene' is a nucleic acid that codes for the “Microorganism” and “microbe' are microscopic unicel expression of an RNA and/or protein that has been introduced 45 lular organisms. (“transformed') into a cell. A transformed cell may be A “natural oil shall mean a predominantly triglyceride oil referred to as a recombinant cell, into which additional exog obtained from an organism, where the oil has not undergone enous gene(s) may be introduced. The exogenous gene may blending with another natural or synthetic oil, or fractionation be from a different species (and so heterologous), or from the so as to substantially alter the fatty acid profile of the triglyc same species (and so homologous), relative to the cell being 50 eride. Here, the term “fractionation” means removing mate transformed. Thus, an exogenous gene can include a homolo rial from the oil in a way that changes its fatty acid profile gous gene that occupies a different location in the genome of relative to the profile produced by the organism, however the cell or is under different control, relative to the endog accomplished. A natural oil encompasses such an oil obtained enous copy of the gene. An exogenous gene may be present in from an organism, where the oil has undergone minimal more than one copy in the cell. An exogenous gene may be 55 processing, including refining, bleaching and/or degumming, maintained in a cell as an insertion into the genome or as an that does not substantially change its triglyceride profile. A episomal molecule. natural oil can also be a "noninteresterified natural oil, which “Exogenously provided refers to a molecule provided to means that the natural oil has not undergone a process in the culture media of a cell culture. which fatty acids have been redistributed in their acyl link “Fatty acid profile' shall mean the distribution of fatty 60 ages to glycerol and remain essentially in the same configu acids in a cellor oil derived from a cell interms of chain length ration as when recovered from the organism. and/or Saturation pattern. In this context the Saturation pattern “Naturally co-expressed with reference to two proteins or can comprise a measure of saturated versus unsaturated acid genes means that the proteins or their genes are co-expressed or a more detailed analysis of the distribution of the positions naturally in a tissue or organism from which they are derived, of double bonds in the various fatty acids of a cell. 65 e.g., because the genes encoding the two proteins are under "Fixed carbon Source' is a molecule(s) containing carbon, the control of a common regulatory sequence or because they typically an organic molecule, that is present at ambient tem are expressed in response to the same stimulus. US 8,846,352 B2 7 8 “Oxidoreductase pathway' are the proteins and/or genes encoding such proteins that transports Xylulose 5-phosphate. encoding them capable of converting Xylose to Xylulose A non-limiting example of a xylose translocator is the XPT 5-phosphase via Xylitol and Xylulose intermediates, includ gene that encodes a xylulose 5-phosphate translocator from ing, without limitation, Xylose reductase (converts Xylose to Arabidopsis. Xylitol), Xylitol dehydrogenase (converts Xylitol to Xylulose), Xylose transporter” is a proteins and genes encoding Such and Xylulokinase (converts Xylulose to Xylulose 5-phos proteins that transports xylose from the cell medium into the phate). Illustrative oxidoreductase pathway genes, include, cell and includes, without limitation, passive transporters and without limitation, the XYL1, XYL2, and XYL3 genes from active transporters and translocators. Passive transporters Pichia stipitis. facilitate the transport of molecule down a concentration gra “Promoter is a nucleic acid control sequence that directs 10 dient (from an area of higher concentration to an area of lower transcription of a nucleic acid. As used herein, a promoter concentration) without energy consumption, and include, includes necessary nucleic acid sequences near the start site without limitation, the Pichia stipitis SUT1, SUT2, and SUT3 of transcription, Such as, in the case of a polymerase II type transporters and the S. cerevisiae HXT transporters. Active promoter, a TATA element. A promoter also optionally transporters consume energy in transporting molecules includes distal enhancer or repressor elements, which can be 15 against a concentration gradient (and so transport from an located as much as several thousand base pairs from the start area of lower concentration to an area of higher concentra site of transcription. tion). Active transporters include, without limitation, primary “Recombinant' is a cell, nucleic acid, protein or vector, active transporters, which use ATP as an energy source, i.e., that has been modified due to the introduction of an exog ATP-binding cassette (ABC) transporters, and secondary enous nucleic acid or the alteration of a native nucleic acid. active transporters, which use energy from chemiosmotic Thus, e.g., recombinant cells express genes that are not found gradients and include, without limitation antiporters and within the native (non-recombinant) form of the cell or symporters, i.e., the GXS1 protein of Candida intermedia, the express native genes differently than those genes are H+-symporter-like proteins (e.g. At5g5925On) from Arabi expressed by a non-recombinant cell. A “recombinant nucleic dopsis, and the XLT1 protein from Trichoderma reesei. acid' is a nucleic acid originally formed in vitro, in general, 25 Xylose utilization gene' is a gene that, when expressed, by the manipulation of nucleic acid, e.g., using polymerases aids the ability of a cell to utilize xylose as a carbon source and endonucleases, or otherwise is in a form not normally (e.g., for producing energy and/or anabolism). Proteins found in nature. Recombinant nucleic acids may be produced, encoded by a xylose utilization gene are referred to herein as for example, to place two or more nucleic acids in operable "xylose utilization enzymes' and include Xylose transporters, linkage. Thus, an isolated nucleic acidoran expression vector 30 Xylose translocators, oxidoreductase pathway proteins, and formed in vitro by ligating DNA molecules that are not nor Xylose isomerase pathway proteins. mally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic II. General acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host 35 Illustrative embodiments of the present invention over cell; however, such nucleic acids, once produced recombi come long standing challenges in using cell cultures to con nantly, although Subsequently replicated intracellularly, are Vert Xylose into high-value cell biomass, by engineering a still considered recombinant for purposes of this invention. Xylose utilization pathway into an organism that previously Similarly, a "recombinant protein’ is a protein made using lacked the ability to metabolize xylose. In various aspects, the recombinant techniques, i.e., through the expression of a 40 present disclosure provides for engineering an active Xylose recombinant nucleic acid. metabolism pathway into an organism, and especially a “Renewable diesel’ is a mixture of alkanes (such as C10:0, microorganism (also referred to herein as a microorganism), C12:0, C14:0, C16:0 and C18:0) produced through hydroge of the kingdom Plantae. Such organisms include microalgae. nation and deoxygenation of lipids. In another aspect, the present disclosure provides for engi "Saccharification' is a process of converting biomass, usu 45 neering an active Xylose metabolism pathway into an organ ally cellulosic or lignocellulosic biomass, into monomeric ism, and especially a microorganism, having both plastids Sugars, such as glucose and Xylose. "Saccharified’ or “depo and a type II fatty acid biosynthesis pathway. lymerized cellulosic material or biomass refers to cellulosic Although others may have obtained ethanol production material or biomass that has been converted into monomeric using Xylose in Saccharomyces, such organisms are not use Sugars through saccharification. 50 ful for commercial scale production of lipid. By contrast, “Sucrose utilization gene' is a gene that, when expressed, oleaginous microorganisms such as oleaginous yeast and ole aids the ability of a cell to utilize Sucrose as an energy (car aginous microalgae can accumulate greater than 20% lipid by bon) source. Proteins encoded by a Sucrose utilization gene dry cell weight, mostly as triglyceride. Although oleaginous are referred to herein as “sucrose utilization enzymes' and yeast are useful in producing lipid, microalgae have a type II include Sucrose transporters, Sucrose invertases, and hexoki 55 fatty acid biosynthesis pathway, which facilitates the use of nases such as glucokinases and fructokinases. genetic engineering to tailor the fatty acid profile of lipid, and Xylose isomerase pathway are the proteins and/or genes especially triglyceride, produced by the cells. See PCT pub encoding them capable of converting Xylose to Xylulose lications WO2011/151149, WO2010/063032, and WO2011/ 5-phosphate via a xylulose intermediate, including, without 15040. In addition to producing lipid, the organisms limitation, Xylose isomerase (converts Xylose to Xylulose) 60 described herein can be used to produce protein, Vitamins, and Xylulokinase (converts Xylulose to Xylulose 5-phos fiber, and other Substances produced naturally, or as a result of phate). Illustrative Xylose isomerase pathway genes include, further genetic engineering. without limitation, the Piromyces sp. gene XylA and the In accordance with an embodiment of the present inven Pichia stipitis gene XYL3. tion, an exogenous nucleic acid is introduced into a cell with Xylulose 5-phosphate translocator”, “xylulose 5-phos 65 a plastid that is operable to produce a xylulose-5-phosphate phate transporter” or "plastidic pentose phosphate transloca transporter (e.g., a xylulose-5-phosphate translocator) pro tor” is a family of plastidic membrane proteins and genes tein that is operable to cause the transport of xylulose-5- US 8,846,352 B2 10 phosphate into a plastid of the cell. In a specific embodiment, ment, when cells of the recombinant organism are cultured on the cell is transformed with exogenous DNA that encodes a a carbon source that is 60% glucose and 40% xylose under Xylulose-5-phosphate transporter in operable linkage with a nitrogen limiting conditions, the cells produce lipid in which plastid targeting sequence. The plastid targeting sequence can the carbonatoms are at least 5, 10, 20, 30, 40, or 50% derived direct the transporter protein to a plastid membrane (prefer from carbonatoms of xylose, as measured by isotopic tracing. ably the inner plastid membrane) so as to effectively transport In yet another embodiment, the cells of the recombinant Xylulose-5-phosphate into a plastid, where it can be metabo organism are capable of consuming at least 90% of the Xylose lized, and potentially into fatty acid. For example, the organ in a culture media having 2.5% Xylose as a sole carbon Source ism can have an endogenous pentose metabolism pathway in in less than or equal to 35, 28, 21, 14 or 7 days. the plastid that metabolizes the xylulose-5-phosphate. As 10 In addition to the introduction of the xylose metabolic shown in the examples below, this has been found to be an pathway, the organism can be genetically engineered by effective method of boosting xylose utilization concurrent introducing genes that are operable to alter the fatty acid with cell growth and/or lipid production. profile of lipid produced by the microorganism. As a result, Optionally, or in addition, the cells may be capable of Xylose may be converted into acylglycerides having an taking up Xylose from the external milieu and converting the 15 altered fatty acid profile. For example, the cells can have a Xylose to Xylulose-5-phosphate. Exogenous genes may be recombinant modification comprising one or more exog needed to produce active proteins to perform these functions. enous genes encoding an active acyl-ACP thioesterase, ketoa For example, and as described in the Examples below, the cell cyl-ACP synthase or a fatty acid desaturase. Alternately, or in may be transformed with DNA operable to encode an active addition, the cells can have a recombinant modification that is Xylose transporter for transporting Xylose from an external operable to reduce or ablate an acyl-ACP thioesterase, ketoa medium into the cell, and a pathway for converting the Xylose cyl-ACP synthase or fatty acid desaturase. Methods for per intoxylulose-5-phosphate. For example, the pathway for con forming Such genetic manipulations are provided in PCT Verting the Xylose into Xylulose-5-phosphate can be a com publications WO2011/151149, WO2010/063032, and bination of a xylulokinase for converting Xylulose to Xylu WO2O11/15040. lose-5-phosphate with a pathway for converting Xylose to 25 In a preferred embodiment, the xylose metabolizing cells Xylulose. The pathway for converting Xylose to Xylulose can are cultivated in a Xylose containing medium. As a result, comprise a Xylose isomerase or a combination of a Xylose biomass is produced. In a specific embodiment, the Xylose is reductase and a Xylitol dehydrogenase. Alternately, Xylose converted into cell biomass, and triglyceride in particular. transport into the cell can rely on the activity of native pro This may be shown by isotope tracing; e.g. using isotopically teins such as hexose transport proteins. 30 labeled Xylose. To produce triglyceride, an oleaginous micro Optionally, in a further refinement of any of the above organism can be cultivated under nutrient limiting conditions, embodiments, the cells may be transformed to express an e.g., under nitrogen limitation. The resulting natural oil is activeXylitoldehydrogenase. This modification may function harvested. Harvesting may include cell lysis to release oil. to reduce or eliminate inhibition of xylose isomerase by xyli The oil may then be used to produce an oleochemical, a tol. 35 lubricant, detergent, fuel, food, oil, cosmetic ingredient, or The various genes may be integrated into a chromosome of other product. For example, the oil may be used to produce the organism, or may be episomal. Preferably, the genes are biodiesel (fatty acid esters) or renewable diesel (a fuel pro stably expressed, and may be in multiple copy. In various duced from the oil via cracking). embodiments, the genes are under the control of a regulatable In an embodiment, the method includes cultivating the or constitutive promoter. After transformation, and optionally 40 cells in a culture medium that comprises a carbon Source that for maintaining stability, the cells may be transformed with a is greater than 50% xylose. For example, the carbon source selectable marker. can be greater than 55%, 60%. 65%, 70%, 75%, 80%, 85%, The cells are capable of propogation and/or lipid accumu 90%, or 95% xylose. lation under heterotrophic growth conditions. Optionally, the Accordingly, a specific embodiment of the present inven cells may be those of an obligate heterotroph such as those of 45 tion comprises a microorganism having a plastid and an oper the microalgae genus, Prototheca. The cells, may be, for able Xylose metabolic pathway resulting from the introduc example, those of Prototheca moriformis. In the Examples tion and expression of exogenous genes. The genes are active below, Prototheca cells are transformed to express an active to convert Xylose to Xylulose-5-phosphate and to transport the xylose metabolic pathway. It should be noted however, that xylulose-5-phosphate into the plastid for further metabolism. the embodiments of the invention are applicable to many 50 The microorganism optionally has a xylose transporter to other species. Preferred species include those of the kingdom transport Xylose from an external milieu into the cell. As a Plantae, the Subkingdom Viridiplantae, the infrakingdom or result of the expression of the exogenous genes, the cell is phyla Chlorophytae, the subphylum Tetraphytina, the class able to grow on pure Xylose and preferably can consume at Trebouxiophyceae, or the order Chlorellales. Specific least 90% of the xylose in a culture media having 2.5%xylose examples include Prototheca moriformis and Chlorella pro 55 as a sole carbon source in, e.g., 21, 14 or 7 days or less. tothecoides. In a preferred embodiment, the resulting recombinant III. Cultivation organism is capable of accumulating at least 10, 20, 30, 40, 50, 60, 70, or 80% triglyceride by dry cell weight when The present invention generally relates to cultivation of cultured using glucose as a carbon Source. Further the organ 60 oleaginous microbes including but not limited to microalgae, ism can be capable of accumulating at least 10, 20, 30, 40, 50. oleaginous yeast, oleaginous fungi, and oleaginous bacteria. 60, 70, or 80% triglyceride by dry cell weight when cultured In some embodiments, microalgae strains, particularly on pure Xylose. For example, the microorganism may be recombinant Chlorella and Prototheca strains, are suitable capable of accumulating at least 50% triglyceride by dry cell for the production of lipid. For the convenience of the reader, weight when cultured on glucose as a carbon Source and is 65 this section is subdivided into subsections. Subsection 1 capable of accumulating at least 20% triglyceride by dry cell describes Prototheca species and strains and how to identify weight when cultured on pure Xylose. In another embodi new Prototheca species and strains and related microalgae by US 8,846,352 B2 11 12 genomic DNA comparison. Subsection 2 describes bioreac least 99%, least 95%, at least 90%, or at least 85% nucleotide tors useful for cultivation. Subsection 3 describes media for identity to at least one of the sequences listed in SEQID NOs: cultivation. Subsection 4 describes oil production in accor 1-9. dance with illustrative cultivation methods of the invention. For sequence comparison to determine percent nucleotide 1. Prototheca Species and Strains or amino acid identity, typically one sequence acts as a ref Prototheca is a remarkable microorganism for use in the erence sequence, to which test sequences are compared. production of lipid, because it can produce high levels of When using a sequence comparison algorithm, test and ref lipid, particularly lipid suitable for fuel production. The lipid erence sequences are input into a computer, Subsequence produced by Prototheca has hydrocarbon chains of shorter coordinates are designated, if necessary, and sequence algo 10 rithm program parameters are designated. The sequence com chain length and a higher degree of Saturation than that pro parison algorithm then calculates the percent sequence iden duced by other microalgae. Moreover, Prototheca lipid is tity for the test sequence(s) relative to the reference sequence, generally free of pigment (low to undetectable levels of chlo based on the designated program parameters. rophyll and certain carotenoids) and in any event contains Optimal alignment of sequences for comparison can be much less pigment than lipid from other microalgae. More 15 conducted, e.g., by the local homology algorithm of Smith & over, recombinant Prototheca cells provided by the invention Waterman, Adv. Appl. Math. 2:482 (1981), by the homology can be used to produce lipid in greater yield and efficiency, alignment algorithm of Needleman & Wunsch, J. Mol. Biol. and with reduced cost, relative to the production of lipid from 48:443 (1970), by the search for similarity method of Pearson other microorganisms based on their increased ability to uti & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by lize Xylose as a carbon Source. In addition, this microalgae computerized implementations of these algorithms (GAP, grows heterotrophically and can be genetically engineered. BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Illustrative Prototheca strains for use in the methods of the Software Package, Genetics Computer Group, 575 Science invention include Prototheca wickerhamii, Prototheca Stag Dr. Madison, Wis.), or by visual inspection (see generally nora (including UTEX 327), Prototheca portoricensis, Pro Ausubel et al., Supra). totheca moriformis (including UTEX strains 1441, 1435), 25 Another example algorithm that is Suitable for determining and Prototheca Zopfi. Species of the genus Prototheca are percent sequence identity and sequence similarity is the obligate heterotrophs. BLAST algorithm, which is described in Altschul et al., J. Species of Prototheca for use in the invention can be iden Mol. Biol. 215:403-410 (1990). Software for performing tified by amplification of certain target regions of the genome. BLAST analyses is publicly available through the National 30 Center for Biotechnology Information. This algorithm For example, identification of a specific Prototheca species or involves first identifying high scoring sequence pairs (HSPs) strain can be achieved through amplification and sequencing by identifying short words of length W in the query sequence, of nuclear and/or chloroplast DNA using primers and meth which either match or satisfy some positive-valued threshold odology using any region of the genome, for example using score T when aligned with a word of the same length in a the methods described in Wu et al., Bot. Bull. Acad. Sin. 35 database sequence. T is referred to as the neighborhood word (2001) 42:115-121 Identification of Chlorella spp. isolates score threshold (Altschul et al., Supra.). These initial neigh using ribosomal DNA sequences. Well established methods borhood word hits act as seeds for initiating searches to find of phylogenetic analysis, Such as amplification and sequenc longer HSPs containing them. The word hits are then ing of ribosomal internal transcribed spacer (ITS1 and ITS2 extended in both directions along each sequence for as far as rDNA), 23S rRNA, 18S rRNA, and other conserved genomic 40 the cumulative alignment score can be increased. Cumulative regions can be used by those skilled in the art to identify scores are calculated using, for nucleotide sequences, the species of not only Prototheca, but other hydrocarbon and parameters M (reward score for a pair of matching residues; lipid producing organisms with similar lipid profiles and pro always >0) and N (penalty score for mismatching residues; duction capability. For examples of methods of identification always <0). For amino acid sequences, a scoring matrix is and classification of algae also see for example Genetics, 45 used to calculate the cumulative score. Extension of the word 2005 August: 170(4):1601-10 and RNA, 2005 April; 11(4): hits in each direction are halted when: the cumulative align 361-4. ment score falls off by the quantity X from its maximum Thus, genomic DNA comparison can be used to identify achieved value; the cumulative score goes to Zero or below Suitable species of microalgae to be used in the present inven due to the accumulation of one or more negative-scoring tion. Regions of conserved genomic DNA, such as but not 50 residue alignments; or the end of either sequence is reached. limited to DNA encoding for 23S rRNA, can be amplified For identifying whether a nucleic acid or polypeptide is from microalgal species and compared to consensus within the scope of the invention, the default parameters of sequences in order to Screen for microalgal species that are the BLAST programs are suitable. The BLASTN program taxonomically related to the preferred microalgae used in the (for nucleotide sequences) uses as defaults a word length (W) present invention. Examples of such DNA sequence compari 55 of 11, an expectation (E) of 10, M-5, N=-4, and a comparison son for species within the Prototheca genus are shown below. of both strands. For amino acid sequences, the BLASTP Genomic DNA comparison can also be useful to identify program uses as defaults a word length (W) of 3, an expecta microalgal species that have been misidentified in a strain tion (E) of 10, and the BLOSUM62 scoring matrix. The collection. Often a strain collection will identify species of TBLATN program (using protein sequence for nucleotide microalgae based on phenotypic and morphological charac 60 sequence) uses as defaults a word length (W) of 3, an expec teristics. The use of these characteristics may lead to miscat tation (E) of 10, and a BLOSUM 62 scoring matrix. (see egorization of the species or the genus of a microalgae. The Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 use of genomic DNA comparison can be a better method of (1989)). categorizing microalgae species based on their phylogenetic In addition to calculating percent sequence identity, the relationship. 65 BLAST algorithm also performs a statistical analysis of the Microalgae for use in the present invention typically have similarity between two sequences (see, e.g., Karlin & Alts genomic DNA sequences encoding for 23S rRNA that have at chul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One US 8,846,352 B2 13 14 measure of similarity provided by the BLAST algorithm is tor throughout the time period. Thus in Some embodiments, the smallest sum probability (P(N)), which provides an indi aqueous culture medium is not flowed through the bioreactor cation of the probability by which a match between two after inoculation. nucleotide or amino acid sequences would occur by chance. Bioreactors equipped with devices Such as spinning blades For example, a nucleic acid is considered similar to a refer and impellers, rocking mechanisms, stir bars, means for pres ence sequence if the Smallest Sum probability in a comparison Surized gas infusion can be used to subject microalgal cul of the test nucleic acid to the reference nucleic acid is less than tures to mixing. Mixing may be continuous or intermittent. about 0.1, more preferably less than about 0.01, and most For example, in some embodiments, a turbulent flow regime preferably less than about 0.001. of gas entry and media entry is not maintained until a desired 10 increase in number of said microalgae has been achieved. Other considerations affecting the selection of microor Bioreactorports can be used to introduce, or extract, gases, ganisms for use in the invention include, in addition to pro Solids, semisolids, and liquids, into the bioreactor chamber duction of suitable lipids or hydrocarbons for production of containing the microalgae. While many bioreactors have oils, fuels, and oleochemicals: (1) high lipid content as a more than one port (for example, one for media entry, and percentage of cell weight; (2) ease of growth; (3) ease of 15 another for sampling), it is not necessary that only one Sub genetic engineering; and (4) ease of biomass processing. In stance enter or leave a port. For example, a port can be used to particular embodiments, the wild-type or genetically engi flow culture media into the bioreactor and later used for neered microorganism yields cells that are at least 40%, at sampling, gas entry, gas exit, or other purposes. Preferably, a least 45%, at least 50%, at least 55%, at least 60%, at least sampling port can be used repeatedly without altering com 65%, or at least 70% or more lipid. Preferred organisms grow promising the axenic nature of the culture. A sampling port heterotrophically (on Sugars in the absence of light). can be configured with a valve or other device that allows the 2. Bioreactor flow of sample to be stopped and started or to provide a means Microorganisms are cultured both for purposes of conduct of continuous sampling. Bioreactors typically have at least ing genetic manipulations and for production of hydrocar one port that allows inoculation of a culture, and Such a port bons (e.g., lipids, fatty acids, aldehydes, alcohols, and 25 can also be used for other purposes such as media orgas entry. alkanes). The former type of culture is conducted on a small Bioreactors ports allow the gas content of the culture of scale and initially, at least, under conditions in which the microalgae to be manipulated. To illustrate, part of the Vol starting microorganism can grow. Culture for purposes of ume of a bioreactor can be gas rather than liquid, and the gas hydrocarbon production is usually conducted on a large scale inlets of the bioreactor to allow pumping of gases into the (e.g., 10,000 L, 40,000 L, 100,000 L or larger bioreactors) in 30 bioreactor. Gases that can be beneficially pumped into a a bioreactor. Prototheca are typically cultured in the methods bioreactor include air, air/CO mixtures, noble gases, such as of the invention in liquid media containing xylose within a argon, and other gases. Bioreactors are typically equipped to bioreactor. In one embodiment, microalgae Such as Prototh enable the user to control the rate of entry of a gas into the eca are cultured in a bioreactor using a fed-batch process. bioreactor. As noted above, increasing gas flow into a biore Typically, the bioreactor does not allow light to enter, and the 35 actor can be used to increase mixing of the culture. microorganisms are cultured heterotrophically, metabolizing Increased gas flow affects the turbidity of the culture as a fixed carbon source, and in the Substantial absence of light. well. Turbulence can be achieved by placing a gas entry port The bioreactor or fermentor is used to culture microalgal below the level of the aqueous culture media so that gas cells through the various phases of their physiological cycle. entering the bioreactor bubbles to the surface of the culture. Bioreactors offer many advantages for use in heterotrophic 40 One or more gas exit ports allow gas to escape, thereby growth and propagation methods. To produce biomass foruse preventing pressure buildup in the bioreactor. Preferably a gas in food, microalgae are preferably fermented in large quanti exit port leads to a “one-way valve that prevents contami ties in liquid. Such as in Suspension cultures as an example. nating microorganisms from entering the bioreactor. Bioreactors such as steel fermentors can accommodate very 3. Media large culture volumes (40,000 liter and greater capacity biore 45 Microalgal culture media typically contains components actors are used in various embodiments of the invention). Such as a fixed nitrogen source, a fixed carbon source, trace Bioreactors also typically allow for the control of culture elements, optionally a buffer for pH maintenance, and phos conditions such as temperature, pH, oxygen tension, and phate (typically provided as a phosphate salt). Other compo carbon dioxide levels. For example, bioreactors are typically nents can include salts such as sodium chloride, particularly configurable, for example, using ports attached to tubing, to 50 for seawater microalgae. Nitrogen sources include organic allow gaseous components, like oxygen or nitrogen, to be and inorganic nitrogen sources, including, for example, with bubbled through a liquid culture. Other culture parameters, out limitation, molecular nitrogen, nitrate, nitrate salts, Such as the pH of the culture media, the identity and concen ammonia (pure or in Salt form, Such as, (NH4)2SO and tration of trace elements, and other media constituents can NH-OH), protein, soybean meal, cornsteep liquor, and yeast also be more readily manipulated using a bioreactor. 55 extract. Examples of trace elements include Zinc, boron, Bioreactors can be configured to flow culture media though cobalt, copper, manganese, and molybdenum in, for example, the bioreactor throughout the time period during which the the respective forms of ZnCl2, HBO, CoCl2.6H2O. microalgae reproduce and increase in number. In some CuCl2.H2O, MnCl2.4H2O and (NH4)Mo.O.4H2O. embodiments, for example, media can be infused into the Solid and liquid growth media are generally available from bioreactor after inoculation but before the cells reach a 60 a wide variety of sources, and instructions for the preparation desired density. In other instances, a bioreactor is filled with of particular media that is suitable for a wide variety of strains culture media at the beginning of a culture, and no more of microorganisms can be found, for example, from the cul culture media is infused after the culture is inoculated. In ture collection of algae (UTEX) maintained by the University other words, the microalgal biomass is cultured in an aqueous of Texas at Austin, 1 University Station A6700, Austin, Tex., medium for a period of time during which the microalgae 65 78712-0183 For example, various fresh water and salt water reproduce and increase in number; however, quantities of media include those described in PCT Pub. No. 2008/151149, aqueous culture medium are not flowed through the bioreac incorporated herein by reference. US 8,846,352 B2 15 16 In a particular example, Proteose Medium is suitable for include Xylose, depolymerized cellulosic material, glycerol, axenic cultures, and a 1 L volume of the medium (pH~6.8) glucose, Sucrose, and Sorghum, each of which is discussed in can be prepared by addition of 1 g of proteose peptone to 1 more detail below. liter of Bristol Medium. Bristol medium comprises 2.94 mM In accordance with the present invention, microorganisms NaNO, 0.17 mM CaCl2.H2O, 0.3 mM MgSO.7HO, 0.43 can be cultured using depolymerized cellulosic biomass as a mM, 1.29 mM KHPO, and 1.43 mM NaCl in an aqueous feedstock. Cellulosic biomass (e.g., stover, Such as corn sto Solution. For 1.5% agar medium, 15 g of agar can be added to Ver) is inexpensive and readily available. Microalgae can 1 L of the solution. The solution is covered and autoclaved, grow on processed cellulosic material, and microalgae of the and then stored at a refrigerated temperature prior to use. invention are especially designed to utilize Xylose as a carbon 10 Source with high efficiency. Cellulosic materials generally Another example is the Prototheca isolation medium (PIM), include about 40-60% cellulose; about 20-40% hemicellu which comprises 10 g/L potassium hydrogen phthalate lose; and 10-30% lignin. (KHP), 0.9 g/L sodium hydroxide, 0.1 g/L magnesium sul Suitable cellulosic materials include residues from herba fate, 0.2 g/L potassium hydrogen phosphate, 0.3 g/L ammo ceous and woody energy crops, as well as agricultural crops, nium chloride, 10 g/L glucose 0.001 g/L thiamine hydrochlo 15 i.e., the plant parts, primarily stalks and leaves, not removed ride, 20 g/L agar, 0.25 g/L 5-fluorocytosine, at a pH in the from the fields with the primary food or fiber product. range of 5.0 to 5.2 (see Pore, 1973, App. Microbiology, 26: Examples include agricultural wastes such as Sugarcane 648-649). Other suitable media for use with the methods of bagasse, rice hulls, corn fiber (including stalks, leaves, husks, the invention can be readily identified by consulting the URL and cobs), wheat Straw, rice Straw, Sugar beet pulp, citrus identified above, or by consulting other organizations that pulp, citrus peels; forestry wastes such as hardwood and maintain cultures of microorganisms, such as SAG, CCAP or Softwood thinnings, and hardwood and softwood residues CCALA. SAG refers to the Culture Collection of Algae at the from timber operations; wood wastes such as saw mill wastes University of Göttingen (Göttingen, Germany), CCAP refers (wood chips, sawdust) and pulp mill waste; urban wastes Such to the culture collection of algae and protozoa managed by the as paper fractions of municipal Solid waste, urban wood waste Scottish Association for Marine Science (Scotland, United 25 and urban green waste such as municipal grass clippings; and Kingdom), and CCALA refers to the culture collection of wood construction waste. Additional cellulosics include algal laboratory at the Institute of Botany (Trebon, Czech dedicated cellulosic crops such as Switchgrass, hybrid poplar Republic). Additionally, U.S. Pat. No. 5,900,370 describes wood, and miscanthus, fiber cane, and fiber Sorghum. Five media formulations and conditions suitable for heterotrophic carbon Sugars that are produced from Such materials include fermentation of Prototheca species. 30 Xylose. For oil production, selection of a fixed carbon source is Cellulosic materials may be treated to increase the effi important, as the cost of the fixed carbon source must be ciency with which the microbe can utilize the sugar(s) con Sufficiently low to make oil production economical. In one tained within the materials. As discussed above, lignocellu embodiment, the fixed carbon source is Xylose. Xylose is a losic biomass is comprised of various fractions, including five-carbon monosaccharide that is found as a component of 35 cellulose, a crystalline polymer of beta 1.4 linked glucose (a hemicellulose. Hemicellulose is a complex polysaccharide six-carbon Sugar), hemicellulose, a more loosely associated that is found in almost all plant cell walls, comprising about polymer predominantly comprised of Xylose (a five-carbon 30% of all plant matter. In addition to xylose, other carbon Sugar) and to a lesser extent mannose, galactose, arabinose, Sources can be included, for example, acetate, floridoside, lignin, a complex aromatic polymer comprised of Sinapyl fructose, galactose, glucuronic acid, glucose, glycerol, lac 40 alcohol and its derivatives, and pectins, which are linear tose, mannose, N-acetylglucosamine, rhamnose, Sucrose, chains of an alpha 1.4 linked polygalacturonic acid. Because and/or Xylose, selection offeedstocks containing those com of the polymeric structure of cellulose and hemicellulose, the pounds, particularly Xylose, is an important aspect of the Sugars (e.g., monomeric glucose and Xylose) in them are not methods of the invention. in a form that can be efficiently used (metabolized) by many Other suitable feedstocks useful in accordance with the 45 microbes. For such microbes, further processing of the cellu methods of the invention include, for example, black liquor, losic biomass to generate the monomeric Sugars that make up corn starch, depolymerized cellulosic material, milk whey, the polymers can be very helpful to ensuring that the cellulo molasses, potato, Sorghum, Sucrose, Sugar beet, Sugar beet sic materials are efficiently utilized as a feedstock (carbon juice, Sugar cane, Sugar cane juice, thick cane juice, rice, and Source). wheat. Carbon Sources can also be provided as a mixture, 50 Cellulose or cellulosic biomass can be subjected to a pro Such as a mixture of Sucrose or Xylose and depolymerized cess, termed “explosion', in which the biomass is treated with Sugar beet pulp, depolymerized cellulosic material and the dilute sulfuric (or other) acid at elevated temperature and like. The one or more carbon source(s) can be Supplied at a pressure. This process conditions the biomass such that it can concentration of at least about 50 uM, at least about 100 uM, be efficiently subjected to enzymatic hydrolysis of the cellu at least about 250 uM, at least about 500 uM, at least about 1 55 losic and hemicellulosic fractions into glucose and Xylose mM, at least about 2.5 mM, at least about 5 mM, at least about monomers. The resulting monomeric Sugars are termed cel 10 mM, at least about 25 mM, at least about 50 mM, at least lulosic Sugars. Cellulosic Sugars can Subsequently be utilized about 100 mM, at least 250 mM, and at least about 500 mM, by microorganisms to produce a variety of metabolites (e.g., of one or more exogenously provided fixed carbon Source(s). lipid). The acid explosion step results inapartial hydrolysis of In some cases, one or more carbon Source(s) can be supplied, 60 the hemicellulose fraction to constitutent monosaccharides. as a feedstock for fed-batch fermentation, at a concentration These sugars can be completely liberated from the biomass of at least about 100 g/L, of at least about 200 g/L, of at least with further treatment. In some embodiments, the further about 300 g/L of at least about 400 g/L, at least about 500 g/L, treatment is a hydrothermal treatment that includes washing at least about 600 g/L, at least about 700 g/L, at least about the exploded material with hot water, which removes con 800 g/L or more or the exogenously provided fixed carbon 65 taminants such as salts. This step is not necessary for cellu source(s) into the fermentation culture. Carbon sources of losic ethanol fermentations due to the more dilute Sugar con particular interest for purposes of the present invention centrations used in Such processes. In other embodiments, the US 8,846,352 B2 17 18 further treatment is additional acid treatment. In still other 20100151538, hereby incorporated by reference. In one embodiments, the further treatment is enzymatic hydrolysis embodiment, the saccharified lignocellulosic Sugars have of the exploded material. These treatments can also be used in been treated to reduce both the toxins (e.g., furfurals and any combination. The type of treatment can affect the type of hydroxymethylfurfurals) and the salt level and then concen Sugars liberated (e.g., five carbon Sugars versus six carbon 5 trated to a concentration of at least 600 g/L or more Sugar Sugars) and the stage at which they are liberated in the pro monomer in the concentrated Sugar stream or fixed carbon cess. As a consequence, different streams of Sugars, whether Source feedstock. In other embodiments, such concentrated they are predominantly five-carbon or six-carbon, can be saccharified lignocellulosic Sugars have a salt level of less created. These enriched five-carbon or six-carbon streams than 700 mM sodium equivalents. In other embodiments, canthus be directed to specific microorganisms with different 10 Such concentrated Saccharified lignocellulosic Sugars have a carbon utilization cabilities. In certain embodiments of the salt level of less than 100 mM sodium equivalents, less than methods of the present invention, five-carbon xylose enriched 200 mM sodium equivalents, less than 300 mM sodium streams are preferred. In other embodiments, different Sugar equivalents, less than 400 mM sodium equivalents, less than streams containing five-carbon Sugars, six-carbon Sugars, or a 500 mM sodium equivalents, less than 600 mM sodium combination of five-carbon/six carbon Sugars that have been 15 equivalents, less than 700 mM sodium equivalents, or less saccharified, can be combined for use in microbial fermenta than 1000 mM sodium equivalents. In still other embodi tion to produce high cell density lipid-producing microbial ments, such concentrated saccharified lignocellulosic Sugar biomass. feedstock has a conductivity of less than 20,000 uS/cm, less Embodiments of the present invention related to lipid pro than 15,000 uS/cm, less than 10,000 uS/cm, less than 7,500 duction may involve fermentation to higher cell densities than uS/cm, less than 5,000 LS/cm, less than 1000 uS/cm, less than what is achieved in ethanol fermentation. Because of the 500 LS/cm, less than 300 LS/cm, or less than 200 LS/cm of higher densities of the cultures for heterotrophic cellulosic oil conductivity. In still other embodiments, such concentrated production, the fixed carbon Source (e.g., the cellulosic saccharified lignocellulosic Sugar feedstock have been derived Sugar stream(s)) is preferably in a concentrated form. enriched for five carbon Sugar monomers, or six carbon Sugar For example, the glucose concentration, or the Xylose con 25 mononers, or a combination of five and six carbon Sugar centration, or the Sum of glucose and Xylose concentration of OOCS. the depolymerized cellulosic material can be at least about In another embodiment of the methods of the invention, the 100 g/liter, at least about 200 g/liter, at least about 300 g/liter, carbon Source includes glycerol. Such as acidulated and non at least about 400 g/liter, at least about 500 g/liter at least acidulated glycerol byproduct from biodiesel transesterifica about 600 g/liter at least about 700 g/liter, at least about 800 30 tion. In one embodiment, the carbon source includes glycerol g/liter or more of glucose or Xylose or a combination of and Xylose. In some cases, all of the glycerol and the Xylose glucose and xylose in the feedstock prior to the cultivation are provided to the microorganism at the beginning of the step, which is optionally a fed batch cultivation in which the fermentation. In some cases, the glycerol and the Xylose are material is fed to the cells over time as the cells grow and provided to the microorganism simultaneously at a predeter accumulate lipid. Various methods for Such saccharification 35 mined ratio. In some cases, the glycerol and the Xylose are fed methods and other methods for processing cellulosics for use to the microbes at a predetermined rate over the course of in microalgal fermentations are described in U.S. Patent fermentation. Application Publication 20100151112. Some microalgae undergo cell division faster in the pres The explosion process treatment of the cellulosic material ence of glycerol than in the presence of another carbon utilizes significant amounts of Sulfuric acid, heat and pres 40 source, such as glucose (see PCT Pub. No. 2008/151149). In sure, thereby liberating by-products of carbohydrates, instances where this is true for glycerol and Xylose, two-stage namely furfurals and hydroxymethylfurfurals. Furfurals and growth processes in which cells are first fed glycerol to rap hydroxymethyl furfurals are produced during hydrolysis of idly increase cell density, and are then fed Xylose to accumu hemicellulose through dehydration of xylose into furfural and late lipids can improve the efficiency with which lipids are water. While this loss of xylose is not desired in many 45 produced. The use of the glycerol byproduct of the transes embodiments of the methods of the present invention, such terification process provides significant economic advantages that either a non-explosion process is employed or the pro when put back into the production process. Other feeding duction of furfurals and hydroxymethyl furfurals is mini methods are provided as well. Such as mixtures of glycerol mized when they are used, these by-products (e.g., furfurals and Xylose. Feeding Such mixtures also captures the same and hydroxymethyl furfurals) can be removed from the sac 50 economic benefits. Thus, the invention provides methods of charified lignocellulosic material prior to introduction into feeding alternative Sugars to microalgae Such as Xylose in the bioreactor, and the conductivities of the cellulosic mate various combinations with glycerol. rials resulting from the explosion process can be reduced by In various embodiments of the methods of the invention, methods described in U.S. Patent Application Publication the carbon Source is Xylose, including a complex feedstock 2O1 OO151112. 55 containing Xylose, Such as a cellulosics-derived material. In In other embodiments, the explosion process itself is one embodiment, the culture medium further includes at least changed so as to avoid the generation of salts at unacceptably one Xylose utilization enzyme. The use of complex feed high levels. For example, a suitable alternative to sulfuric acid stocks containing Xylose can provide significant cost savings (or other acid) explosion of the cellulosic biomass is mechani in the production of hydrocarbons and other oils. cal pulping to render the cellulosic biomass receptive to enzy 60 4. Oil Production matic hydrolysis (saccharification). In still other embodi For the production of oil in accordance with the methods of ments, native strains of microorganisms resistant to high the invention, it is preferable to culture cells in the dark (or in levels of salts or genetically engineered strains with resis the Substantial absence of light), as is the case, for example, tance to high levels of salts are used. A non-limiting example when using extremely large (40,000 liter and higher) fermen of a method of treating cellulosic Sugars so that it is suitable 65 tors that do not allow light to strike the culture. Obligate for use as a concentrated feedstock for the present invention is heterotroph species Such as species of Prototheca are grown described in U.S. Patent Application Publication and propagated for the production of oil in a medium con US 8,846,352 B2 19 20 taining a fixed carbon Source and in the absence of light. Such cofactor(s) are provided to the culture by including in the growth is known as heterotrophic growth. culture a microbe (e.g., microalgae) containing an exogenous As an example, an inoculum of lipid-producing microalgal gene encoding the cofactor(s). Alternatively, cofactor(s) may cells are introduced into the medium; there is a lag period (lag be provided to a culture by including a microbe (e.g., microal phase) before the cells begin to propagate. Following the lag gae) containing an exogenous gene that encodes a protein that period, the propagation rate increases steadily and enters the participates in the synthesis of the cofactor. In certain log, or exponential, phase. The exponential phase is in turn embodiments, suitable cofactors include any vitamin followed by a slowing of propagation due to decreases in required by a lipid pathway enzyme, such as, for example: nutrients such as nitrogen, increases in toxic Substances, and biotin, pantothenate. Genes encoding cofactors Suitable for quorum sensing mechanisms. After this slowing, propagation 10 stops, and the cells enter a stationary phase or steady growth use in the invention or that participate in the synthesis of Such state, depending on the particular environment provided to cofactors are well known and can be introduced into microbes the cells. For obtaining lipid rich biomass, the culture is (e.g., microalgae), using contructs and techniques such as typically harvested well after the end of the exponential those described above. phase, which may be terminated early by allowing nitrogen or 15 The specific examples of bioreactors, culture conditions, another key nutrient (other than carbon) to become depleted, and heterotrophic growth and propagation methods described forcing the cells to convert the carbon Sources, present in herein can be combined in any Suitable manner to improve excess, to lipid. Culture condition parameters can be manipu efficiencies of microbial growth and lipid and/or protein pro lated to optimize total oil production, the combination of lipid duction. species produced, and/or production of a specific oil. Microalgal biomass with a high percentage of oil/lipid As discussed above, a bioreactor or fermentor is used to accumulation by dry weight has been generated using differ allow cells to undergo the various phases of their growth ent methods of culture, which are known in the art (see PCT cycle. As an example, an inoculum of lipid-producing cells Pub. No. 2008/151149). Microalgal biomass generated by the can be introduced into a medium followed by a lag period (lag culture methods described herein and useful in accordance phase) before the cells begin growth. Following the lag 25 with the present invention comprises at least 10% microalgal period, the growth rate increases steadily and enters the log, oil by dry weight. In some embodiments, the microalgal or exponential, phase. The exponential phase is in turn fol biomass comprises at least 25%, at least 50%, at least 55%, at lowed by a slowing of growth due to decreases in nutrients least 60% microalgal oil, or at least 70% microalgal oil by dry and/or increases in toxic Substances. After this slowing, weight. In some embodiments, the microalgal biomass con growth stops, and the cells enter a stationary phase or steady 30 tains from 10-90% microalgal oil, from 25-75% microalgal state, depending on the particular environment provided to oil, from 40-75% microalgal oil, or from 50-70% microalgal the cells. Lipid production by cells disclosed herein can occur oil by dry weight. during the log phase or thereafter, including the stationary The microalgal oil of the biomass described herein, or phase wherein nutrients are Supplied, or still available, to extracted from the biomass for use in the methods and com allow the continuation of lipid production in the absence of 35 positions of the present invention can comprise glycerolipids cell division. with one or more distinct fatty acid ester side chains. Glyc Preferably, microorganisms grown using conditions erolipids are comprised of a glycerol molecule esterified to described herein and known in the art comprise at least about one, two or three fatty acid molecules, which can be of vary 20% by weight of lipid, at least about 40% by weight, at least ing lengths and have varying degrees of saturation. The length about 50% by weight, at least about 60% by weight, and at 40 and Saturation characteristics of the fatty acid molecules (and least about 70% by weight. Process conditions can be the microalgal oils) can be manipulated to modify the prop adjusted to increase the yield of lipids suitable for a particular erties or proportions of the fatty acid molecules in the use and/or to reduce production cost. For example, in certain microalgal oils of the present invention via culture conditions embodiments, a microalgae is cultured in the presence of a or via lipid pathway engineering, as described in more detail limiting concentration of one or more nutrients, such as, for 45 in Section V, below. Thus, specific blends of algal oil can be example, nitrogen, phosphorous, or Sulfur, while providing prepared either within a single species of algae by mixing an excess of fixed carbon energy Such as glucose. Nitrogen together the biomass or algal oil from two or more species of limitation tends to increase microbial lipid yield over micro microalgae, or by blending algal oil of the invention with oils bial lipid yield in a culture in which nitrogen is provided in from other sources Such as Soy, rapeseed, canola, palm, palm excess. In particular embodiments, the increase in lipid yield 50 kernel, coconut, corn, waste vegetable, Chinese tallow, olive, is at least about: 10%, 50%, 100%, 200%, or 500% greater sunflower, cottonseed, chicken fat, beef tallow, porcine tal than obtained under nitrogen replete conditions. The microbe low, microalgae, macroalgae, microbes, Cuphea, flax, peanut, can be cultured in the presence of a limiting amount of a choice white grease, lard, Camelina sativa, mustard seed, nutrient for a portion of the total culture period or for the cashew nut, oats, lupine, kenaf, calendula, help, coffee, lin entire period. In particular embodiments, the nutrient concen 55 seed (flax), hazelnut, euphorbia, pumpkin seed, coriander, tration is cycled between a limiting concentration and a non camelia, Sesame, safflower, rice, tung tree, cocoa, copra, limiting concentration at least twice during the total culture pium poppy, castor beans, pecan, jojoba, macadamia, Brazil period. Lipid content of cells can be increased by continuing nuts, avocado, petroleum, or a distillate fraction of any of the the culture for increased periods of time while providing an preceding oils. excess of carbon, but limiting or no nitrogen. 60 The oil composition, i.e., the properties and proportions of In another embodiment, lipid yield is increased by cultur the fatty acid constituents of the glycerolipids, can also be ing a lipid-producing microbe (e.g., microalgae) in the pres manipulated by combining biomass or oil from at least two ence of one or more cofactor(s) for a lipid pathway enzyme distinct species of microalgae. In some embodiments, at least (e.g., a fatty acid synthetic enzyme). Generally, the concen two of the distinct species of microalgae have different glyc tration of the cofactor(s) is sufficient to increase microbial 65 erolipid profiles. The distinct species of microalgae can be lipid (e.g., fatty acid) yield over microbial lipid yield in the cultured together or separately as described herein, prefer absence of the cofactor(s). In a particular embodiment, the ably under heterotrophic conditions, to generate the respec US 8,846,352 B2 21 22 tive oils. Different species of microalgae can contain different troporation (Fromm et al., Proc. Natl. Acad. Sci. (USA) percentages of distinct fatty acid constituents in the cells (1985) 82:5824 5828); use of a laser beam, microinjection or glycerolipids. any other method capable of introducing DNA into a microal Generally, wild type Prototheca strains have very little or gae can also be used for transformation of a Prototheca cell. no fatty acids with the chain length C8-C14. For example, 2. Homologous Recombination Methods Prototheca moriformis (UTEX 1435), Prototheca krugani Homologous recombination refers to the ability of comple (UTEX 329), Prototheca stagnora (UTEX 1442) and Pro mentary DNA sequences in a host cell to align and exchange totheca Zopfii (UTEX 1438) contains no (or undectable regions of homology. Transgenic DNA ('donor') containing amounts) C8 fatty acids, between 0-0.01% C10 fatty acids, sequences homologous to the genomic sequences being tar between 0.03-2.1% C12 fatty acids and between 1.0-1.7% 10 geted (“template') is introduced into the host cell and then C14 fatty acids. undergoes recombination into the genome at the site of the Microalgal oil can also include other constituents produced corresponding genomic homologous sequences. The mecha by the microalgae, or incorporated into the microalgal oil nistic steps of this process, in most cases, include: (1) pairing from the culture medium. These other constituents can be of homologous DNA segments; (2) introduction of double present in varying amount depending on the culture condi 15 stranded breaks into the donor DNA molecule; (3) invasion of tions used to culture the microalgae, the species of microal the template DNA molecule by the free donor DNA ends gae, the extraction method used to recover microalgal oil followed by DNA synthesis; and (4) resolution of double from the biomass and other factors that may affect microalgal strand break repair events that result in final recombination oil composition. Non-limiting examples of Such constituents products. include carotenoids, present from 0.1-0.4 micrograms/ml. The ability to carry out homologous recombination in a chlorophyll present from 0-0.02 milligrams/kilogram of oil, host organism has many practical implications for what can gamma tocopherol present from 0.4–0.6 milligrams/100 be carried out at the molecular genetic level and is useful in grams of oil, and total tocotrienols present from 0.2-0.5 mil the generation of an oleaginous microbe that can produced ligrams/gram of oil. tailored oils. By its very nature homologous recombination is The other constituents can include, without limitation, 25 a precise gene targeting event; hence, most transgenic lines phospholipids, tocopherols, tocotrienols, carotenoids (e.g., generated with the same targeting sequence will be essen alpha-carotene, beta-carotene, lycopene, etc.), Xanthophylls tially identical in terms of phenotype, necessitating the (e.g., lutein, Zeaxanthin, alpha-cryptoxanthin and beta-cry screening of far fewer transformation events. Homologous toxanthin), and various organic or inorganic compounds. recombination also targets gene insertion events into the host In some cases, the oil extracted from Prototheca species 30 chromosome, resulting in excellent genetic stability, even in comprises no more than 0.02 mg/kg chlorophyll. In some the absence of genetic selection. Because different chromo cases, the oil extracted from Prototheca species comprises no somal loci can impact gene expression, even from heterolo more than 0.4 mcg/ml total carotenoids. In some cases the gous promoters/UTRS, homologous recombination can be a Prototheca oil comprises between 0.40-0.60 milligrams of method of querying loci in an unfamiliar genome environ gamma tocopherol per 100 grams of oil. In other cases, the 35 ment and to assess the impact of these environments on gene Prototheca oil comprises between 0.2-0.5 milligrams of total expression. tocotrienols per gram of oil. Particularly useful genetic engineering applications using homologous recombination is to co-opt specific host regula IV. Genetic Engineering Methods and Materials tory elements such as promoters/UTRs to drive heterologous 40 gene expression in a highly specific fashion. For example, Embodiments of the present invention provide methods precise ablation of the endogenous Stearoyl ACP desaturase and materials for genetically modifying cells and recombi gene with a heterologous C12:0 specific FATB (thioesterase) nant host cells useful in the methods of the present invention, gene cassette and Suitable selective marker, might be including but not limited to recombinant oleaginous micro expected to dramatically decrease endogenous levels of organisms, microalgae, and oleaginous microalgae Such as 45 C18:1 fatty acids concomitant with increased levels of the Prototheca moriformis, Prototheca Zopfii, Prototheca kru C12:0 fatty acids. See U.S. Patent Application Publication gani, and Prototheca stagnora host cells. The description of 2O1 OO151112. these methods and materials is divided into subsections for Because homologous recombination is a precise gene tar the convenience of the reader. In Subsection 1, transformation geting event, it can be used to modify any nucleotide(s) within methods are described. In Subsection 2, homologous recom 50 a gene or region of interest precisely, so long as Sufficient bination methods are described. In subsection 3, expression flanking regions have been identified. Therefore, homologous vectors and vector components are described. recombination can be used as a means to modify regulatory 1. Transformation Methods sequences impacting gene expression of RNA and/or pro Cells can be transformed by any suitable technique includ teins. It can also be used to modify protein coding regions in ing, e.g., biolistics, electroporation (see Maruyama et al. 55 an effort to modify enzyme activities such as Substrate speci (2004), Biotechnology Techniques 8:821-826), glass bead ficity, affinities and Km, and thus affecting the desired change transformation and silicon carbide whisker transformation. in metabolism of the host cell. Homologous recombination Another method that can be used involves forming proto provides a powerful means to manipulate the gost genome plasts and using CaCl2 and polyethylene glycol (PEG) to resulting in gene targeting, gene conversion, gene deletion, introduce recombinant DNA into microalgal cells (see Kim et 60 gene duplication, gene inversion, and the exchange of gene al. (2002), Mar: Biotechnol. 4:63-73, which reports the use of expression regulatory elements such as promoters, enhancers this method for the transformation of Chorella ellipsoidea). and 3'UTRS. Co-transformation of microalgae can be used to introduce Homologous recombination can be achieved by using tar two distinct vector molecules into a cell simultaneously (see geting constructs containing pieces of endogenous sequences for example Protist 2004 December; 155(4):381-93). 65 to “target the gene or region of interest within the endog Biolistic methods (see, for example, Sanford, Trends In enous host cell genome. Such targeting sequences can either Biotech. (1988) 6:299 302, U.S. Pat. No. 4,945,050; elec be located 5' of the gene or region of interest, 3' of the gene/ US 8,846,352 B2 23 24 region of interest, or can flank the gene/region of interest. from Chlamydomonas reinhardtii, used in the Examples Such targeting constructs can be transformed into the host cell below, and viral promoters, such as cauliflower mosaic virus either as a supercoiled plasmid DNA with additional vector (CMV) and chlorella virus, which have been shown to be backbone, a PCR product with no vector backbone, or as a active in multiple species of microalgae (see for example linearized molecule. In some cases, it may be advantageous to Plant Cell Rep. 2005 March; 23(10-11):727-35; J. Microbiol. first expose the homologous sequences within the transgenic 2005 August; 43(4):361-5; Mar Biotechnol (NY). 2002 Janu DNA (donor DNA) with a restriction enzyme. This step can ary; 4(1):63-73). Another promoter that is suitable for use for increase the recombination efficiency and decrease the occur expression of exogenous genes in Prototheca is the Chlorella rence of undesired events. Other methods of increasing Sorokiniana glutamate dehydrogenase promoter/5'UTR recombination efficiency include using PCR to generate 10 (SEQID NO: 10). Optionally, at least 10, 20,30, 40, 50, or 60 transforming transgenic DNA containing linear ends nucleotides or more of these sequences containing a promoter homologous to the genomic sequences being targeted. are used. Illustrative promoters useful for expression of exog 3. Vectors and Vector Components enous genes in Prototheca are listed in the sequence listing of Vectors for transformation of microorganisms in accor this application, such as the promoter of the Chlorella HUP1 dance with the present invention can be prepared by known 15 gene (SEQ ID NO:11) and the Chlorella ellipsoidea nitrate techniques familiar to those skilled in the art in view of the reductase promoter (SEQ ID NO:12). Chlorella virus pro disclosure herein. A vector typically contains one or more moters can also be used to express genes in Prototheca. Such genes, in which each gene codes for the expression of a as SEQ ID NOs: 1-7 of U.S. Pat. No. 6,395,965. Additional desired product (the gene product) and is operably linked to promoters active in Prototheca can be found, for example, in (or contains) one or more control sequences that regulate gene Biochem Biophys Res Commun. 1994 Oct. 14; 204(1):187 expression or target the gene product to a particular location 94; Plant Mol. Biol. 1994 October; 26(1):85-93; Virology. in the recombinant cell. To aid the reader, this subsection is 2004 Aug. 15: 326(1): 150-9; and Virology. 2004 Jan. 5; 318 divided into subsections. Subsection. A describes control (1):214-23. sequences typically contained on vectors as well as control A promoter can generally be characterized as either con sequences for use particularly in microalgae Such as Proto 25 stitutive or inducible. Constitutive promoters are generally heca. Subsection B describes genes typically contained in active or function to drive expression at all times (or at certain vectors as well as codon optimization methods and genes times in the cell life cycle) at the same level. Inducible pro prepared using them for use particularly in microalgae, Such moters, conversely, are active (or rendered inactive) or are as Prototheca. significantly up- or down-regulated only in response to a A. Control Sequences 30 stimulus. Both types of promoters find application in the Control sequences are nucleic acids that regulate the methods of the invention. Inducible promoters useful in the expression of a coding sequence or direct a gene product to a invention include those that mediate transcription of an oper particular location in or outside a cell. Control sequences that ably linked gene in response to a stimulus, Such as an exog regulate expression include, for example, promoters that enously provided Small molecule (e.g., glucose, as in SEQID regulate transcription of a coding sequence and terminators 35 NO:11), temperature (heat or cold), lack of nitrogen in culture that terminate transcription of a coding sequence. Another media, etc. Suitable promoters can activate transcription of an control sequence is a 3' untranslated sequence located at the essentially silent gene or upregulate, preferably Substantially, end of a coding sequence that encodes a polyadenylation transcription of an operably linked gene that is transcribed at signal. Control sequences that direct gene products to particu a low level. lar locations include those that encode signal peptides, which 40 Inclusion of termination region control sequence is direct the protein to which they are attached to a particular optional, and if employed, then the choice is be primarily one location in or outside the cell. of convenience, as the termination region is relatively inter Thus, an exemplary vector design for expression of an changeable. The termination region may be native to the exogenous gene in a microalgae contains a coding sequence transcriptional initiation region (the promoter), may be native for a desired gene product (for example, a selectable marker, 45 to the DNA sequence of interest, or may be obtainable from a lipid pathway modification enzyme, or a Xylose utilization another source. See, for example, Chen and Orozco, Nucleic enzyme) in operable linkage with a promoter active in Acids Res. (1988) 16:8411. microalgae. Alternatively, if the vector does not contain a The present invention also provides control sequences and promoter in operable linkage with the coding sequence of recombinant genes and vectors containing them that provide interest, the coding sequence can be transformed into the cells 50 for the compartmentalized expression of a gene of interest. Such that it becomes operably linked to an endogenous pro Organelles for targeting are chloroplasts, plastids, mitochon moter at the point of vector integration. dria, and endoplasmic reticulum. In addition, the present Many promoters are active in microalgae, including pro invention provides control sequences and recombinant genes moters that are endogenous to the algae being transformed, as and vectors containing them that provide for the Secretion of well as promoters that are not endogenous to the algae being 55 a protein outside the cell. transformed (i.e., promoters from other algae, promoters Proteins expressed in the nuclear genome of Prototheca from higher plants, and promoters from plant viruses or algae can be targeted to the plastid using plastid targeting signals. viruses). Illustrative exogenous and/or endogenous promot Plastid targeting sequences endogenous to Chlorella are ers that are active in microalgae (as well as antibiotic resis known, such as genes in the Chlorella nuclear genome that tance genes functional in microalgae) are described in PCT 60 encode proteins that are targeted to the plastid; see for Pub. No. 2008/151149 and references cited therein). See also example GenBank Accession numbers AY646.197 and U.S. Patent Application Publication 20100151112. AF499684, and in one embodiment, such control sequences The promoter used to express an exogenous gene can be the are used in the vectors of the present invention to target promoter naturally linked to that gene or can be a heterolo expression of a protein to a Prototheca plastid. gous promoter. Some promoters are active in more than one 65 The use of algal plastid targeting sequences to target het species of microalgae. Other promoters are species-specific. erologous proteins to the correct compartment in the host cell Illustrative promoters include promoters such as B-tubulin has been described. See Examples 11 and 12 of U.S. Patent US 8,846,352 B2 25 26 Application Publication 20100151112. In one embodiment sitivity of microalgae to antibiotics are well known. For the native plastid targeting sequence of the heterologous pro example, Mol Gen Genet. 1996 Oct. 16:252(5):572-9. tein is used to traffic the heterologous protein to the plastid/ For purposes of the present invention, the expression vec chloroplast. In some embodiment, the native plastid targeting torused to prepare a recombinant host cell of embodiments of sequences are replaced with heterologous plastid targeting the invention can include at least two, and often three, genes, sequences in order to increase the trafficking of the recombi if one of the genes is a selectable marker. For example, a nant or heterologous proteins into the plastid/chloroplast. In genetically engineered Prototheca of the invention can be Some embodiments, native plastid targeting sequences are made by transformation with vectors of the invention that replaced with plastid targeting sequences of microalgal pro comprise, in addition to a selectable marker, one or more 10 exogenous genes, such as, for example, a xylose utilization teins. Sequences were BLASTed and analyzed for homology gene or a Xylose utilization gene and an acyl ACP to known proteins that traffic to the plastid/chloroplast. The thioesterase gene. One or both genes can be expressed using cDNAS encoding these proteins were cloned and plastid tar an inducible promoter, which allow the relative timing of geting sequences were isolated from these cDNAs. Such algal expression of these genes to be controlled to enhance the lipid plastid targeting sequences can be used in expression vectors 15 yield and conversion to fatty acid esters. Expression of the useful in the cells and methods of the invention. two or more exogenous genes may be under control of the In another embodiment of the present invention, the same inducible promoter or under control of different induc expression of a polypeptide in Prototheca is targeted to the ible (or constitutive) promoters. In the latter situation, expres endoplasmic reticulum. The inclusion of an appropriate sion of a first exogenous gene can be induced for a first period retention or sorting signal in an expression vector ensure that of time (during which expression of a second exogenous gene proteins are retained in the endoplasmic reticulum (ER) and may or may not be induced) and expression of a second do not go downstream into Golgi. For example, the exogenous gene can be induced for a second period of time IMPACTVECTOR1.3 vector, from Wageningen UR-Plant (during which expression of a first exogenous gene may or Research International, includes the well known KDEL may not be induced). retention or sorting signal. With this vector, ER retention has 25 In other embodiments, the two or more exogenous genes a practical advantage in that it has been reported to improve (in addition to any selectable marker) are a Xylose utilization expression levels 5-fold or more. The main reason for this gene, a fatty acyl-ACP thioesterase, a keto-acyl-ACP syn appears to be that the ER contains lower concentrations and/ thase, a steroyl-ACP desturase, a fatty acid desaturase, and a or different proteases responsible for post-translational deg fatty acyl-CoA/aldehyde reductase, the combined action of radation of expressed proteins than are present in the cyto 30 which yields an alcohol product. Further provided are other plasm. ER retention signals functional in green microalgae combinations of exogenous genes in addition to the Xylose are known. For example, see Proc Natl AcadSci USA. 2005 utilization gene, including without limitation, a fatty acyl Apr. 26; 102(17):6225-30. ACP thioesterase and a fatty acyl-CoA reductase to generate In another embodiment of the present invention, a polypep aldehydes. In one embodiment, the vector provides, in addi tide is targeted for secretion outside the cell into the culture 35 tion to the Xylose utilization gene, for the combination of a media. See Hawkins et al., Current Microbiology Vol. 38 fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, and (1999), pp. 335-341 for examples of secretion signals active a fatty aldehyde decarbonylase to generate alkanes. In each of in Chlorella that can be used, inaccordance with the methods these embodiments, one or more of the exogenous genes can of the invention, in Prototheca. be expressed using an inducible promoter. B. Genes and Codon Optimization 40 Other illustrative vectors that express two or more exog Typically, a gene includes a promoter, coding sequence, enous genes include those encoding both a Xylose utilization and termination control sequences. When assembled by enzyme and a selectable marker. The recombinant Prototheca recombinant DNA technology, a gene may be termed an transformed with vectors of the invention can be used to expression cassette and may be flanked by restriction sites for produce lipids at lower manufacturing cost due to the engi convenient insertion into a vector that is used to introduce the 45 neered ability to use Xylose as a carbon Source. Insertion of recombinant gene into a host cell. The expression cassette can exogenous genes as described above can be combined with be flanked by DNA sequences from the genome or other the disruption of polysaccharide biosynthesis through nucleic acid target to facilitate stable integration of the directed and/or random mutagenesis, which steers even expression cassette into the genome by homologous recom greater carbon flux into lipid production. Individually and in bination. Alternatively, the vector and its expression cassette 50 combination, trophic conversion, engineering to alter lipid may remain unintegrated, in which case, the vector typically production, and treatment with exogenous enzymes alter the includes an origin of replication, which is capable of provid lipid composition produced by a microorganism. The alter ing for replication of the heterologous vector DNA. ation can be a change in the amount of lipids produced, the A common gene present on a vector is a gene that codes for amount of one or more hydrocarbon species produced relative a protein, the expression of which allows the recombinant cell 55 to other lipids, and/or the types of lipid species produced in containing the protein to be differentiated from cells that do the microorganism. For example, microalgae can be engi not express the protein. Such a gene, and its corresponding neered to produce a higher amount and/or percentage of gene product, is called a selectable marker. Selectable mark TAGS. ers can be employed in a transgene construct useful for trans For optimal expression of a recombinant protein, it is ben forming Prototheca. Examples of suitable selectable markers 60 eficial to employ coding sequences that produce mRNA with include the G418 resistance gene, the nitrate reductase gene codons preferentially used by the host cell to be transformed. (see Dawson et al. (1997), Current Microbiology 35:356 Thus, proper expression of transgenes can require that the 362), the hygromycin phosphotransferase gene (HPT: see codon usage of the transgene matches the specific codon bias Kim et al. (2002), Mar. Biotechnol. 4:63-73), the neomycin of the organism in which the transgene is being expressed. phosphotransferase gene, and the ble gene, which confers 65 The precise mechanisms underlying this effect are many, but resistance to phleomycin (Huang et al. (2007), Appl. Micro include the proper balancing of available aminoacylated biol. Biotechnol. 72: 197-205). Methods of determining sen tRNA pools with proteins being synthesized in the cell, US 8,846,352 B2 27 28 coupled with more efficient translation of the transgenic mes In some embodiments, the recombinant oleaginous microbe senger RNA (mRNA) when this need is met. When codon is a microalgae. In other embodiments, the recombinant ole usage in the transgene is not optimized, available tRNA pools aginous microbe is a microalgae of the genus Prototheca. In are not sufficient to allow for efficient translation of the het one embodiment, the recombinant oleaginous microbe is erologous mRNA resulting in ribosomal stalling and termi genetically engineered to contain one or more exogenous nation and possible instability of the transgenic mRNA. genes that are involved in Xylose utilization. Genes involved The present invention provides codon-optimized nucleic in xylose utilization are described in detail below. It is con acids useful for the Successful expression of recombinant templated in one or more embodiments of the invention that proteins in Prototheca. Codon usage in Prototheca species the genes involved in Xylose utilization can be used alone, or was analyzed by studying cDNA sequences isolated from 10 in combination with other genes that involve Xylose utiliza tion, without limit to the number or combination(s) of such Prototheca moriformis. This analysis represents the interro genes. It is contemplated in one or more embodiments of the gation over 24,000 codons and resulted in Table 1 below. invention that genes involved in Xylose utilization can be used in combination with other exogenous genes that involve uti TABLE 1. 15 lization of other carbon sources or lipid production, without Preferred codon usage in Prototheca strains. limit to the number or combination(s) of Such genes. In one embodiment, the recombinant cell of the invention Ala GCG 345 (0.36) Asn AAT 8 (0.04) GCA 66 (0.07) AAC 201 (0.96) further contains one or more exogenous Xylose utilization GCT 101 (0.11) Pro CCG 161 (0.29) genes. There are at least two different pathways utilized by GCC 442 (0.46) CCA 49 (0.09) eukaryotic organisms to metabolize Xylose. One such path Cys TGT 12 (0.10) CCT 71 (0.13) way is the oxo-reductive (oxidoreductase) pathway or aldo TGC 105 (0.90) CCC 267 (0.49) keto reductase pathway. In the first step in this pathway, Asp GAT 43 (0.12) Gln CAG 226 (0.82) GAC 316 (0.88) CAA 48 (0.18) D-xylose is reduced into xylitol by xylose reductase. Xylitol Glu GAG 377 (0.96) Arg AGG 33 (0.06) is then oxidized into Xylulose by Xylitol dehydrogenase. GAA 14 (0.04) AGA 14 (0.02) 25 Xylulose is then converted into xylulose-5-phosphate by Phe TTT 89 (0.29) CGG 102 (0.18) Xylulose kinase and then metabolized via the pentose phos TTC 216 (0.71) CGA 49 (0.08) Gly GGG 92 (0.12) CGT 51 (0.09) phate pathway. Another pathway that is utilized to metabolize GGA 56 (0.07) CGC 331 (0.57) Xylose is the Xylose isomerase pathway. In the first step in the GGT 76 (0.10) Ser AGT 16 (0.03) Xylose isomerase pathway, Xylose is isomerized into Xylulose GGC 559 (0.71) AGC 123 (0.22) 30 by Xylose isomerase. Xylulose is then converted into Xylu His CAT 42 (0.21) TCG 152 (0.28) CAC 154 (0.79) TCA 31 (0.06) lose-5-phosphate by Xyulose kinase. Xylulose-5-phosphate is Ile ATA 4 (0.01) TCT 55 (0.10) then metabolized via the pentose phosphate pathway. ATT 30 (0.08) TCC 173 (0.31) In some organisms, one or more of these pathways exist ATC 338 (0.91) Thr ACG 184 (0.38) Lys AAG 284 (0.98) ACA 24 (0.05) and are utilized in concert. In some organisms, both the oXo AAA 7 (0.02) ACT 21 (0.05) 35 reductive pathway and the Xylose isomerase pathways exist. Leu TTG 26 (0.04) ACC 249 (0.52) In Such organisms, the production of xylitol may inhibit TTA 3 (0.00) Val GTG 308 (0.50) Xylose isomerase activity. In such cases, Xylitol dehydroge CTG 447 (0.61) GTA 9 (0.01) CTA 20 (0.03) GTT 35 (0.06) nase is expressed to relieve Such inhibition. CTT 45 (0.06) GTC 262 (0.43) In organisms that do not utilize Xylose, or are unable to CTC 190 (0.26) Trp TGG 107 (1.00) 40 utilize xylose in an efficient manner, one or both of the above Met ATG 191 (1.00) Tyr TAT 10 (0.05) pathways may not exist or only exist partially in these organ TAC 180 (0.95) isms. Genetically engineering Such organisms to efficiently Stop TGA/TAG/TAA utilize Xylose may encompass the introduction of one or several (or all) members of one or both of these pathways. The In other embodiments, the gene in the recombinant vector 45 introduction of a transporter protein may be necessary to has been codon-optimized with reference to a microalgal transport Xylose or more efficiently transport Xylose into the strain other thana Prototheca strain. For example, methods of cell from the extracellular environment. A transporter protein, recoding genes for expression in microalgae are described in including a transporter protein that has specificity for pentose U.S. Pat. No. 7,135,290. Additional information for codon Sugars such as Xylose, is a family of cell membrane proteins optimization is available, e.g., at the codon usage database of 50 and genes encoding Such proteins that transport Sugars (such GenBank. as Xylose) from the extracellular environment, such as the While the methods and materials of the inventionallow for culture medium, into the cytosol of the cell. A transporter may the introduction of any exogenous gene into Prototheca, be active or passive. Passive transporters facilitate the trans genes relating to Xylose utilization and lipid pathway modi port of molecule(s) down a concentration gradient (from an fication are of particularinterest, as discussed in the following 55 area of higher concentration to an area of lower concentra sections. tion) without energy consumption. In some embodiments, the transporter is a pentose transporter, having specificity for V. Xylose Utilization pentose Sugars. In other embodiments, the transporter is a transporter derived from Pichia stipitis. In still other embodi An embodiment of the present invention also provides 60 ments, the transporter is derived from Pichia stipitis SUT1 recombinant oleaginous microbes that have been modified to (without limitation, GenBank Accession number alter the ability of the microbe to metabolize different feed XP 001387, hereby incorporated by this reference) (SEQID stocks that include pentose Sugars such as Xylose, which is NO: 37), SUT2, and/or SUT3 transporters or from the S. abundant in cellulosic sources. Such recombinant oleaginous cerevisiae HXT transporters. Active transporters consume microbes include those that have plastids and type II fatty acid 65 energy in transporting molecules, usually against a concen synthesis pathways, are of the kingdom Plantae, microalgae, tration gradient. Active transporters include, without limita oleaginous yeast, oleaginous bacteria and oleaginous fungi. tion, primary active transporters, which use ATP as an energy US 8,846,352 B2 29 30 Source, i.e., ATP-binding cassette (ABC) transporters, and into Prototheca. Example 8 demonstrates the lipid production secondary active transporters, which use energy from chemi capability of several selected strains. osmotic gradients and include, without limitation antiporters In some embodiments, the cell may further contain one or and symporters, i.e., the GXS1 protein (SEQ ID NO:39) of more exogenous Sucrose utilization genes, as described in Candida intermedia (without limitation, GenBank Accession 5 U.S. Patent Application Publication 20100151112. Such a number CAI44932.1, hereby incorporated by this reference), cell can metabolize both Xylose and Sucrose. and the H+-symporter-like proteins (e.g., SEQID NO: 41). In some embodiments, the transporter is derived from the VI. Lipid Pathway Engineering At5g5925On gene from Arabidopsis. In other embodiments, 10 In addition to altering the ability of Prototheca to utilize the transporter is derived from the gene encoding the XLT1 feedstocks such as Xylose-containing feedstocks, the present protein (SEQID NO: 43) from Trichoderma reesei. invention also provides recombinant Prototheca that have Additionally, the introduction of a transporter protein (e.g., been further modified to alter the properties and/or propor Xylulose-5-phosphate translocator) may also be necessary or tions of lipids produced. The pathway can further, or alterna preferred in order to distribute the correct substrate into the 15 tively, be modified to alter the properties and/or proportions correct Subcompartment of the microbe. Pentose Sugar of various lipid molecules produced through enzymatic pro metabolism is thought to occur through the pentose phos cessing of lipids and intermediates in the fatty acid pathway. phate pathway, which occurs mainly in the plastid. Such In various embodiments, the recombinant Prototheca cells of translocators are a family of plastidic membrane proteins and the invention have, relative to their untransformed counter genes encoding Such proteins that transport hexose, triose, or parts, optimized lipid yield per unit volume and/or per unit pentose or phosphate versions of these molecules from the time, carbon chain length (e.g., for renewable diesel produc cytosol to the plastid. In one embodiment, such a translocator tion or for industrial chemicals applications requiring lipid has a preference for pentose or pentose phosphate molecules feedstock), reduced number of double or triple bonds, option over hexose/hexose phosphate or triose?triose phosphate. In ally to Zero, and increasing the hydrogen:carbon ratio of a another embodiment, the translocator is derived from a gene 25 particular species of lipid or of a population of distinct lipid. encoding a xylulose 5-phosphate translocator protein. In In particular embodiments, one or more key enzymes that another embodiment, the translocator is derived from the control metabolism to fatty acid synthesis have been “up XPT gene from Arabidopsis. In some cases, the translocator regulated' or “down-regulated to improve lipid production. is a chimeric protein comprising a transit peptide endogenous Up-regulation can be achieved, for example, by transforming to the microbe being engineered to utilize xylose. For 30 cells with expression constructs in which a gene encoding the example, chimeric translocators with a transit peptide endog enzyme of interest is expressed, e.g., using a strong promoter enous to Prototheca (e.g., SEQID NOs: 45, 47 and 49) are and/or enhancer elements that increase transcription. Such useful in some embodiments. constructs can include a selectable marker Such that the trans In some embodiments, the heterologous exogenous gene formants can be subjected to selection, which can result in that is introduced into the microorganism is a part of the 35 maintenance or amplification of the construct and an increase oXo-reductive or aldo keto reductase pathway. In some cases, in the expression level of the encoded enzyme. Examples of the heterologous exogenous gene is a xylose reductase. In enzymes Suitable for up-regulation according to the methods other cases, the heterologous exogenous gene is a xylitol of the invention are described in U.S. Patent Application dehydrogenase. In other embodiments, the xylitol dehydro Publication 20100151112. genase is the XYL2 gene from Pichia stipitis. 40 Up- and/or down-regulation of genes can be applied to In other embodiments, the heterologous exogenous gene global regulators controlling the expression of the genes of that is introduced into the microorganism is part of the Xylose the fatty acid biosynthetic pathways. Accordingly, one or isomerase pathway. In some cases, the heterologous exog more global regulators of fatty acid synthesis can be up- or enous gene is a xylose isomerase. In some embodiments, the down-regulated, as appropriate, to inhibit or enhance, respec xylose isomerase is encoded by the XYLA gene from Piro 45 tively, the expression of a plurality of fatty acid synthetic myces sp. In other embodiments, the heterologous exogenous genes and, ultimately, to increase lipid production. Examples gene is a xylulose kinase. In some embodiments, the Xylulose include sterol regulatory element binding proteins (SREBPs), kinase is encoded by the XYL3 gene from Pichia stipitis. such as SREBP-1a and SREBP-1c (for examples see Gen In some embodiments, the heterologous exogenous gene bank accession numbers NP 035610 and Q9WTN3). encodes a translocator protein. In other embodiments, such 50 Embodiments of the present invention also provide recom translocator is a pentose phosphate translocator. In still other binant microalgal cells that have been modified to contain one embodiments, the pentose phosphate translocator is encoded or more exogenous genes encoding lipid modification by the XPT gene from Arabidopsis thaliana. In still other enzymes such as, for example, fatty acyl-ACP thioesterases, embodiments, the genetically engineered microorganism fatty acyl-CoA/aldehyde reductases, fatty acyl-CoA reduc comprises one or more of the foregoing exogenous heterolo 55 tases, fatty aldehyde decarbonylase, fatty aldehyde reduc gous genes. In other embodiments, the genetically engineered tases, and squalene synthases (see U.S. Patent Application microorganism comprises all of the foregoing exogenous het Publication 20100151112). erologous genes. Thus, in particular embodiments, microbes of the present Example 3 below demonstrates the successful introduction invention are genetically engineered to express a Xylose uti of different members of the xylose isomerase pathway pro 60 lization gene as well as one or more exogenous genes selected teins, oxo-reductive pathway enzymes, and a Xylose translo from an acyl-ACP thioesterase, an acyl-CoA/aldehyde reduc cator into microalgae to produce a genetically engineered tase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a microalgae cells that can utilize Xylose as a fixed carbon fatty aldehyde decarbonylase, or a naturally co-expressed Source when grown under heterotrophic conditions. acyl carrier protein. Suitable expression methods are Example 7 demonstrates the introduction of various com 65 described above with respect to the expression of a lipase binations of Xylose transporters, Xylose oxidoreductase/ gene, including, among other methods, inducible expression isomerase enzymes, and Xylose translocators (see Table 6) and compartmentalized expression. A fatty acyl-ACP US 8,846,352 B2 31 32 thioesterase cleaves a fatty acid from an acyl carrier protein In some embodiments, the cell has been genetically engi (ACP) during lipid synthesis. Through further enzymatic pro neered and/or selected to express a global regulator of fatty cessing, the cleaved fatty acid is then combined with a coen acid synthesis at an altered level compared to the wild-type Zyme to yield an acyl-CoA molecule. This acyl-CoA is the cell, whereby the expression levels of a plurality of fatty acid substrate for the enzymatic activity of a fatty acyl-CoA reduc synthetic genes are altered compared to the wild-type cell. In tase to yield an aldehyde, as well as for a fatty acyl-CoA/ Some cases, the lipid pathway enzyme comprises an enzyme aldehyde reductase to yield an alcohol. The aldehyde pro that modifies a fatty acid. In some cases, the lipid pathway duced by the action of the fatty acyl-CoA reductase identified enzyme is selected from a Stearoyl-ACP desaturase and a above is the substrate for further enzymatic activity by either glycerolipid desaturase. 10 In other embodiments, the present invention is directed to a fatty aldehyde reductase to yield an alcohol, or a fatty an oil-producing microbe containing one or more exogenous aldehyde decarbonylase to yield an alkane or alkene. genes, wherein the exogenous genes encode protein(s) In some embodiments, fatty acids, glycerolipids, or the selected from Xylose utilization enzymes as well as proteins corresponding primary alcohols, aldehydes, alkanes or alk selected from the group consisting of a fatty acyl-ACP enes, generated by the methods described herein, contain 8, 15 thioesterase, a fatty acyl-CoA reductase, a fatty aldehyde 10, 12, or 14 carbon atoms. Preferred fatty acids for the reductase, a fatty acyl-CoA/aldehyde reductase, a fatty alde production of diesel, biodiesel, renewable diesel, or jet fuel, hyde decarbonylase, and an acyl carrier protein. In one or the corresponding primary alcohols, aldehydes, alkanes embodiment, the exogenous gene is in operable linkage with and alkenes, for industrial applications contain 8 to 14 carbon a promoter, that is inducible or repressible in response to a atoms. In certain embodiments, the above fatty acids, as well stimulus. In some cases, the stimulus is selected from the as the other corresponding hydrocarbon molecules, are Satu group consisting of an exogenously provided Small molecule, rated (with no carbon-carbon double or triple bonds); mono heat, cold, and limited or no nitrogen in the culture media. In unsaturated (single double bond); poly unsaturated (two or Some cases, the exogenous gene is expressed in a cellular more double bonds); are linear (not cyclic) or branched. For compartment. In some embodiments, the cellular compart fuel production, greater Saturation is preferred. 25 ment is selected from the group consisting of a chloroplast, a The enzymes described directly above have a preferential plastid and a mitochondrion. In some embodiments the specificity for hydrolysis of a Substrate containing a specific microbe is Prototheca moriformis, Prototheca krugani, Pro number of carbon atoms. For example, a fatty acyl-ACP totheca stagnora or Prototheca Zopfi. Various exogenous thioesterase may have a preference for cleaving a fatty acid gene and gene combinations for lipid pathway modification having 12 carbonatoms from the ACP. In some embodiments, 30 are described in U.S. Patent Application Publication the ACP and the length-specific thioesterase may have an 2O1 OO151112. affinity for one another that makes them particularly useful as An embodiment of the present invention also provides a combination (e.g., the exogenous ACP and thioesterase methods for producing an alcohol comprising culturing a genes may be naturally co-expressed in a particular tissue or population of recombinant Prototheca cells in a culture organism from which they are derived). Therefore, in various 35 medium, wherein the cells contain a first exogenous gene embodiments, a recombinant cell of an embodiment of the encoding a Xylose utilization enzyme; a second exogenous invention can contain an exogenous gene that encodes a pro gene encoding a fatty acyl-ACP thioesterase; and a third tein with specificity for catalyzing an enzymatic activity (e.g., exogenous gene encoding a fatty acyl-CoA/aldehyde reduc cleavage of a fatty acid from an ACP, reduction of an acyl tase, and the cells synthesize a fatty acid linked to an acyl CoA to an aldehyde or an alcohol, or conversion of an alde 40 carrier protein (ACP), the fatty acyl-ACP thioesterase cata hyde to an alkane, or the gene product of a KAS, SAD, or FAD lyzes the cleavage of the fatty acid from the ACP to yield, gene) with regard to the number of carbon atoms and satura through further processing, a fatty acyl-CoA, and the fatty tion pattern contained in the substrate. See U.S. Patent Appli acyl-CoA/aldehyde reductase catalyzes the reduction of the cation Publication 20100151112 for various vectors and acyl-CoA to an alcohol. methods for expressing an exogenous thioesterase. Thus, in 45 An embodiment of the present invention also provides an embodiment of the present invention a genetically modi methods of producing an aldehyde in a Prototheca cell. In one fied cell both metabolizes xylose and produces an altered fatty embodiment, the method comprises culturing a population of acid profile (i.e. distribution of chain length and/or Saturation Prototheca cells in a culture medium, wherein the cells con pattern in fatty acids produced by the cell). tain a first exogenous gene encoding a Xylose utilization Thus, the present invention provides a cell (and in specific 50 enzyme; a second exogenous gene encoding a fatty acyl-ACP embodiments, a Prototheca cell) that has been genetically thioesterase; and a third exogenous gene encoding a fatty engineered to express a Xylose utilization enzyme and one or acyl-CoA reductase, and the cells synthesize a fatty acid more lipid pathway enzymes at an altered level compared to linked to an acyl carrier protein (ACP), the fatty acyl-ACP a wild-type cell of the same species. In some cases, the cell thioesterase catalyzes the cleavage of the fatty acid from the produces more lipid compared to the wild-type cell when 55 ACP to yield, through further processing, a fatty acyl-CoA, both cells are grown under the same conditions. In some and the fatty acyl-CoA reductase catalyzes the reduction of cases, the cell has been genetically engineered and/or selected the acyl-CoA to an aldehyde. to express a lipid pathway enzyme at a higher level than the An embodiment of the present invention also provides wild-type cell. In some cases, the lipid pathway enzyme is methods of producing a fatty acid molecule having a specified selected from the group consisting of pyruvate dehydroge 60 carbon chain length in a Prototheca cell that contains an nase, acetyl-CoA carboxylase, acyl carrier protein, and glyc exogenous Xylose utilization gene. In one embodiment, the erol-3 phosphate acyltransferase. In some cases, the cell has method comprises culturing a population of lipid-producing been genetically engineered and/or selected to express a lipid Prototheca cells in a culture medium, wherein the microbes pathway enzyme at a lower level than the wild-type cell. In at contain an exogenous gene encoding an exogenous Xylose least one embodiment in which the cell expresses the lipid 65 utilization gene and a fatty acyl-ACP thioesterase having an pathway enzyme at a lower level, the lipid pathway enzyme activity specific or preferential to a certain carbon chain comprises citrate synthase. length, Such as 8, 10, 12 or 14 carbon atoms, and wherein the US 8,846,352 B2 33 34 microbes synthesize a fatty acid linked to an acyl carrier lipids and/or hydrocarbons. Below are described a number of protein (ACP) and the thioesterase catalyzes the cleavage of lysis techniques. These techniques can be used individually the fatty acid from the ACP when the fatty acid has been or in combination. synthesized to the specific carbon chain length. In one embodiment of the present invention, the step of In the various embodiments described above, the cell can lysing a microorganism comprises heating of a cellular Sus contain at least one exogenous gene encoding a lipid pathway pension containing the microorganism. In this embodiment, enzyme. In some cases, the lipid pathway enzyme is selected the fermentation broth containing the microorganisms (or a from the group consisting of a stearoyl-ACP desaturase, a Suspension of microorganisms isolated from the fermentation glycerolipid desaturase, apyruvate dehydrogenase, an acetyl broth) is heated until the microorganisms, i.e., the cell walls 10 and membranes of microorganisms degrade or breakdown. CoA carboxylase, an acyl carrier protein, and a glycerol-3 Typically, temperatures applied are at least 50° C. Higher phosphate acyltransferase. In other cases, the cell contains a temperatures, such as at least 60° C., at least 70° C., at least lipid modification enzyme selected from the group consisting 80°C., at least 90° C., at least 100°C., at least 110°C., at least of a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde 120° C., at least 130° C. or higher are used for more efficient reductase, a fatty acyl-CoA reductase, a fatty aldehyde reduc 15 cell lysis. Lysing cells by heat treatment can be performed by tase, a fatty aldehyde decarbonylase, and/or an acyl carrier boiling the microorganism. Alternatively, heat treatment protein. (without boiling) can be performed in an autoclave. The heat treated lysate may be cooled for further treatment. Cell dis VII. Production of Fuels and Chemicals or Other ruption can also be performed by Steam treatment, i.e., Products through addition of pressurized steam. Steam treatment of microalgae for cell disruption is described, for example, in For the production of fuel in accordance with the embodi U.S. Pat. No. 6,750,048. In some embodiments, steam treat ments of the invention lipids produced by cells of the inven ment may be achieved by sparging steam into the fermentor tion are harvested, or otherwise collected, by any convenient and maintaining the broth at a desired temperature for less means. Lipids can be isolated by whole cell extraction. The 25 than about 90 minutes, preferably less than about 60 minutes, cells are first disrupted, and then intracellular and cell mem and more preferably less than about 30 minutes. brane/cell wall-associated lipids as well as extracellular In another embodiment of the present invention, the step of hydrocarbons can be separated from the cell mass. Such as by lysing a microorganism comprises adding a base to a cellular use of centrifugation as described above. Intracellular lipids Suspension containing the microorganism. The base should produced in microorganisms are, in some embodiments, 30 be strong enough to hydrolyze at least a portion of the pro extracted after lysing the cells of the microorganism. Once teinaceous compounds of the microorganisms used. Bases extracted, the lipids are further refined to produce oils, fuels, which are useful for solubilizing proteins are known in the art or oleochemicals. of chemistry. Exemplary bases which are useful in the meth After completion of culturing, the microorganisms can be ods of the present invention include, but are not limited to, separated from the fermentation broth. Optionally, the sepa 35 hydroxides, carbonates and bicarbonates of lithium, Sodium, ration is effected by centrifugation to generate a concentrated potassium, calcium, and mixtures thereof. A preferred base is paste. The biomass can then optionally be washed with a KOH. Base treatment of microalgae for cell disruption is washing solution (e.g., DI water) to get rid of the fermentation described, for example, in U.S. Pat. No. 6,750,048. broth and debris. Optionally, the washed microbial biomass In another embodiment of the present invention, the step of may also be dried (oven dried, lyophilized, etc.) prior to cell 40 lysing a microorganism comprises adding an acid to a cellular disruption. Alternatively, cells can be lysed without separa Suspension containing the microorganism. Acid lysis can be tion from some or all of the fermentation broth when the effected using an acid at a concentration of 10-500 mN or fermentation is complete. For example, the cells can be at a preferably 40-160 mN. Acid lysis is preferably performed at ratio of less than 1:1 V:V cells to extracellular liquid when the above room temperature (e.g., at 40-160°, and preferably a cells are lysed. 45 temperature of 50-130°. For moderate temperatures (e.g., Microorganisms containing a lipid can be lysed to produce room temperature to 100° C. and particularly room tempera a lysate. As detailed herein, the step of lysing a microorgan ture to 65°, acid treatment can usefully be combined with ism (also referred to as cell lysis) can be achieved by any Sonication or other cell disruption methods. convenient means, including heat-induced lysis, adding a In another embodiment of the present invention, the step of base, adding an acid, using enzymes such as proteases and 50 lysing a microorganism comprises lysing the microorganism polysaccharide degradation enzymes, using ultrasound, by using an enzyme. Preferred enzymes for lysing a micro mechanical lysis, using osmotic shock, infection with a lytic organism are proteases and polysaccharide-degrading virus, and/or expression of one or more lytic genes. Lysis is enzymes Such as hemicellulase (e.g., hemicellulase from performed to release intracellular molecules which have been Aspergillus niger; Sigma Aldrich, St. Louis, Mo.; #H2125), produced by the microorganism. Each of these methods for 55 pectinase (e.g., pectinase from Rhizopus sp., Sigma Aldrich, lysing a microorganism can be used as a single method or in St. Louis, Mo.; iP2401), Mannaway 4.0 L (Novozymes), combination simultaneously or sequentially. The extent of cellulase (e.g., cellulose from Trichoderma viride; Sigma cell disruption can be observed by microscopic analysis. Aldrich, St. Louis, Mo.; #C9422), and driselase (e.g., drise Using one or more of the methods described herein, typically lase from Basidiomycetes sp.: Sigma Aldrich, St. Louis, Mo.; more than 70% cell breakage is observed. Preferably, cell 60 HD9515. breakage is more than 80%, more preferably more than 90% In other embodiments of the present invention, lysis is and most preferred about 100%. accomplished using an enzyme such as, for example, a cel In particular embodiments, the microorganism is lysed lulase Such as a polysaccharide-degrading enzyme, option after growth, for example to increase the exposure of cellular ally from Chlorella or a Chlorella virus, or a proteases, such lipid and/or hydrocarbon for extraction or further processing. 65 as Streptomyces griseus protease, chymotrypsin, proteinase The timing of lipase expression (e.g., via an inducible pro K, proteases listed in Degradation of Polylactide by Commer moter) or cell lysis can be adjusted to optimize the yield of cial Proteases, Oda Y et al., Journal of Polymers and the US 8,846,352 B2 35 36 Environment, Volume 8, Number 1, January 2000, pp. 29-32 the lytic gene to lyse the cells. In one embodiment, the lytic (4), Alcalase 2.4 FG (Novozymes), and Flavourzyme 100 L gene encodes a polysaccharide-degrading enzyme. In certain (Novozymes). Any combination of a protease and a polysac other embodiments, the lytic gene is a gene from a lytic virus. charide-degrading enzyme can also be used, including any Thus, for example, alytic gene from a Chlorella virus can be combination of the preceding proteases and polysaccharide expressed in an algal cell; see Virology 260, 308-315 (1999); degrading enzymes. FEMS Microbiology Letters 180 (1999) 45-53; Virology 263, Lysis can be performed using an expeller press. In this 376-387 (1999); and Virology 230,361-368 (1997). Expres process, biomass is forced through a screw-type device at sion of lytic genes is preferably done using an inducible high pressure, lysing the cells and causing the intracellular promoter, such as a promoter active in microalgae that is lipid to be released and separated from the protein and fiber 10 induced by a stimulus such as the presence of a small mol (and other components) in the cell. See PCT patent applica ecule, light, heat, and other stimuli. tion number U.S. Ser. No. 10/031,108, incorporated herein by Various methods are available for separating lipids from reference. cellular lysates produced by the above methods. For example, In some cases, the step of lysing a microorganism is per lipids and lipid derivatives such as fatty aldehydes, fatty alco formed by using ultrasound, i.e., Sonication. Thus, cells can 15 hols, and hydrocarbons such as alkanes, can be extracted with also by lysed with high frequency Sound. The Sound can be a hydrophobic solvent such as hexane (see Frenz et al. 1989, produced electronically and transported through a metallic tip Enzyme Microb. Technol., 11:717). Lipids and lipid deriva to an appropriately concentrated cellular Suspension. This tives can also be extracted using liquefaction (see for example Sonication (or ultrasonication) disrupts cellular integrity Sawayama et al. 1999, Biomass and Bioenergy 17:33-39 and based on the creation of cavities in cell Suspension. Inoue et al. 1993, Biomass Bioenergy 6(4):269-274); oil liq In some cases, the step of lysing a microorganism is per uefaction (see for example Minowa et al. 1995, Fuel 74(12): formed by mechanical lysis. Cells can be lysed mechanically 1735-1738); and supercritical CO extraction (see for and optionally homogenized to facilitate hydrocarbon (e.g., example Mendes et al. 2003, Inorganica Chimica Acta 356: lipid) collection. For example, a pressure disrupter can be 328-334). Miao and Wu describe a protocol of the recovery of used to pump a cell containing slurry through a restricted 25 microalgal lipid from a culture of Chlorella prototheocoides orifice valve. High pressure (up to 1500 bar) is applied, fol in which the cells were harvested by centrifugation, washed lowed by an instant expansion through an exiting nozzle. Cell with distilled water and dried by freeze drying. The resulting disruption is accomplished by three different mechanisms: cell powder was pulverized in a mortar and then extracted impingement on the valve, high liquid shearin the orifice, and with n-hexane. Miao and Wu, Biosource Technology (2006) Sudden pressure drop upon discharge, causing an explosion of 30 97:841-846. the cell. The method releases intracellular molecules. Alter Thus, lipids, lipid derivatives and hydrocarbons generated natively, a ball mill can be used. In a ball mill, cells are by the microorganisms of the present invention can be recov agitated in Suspension with Small abrasive particles, such as ered by extraction with an organic solvent. In some cases, the beads. Cells break because of shear forces, grinding between preferred organic solvent is hexane. Typically, the organic beads, and collisions with beads. The beads disrupt the cells 35 solvent is added directly to the lysate without prior separation to release cellular contents. Cells can also be disrupted by of the lysate components. In one embodiment, the lysate shear forces, such as with the use of blending (Such as with a generated by one or more of the methods described above is high speed or Waring blender as examples), the french press, contacted with an organic solvent for a period of time suffi or even centrifugation in case of weak cell walls, to disrupt cient to allow the lipid and/or hydrocarbon components to cells. 40 form a solution with the organic solvent. In some cases, the In some cases, the step of lysing a microorganism is per solution can then be further refined to recover specific desired formed by applying an osmotic shock. lipid or hydrocarbon components. Hexane extraction meth In some cases, the step of lysing a microorganism com ods are well known in the art. prises infection of the microorganism with a lytic virus. A Lipids and lipid derivatives such as fatty aldehydes, fatty wide variety of viruses are known to lyse microorganisms 45 alcohols, and hydrocarbons such as alkanes produced by cells Suitable for use in the present invention, and the selection and as described herein can be modified by the use of one or more use of a particularlytic virus for a particular microorganism is enzymes, including a lipase, as described above. When the within the level of skill in the art. For example, paramecium hydrocarbons are in the extracellular environment of the cells, bursaria chlorella virus (PBCV-1) is the prototype of a group the one or more enzymes can be added to that environment (family Phycodnaviridae, genus Chlorovirus) of large, icosa 50 under conditions in which the enzyme modifies the hydrocar hedral, plaque-forming, double-stranded DNA viruses that bon or completes its synthesis from a hydrocarbon precursor. replicate in, and lyse, certain unicellular, eukaryotic chlo Alternatively, the hydrocarbons can be partially, or com rella-like green algae. Accordingly, any Susceptible microal pletely, isolated from the cellular material before addition of gae can be lysed by infecting the culture with a suitable one or more catalysts such as enzymes. Such catalysts are Chlorella virus. Methods of infecting species of Chlorella 55 exogenously added, and their activity occurs outside the cell with a chlorella virus are known. See for example Adv. Virus or in vitro. Res. 2006; 66:293-336; Virology, 1999 Apr. 25: 257(1): 15 Thus, lipids and hydrocarbons produced by cells in vivo, or 23: Virology, 2004 Jan. 5; 318(1):214-23; Nucleic Acids enzymatically modified in vitro, as described herein can be Symp. Ser: 2000; (44):161-2; J. Virol. 2006 March: 80(5): optionally further processed by conventional means. The pro 2437-44; and Annu. Rev. Microbiol. 1999:53:447-94. 60 cessing can include “cracking to reduce the size, and thus In some cases, the step of lysing a microorganism com increase the hydrogen:carbon ratio, of hydrocarbon mol prises autolysis. In this embodiment, a microorganism ecules. Catalytic and thermal cracking methods are routinely according to the invention is genetically engineered to pro used in hydrocarbon and triglyceride oil processing. Catalytic duce a lytic protein that will lyse the microorganism. This methods involve the use of a catalyst, Such as a solid acid lytic gene can be expressed using an inducible promoter so 65 catalyst. For example, the catalyst can be silica-alumina or a that the cells can first be grown to a desirable density in a Zeolite, which result in the heterolytic, or asymmetric, break fermentor, followed by induction of the promoter to express age of a carbon-carbon bond to result in a carbocation and a US 8,846,352 B2 37 38 hydride anion. These reactive intermediates then undergo tillate material derived from biomass or otherwise that meets either rearrangement or hydride transfer with another hydro the appropriate ASTM specification can be defined as diesel carbon. The reactions canthus regenerate the intermediates to fuel (ASTM D975),jet fuel (ASTM D1655), or as biodiesel if result in a self-propagating chain mechanism. Hydrocarbons it is a fatty acid methyl ester (ASTM D6751). can also be processed to reduce, optionally to Zero, the num- 5 After extraction, lipid and/or hydrocarbon components ber of carbon-carbon double, or triple, bonds therein. Hydro recovered from the microbial biomass described herein can carbons can also be processed to remove or eliminate a ring or be subjected to chemical treatment to manufacture a fuel for cyclic structure therein. Hydrocarbons can also be processed use in diesel vehicles and jet engines. to increase the hydrogen:carbon ratio. This can include the Biodiesel is a liquid which varies in color between addition of hydrogen (“hydrogenation') and/or the “crack- 10 golden and dark brown—depending on the production feed ing of hydrocarbons into smaller hydrocarbons. stock. It is practically immiscible with water, has a high Thermal methods involve the use of elevated temperature boiling point and low vapor pressure. Biodiesel refers to a and pressure to reduce hydrocarbon size. An elevated tem diesel-equivalent processed fuel for use in diesel-engine perature of about 800° C. and pressure of about 700 kPa can vehicles. Biodiesel is biodegradable and non-toxic. An addi be used. These conditions generate “light,” a term that is 15 tional benefit of biodiesel over conventional diesel fuel is Sometimes used to refer to hydrogen-rich hydrocarbon mol lower engine wear. Typically, biodiesel comprises C14-C18 ecules (as distinguished from photon flux), while also gener alkyl esters. Various processes convert biomass or a lipid ating, by condensation, heavier hydrocarbon molecules produced and isolated as described herein to diesel fuels. A which are relatively depleted of hydrogen. The methodology preferred method to produce biodiesel is by transesterifica provides homolytic, or symmetrical, breakage and produces 20 tion of a lipid as described herein. A preferred alkyl ester for alkenes, which may be optionally enzymatically Saturated as use as biodiesel is a methyl ester or ethyl ester. described above. Biodiesel produced by a method described herein can be Catalytic and thermal methods are standard in plants for used alone or blended with conventional diesel fuel at any hydrocarbon processing and oil refining. Thus hydrocarbons concentration in most modern diesel-engine vehicles. When produced by cells as described herein can be collected and 25 blended with conventional diesel fuel (petroleum diesel), processed or refined via conventional means. See Hillen et al. biodiesel may be present from about 0.1% to about 99.9%. (Biotechnology and Bioengineering, Vol. XXIV: 193-205 Much of the world uses a system known as the “B” factor to (1982)) for a report on hydrocracking of microalgae-pro state the amount of biodiesel in any fuel mix. For example, duced hydrocarbons. In alternative embodiments, the fraction fuel containing 20% biodiesel is labeled B20. Pure biodiesel is treated with another catalyst, such as an organic compound, 30 is referred to as B100. heat, and/or an inorganic compound. For processing of lipids Biodiesel can also be used as a heating fuel in domestic and into biodiesel, a transesterification process can be used as commercial boilers. Existing oil boilers may contain rubber described in Section V herein. parts and may require conversion to run on biodiesel. The Hydrocarbons produced via methods of the present inven conversion process is usually relatively simple, involving the tion are useful in a variety of industrial applications. For 35 exchange of rubber parts for synthetic parts due to biodiesel example, the production of linear alkylbenzene Sulfonate being a strong solvent. Due to its strong solvent power, burn (LAS), an anionic Surfactant used in nearly all types of deter ing biodiesel will increase the efficiency of boilers. Biodiesel gents and cleaning preparations, utilizes hydrocarbons gen can be used as an additive informulations of diesel to increase erally comprising a chain of 10-14 carbon atoms. See, for the lubricity of pure Ultra-Low Sulfur Diesel (ULSD) fuel, example, U.S. Pat. Nos. 6,946,430; 5,506.201; 6,692,730; 40 which is advantageous because it has virtually no Sulfur con 6,268,517; 6,020,509; 6,140,302; 5,080,848; and 5,567,359. tent. Biodiesel is a better solvent than petrodiesel and can be Surfactants, such as LAS, can be used in the manufacture of used to break down deposits of residues in the fuel lines of personal care compositions and detergents, such as those vehicles that have previously been run on petrodiesel. described in U.S. Pat. Nos. 5,942,479; 6,086,903; 5,833,999; Biodiesel can be produced by transesterification of triglyc 6,468,955; and 6,407,044. 45 erides contained in oil-rich biomass. Thus, in another aspect Increasing interest is directed to the use of hydrocarbon of the present invention a method for producing biodiesel is components of biological origin in fuels, such as biodiesel, provided. In a preferred embodiment, the method for produc renewable diesel, and jet fuel, because renewable biological ing biodiesel comprises the steps of (a) cultivating a lipid starting materials that may replace starting materials derived containing microorganism using methods disclosed herein from fossil fuels are available, and the use thereof is desirable. 50 (b) lysing a lipid-containing microorganism to produce a There is an urgent need for methods for producing hydrocar lysate, (c) isolating lipid from the lysed microorganism, and bon components from biological materials. The present (d) transesterifying the lipid composition, whereby biodiesel invention fulfills this need by providing methods for produc is produced. Methods for growth of a microorganism, lysing tion of biodiesel, renewable diesel, and jet fuel using the a microorganism to produce a lysate, treating the lysate in a lipids generated by the methods described hereinas a biologi- 55 medium comprising an organic solvent to form a heteroge cal material to produce biodiesel, renewable diesel, and jet neous mixture and separating the treated lysate into a lipid fuel. composition have been described above and can also be used Traditional diesel fuels are petroleum distillates rich in in the method of producing biodiesel. paraffinic hydrocarbons. They have boiling ranges as broad as The lipid profile of the biodiesel is usually highly similar to 370° to 780° F., which are suitable for combustion in a com- 60 the lipid profile of the feedstock oil. Other oils provided by the pression ignition engine, Such as a diesel engine vehicle. The methods and compositions of the invention can be subjected American Society of Testing and Materials (ASTM) estab to transesterification to yield biodiesel with lipid profiles lishes the grade of diesel according to the boiling range, along including (a) at least 4% C8-C14; (b) at least 0.3% C8; (c) at with allowable ranges of other fuel properties, such as cetane least 2% C10; (d) at least 2% C12; and (3) at least 30% number, cloud point, flash point, Viscosity, aniline point, Sul- 65 C8-C14. fur content, water content, ash content, copper strip corro Lipid compositions can be subjected to transesterification Sion, and carbon residue. Technically, any hydrocarbon dis to yield long-chain fatty acid esters useful as biodiesel. Pre US 8,846,352 B2 39 40 ferred transesterification reactions are outlined below and alcohol having a carbon atom number not less than 3 for a include base catalyzed transesterification and transesterifica period of time, preferably from 0.5-48 hours, and more pref tion using recombinant lipases. In a base-catalyzed transes erably from 0.5-1.5 hours. Some suitable methods also terification process, the triacylglycerides are reacted with an include washing a deactivated immobilized lipase with an alcohol, such as methanol or ethanol, in the presence of an 5 alcohol having a carbonatom number not less than 3 and then alkaline catalyst, typically potassium hydroxide. This reac immersing the deactivated immobilized lipase in a vegetable tion forms methyl or ethyl esters and glycerin (glycerol) as a oil for 0.5-48 hours. byproduct. In particular embodiments, a recombinant lipase is Animal and plant oils are typically made of triglycerides expressed in the same microorganisms that produce the lipid 10 on which the lipase acts. Suitable recombinant lipases include which are esters of free fatty acids with the trihydric alcohol, those listed above in and/or having GenBank Accession num glycerol. In transesterification, the glycerol in a triacylglyc bers listed in Table 7 of U.S. Patent Application Publication eride (TAG) is replaced with a short-chain alcohol such as 20100151112, or a polypeptide that has at least 70% amino methanol or ethanol. A typical reaction scheme is as follows: acid identity with one of the lipases listed in that Table 7 and 15 that exhibits lipase activity. In additional embodiments, the enzymatic activity is present in a sequence that has at least O-OCR cat.base about 75%, at least about 80%, at least about 85%, at least O OCR2 -- about 90%, at least about 95%, or at least about 99% identity 3 EtOH with one of the above described sequences, all of which are O-OCR 2O hereby incorporated by reference as if fully set forth. DNA Triglyceride encoding the lipase and selectable marker is preferably RCOOEt + R2COOEt + R3COOEt + CH3(OH) codon-optimized cDNA. Methods of recoding genes for Ethyl esters of fatty acids Glycerol expression in microalgae are described herein and in U.S. Pat. No. 7,135,290. 25 The common international standard for biodiesel is EN In this reaction, the alcohol is deprotonated with a base to 14214. ASTM D6751 is the most common biodiesel standard make it a stronger nucleophile. Commonly, ethanol or metha referenced in the United States and Canada. Germany uses nol is used in vast excess (up to 50-fold). Normally, this DIN EN 14214 and the UK requires compliance with BS EN reaction will proceed either exceedingly slowly or not at all. 14214. Basic industrial tests to determine whether the prod Heat, as well as an acid or base can be used to help the reaction 30 ucts conform to these standards typically include gas chro proceed more quickly. The acid or base are not consumed by matography, HPLC, and others. Biodiesel meeting the quality the transesterification reaction, thus they are not reactants but standards is very non-toxic, with a toxicity rating (LDs) of catalysts. Almost all biodiesel has been produced using the greater than 50 mL/kg. base-catalyzed technique as it requires only low temperatures Although biodiesel that meets the ASTM standards has to and pressures and produces over 98% conversion yield (pro- 35 be non-toxic, there can be contaminants which tend to crys vided the starting oil is low in moisture and free fatty acids). tallize and/or precipitate and fall out of solution as sediment. Transesterification has also been carried out, as discussed Sediment formation is particularly a problem when biodiesel above, using an enzyme. Such as a lipase instead of a base. is used at lower temperatures. The sediment or precipitates Lipase-catalyzed transesterification can be carried out, for may cause problems such as decreasing fuel flow, clogging example, at a temperature between the room temperature and 40 fuel lines, clogging filters, and the like. Processes are well 80° C., and a mole ratio of the TAG to the lower alcohol of known in the art that specifically deal with the removal of greater than 1:1, preferably about 3:1. Lipases suitable for use these contaminants and sediments in biodiesel in order to in transesterification include, but are not limited to, those produce a higher quality product. Examples for Such pro listed in Table 7. Other examples of lipases useful for trans cesses include, but are not limited to, pretreatment of the oil to esterification are found in, e.g. U.S. Pat. Nos. 4.798.793; 45 remove contaminants such as phospholipids and free fatty 4,940,845; 5,156,963; 5,342,768; 5,776,741 and WO89/ acids (e.g., degumming, caustic refining and silica adsorbant 01032. Such lipases include, but are not limited to, lipases filtration) and cold filtration. Cold filtration is a process that produced by microorganisms of Rhizopus, Aspergillus, Can was developed specifically to remove any particulates and dida, Mucor, Pseudomonas, Rhizomucor, Candida, and sediments that are present in the biodiesel after production. Humicola and pancreas lipase. Lipases Suitable for use in 50 This process cools the biodiesel and filters out any sediments transesterification are described in Table 7 of U.S. Patent or precipitates that mightform when the fuel is used at a lower Application Publication 20100151112. temperature. Such a process is well known in the art and is One challenge to using a lipase for the production of fatty described in US Patent Application Publication No. 2007 acid esters suitable for biodiesel is that the price of lipase is 0.175091. Suitable methods may include cooling the biodiesel much higher than the price of sodium hydroxide (NaOH) 55 to a temperature of less than about 38°C. so that the impuri used by the strong base process. This challenge has been ties and contaminants precipitate out as particulates in the addressed by using an immobilized lipase, which can be biodiesel liquid. Diatomaceous earth or other filtering mate recycled. However, the activity of the immobilized lipase rial may then added to the cooled biodiesel to form a slurry, must be maintained after being recycled for a minimum num which may then filtered through a pressure leaf or other type ber of cycles to allow a lipase-based process to compete with 60 offilter to remove the particulates. The filtered biodiesel may the strong base process interms of the production cost. Immo then be run through a polish filter to remove any remaining bilized lipases are subject to poisoning by the lower alcohols sediments and diatomaceous earth, so as to produce the final typically used in transesterification. U.S. Pat. No. 6,398,707 biodiesel product. (issued Jun. 4, 2002 to Wu et al.) describes methods for Example 14 of U.S. Patent Application Publication enhancing the activity of immobilized lipases and regenerat- 65 20100151112 describes the production of biodiesel using ing immobilized lipases having reduced activity. Some Suit triglyceride oil from Prototheca moriformis. The Cold Soak able methods include immersing an immobilized lipase in an Filterability by the ASTM D6751A1 method of the biodiesel US 8,846,352 B2 41 42 produced in that Example 14 was 120 seconds for a volume of and U.S. Pat. Nos. 7,238,277; 6,630,066: 6,596,155; 6,977, 300 ml. This test involves filtration of 300 ml of B100, which 322; 7,041,866; 6,217,746; 5,885,440; 6,881,873. is chilled to 40° F. for 16 hours, allowed to warm to room In one embodiment of the method for producing renewable temp, and filtered under vacuum using 0.7 micron glass fiber diesel, treating the lipid to produce an alkane is performed by filter with stainless steel support. Oils of the invention can be 5 hydrotreating of the lipid composition. In hydrothermal pro transesterified to generate biodiesel with a cold soak time of cessing, typically, biomass is reacted in water at an elevated less than 120 seconds, less than 100 seconds, and less than 90 temperature and pressure to form oils and residual Solids. seconds. Conversion temperatures are typically 300° to 660° F., with Subsequent processes may also be used if the biodiesel will pressure sufficient to keep the waterprimarily as a liquid, 100 be used in particularly cold temperatures. Such processes 10 to 170 standard atmospheres (atm). Reaction times are on the order of 15 to 30 minutes. After the reaction is completed, the include winterization and fractionation. Both processes are organics are separated from the water. Thereby a distillate designed to improve the cold flow and winter performance of suitable for diesel is produced. the fuel by lowering the cloud point (the temperature at which In some methods of making renewable diesel, the first step the biodiesel starts to crystallize). There are several 15 of treating a triglyceride is hydroprocessing to Saturate approaches to winterizing biodiesel. One approach is to blend double bonds, followed by deoxygenation at elevated tem the biodiesel with petroleum diesel. Another approach is to perature in the presence of hydrogen and a catalyst. In some use additives that can lower the cloud point of biodiesel. methods, hydrogenation and deoxygenation occur in the Another approach is to remove Saturated methyl esters indis same reaction. In other methods, deoxygenation occurs criminately by mixing in additives and allowing for the crys before hydrogenation. Isomerization is then optionally per tallization of Saturates and then filtering out the crystals. formed, also in the presence of hydrogen and a catalyst. Fractionation selectively separates methyl esters into indi Naphtha components are preferably removed through distil vidual components or fractions, allowing for the removal or lation. For examples, see U.S. Pat. No. 5,475,160 (hydroge inclusion of specific methyl esters. Fractionation methods nation of triglycerides); U.S. Pat. No. 5,091,116 (deoxygen include urea fractionation, solvent fractionation and thermal 25 ation, hydrogenation and gas removal); U.S. Pat. No. 6,391, distillation. 815 (hydrogenation); and U.S. Pat. No. 5,888,947 Another valuable fuel provided by the methods of the (isomerization). present invention is renewable diesel, which comprises One suitable method for the hydrogenation of triglycerides alkanes, such as C10:0, C12:0, C14:0, C16:0 and C18:0 and includes preparing an aqueous solution of copper, Zinc, mag thus, are distinguishable from biodiesel. High quality renew 30 nesium and lanthanum salts and another Solution of alkali able diesel conforms to the ASTM D975 standard. The lipids metal or preferably, ammonium carbonate. The two solutions produced by the methods of the present invention can serve as may be heated to a temperature of about 20° C. to about 85° feedstock to produce renewable diesel. Thus, in another C. and metered together into a precipitation container at rates aspect of the present invention, a method for producing Such that the pH in the precipitation container is maintained renewable diesel is provided. Renewable diesel can be pro 35 between 5.5 and 7.5 in order to form a catalyst. Additional duced by at least three processes: hydrothermal processing water may be used either initially in the precipitation con (hydrotreating); hydroprocessing; and indirect liquefaction. tainer or added concurrently with the salt solution and pre These processes yield non-ester distillates. During these pro cipitation Solution. The resulting precipitate may then be cesses, triacylglycerides produced and isolated as described thoroughly washed, dried, calcined at about 300° C. and herein, are converted to alkanes. 40 activated in hydrogen at temperatures ranging from about In one embodiment, the method for producing renewable 100° C. to about 400°C. One or more triglycerides may then diesel comprises (a) cultivating a lipid-containing microor be contacted and reacted with hydrogen in the presence of the ganism using methods disclosed herein (b) lysing the micro above-described catalyst in a reactor. The reactor may be a organism to produce a lysate, (c) isolating lipid from the lysed trickle bed reactor, fixed bed gas-solid reactor, packed bubble microorganism, and (d) deoxygenating and hydrotreating the 45 column reactor, continuously stirred tank reactor, a slurry lipid to produce an alkane, whereby renewable diesel is pro phase reactor, or any other Suitable reactor type known in the duced. Lipids suitable for manufacturing renewable diesel art. The process may be carried out either batchwise or in can be obtained via extraction from microbial biomass using continuous fashion. Reaction temperatures are typically in an organic solvent Such as hexane, or via other methods. Such the range of from about 170° C. to about 250° C. while as those described in U.S. Pat. No. 5,928,696. Some suitable 50 reaction pressures are typically in the range of from about 300 methods may include mechanical pressing and centrifuging. psig to about 2000 psig. Moreover, the molar ratio of hydro See PCT patent application U.S. Ser. No. 10/031,108, incor gen to triglyceride in the process of the present invention is porated herein by reference. typically in the range of from about 20:1 to about 700:1. The In some methods, the microbial lipid is first cracked in process is typically carried out at a weight hourly space Veloc conjunction with hydrotreating to reduce carbon chain length 55 ity (WHSV) in the range of from about 0.1 hr' to about 5 and saturate double bonds, respectively. The material is then hr'. One skilled in the art will recognize that the time period isomerized, also in conjunction with hydrotreating. The required for reaction will vary according to the temperature naptha fraction can then be removed through distillation, used, the molar ratio of hydrogen to triglyceride, and the followed by additional distillation to vaporize and distill com partial pressure of hydrogen. The products produced by Such ponents desired in the diesel fuel to meet an ASTM D975 60 hydrogenation processes include fatty alcohols, glycerol, standard while leaving components that are heavier than traces of paraffins and unreacted triglycerides. These prod desired for meeting the D975 standard. Hydrotreating, hydro ucts are typically separated by conventional means such as, cracking, deoxygenation and isomerization methods of for example, distillation, extraction, filtration, crystallization, chemically modifying oils, including triglyceride oils, are and the like. well known in the art. See for example European patent 65 Petroleum refiners use hydroprocessing to remove impu applications EP1741768 (A1); EP1741767 (A1); EP1682466 rities by treating feeds with hydrogen. Hydroprocessing con (A1); EP1640437 (A1); EP1681337 (A1); EP1795576 (A1); version temperatures are typically 300° to 700°F. Pressures US 8,846,352 B2 43 44 are typically 40 to 100 atm. The reaction times are typically Other oils provided by the methods and compositions of on the order of 10 to 60 minutes. Solid catalysts are employed the invention can be subjected to combinations of hydrotreat to increase certain reaction rates, improve selectivity for cer ing, isomerization, and other covalent modification including tain products, and optimize hydrogen consumption. oils with lipid profiles including (a) at least 4% C8-C14; (b) at Suitable methods for the deoxygenation of an oil includes 5 least 0.3% C8; (c) at least 2% C10; (d) at least 2% C12; and heating an oil to a temperature in the range of from about 350° (3) at least 30% C8-C14. F. to about 550°F. and continuously contacting the heated oil A traditional ultra-low sulfur diesel can be produced from with nitrogen under at least pressure ranging from about any form of biomass by a two-step process. First, the biomass atmospheric to above for at least about 5 minutes. is converted to a syngas, a gaseous mixture rich in hydrogen Suitable methods for isomerization include using alkali 10 and carbon monoxide. Then, the Syngas is catalytically con isomerization and other oil isomerization known in the art. Verted to liquids. Typically, the production of liquids is Hydrotreating and hydroprocessing ultimately lead to a accomplished using Fischer-Tropsch (FT) synthesis. This reduction in the molecular weight of the triglyceride feed. technology applies to coal, natural gas, and heavy oils. Thus, The triglyceride molecule is reduced to four hydrocarbon in yet another preferred embodiment of the method for pro molecules under hydroprocessing conditions: a propane mol- 15 ducing renewable diesel, treating the lipid composition to ecule and three heavier hydrocarbon molecules, typically in produce an alkane is performed by indirect liquefaction of the the C8 to C18 range. lipid composition. Thus, in one embodiment, the product of one or more The present invention also provides methods to produce jet chemical reaction(s) performed on lipid compositions of the fuel. Jet fuel is clear to straw colored. The most common fuel invention is an alkane mixture that comprises ASTM D975 is an unleaded/paraffin oil-based fuel classified as Aeroplane renewable diesel. Production of hydrocarbons by microor A-1, which is produced to an internationally standardized set ganisms is reviewed by Metzger et al. Appl Microbiol Bio of specifications. Jet fuel is a mixture of a large number of technol (2005) 66: 486-496 and A Look Back at the U.S. different hydrocarbons, possibly as many as a thousand or Department of Energy’s Aquatic Species Program: Biodiesel more. The range of their sizes (molecular weights or carbon from Algae, NREL/TP-580-24190, John Sheehan, Terri 25 numbers) is restricted by the requirements for the product, for Dunahay, John Benemann and Paul Roessler (1998). example, freezing point or Smoke point. Kerosone-type The distillation properties of a diesel fuel is described in Aeroplane fuel (including Jet A and Jet A-1) has a carbon terms of T10-T90 (temperature at 10% and 90%, respectively, number distribution between about 8 and 16 carbon numbers. volume distilled). Renewable diesel can be produced from Wide-cut or naphta-type Aeroplane fuel (including Jet B) Prototheca moriformis triglyceride oil as described in 30 typically has a carbon number distribution between about 5 Example 14 of U.S. Patent Application Publication and 15 carbons. 20100151112. The T10-T90 of the material produced in that Both Aeroplanes (Jet A and Jet B) may contain a number of Example 14 was 57.9°C. Methods of hydrotreating, isomer additives. Useful additives include, but are not limited to, ization, and other covalent modification of oils disclosed antioxidants, antistatic agents, corrosion inhibitors, and fuel herein, as well as methods of distillation and fractionation 35 system icing inhibitor (FSII) agents. Antioxidants prevent (such as cold filtration) disclosed herein, can be employed to gumming and usually, are based on alkylated phenols, for generate renewable diesel compositions with other T10-T90 example, AO-30, AO-31, or AO-37. Antistatic agents dissi ranges, such as 20, 25, 30, 35, 40, 45, 50, 60 and 65° C. using pate static electricity and prevent sparking. Stadis 450 with triglyceride oils produced according to the methods disclosed dinonylnaphthylsulfonic acid (DINNSA) as the active ingre herein. 40 dient is an example. Corrosion inhibitors, e.g., DCI-4A, can The T10 of the material produced in Example 14 of U.S. be used for civilian and military fuels and DCI-6A is used for Patent Application Publication 20100151112 was 242.1° C. military fuels. FSII agents include, e.g., Di-EGME. Methods of hydrotreating, isomerization, and other covalent In one embodiment of the invention, a jet fuel is produced modification of oils disclosed herein, as well as methods of by blending algal fuels with existing jet fuel. The lipids pro distillation and fractionation (such as cold filtration) dis 45 duced by the methods of the present invention can serve as closed herein, can be employed to generate renewable diesel feedstock to produce jet fuel. Thus, in another aspect of the compositions with other T10 values, such as T10between 180 present invention, a method for producing jet fuel is provided. and 295, between 190 and 270, between 210 and 250, Herewith two methods for producing jet fuel from the lipids between 225 and 245, and at least 290. produced by the methods of the present invention are pro The T90 of the material produced in Example 14 of U.S. 50 vided: fluid catalytic cracking (FCC); and hydrodeoxygen Patent Application Publication 20100151112 was 300° C. ation (HDO). Methods of hydrotreating, isomerization, and other covalent Fluid Catalytic Cracking (FCC) is one method which is modification of oils disclosed herein, as well as methods of used to produce olefins, especially propylene from heavy distillation and fractionation (such as cold filtration) dis crude fractions. The lipids produced by the method of the closed herein can be employed to generate renewable diesel 55 present invention can be converted to olefins. The process compositions with other T90 values, such as T90 between 280 involves flowing the lipids produced through an FCC Zone and 380, between 290 and 360, between 300 and 350, and collecting a product stream comprised of olefins, which is between 310 and 340, and at least 290. useful as a jet fuel. The lipids produced are contacted with a The FBP of the material produced in Example 14 U.S. cracking catalyst at cracking conditions to provide a product Patent Application Publication 20100151112 was 300° C. 60 stream comprising olefins and hydrocarbons useful as jet fuel. Methods of hydrotreating, isomerization, and other covalent In one embodiment, the method for producing jet fuel modification of oils disclosed herein, as well as methods of comprises (a) cultivating a lipid-containing microalgae in distillation and fractionation (such as cold filtration) dis Xylose-containing media using Prototheca cells and methods closed herein, can be employed to generate renewable diesel disclosed herein, (b) lysing the lipid-containing microorgan compositions with other FBP values, such as FBP between 65 ism to produce a lysate, (c) isolating lipid from the lysate, and 290 and 400, between 300 and 385, between 310 and 370, (d) treating the lipid composition, whereby jet fuel is pro between 315 and 360, and at least 300. duced. In one embodiment of the method for producing a jet US 8,846,352 B2 45 46 fuel, the lipid composition can be flowed through a fluid ture of about 149° C. to about 316°C. (300°F. to 600°F). The catalytic cracking Zone, which, in one embodiment, may catalyst is flowed from a blending vessel to the riser where it comprise contacting the lipid composition with a cracking contacts the lipid composition for a time of abort 2 seconds or catalyst at cracking conditions to provide a product stream less. comprising C-C olefins. The blended catalyst and reacted lipid composition vapors In certain embodiments of this method, it may be desirable are then discharged from the top of the riser through an outlet to remove any contaminants that may be present in the lipid and separated into a cracked product vapor stream including composition. Thus, prior to flowing the lipid composition olefins and a collection of catalyst particles covered with through a fluid catalytic cracking Zone, the lipid composition Substantial quantities of coke and generally referred to as is pretreated. Pretreatment may involve contacting the lipid 10 “coked catalyst.” In an effort to minimize the contact time of composition with an ion-exchange resin. The ion exchange the lipid composition and the catalyst which may promote resin is an acidic ion exchange resin, such as AmberlystTM-15 further conversion of desired products to undesirable other and can be used as a bed in a reactor through which the lipid products, any arrangement of separators such as a Swirl arm composition is flowed, either upflow or downflow. Other pre arrangement can be used to remove coked catalyst from the treatments may include mild acid washes by contacting the 15 product stream quickly. The separator, e.g. Swirl arm separa lipid composition with an acid, such as Sulfuric, acetic, nitric, tor, is located in an upper portion of a chamber with a strip or hydrochloric acid. Contacting is done with a dilute acid ping Zone situated in the lower portion of the chamber. Cata Solution usually at ambient temperature and atmospheric lyst separated by the Swirl arm arrangement drops down into pressure. the stripping Zone. The cracked product vapor stream com The lipid composition, optionally pretreated, is flowed to prising cracked hydrocarbons including light olefins and an FCC Zone where the hydrocarbonaceous components are Some catalyst exit the chamber via a conduit which is in cracked to olefins. Catalytic cracking is accomplished by communication with cyclones. The cyclones remove remain contacting the lipid composition in a reaction Zone with a ing catalyst particles from the product vapor stream to reduce catalyst composed offinely divided particulate material. The particle concentrations to very low levels. The product vapor reaction is catalytic cracking, as opposed to hydrocracking, 25 stream then exits the top of the separating vessel. Catalyst and is carried out in the absence of added hydrogen or the separated by the cyclones is returned to the separating vessel consumption of hydrogen. As the cracking reaction proceeds, and then to the stripping Zone. The stripping Zone removes Substantial amounts of coke are deposited on the catalyst. The adsorbed hydrocarbons from the surface of the catalyst by catalyst is regenerated at high temperatures by burning coke counter-current contact with steam. from the catalyst in a regeneration Zone. Coke-containing 30 Low hydrocarbon partial pressure operates to favor the catalyst, referred to herein as “coked catalyst”, is continually production of light olefins. Accordingly, the riser pressure is transported from the reaction Zone to the regeneration Zone to set at about 172 to 241 kPa (25 to 35 psia) with a hydrocarbon be regenerated and replaced by essentially coke-free regen partial pressure of about 35 to 172 kPa (5 to 25 psia), with a erated catalyst from the regeneration Zone. Fluidization of the preferred hydrocarbon partial pressure of about 69 to 138 kPa catalyst particles by various gaseous streams allows the trans 35 (10 to 20 psia). This relatively low partial pressure for hydro port of catalyst between the reaction Zone and regeneration carbon is achieved by using steam as a diluent to the extent Zone. Methods for cracking hydrocarbons, such as those of that the diluent is 10 to 55 wt-% of lipid composition and the lipid composition described herein, in a fluidized stream preferably about 15 wt-% of lipid composition. Other diluents of catalyst, transporting catalyst between reaction and regen Such as dry gas can be used to reach equivalent hydrocarbon eration Zones, and combusting coke in the regenerator are 40 partial pressures. well known by those skilled in the art of FCC processes. The temperature of the cracked stream at the riser outlet Exemplary FCC applications and catalysts useful for crack will be about 510° C. to 621° C. (950 F. to 1150°F). How ing the lipid composition to produce C-Cs olefins are ever, riser outlet temperatures above 566° C. (1050°F) make described in U.S. Pat. Nos. 6,538,169 and 7,288,685, which more dry gas and more olefins. Whereas, riser outlet tempera are incorporated in their entirety herein by reference. 45 tures below 566° C. (1050 F) make less ethylene and pro Suitable FCC catalysts generally comprise at least two pylene. Accordingly, it is preferred to run the FCC process at components that may or may not be on the same matrix. In a preferred temperature of about 566° C. to about 630°C., Some embodiments, both components may be circulated preferred pressure of about 138 kPa to about 240 kPa (20 to 35 throughout the entire reaction vessel. The first component psia). Another condition for the process is the catalyst to lipid generally includes any of the well-known catalysts that are 50 composition ratio which can vary from about 5 to about 20 used in the art of fluidized catalytic cracking, Such as an active and preferably from about 10 to about 15. amorphous clay-type catalyst and/or a high activity, crystal In one embodiment of the method for producing a jet fuel, line molecular sieve. Molecular sieve catalysts may some the lipid composition is introduced into the lift section of an times be preferred over amorphous catalysts because of their FCC reactor. The temperature in the lift section will be very much-improved selectivity to desired products. In some pre 55 hot and range from about 700° C. (1292°F) to about 760° C. ferred embodiments, zeolites may be used as the molecular (1400°F.) with a catalyst to lipid composition ratio of about sieve in the FCC processes. Preferably, the first catalyst com 100 to about 150. It is anticipated that introducing the lipid ponent comprises a large pore Zeolite. Such as an Y-type composition into the lift section will produce considerable Zeolite, an active alumina material, a binder material, com amounts of propylene and ethylene. prising either silica or alumina and an inert filler Such as 60 In another embodiment of the method for producing a jet kaolin. fuel using the lipid composition or the lipids produced as In one embodiment, cracking the lipid composition of the described herein, the structure of the lipid composition or the present invention, takes place in the riser section or, alterna lipids is broken by a process referred to as hydrodeoxygen tively, the lift section, of the FCC Zone. The lipid composition ation (HDO). HDO means removal of oxygen by means of is introduced into the riser by a nozzle resulting in the rapid 65 hydrogen, that is, oxygen is removed while breaking the vaporization of the lipid composition. Before contacting the structure of the material. Olefinic double bonds are hydroge catalyst, the lipid composition will ordinarily have a tempera nated and any Sulphur and nitrogen compounds are removed. US 8,846,352 B2 47 48 Sulphur removal is called hydrodesulphurization (HDS). Pre gen is returned back to the first catalyst bed and/or between treatment and purity of the raw materials (lipid composition the catalyst beds to make up for the withdrawn gas stream. or the lipids) contribute to the service life of the catalyst. Water is removed from the condensed liquid. The liquid is Generally in the HDO/HDS step, hydrogen is mixed with passed to the first catalyst bed or between the catalyst beds. the feed stock (lipid composition or the lipids) and then the After the HDO step, the product is subjected to an isomer mixture is passed through a catalyst bed as a co-current flow, ization step. It is Substantial for the process that the impurities either as a single phase or a two phase feed stock. After the are removed as completely as possible before the hydrocar HDO/MDS step, the product fraction is separated and passed bons are contacted with the isomerization catalyst. The to a separate isomerzation reactor. An isomerization reactor isomerization step comprises an optional stripping step, for biological starting material is described in the literature 10 wherein the reaction product from the HDO step may be (FI 100 248) as a co-current reactor. purified by Stripping with water vapour or a suitable gas Such The process for producing a fuel by hydrogenating a hydro as light hydrocarbon, nitrogen or hydrogen. The optional carbon feed, e.g., the lipid composition or the lipids herein, stripping step is carried out in counter-current manner in a can also be performed by passing the lipid composition or the unit upstream of the isomerization catalyst, wherein the gas lipids as a co-current flow with hydrogen gas through a first 15 and liquid are contacted with each other, or before the actual hydrogenation Zone, and thereafter the hydrocarbon effluent isomerization reactor in a separate stripping unit utilizing is further hydrogenated in a second hydrogenation Zone by counter-current principle. passing hydrogen gas to the second hydrogenation Zone as a After the Stripping step the hydrogen gas and the hydroge counter-current flow relative to the hydrocarbon effluent. nated lipid composition or lipids herein, and optionally an Exemplary HDO applications and catalysts useful for crack n-paraffin mixture, are passed to a reactive isomerization unit ing the lipid composition to produce C-C olefins are comprising one or several catalystbed(s). The catalyst beds of described in U.S. Pat. No. 7,232,935, which is incorporated in the isomerization step may operate either in co-current or its entirety by reference. COunter-Current manner. Typically, in the hydrodeoxygenation step, the structure of It is important for the process that the counter-current flow the biological component, such as the lipid composition or 25 principle is applied in the isomerization step. In the isomer lipids herein, is decomposed, oxygen, nitrogen, phosphorus ization step this is done by carrying out either the optional and Sulphur compounds, and light hydrocarbons as gas are stripping step or the isomerization reaction step or both in removed, and the olefinic bonds are hydrogenated. In the counter-current manner. In the isomerization step, the pres second step of the process, i.e. in the so-called isomerization sure varies in the range of 20 150 bar, preferably in the range step, isomerzation is carried out for branching the hydrocar 30 of 20 100 bar, the temperature being between 200 and 500° bon chain and improving the performance of the paraffin at C., preferably between 300 and 400°C. In the isomerization low temperatures. step, isomerization catalysts known in the art may be used. In the first step, i.e. HDO step, of the cracking process, Suitable isomerization catalysts contain molecular sieve and/ hydrogen gas and the lipid composition or lipids herein which or a metal from Group VII and/or a carrier. Preferably, the are to be hydrogenated are passed to a HDO catalyst bed 35 isomerization catalyst contains SAPO-11 or SAPO41 or system either as co-current or counter-current flows, said ZSM-22 or ZSM-23 or ferrierite and Pt, Pdor Niand Al-O or catalyst bed system comprising one or more catalyst bed(s), SiO. Typical isomerization catalysts are, for example, preferably 1-3 catalyst beds. The HDO step is typically oper Pt/SAPO-11/Al-O, Pt/ZSM-22/Al-O, Pt/ZSM-23/Al-O, ated in a co-current manner. In case of a HDO catalyst bed and Pt/SAPO-11/SiO. The isomerization step and the HDO system comprising two or more catalyst beds, one or more of 40 step may be carried out in the same pressure vessel or in the beds may be operated using the counter-current flow separate pressure vessels. Optional prehydrogenation may be principle. In the HDO step, the pressure varies between 20 carried out in a separate pressure vessel or in the same pres and 150 bar, preferably between 50 and 100 bar, and the sure vessel as the HDO and isomerization steps. temperature varies between 200 and 500°C., preferably in the Thus, in one embodiment, the product of the one or more range of 300-400°C. In the HDO step, known hydrogenation 45 chemical reactions is an alkane mixture that comprises catalysts containing metals from Group VII and/or VIB of the ASTM D1655 jet fuel. In some embodiments, the composi Periodic System may be used. Preferably, the hydrogenation tion conforming to the specification of ASTM 1655 jet fuel catalysts are supported Pd, Pt, Ni, NiMo or a CoMo catalysts, has a sulfur content that is less than 10 ppm. In other embodi the support being alumina and/or silica. Typically, NiMo/ ments, the composition conforming to the specification of Al-O and CoMo, Al2O catalysts are used. 50 ASTM 1655 jet fuel has a T10 value of the distillation curve Prior to the HDO step, the lipid composition or lipids of less than 205°C. In another embodiment, the composition herein may optionally be treated by prehydrogenation under conforming to the specification of ASTM 1655 jet fuel has a milder conditions thus avoiding side reactions of the double final boiling point (FBP) of less than 300° C. In another bonds. Such prehydrogenation is carried out in the presence embodiment, the composition conforming to the specifica of a prehydrogenation catalyst attemperatures of 50 400° C. 55 tion of ASTM 1655 jet fuel has a flash point of at least 38°C. and at hydrogen pressures of 1 200 bar, preferably at a tem In another embodiment, the composition conforming to the perature between 150 and 250° C. and at a hydrogen pressure specification of ASTM 1655 jet fuel has a density between between 10 and 100 bar. The catalyst may contain metals 775 K/M and 840 K/M. In yet another embodiment, the from Group VIII and/or VIB of the Periodic System. Prefer composition conforming to the specification of ASTM 1655 ably, the prehydrogenation catalyst is a supported Pd, Pt, Ni, 60 jet fuel has a freezing point that is below -47°C. In another NiMo or a CoMo catalyst, the support being alumina and/or embodiment, the composition conforming to the specifica silica. tion of ASTM 1655 jet fuel has a net Heat of Combustion that A gaseous stream from the HDO step containing hydrogen is at least 42.8 MJ/K. In another embodiment, the composi is cooled and then carbon monoxide, carbon dioxide, nitro tion conforming to the specification of ASTM 1655 jet fuel gen, phosphorus and Sulphur compounds, gaseous light 65 has a hydrogen content that is at least 13.4 mass %. In another hydrocarbons and other impurities are removed therefrom. embodiment, the composition conforming to the specifica After compressing, the purified hydrogen or recycled hydro tion of ASTM 1655 jet fuel has athermal stability, as tested by US 8,846,352 B2 49 50 quantitative gravimetric JFTOT at 260° C., that is below 3 Generally, enzymatic oil splitting methods use enzymes, mm of Hg. In another embodiment, the composition con lipases, as biocatalysts acting on a water/oil mixture. Enzy forming to the specification of ASTM 1655 jet fuel has an matic splitting then splits the oil or fat, respectively, is into existent gum that is below 7 mg/dl. glycerol and free fatty acids. The glycerol may then migrate Thus, the present invention discloses a variety of methods into the water phase whereby the organic phase is enriched in which chemical modification of microalgal lipid is under with free fatty acids. taken to yield products useful in a variety of industrial and The enzymatic splitting reactions generally take place at other applications. Examples of processes for modifying oil the phase boundary between organic and aqueous phase, produced by the methods disclosed herein include, but are not where the enzyme is present only at the phase boundary. limited to, hydrolysis of the oil, hydroprocessing of the oil, 10 Triglycerides that meet the phase boundary then contribute to and esterification of the oil. The modification of the microal or participate in the splitting reaction. As the reaction pro gal oil produces basic oleochemicals that can be further modi ceeds, the occupation density or concentration of fatty acids fied into selected derivative oleochemicals for a desired func still chemically bonded as glycerides, in comparison to free tion. In a manner similar to that described above with fatty acids, decreases at the phase boundary so that the reac reference to fuel producing processes, these chemical modi 15 tion is slowed down. In certain embodiments, enzymatic fications can also be performed on oils generated from the splitting may occur at room temperature. One of ordinary microbial cultures described herein. Examples of basic ole skill in the art knows the suitable conditions for splitting oil ochemicals include, but are not limited to, soaps, fatty acids, into the desired fatty acids. fatty acid methyl esters, and glycerol. Examples of derivative By way of example, the reaction speed can be accelerated oleochemicals include, but are not limited to, fatty nitriles, by increasing the interface boundary Surface. Once the reac esters, dimer acids, quats, Surfactants, fatty alkanolamides, tion is complete, free fatty acids are then separated from the fatty alcohol Sulfates, resins, emulsifiers, fatty alcohols, ole organic phase freed from enzyme, and the residue which still fins, and higher alkanes. contains fatty acids chemically bonded as glycerides is fed Hydrolysis of the fatty acid constituents from the glycero back or recycled and mixed with fresh oil or fat to be sub lipids produced by the methods of the invention yields free 25 jected to splitting. In this manner, recycled glycerides are then fatty acids that can be derivatized to produce other useful Subjected to a further enzymatic splitting process. In some chemicals. Hydrolysis occurs in the presence of water and a embodiments, the free fatty acids are extracted from an oil or catalyst which may be either an acid or a base. The liberated fat partially split in Such a manner. In that way, if the chemi free fatty acids can be derivatized to yield a variety of prod cally bound fatty acids (triglycerides) are returned or fed back ucts, as reported in the following: U.S. Pat. No. 5,304,664 30 into the splitting process, the enzyme consumption can be (Highly sulfated fatty acids); U.S. Pat. No. 7,262,158 drastically reduced. (Cleansing compositions); U.S. Pat. No. 7,115,173 (Fabric The splitting degree is determined as the ratio of the mea softener compositions); U.S. Pat. No. 6,342,208 (Emulsions sured acid value divided by the theoretically possible acid for treating skin); U.S. Pat. No. 7.264,886 (Water repellant value which can be computed for a given oil or fat. Preferably, compositions); U.S. Pat. No. 6,924.333 (Paint additives): 35 the acid value is measured by means of titration according to U.S. Pat. No. 6,596,768 (Lipid-enriched ruminant feedstock); standard common methods. Alternatively, the density of the and U.S. Pat. No. 6,380,410 (Surfactants for detergents and aqueous glycerol phase can be taken as a measure for the cleaners). splitting degree. With regard to hydrolysis, in one embodiment of the inven In one embodiment, the slitting process as described herein tion, a triglyceride oil is optionally first hydrolyzed in a liquid 40 is also suitable for splitting the mono-, di- and triglyceride medium such as water or Sodium hydroxide so as to obtain that are contained in the so-called soap-stock from the alkali glycerol and Soaps. There are various Suitable triglyceride refining processes of the produced oils. In this manner, the hydrolysis methods, including, but not limited to, Saponifica soap-stock can be quantitatively converted without prior tion, acid hydrolysis, alkaline hydrolysis, enzymatic hydroly saponification of the neutral oils into the fatty acids. For this sis (referred herein as splitting), and hydrolysis using hot 45 purpose, the fatty acids being chemically bonded in the soaps compressed water. One skilled in the art will recognize that a are released, preferably before splitting, through an addition triglyceride oil need not be hydrolyzed to produce an ole of acid. In certain embodiments, a buffer solution is used in ochemical; rather, the oil may be converted directly to the addition to water and enzyme for the splitting process. desired oleochemical by other known processes. For In one embodiment, oils produced in accordance with the example, the triglyceride oil may be directly converted to a 50 methods of the invention can also be subjected to saponifica methyl ester fatty acid through esterification. tion as a method of hydrolysis. Animal and plant oils are In some embodiments, catalytic hydrolysis of the oil pro typically made of triacylglycerols (TAGs), which are esters of duced by methods disclosed herein occurs by splitting the oil fatty acids with the trihydric alcohol, glycerol. In an alkaline into glycerol and fatty acids. As discussed above, the fatty hydrolysis reaction, the glycerolina TAG is removed, leaving acids may then be further processed through several other 55 three carboxylic acid anions that can associate with alkali modifications to obtained derivative oleochemicals. For metal cations such as Sodium or potassium to produce fatty example, in one embodiment the fatty acids may undergo an acid salts. In this scheme, the carboxylic acid constituents are amination reaction to produce fatty nitrogen compounds. In cleaved from the glycerol moiety and replaced with hydroxyl another embodiment, the fatty acids may undergo oZonolysis groups. The quantity of base (e.g., KOH) that is used in the to produce mono- and dibasic-acids. 60 reaction is determined by the desired degree of Saponifica In other embodiments hydrolysis may occur via the, split tion. If the objective is, for example, to produce a Soap product ting of oils produced herein to create oleochemicals. In some that comprises some of the oils originally present in the TAG preferred embodiments of the invention, a triglyceride oil composition, an amount of base insufficient to convert all of may be split before other processes is performed. One skilled the TAGs to fatty acid salts is introduced into the reaction in the art will recognize that there are many suitable triglyc 65 mixture. Normally, this reaction is performed in an aqueous eride splitting methods, including, but not limited to, enzy solution and proceeds slowly, but may be expedited by the matic splitting and pressure splitting. addition of heat. Precipitation of the fatty acid salts can be US 8,846,352 B2 51 52 facilitated by addition of salts, such as water-soluble alkali functionality of the catalyst. The hydrogenation catalysts may metal halides (e.g., NaCl or KCl), to the reaction mixture. be prepared by methods known to those of ordinary skill in Preferably, the base is an alkali metal hydroxide, such as the art. NaOH or KOH. Alternatively, other bases, such as alkanola In some embodiments the hydrogenation catalyst includes mines, including for example triethanolamine and aminom a Supported Group VIII metal catalyst and a metal sponge ethylpropanol, can be used in the reaction scheme. In some material (e.g., a sponge nickel catalyst). Raney nickel pro cases, these alternatives may be preferred to produce a clear vides an example of an activated Sponge nickel catalyst Suit Soap product. able for use in this invention. In other embodiment, the hydro In some methods, the first step of chemical modification genation reaction in the invention is performed using a may be hydroprocessing to Saturate double bonds, followed 10 catalyst comprising a nickel-rhenium catalyst or a tungsten by deoxygenation at elevated temperature in the presence of modified nickel catalyst. One example of a suitable catalyst hydrogen and a catalyst. In other methods, hydrogenation and for the hydrogenation reaction of the invention is a carbon deoxygenation may occur in the same reaction. In still other Supported nickel-rhenium catalyst. methods deoxygenation occurs before hydrogenation. In an embodiment, a Suitable Raney nickel catalyst may be Isomerization may then be optionally performed, also in the 15 prepared by treating an alloy of approximately equal amounts presence of hydrogen and a catalyst. Finally, gases and naph by weight of nickel and aluminum with an aqueous alkali tha components can be removed if desired. For example, see Solution, e.g., containing about 25 weight '% of Sodium U.S. Pat. No. 5,475,160 (hydrogenation of triglycerides); hydroxide. The aluminum is selectively dissolved by the U.S. Pat. No. 5,091,116 (deoxygenation, hydrogenation and aqueous alkali solution resulting in a sponge shaped material gas removal); U.S. Pat. No. 6.391.815 (hydrogenation); and comprising mostly nickel with minor amounts of aluminum. U.S. Pat. No. 5,888,947 (isomerization). The initial alloy includes promoter metals (i.e., molybdenum In some embodiments of the invention, the triglyceride oils or chromium) in the amount such that about 1 to 2 weight% are partially or completely deoxygenated. The deoxygenation remains in the formed sponge nickel catalyst. In another reactions form desired products, including, but not limited to, embodiment, the hydrogenation catalyst is prepared using a fatty acids, fatty alcohols, polyols, ketones, and aldehydes. In solution of ruthenium(III) nitrosylnitrate, ruthenium (III) general, without being limited by any particular theory, the 25 chloride in water to impregnate a suitable Support material. deoxygenation reactions involve a combination of various The solution is then dried to form a solid having a water different reaction pathways, including without limitation: content of less than about 1% by weight. The solid may then hydrogenolysis, hydrogenation, consecutive hydrogenation be reduced at atmospheric pressure in a hydrogen stream at hydrogenolysis, consecutive hydrogenolysis-hydrogenation, 300° C. (uncalcined) or 400° C. (calcined) in a rotary ball and combined hydrogenation-hydrogenolysis reactions, 30 furnace for 4 hours. After cooling and rendering the catalyst resulting in at least the partial removal of oxygen from the inert with nitrogen, 5% by Volume of oxygen in nitrogen is fatty acidor fatty acid ester to produce reaction products, such passed over the catalyst for 2 hours. as fatty alcohols, that can be easily converted to the desired In certain embodiments, the catalyst described includes a chemicals by further processing. For example, in one embodi catalyst Support. The catalyst Support stabilizes and Supports ment, a fatty alcohol may be converted to olefins through FCC the catalyst. The type of catalyst Support used depends on the reaction or to higher alkanes through a condensation reaction. 35 chosen catalyst and the reaction conditions. Suitable Supports One such chemical modification is hydrogenation, which for the invention include, but are not limited to, carbon, silica, is the addition of hydrogen to double bonds in the fatty acid silica-alumina, Zirconia, titania, ceria, Vanadia, nitride, boron constituents of glycerolipids or of free fatty acids. The hydro nitride, heteropolyacids, hydroxyapatite, Zinc oxide, chro genation process permits the transformation of liquid oils into mia, Zeolites, carbon nanotubes, carbon fullerene and any semi-solid or solid fats, which may be more suitable for 40 combination thereof. specific applications. The catalysts used in this invention can be prepared using Hydrogenation of oil produced by the methods described conventional methods known to those in the art. Suitable herein can be performed in conjunction with one or more of methods may include, but are not limited to, incipient wet the methods and/or materials provided herein, as reported in ting, evaporative impregnation, chemical vapor deposition, the following: U.S. Pat. No. 7,288,278 (Food additives or 45 wash-coating, magnetron Sputtering techniques, and the like. medicaments); U.S. Pat. No. 5,346,724 (Lubrication prod The conditions for which to carry out the hydrogenation ucts); U.S. Pat. No. 5,475,160 (Fatty alcohols); U.S. Pat. No. reaction will vary based on the type of starting material and 5,091,116 (Edible oils); U.S. Pat. No. 6,808,737 (Structural the desired products. One of ordinary skill in the art, with the fats for margarine and spreads); U.S. Pat. No. 5.298,637 benefit of this disclosure, will recognize the appropriate reac (Reduced-calorie fat substitutes); U.S. Pat. No. 6,391,815 50 tion conditions. In general, the hydrogenation reaction is (Hydrogenation catalyst and sulfur adsorbent); U.S. Pat. Nos. conducted at temperatures of 80° C. to 250° C., and prefer 5,233,099 and 5,233,100 (Fatty alcohols); U.S. Pat. No. ably at 90° C. to 200° C., and most preferably at 100° C. to 4.584,139 (Hydrogenation catalysts); U.S. Pat. No. 6,057, 150°C. In some embodiments, the hydrogenation reaction is 375 (Foam suppressing agents); and U.S. Pat. No. 7,118.773 conducted at pressures from 500 KPa to 14000 KPa. (Edible emulsion spreads). The hydrogen used in the hydrogenolysis reaction of the One skilled in the art will recognize that various processes 55 current invention may include external hydrogen, recycled may be used to hydrogenate oil. One suitable method includes hydrogen, in situ generated hydrogen, and any combination contacting the oil with hydrogen or hydrogen mixed with a thereof. As used herein, the term “external hydrogen refers Suitable gas and a catalyst under conditions sufficient in a to hydrogen that does not originate from the biomass reaction hydrogenation reactor to form a hydrogenated product. The itself, but rather is added to the system from another source. hydrogenation catalyst generally can include Cu, Re, Ni, Fe, 60 In some embodiments of the invention, it is desirable to Co, Ru, Pd, Rh, Pt, Os, Ir, and alloys or any combination convert the starting triglyceride or fatty acid to a smaller thereof, either alone or with promoters such as W. Mo, Au, molecule that will be more readily converted to desired higher Ag, Cr, Zn, Mn, Sn, B. P. Bi, and alloys or any combination hydrocarbons. One suitable method for this conversion is thereof. Other effective hydrogenation catalyst materials through a hydrogenolysis reaction. Various processes are include either supported nickel or ruthenium modified with 65 known for performing hydrogenolysis of oil. One Suitable rhenium. In an embodiment, the hydrogenation catalyst also method includes contacting oil with hydrogen or hydrogen includes any one of the Supports, depending on the desired mixed with a suitable gas and a hydrogenolysis catalyst in a US 8,846,352 B2 53 54 hydrogenolysis reactor under conditions Sufficient to form a any combination thereof. In some embodiments, the conden reaction product comprising Smaller molecules or polyols. As sation catalyst can also include a modifier. Suitable modifiers used herein, the term “smaller molecules or polyols includes include La, Y. Sc, P. B., Bi, Li, Na, K, Rb, Cs, Mg, Ca, Sr., Ba, any molecule that has a Smaller molecular weight, which can and any combination thereof. In some embodiments, the con include a smaller number of carbon atoms or oxygen atoms 5 densation catalyst can also include a metal. Suitable metals than the starting oil. In an embodiment, the reaction products include Cu, Ag, Au, Pt, Ni, Fe, Co, Ru, Zn, Cd, Ga, In, Rh, Pd, include Smaller molecules that include polyols and alcohols. Ir, Re, Mn, Cr, Mo, W. Sn, Os, alloys, and any combination One of ordinary skill in the art can readily select the appro thereof. priate method by which to carry out the hydrogenolysis reac In certain embodiments, the catalyst described in the con tion. 10 densation reaction may include a catalyst Support as In some embodiments, a 5 and/or 6 carbon Sugar or Sugar described above for the hydrogenation reaction. In certain alcohol may be converted to propylene glycol, ethylene gly embodiments, the condensation catalyst is self-supporting. col, and glycerol using a hydrogenolysis catalyst. The hydro As used herein, the term “self-supporting means that the genolysis catalyst may include Cr, Mo, W. Re, Mn, Cu, Cd, catalyst does not need another material to serve as Support. In Fe, Co, Ni, Pt, Pd, Rh, Ru, Ir, Os, and alloys or any combi 15 other embodiments, the condensation catalyst in used in con nation thereof, either alone or with promoters such as Au, Ag, junction with a separate Support Suitable for Suspending the Cr, Zn, Mn, Sn, Bi, B, O, and alloys or any combination catalyst. In an embodiment, the condensation catalyst Support thereof. The hydrogenolysis catalyst may also include a car is silica. bonaceous pyropolymer catalyst containing transition metals The conditions under which the condensation reaction (e.g., chromium, molybdemum, tungsten, rhenium, manga occurs will vary based on the type of starting material and the nese, copper, cadmium) or Group VIII metals (e.g., iron, desired products. One of ordinary skill in the art, with the cobalt, nickel, platinum, palladium, rhodium, ruthenium, iri benefit of this disclosure, will recognize the appropriate con dium, and osmium). In certain embodiments, the hydro ditions to use to carry out the reaction. In some embodiments, genolysis catalyst may include any of the above metals com the condensation reaction is carried out at a temperature at bined with an alkaline earth metal oxide or adhered to a which the thermodynamics for the proposed reaction are catalytically active Support. In certain embodiments, the cata 25 favorable. The temperature for the condensation reaction will lyst described in the hydrogenolysis reaction may include a vary depending on the specific starting polyol or alcohol. In catalyst Support as described above for the hydrogenation Some embodiments, the temperature for the condensation reaction. reaction is in a range from 80°C. to 500° C., and preferably The conditions for which to carry out the hydrogenolysis from 125° C. to 450° C., and most preferably from 125° C. to reaction will vary based on the type of starting material and 30 250° C. In some embodiments, the condensation reaction is the desired products. One of ordinary skill in the art, with the conducted at pressures in a range between 0 KPato 9000 KPa, benefit of this disclosure, will recognize the appropriate con and preferably in a range between 0 KPa and 7000 KPa, and ditions to use to carry out the reaction. In general, they hydro even more preferably between 0 KPa and 5000 KPa. genolysis reaction is conducted attemperatures of 110° C. to The higher alkanes formed by certain methods of the 300° C., and preferably at 170° C. to 220° C., and most 35 invention include, but are not limited to, branched or straight preferably at 200° C. to 225°C. In some embodiments, the chain alkanes that have from 4 to 30 carbonatoms, branched hydrogenolysis reaction is conducted under basic conditions, or straight chain alkenes that have from 4 to 30 carbonatoms, preferably at a pH of8 to 13, and even more preferably at a pH cycloalkanes that have from 5 to 30 carbon atoms, cycloalk of 10 to 12. In some embodiments, the hydrogenolysis reac enes that have from 5 to 30 carbon atoms, aryls, fused aryls, tion is conducted at pressures in a range between 60 KPa and alcohols, and ketones. Suitable alkanes include, but are not 16500 KPa, and preferably in a range between 1700 KPa and 40 limited to, butane, pentane, pentene, 2-methylbutane, hexane, 14000 KPa, and even more preferably between 4800 KPa and hexene, 2-methylpentane, 3-methylpentane, 2,2-dimethylbu 11OOO KPa. tane, 2,3-dimethylbutane, heptane, heptene, octane, octene, The hydrogen used in the hydrogenolysis reaction of the 2,2,4-trimethylpentane, 2,3-dimethylhexane, 2,3,4-trimeth current invention can include external hydrogen, recycled ylpentane, 2,3-dimethylpentane, nonane, nonene, decane, hydrogen, in situ generated hydrogen, and any combination 45 decene, undecane, undecene, dodecane, dodecene, tridecane, thereof. tridecene, tetradecane, tetradecene, pentadecane, penta In some embodiments, the reaction products discussed decene, nonyldecane, nonyldecene, eicosane, eicosene, above may be converted into higher hydrocarbons through a uneicosane, uneicosene, doeicosane, doeicosene, trieicosane, condensation reaction in a condensation reactor. In Such trieicosene, tetraeicosane, tetraeicosene, and isomers thereof. embodiments, condensation of the reaction products occurs 50 Some of these products may be suitable for use as fuels. in the presence of a catalyst capable of forming higher hydro In some embodiments, the cycloalkanes and the cycloalk carbons. While not intending to be limited by theory, it is enes are unsubstituted. In other embodiments, the cycloal believed that the production of higher hydrocarbons proceeds kanes and cycloalkenes are mono-Substituted. In still other through a stepwise addition reaction including the formation embodiments, the cycloalkanes and cycloalkenes are multi of carbon-carbon, or carbon-oxygen bond. The resulting substituted. In the embodiments comprising the substituted reaction products include any number of compounds contain 55 cycloalkanes and cycloalkenes, the Substituted group ing these moieties, as described in more detail below. includes, without limitation, a branched or straight chain In certain embodiments, suitable condensation catalysts alkyl having 1 to 12 carbon atoms, a branched or straight include an acid catalyst, a base catalyst, or an acid/base cata chain alkylene having 1 to 12 carbonatoms, a phenyl, and any lyst. As used herein, the term “acid/base catalyst” refers to a combination thereof. Suitable cycloalkanes and cycloalkenes catalyst that has both an acid and a base functionality. In some 60 include, but are not limited to, cyclopentane, cyclopentene, embodiments the condensation catalyst can include, without cyclohexane, cyclohexene, methyl-cyclopentane, methyl-cy limitation, Zeolites, carbides, nitrides, Zirconia, alumina, clopentene, ethyl-cyclopentane, ethyl-cyclopentene, ethyl silica, aluminosilicates, phosphates, titanium oxides, Zinc cyclohexane, ethyl-cyclohexene, isomers and any combina oxides, Vanadium oxides, lanthanum oxides, yttrium oxides, tion thereof. Scandium oxides, magnesium oxides, cerium oxides, barium 65 In some embodiments, the aryls formed are unsubstituted. oxides, calcium oxides, hydroxides, heteropolyacids, inor In another embodiment, the aryls formed are mono-Substi ganic acids, acid modified resins, base modified resins, and tuted. In the embodiments comprising the Substituted aryls, US 8,846,352 B2 55 56 the Substituted group includes, without limitation, a branched digestible fat substitutes); U.S. Pat. No. 4,288,378 (Peanut or straight chain alkyl having 1 to 12 carbon atoms, a butter stabilizer); U.S. Pat. No. 5,391,383 (Edible spray oil); branched or straight chain alkylene having 1 to 12 carbon U.S. Pat. No. 6,022.577 (Edible fats for food products); U.S. atoms, a phenyl, and any combination thereof. Suitable aryls Pat. No. 5,434.278 (Edible fats for food products); U.S. Pat. for the invention include, but are not limited to, benzene, No. 5,268,192 (Low calorie nut products); U.S. Pat. No. toluene, Xylene, ethylbenzene, para Xylene, meta Xylene, and 5,258,197 (Reduce calorie edible compositions); U.S. Pat. any combination thereof. No. 4,335,156 (Edible fat product); U.S. Pat. No. 7,288,278 The alcohols produced in certain methods of the invention (Food additives or medicaments); U.S. Pat. No. 7,115,760 have from 4 to 30 carbon atoms. In some embodiments, the (Fractionation process); U.S. Pat. No. 6,808.737 (Structural alcohols are cyclic. In other embodiments, the alcohols are 10 fats); U.S. Pat. No. 5,888,947 (Engine lubricants); U.S. Pat. branched. In another embodiment, the alcohols are straight No. 5,686,131 (Edible oil mixtures); and U.S. Pat. No. 4,603, chained. Suitable alcohols for the invention include, but are 188 (Curable urethane compositions). not limited to, butanol, pentanol, hexanol, heptanol, octanol, In one embodiment inaccordance with the invention, trans nonanol, decanol, undecanol, dodecanol, tridecanol, tetrade esterification of the oil, as described above, is followed by canol, pentadecanol, hexadecanol, heptyldecanol, octylde 15 reaction of the transesterified product with polyol, as reported canol, nonyldecanol, eicosanol, uneicosanol, doeicosanol, in U.S. Pat. No. 6,465.642, to produce polyol fatty acid poly trieicosanol, tetraeicosanol, and isomers thereof. esters. Such an esterification and separation process may The ketones produced in certain methods of the invention comprise the steps as follows: reacting a lower alkyl ester have from 4 to 30 carbon atoms. In an embodiment, the with polyol in the presence of soap; removing residual Soap ketones are cyclic. In another embodiment, the ketones are from the product mixture; water-washing and drying the branched. In another embodiment, the ketones are straight product mixture to remove impurities; bleaching the product chained. Suitable ketones for the invention include, but are mixture for refinement; separating at least a portion of the not limited to, butanone, pentanone, hexanone, heptanone, unreacted lower alkyl ester from the polyol fatty acid poly octanone, nonanone, decanone, undecanone, dodecanone, ester in the product mixture; and recycling the separated tridecanone, tetradecanone, pentadecanone, hexadecanone, 25 unreacted lower alkyl ester. heptyldecanone, octyldecanone, nonyldecanone, eicosanone, Transesterification can also be performed on microbial uneicosanone, doeicosanone, trieicosanone, tetraeicosanone, biomass with short chain fatty acid esters, as reported in U.S. and isomers thereof. Pat. No. 6,278,006. In general, transesterification may be Another chemical modification that may be performed on performed by adding a short chain fatty acid ester to an oil in microalgal oil produced by the methods of the invention is 30 the presence of a Suitable catalyst and heating the mixture. In interesterification. Naturally produced glycerolipids do not some embodiments, the oil comprises about 5% to about 90% have a uniform distribution offatty acid constituents. In the of the reaction mixture by weight. In some embodiments, the context of oils, interesterification refers to the exchange of short chain fatty acid esters can be about 10% to about 50% of acyl radicals between two esters of different glycerolipids. the reaction mixture by weight. Non-limiting examples of The interesterification process provides a mechanism by 35 catalysts include base catalysts, Sodium methoxide, acid cata which the fatty acid constituents of a mixture of glycerolipids lysts including inorganic acids Such as Sulfuric acid and acidi can be rearranged to modify the distribution pattern. Interes fied clays, organic acids such as methane Sulfonic acid, ben terification is a well-known chemical process, and generally Zenesulfonic acid, and toluenesulfonic acid, and acidic resins comprises heating (to about 200°C.) a mixture of oils for a Such as Amberlyst 15. Metals such as Sodium and magne period (e.g., 30 minutes) in the presence of a catalyst, such as 40 sium, and metal hydrides also are useful catalysts. an alkali metal or alkali metal alkylate (e.g., Sodium methox Another such chemical modification is hydroxylation, ide). This process can be used to randomize the distribution which involves the addition of water to a double bond result pattern of the fatty acid constituents of an oil mixture, or can ing in Saturation and the incorporation of a hydroxyl moiety. be directed to produce a desired distribution pattern. This The hydroxylation process provides a mechanism for con method of chemical modification of lipids can be performed 45 Verting one or more fatty acid constituents of a glycerolipid to on materials provided herein, such as microbial biomass with a hydroxy fatty acid. Hydroxylation can be performed, for a percentage of dry cell weight as lipid at least 20%. example, via the method reported in U.S. Pat. No. 5,576,027. Directed interesterification, in which a specific distribution Hydroxylated fatty acids, including castor oil and its deriva pattern of fatty acids is sought, can be performed by main tives, are useful as components in several industrial applica taining the oil mixture at a temperature below the melting 50 tions, including food additives, Surfactants, pigment wetting point of some TAGs which might occur. This results in selec agents, defoaming agents, waterproofing additives, plasticiz tive crystallization of these TAGs, which effectively removes ing agents, cosmetic emulsifying and/or deodorant agents, as them from the reaction mixture as they crystallize. The pro well as in electronics, pharmaceuticals, paints, inks, adhe cess can be continued until most of the fatty acids in the oil sives, and lubricants. One example of how the hydroxylation have precipitated, for example. A directed interesterification 55 of a glyceride may be performed is as follows: fat may be process can be used, for example, to produce a product with a heated, preferably to about 30-50° C. combined with heptane lower calorie content via the substitution of longer-chain fatty and maintained at temperature for thirty minutes or more; acids with shorter-chain counterparts. Directed interesterifi acetic acid may then be added to the mixture followed by an cation can also be used to produce a product with a mixture of aqueous Solution of Sulfuric acid followed by an aqueous fats that can provide desired melting characteristics and struc 60 hydrogen peroxide Solution which is added in Small incre tural features sought in food additives or products (e.g., mar ments to the mixture over one hour, after the aqueous hydro garine) without resorting to hydrogenation, which can pro gen peroxide, the temperature may then be increased to at duce unwanted trans isomers. least about 60° C. and stirred for at least six hours; after the Interesterification of oils produced by the methods stirring, the mixture is allowed to settle and a lower aqueous described herein can be performed in conjunction with one or 65 layer formed by the reaction may be removed while the upper more of the methods and/or materials, or to produce products, heptane layer formed by the reaction may be washed with hot as reported in the following: U.S. Pat. No. 6,080,853 (Non water having a temperature of about 60° C.; the washed US 8,846,352 B2 57 58 heptane layer may then be neutralized with an aqueous potas g/L MgSO4.7H2O, 0.25 g/L Citric Acid monohydrate, 0.025 sium hydroxide solution to a pH of about 5 to 7 and then g/L CaCl2.H2O, 2 g/L yeast extract) plus 2% glucose and removed by distillation under vacuum; the reaction product grown for 7 days at 28°C. with agitation (200 rpm) in a 6-well may then be dried under vacuum at 100° C. and the dried plate. Dry cell weights were determined by centrifuging 1 ml product steam-deodorized under vacuum conditions and fil 5 of culture at 14,000 rpm for 5 minina pre-weighed Eppendorf tered at about 50° to 60° C. using diatomaceous earth. tube. The culture Supernatant was discarded and the resulting Hydroxylation of microbial oils produced by the methods cell pellet washed with 1 ml of deionized water. The culture described herein can be performed in conjunction with one or was again centrifuged, the Supernatant discarded, and the cell more of the methods and/or materials, or to produce products, pellets placed at -80° C. until frozen. Samples were then 10 lyophilized for 24 hrs and dry cell weights calculated. as reported in the following: U.S. Pat. No. 6,590,113 (Oil For determination of total lipid in cultures, 3 ml of culture based coatings and ink); U.S. Pat. No. 4,049,724 (Hydroxy was removed and Subjected to analysis using an Ankom sys lation process); U.S. Pat. No. 6,113,971 (Olive oil butter): tem (Ankom Inc., Macedon, N.Y.) according to the manufac U.S. Pat. No. 4,992,189 (Lubricants and lube additives); U.S. turer's protocol. Samples were subjected to solvent extraction Pat. No. 5,576,027 (Hydroxylated milk); and U.S. Pat. No. with an Amkom XT 10 extractor according to the manufac 6,869,597 (Cosmetics). 15 turer's protocol. Total lipid was determined as the difference Hydroxylated glycerolipids can be converted to estolides. in mass between acid hydrolyzed dried samples and solvent Estolides consist of a glycerolipid in which a hydroxylated extracted, dried samples. Percent oil dry cell weight measure fatty acid constituent has been esterified to another fatty acid ments are shown in Table 2. molecule. Conversion of hydroxylated glycerolipids to estolides can be carried out by warming a mixture of glyc TABLE 2 erolipids and fatty acids and contacting the mixture with a mineral acid, as described by Isbell et al., JAOCS 71(2): 169 Percent oil by dry cell weight 174 (1994). Estolides are useful in a variety of applications, including without limitation those reported in the following: Species Strain % Oil U.S. Pat. No. 7,196,124 (Elastomeric materials and floor 25 Prototheca stagnora UTEX327 13.14 coverings); U.S. Pat. No. 5,458,795 (Thickened oils for high Prototheca moriformis UTEX 1441 18.02 temperature applications); U.S. Pat. No. 5,451.332 (Fluids Prototheca moriformis UTEX 1435 27.17 for industrial applications); U.S. Pat. No. 5,427,704 (Fuel additives); and U.S. Pat. No. 5,380,894 (Lubricants, greases, Microalgae samples from the strains listed in Table 22 of plasticizers, and printing inks). 30 U.S. Patent Application Publication 20100151112 were Other chemical reactions that can be performed on micro bial oils include reacting triacylglycerols with a cyclopro genotyped. Genomic DNA was isolated from algal biomass panating agent to enhance fluidity and/or oxidative stability, as follows. Cells (approximately 200 mg) were centrifuged as reported in U.S. Pat. No. 6,051,539; manufacturing of from liquid cultures 5 minutes at 14,000xg. Cells were then waxes from triacylglycerols, as reported in U.S. Pat. No. resuspended insterile distilled water, centrifuged 5 minutes at 6,770,104; and epoxidation of triacylglycerols, as reported in 35 14,000xg and the Supernatant discarded. A single glass bead “The effect of fatty acid composition on the acrylation kinet ~2 mm in diameter was added to the biomass and tubes were ics of epoxidized triacylglycerols. Journal of the American placed at -80° C. for at least 15 minutes. Samples were Oil Chemists’ Society, 79:1, 59-63, (2001) and Free Radical removed and 150 ul of grinding buffer (1% Sarkosyl, 0.25 M Biology and Medicine, 37:1, 104-114 (2004). Sucrose, 50 mM. NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH The generation of oil-bearing microbial biomass for fuel 40 8.0, RNase A 0.5ug/ul) was added. Pellets were resuspended and chemical products as described above results in the pro by vortexing briefly, followed by the addition of 40 ul of 5M duction of delipidated biomass meal. Delipidated meal is a NaCl. Samples were vortexed briefly, followed by the addi byproduct of preparing algal oil and is useful as animal feed tion of 66 ul of 5% CTAB (Cetyl trimethylammonium bro for farm animals, e.g., ruminants, poultry, Swine and aquac mide) and a final brief vortex. Samples were next incubated at ulture. The resulting meal, although of reduced oil content, 45 65° C. for 10 minutes after which they were centrifuged at still contains high quality proteins, carbohydrates, fiber, ash, 14,000xg for 10 minutes. The supernatant was transferred to residual oil and other nutrients appropriate for an animal feed. a fresh tube and extracted once with 300 ul of Phenol:Chlo Because the cells are predominantly lysed by the oil separa roform:Isoamyl alcohol 12:12:1, followed by centrifugation tion process, the delipidated meal is easily digestible by Such for 5 minutes at 14,000xg. The resulting aqueous phase was animals. Delipidated meal can optionally be combined with 50 transferred to a fresh tube containing 0.7 vol of isopropanol other ingredients, such as grain, in an animal feed. Because delipidated meal has a powdery consistency, it can be pressed (~ 190 ul), mixed by inversion and incubated at room tem into pellets using an extruder or expander or another type of perature for 30 minutes or overnight at 4°C. DNA was recov machine, which are commercially available. ered via centrifugation at 14,000xg for 10 minutes. The The invention, having been described in detail above, is resulting pellet was then washed twice with 70% ethanol, exemplified in the following examples, which are offered to 55 followed by a final wash with 100% ethanol. Pellets were air illustrate, but not to limit, the claimed invention. dried for 20-30 minutes at room temperature followed by resuspensionin 50 ul of 10 mM TrisCl, 1 mM EDTA (pH 8.0). VIII. Examples Five ul of total algal DNA, prepared as described above, was diluted 1:50 in 10 mM Tris, pH 8.0. PCR reactions, final Example 1 60 volume 20 ul, were set up as follows. Tenul of 2x iProof HF master mix (BIO-RAD) was added to 0.4 ul primer SZ02613 Methods for Culturing Prototheca (5'-TGTTGAAGAATGAGCCGGCGAC-3 (SEQ ID NO:13) at 10 mM stock concentration). This primer sequence Prototheca strains were cultivated to achieve a high per runs from position 567-588 in Gen Bank accession no. centage of oil by dry cell weight. Cryopreserved cells were 65 L43357 and is highly conserved in higher plants and algal thawed at room temperature and 500 ul of cells were added to plastid genomes. This was followed by the addition of 0.4 ul 4.5 ml of medium (4.2 g/L KHPO, 3.1 g/L NaH2PO, 0.24 primer SZ02615 (5'-CAGTGAGCTATTACGCACTC-3' US 8,846,352 B2 59 60 (SEQID NO:14) at 10 mM stock concentration). This primer mixture was incubated in ice for another 1 min. After vortex sequence is complementary to position 1112-1093 in Gen ing, DNA-coated particles were pelleted by spinning at Bank accession no. L43357 and is highly conserved in higher 10,000 rpm in an Eppendorf 5415C microfuge for 10 sec plants and algal plastid genomes. Next, 5 Jul of diluted total onds. The gold pellet was washed once with 500 ul of cold DNA and 3.2 LuldHO were added. PCR reactions were run as 100% ethanol, pelleted by brief spinning in the microfuge, follows: 98°C. 45"; 98°C., 8";53°C., 12"; 72°C. 20" for 35 and resuspended with 50 ul of ice-cold ethanol. After a brief cycles followed by 72°C. for 1 min and holding at 25°C. For (1-2 sec) sonication, 10 ul of DNA-coated particles were purification of PCR products, 20 ul of 10 mM Tris, pH 8.0, immediately transferred to the carrier membrane. was added to each reaction, followed by extraction with 40 ul Prototheca strains were grown in proteose medium (2 g/L of Phenol:Chloroform:isoamyl alcohol 12:12:1, vortexing 10 and centrifuging at 14,000xg for 5 minutes. PCR reactions yeast extract, 2.94 mM NaNO, 0.17 mM CaC12H2O, 0.3 were applied to S-400 columns (GE Healthcare) and centri mM MgSO.7HO, 0.4 mM KHPO, 1.28 mM KHPO, fuged for 2 minutes at 3,000xg. Purified PCR products were 0.43 mM NaCl) on a gyratory shaker until it reaches a cell subsequently TOPO cloned into PCR8/GW/TOPO and posi density of 2x10 cells/ml. The cells were harvested, washed tive clones selected for on LB/Spec plates. 15 once with sterile distilled water, and resuspended in 50 ul of Purified plasmid DNA was sequenced in both directions medium. 1x10" cells were spread in the center third of a using M13 forward and reverse primers. In total, twelve Pro non-selective proteose media plate. The cells were bom totheca strains were selected to have their 23S rRNA DNA barded with the PDS-1000/He Biolistic Particle Delivery sys sequenced and the sequences are listed in the Sequence List tem (Bio-Rad). Rupture disks (1100 and 1350 psi) were used, ing. A Summary of the strains and Sequence Listing Numbers and the plates are placed 9 and 12 cm below the screen/ is included below. The sequences were analyzed for overall macrocarrier assembly. The cells were allowed to recover at divergence from the UTEX 1435 (SEQID NO: 5) sequence. 25° C. for 12-24 h. Upon recovery, the cells were scraped Two pairs emerged (UTEX 329/UTEX 1533 and UTEX 329/ from the plates with a rubber spatula, mixed with 100 ul of UTEX 1440) as the most divergent. In both cases, pairwise medium and spread on plates containing the appropriate anti alignment resulted in 75.0% pairwise sequence identity. The 25 biotic selection. After 7-10 days of incubation at 25° C. percent sequence identity to UTEX 1435 is also included colonies representing transformed cells were visible on the below. plates from 1100 and 1350 psi rupture discs and from 9 and 12 cm distances. Colonies were picked and spotted on selective agar plates for a second round of selection. Species Strain % nt identity SEQID NO. 30 Example 3 Prototheca kruegani UTEX 329 75.2 SEQID NO: 1 Prototheca wickerhanii UTEX 1440 99 SEQID NO: 2 Prototheca stagnora UTEX 1442 75.7 SEQID NO:3 Generation of a Microalgal Strain Capable of Prototheca moriformis UTEX 288 75.4 SEQID NO:4 Metabolizing Xylose Prototheca moriformis UTEX 1439; 100 SEQID NO: 5 1441; 1435: 35 1437 Methods and effects of expressing a heterologous exog Prototheca wikerhamii UTEX 1533 99.8 SEQID NO: 6 enous gene in Prototheca species, including, for example, Prototheca moriformis UTEX 1434 75.9 SEQID NO: 7 codon optimization and chromosomal recombination sites, Prototheca Zopfi UTEX 1438 75.7 SEQID NO: 8 have been previously described in PCT Application No. PCT/ Prototheca moriformis UTEX 1436 88.9 SEQID NO: 9 40 US2009/66142, hereby incorporated by reference, for these teachings. This example demonstrates the generation of a Lipid samples from a subset of the above-listed strains transgenic strain derived from Prototheca moriformis (UTEX were analyzed for lipid profile using HPLC. Results are 1435) containing exogenous genes for the metabolism and shown below in Table 3. transport of Xylose. The resulting transgenic Prototheca TABLE 3 Diversity of lipid chains in microalgal species Strain C14:O C16:O C16:1 C18:O C18:1 C18:2 C18:3 UTEX O 12.O1 O O S.O.33 17.14 O 327 UTEX 141 29.44 O.70 3.05 57.72 12.37 0.97 1441 UTEX 109 25.77 O 2.75 54.01 11.90 244 1435 Example 2 strain generated had the ability to grow on media containing Xylose as the sole carbon Source. As discussed above, the Methods for Transforming Prototheca ability to metabolize Xylose is important because Xylose, a 60 pentose, is a significant component of cellulosic-derived A. General Method for Biolistic Transformation of Pro feedstocks. Xylose is the major Sugar monomer component of totheca hemicellulose, which, along with cellulose, forms the major S550d gold carriers from Seashell Technology were pre structural components of most plant matter. pared according to the protocol from manufacturer. Linear Preliminary results showed that Prototheca moriformis ized plasmid (20 g) was mixed with 50 ul of binding buffer 65 (UTEX 1435) can convert Small amounts of xylose to xylitol, and 60 ul (30 mg) of S550d gold carriers and incubated in ice as indicated by analytical measurements of the Sugar levels in for 1 min Precipitation buffer (100 ul) was added, and the liquid media over time. Prototheca moriformis (UTEX 1435) US 8,846,352 B2 61 62 was cultured in the media and conditions as described in nitrate reductase 3'UTR (SEQID NO: 18); (2) Pichia stipitis Example 1 above in 4% xylose with 0.25% glucose as carbon XYL3 under the control of the Chlorella protothecoides EF1a Sources in the medium. Also included were a no-cells-added promoter and 3'UTR; and (3) Piromyces sp. XylA under the negative control and a 4% glucose condition as a positive control of the Chlorella protothecoides actin promoter and control for growth and a negative control for xylitol produc EF1a 3'UTR. Positive clones were selected on sucrose-con tion. Table 4 shows that no Xylitol was produced in the nega taining medium and plates and expression of the transgenes tive control condition, whereas Xylose containing medium were confirmed using rabbit polyclonal antibodies generated with Prototheca cells produced detectable amounts of xylitol against XylA or XYL3 in Western blots. in the culture supernatant after 5 days in culture. There was robust growth for cells in the glucose only condition, but no 10 detectable level of xylitol was produced. Element SEQID NO: 5' Prototheca moriformis KE858 homologous 16 TABLE 4 recombination targeting sequence 3' Prototheca moriformis KE858 homologous 17 Day 5 15 recombination targeting sequence S. cerevisae Suc2 invertase cassette 18 xylose C. protothecoides actin promoter 19 Strain Media OD750 (g/L) xylitol (gL) Codon optimized Pichia stipitis XYL3 coding region 2O no cells 4% xylose + 0.25% glucose O.O 41.O none detected C. protothecoides EF1a 3'UTR 21 control Chiorella protothecoides EF1a promoter 22 UTEX 4% xylose + 0.25% glucose 2.4 37.7 detected at Codon optimized Piromyces sp. XylA coding region 23 1435 <2.5 g/L C. protothecoides EF1a 3'UTR 21 UTEX 4% glucose control 15.4 none none detected 1435 detected Once the XylA/XYL3 recipient strain was generated and confirmed using Western blots, the strain was then retrans This result may indicate that xylose can be converted into 25 formed with one of two cassettes. The first cassette was an Xylitol via an endogenous aldo-keto reductase enzymatic integrative transformation vector that carried a gene confer activity in Prototheca moriformis. Enzymes belonging to this ring resistance to the antibiotic G418 and an additional phos family of NAPDH-dependent oxidoreductases reside within phate/pentose phosphate translocator gene XPT from Arabi the confines of the plasma membrane, and if such an enzyme doposis thaliana with the plastid transit peptide replaced with is responsible for the conversion of xylose to xylitol, then the 30 a transit peptide from an orthologous gene belonging to the Xylose in the culture medium must be entering into the cell via same translocator family from Prototheca moriformis. The an endogenous Sugar transporter. primary amino acid sequence (including the replaced transit In organisms that can metabolize Xylose, Xylose is con peptide SEQ ID NO: 25) is listed in SEQ ID NO: 24. An verted into xylulose-5-phosphate, which is further metabo alternative plastid transit peptide SEQ ID NO: 26 was also lized via the pentose phosphate pathway. Prototheca mori 35 tried and was equally successful. The second cassette was an formis may be capable of taking up Xylose from its episomal (non-integrative) transformation vector containing surrounding, but is deficient in its ability to efficiently convert a gene conferring resistance to the antibiotic G418 and an xylose intoxylulose-5-phosphate. The results shown in Table additional phosphate/pentose phosphate translocator gene 4 above Suggest that this may be the case. To test this hypoth XPT from Arabidoposis thaliana with the plastid transit pep esis, the present invention provides methods and reagents for 40 tide replaced with a transit peptide from an orthologous gene expressing Xylose isomerase to first convert Xylose into Xylu belonging to the same translocator family from Prototheca lose and xylulokinase to convert the xylulose to xylulose-5- moriformis and the Pichia stipitis XYL2 gene, a xylitol dehy phosphate. An exemplary illustration of Such transgenic drogenase. In yeast (such as Pichia), XylA can be inhibited to microalgal cells is described below using Prototheca mori some extent by xylitol. The preliminary experiment described formis (UTEX 1435). 45 above examining culture Supernatant with non-transformed A transgenic recipient strain of Prototheca moriformis Prototheca cells showed detectable, low levels of xylitol in expressing the Xylose isomerase (XylA) gene from Piromy the medium, indicative of an endogenous reductase activity ces sp. and thexylulokinase (XYL3) gene from Pichia stipitis generating Xylitol directly from Xylose. This second construct and the invertase gene from S. cerevisiae (suc2, a selectable containing the XYL2 Xylitol dehydrogenase provides meth marker) was generated using the methodology and transfor 50 ods for alleviating such xyitol inhibition on XylA. Relevant mations previously described in PCT Patent Publication Nos. portions of each of these two vectors are listed as SEQID NO: WO 2010/063031 and WO 2010/063032, each of which is 32 and SEQ ID NO. 23, respectively, along with individual incorporated herein by reference for their teachings of these cassette components below. The XPT alone integrative cas methods. Positive clones were selected on Sucrose containing sette contained the 5' and 3' Prototheca moriformis 6S medium and plates. The primary amino acid sequence of the 55 homologous recombination targeting sequence and a neomy Piromyces sp. XylA (Gen Bank Accession No. CAB76571, cin resistance gene under the control of the C. reinhardtii hereby incorporated by reference) is listed as SEQID NO: 15. beta-tubulin promoter and Chlorella vulgaris nitrate reduc The primary amino acid sequence of Pichia stipitis XYL3 tase 3'UTR and the A. thaliana XPT gene with the replaced (GenBank Accession No. CAA39066.1, hereby incorporated transit peptide sequence (as described above) under the con by reference) is listed as SEQID NO:51. Relevantportions of 60 trol of the Chlorella protothecoides actin promoter and EF1a the Piromyces sp. XylA/Pichia stipitis XYL3 transformation 3'UTR. The episomal cassette contained a neomycin resis cassette are listed below. This cassette contained 5' and 3' tance gene under the control of C. Sorokiniana Gdh promoter Prototheca moriformis KE858 homologous recombination and C. vulgaris nitrate reductase 3'UTR; the Pichia stipitis targeting sequences for genomic integration and are listed as XYL2 gene under the control of the C. protothecoides actin SEQID NOs: 16 and 17. Also contained are (1) S. cerevisae 65 promoter and EF1a 3'UTR; and the A. thaliana XPT gene Suc2 Sucrose invertase gene under the control of the C. rein under the control of C. reinhardtii beta-tubulin promoter and hardtii beta-tubulin promoter and the Chlorella vulgaris C. protothecoides EF1a 3'UTR. US 8,846,352 B2 64 source. Growth (measured by OD750 readings) and xylose Element SEQ ID NO: consumption (measuring the amount of Xylose remaining in the media) were measured at various time points during cul Integrative Cassette ture. No growth or xylose consumption was observed in the 5' Prototheca moriformis 6S homologous SEQ ID NO: 27 parental recipient Strain, while both transgenic strains showed recombination targeting sequence growth and Xylose consumption. Table 5 summarizes the growth and Xylose consumption results. TABLE 5 Growth and Xylose Consumption Results. xylose xylose Genes expressed Carbon source OD750 (g/L) OD750 (g/L) Day 6 Day 8 no cells 2% xylose + 0.5% glucose O.O 21.3 O.O 2O.S control XylA,XYL3 2% xylose + 0.5% glucose 4.7 20.4 S.1 17.9 XylA,XYL3 2% xylose + 0.5% glucose 4.4 21.3 4.6 19.1 XylA,XYL3, XPT, 2% xylose + 0.5% glucose 6.8 15 8.2 9.1 XYL2 (duplicate 1) XylA,XYL3, XPT, 2% xylose + 0.5% glucose 6.3 16 7.4 11.4 XYL2 (duplicate 2) Ole 2% xylose O.O 23.9 O.O 23.9 XylA,XYL3 2% xylose O.O 23.9 O.1 21.7 XylA,XYL3, XPT, 2% xylose 1.O 21.2 2.6 19.3 XYL2 Day 7 Day 25 no cells control 2% xylose + 0.5% O 24.2 O 25.7 glucose XylA,XYL3 2% xylose + 0.5% 5.2 21.6 4.8 20.4 glucose XylA,XYL3, XPT 2% xylose + 0.5% S.6 18.3 10.6 not glucose detected -continued 35 Example 4 Element SEQ ID NO: Generation of Engineered Microalgal Strains 3' Prototheca moriformis 6S homologous SEQ ID NO:28 Expressing Transporters for Transport of Xylose into recombination targeting sequence he C 1 Neomycin cassette (C. reinhardtii B-tubulin promoter SEQID NO: 29 the Cytoplasm and C. vulgaris nitrate reductase 3'UTR) 40 C. protothecoides actin promoter SEOID NO: 19 Codon-optimized A. thaliana XPT coding region SEOID NO:30 C. protothecoides EF1a 3'UTR SEOID NO: 21 Four xylose-specific transporters (SUT1, GXS1, sym Nonintegrative Cassette porter, and XLT1, SEQ ID NOs: 37, 39, 41 and 43, respec Neomycin cassette (C. Sorokinana Gdh promoter and SEQID NO:31 45 tively), some of which transport Xylose actively and some of C. protothecoidesvulgaris nitrate actinreductase promoter 3'UTR) SEQ ID NO: 19 which allow passive diffusion of xylose into the cell, were Codon-optimized P. stipitis XYL2 coding region SEQ ID NO:32 expressed as transgenes in Prototheca moriformis. The gene C. reinhardtii B-tubulin promoter SEQ ID NO:33 for each Xylose transporter was codon optimized (the codon Codon-optimized A. thaliana XPT coding region SEQ ID NO:30 C. protothecoides EF1a 3'UTR SEQ ID NO:21 50 optimized coding regions are shown at SEQID NOs: 36,38, 40 and 42, respectively) to allow for maximal expression in Positive clones from both transformations were selected Prototheca moriformis and inserted into the genome of the using Sucrose-containing media/plates with the addition of 50 microalgal cells under the control of the Chlorella prototh ug/ml G418. ecoides actin promoter and EF1a 3'UTR. Expression of each Of the many positive clones from the transformation, two 55 of the four genes was confirmed, as was their insertion and strains, one from each transformation, were selected for more localization to the cell membrane. Confirmation of localiza detailed characterization. Both strains were grown on Solid media plates containing 2% Xylose as the sole carbon Source. tion to the cell membrane included fusing DNA sequences After 3-4 weeks of incubation, growth was measured for these encoding a fluorescent protein in-frame to the carboxyl ter transgenic strains and growth was observed for both trans 60 minus of each of the transporter transgenes and observing genic strains, but not for the parental recipient strain contain cells expressing the constructs utilizing fluorescence micros ing only the XylA and XYL3 transgenes. Confirmation of copy. these results was performed to test xylose consumption using Micrographs of cells expressing each of the four transport a liquid media environment. Liquid cultures containing both transgenic strains (XylA, XYL3, XPT and XYL2; XylA 65 ers with a fluorescent protein fused to their carboxyl terminus XYL3 and XPT) and the parental recipient strain (XylA and showed that the transporters were expressed and localized to XYL3 only) were set up using 2% xylose as the sole carbon the cell membrane. US 8,846,352 B2 65 66 Example 5 ized to the inner membrane of plastids; this was determined by fusing a fluorescent protein to the carboxyl terminus of the Generation of Engineered Microalgal Strains translocator chimera and examining the cells expressing the Expressing Enzymes for Conversion of Xylose to fusion utilizing fluorescence microscopy. The fluorescent sig Xylulose-5-Phosphate nal appeared to be concentrated around the organellar-like structures within the cell, consistent with localization in the Prototheca moriformis was engineered to express codon inner membrane of plastids. The three chimeric translocators optimized genes encoding oxido-reductase pathway enzymes GPT-A-XPT, GPT-F-XPT, and S106SAD-XPT are shown in XYL1, XYL2, or XYL3 (SEQ ID NOs: 34, 32, and 20, SEQ ID NOs: 45, 47 and 49, respectively. respectively), or isomerase pathway enzymes XylA or XYL3 10 (SEQ ID NO: 23 and 20, respectively) which correspond to Example 7 two alternative pathways for the biochemical conversion of xylose to xylulose-5-phosphate. The two pathways share the Generation of Engineered Microalgal Strains same terminal enzyme (XYL3). Expression of each gene was Expressing Combinations of Genes for Xylose confirmed at the RNA level. In addition, antibodies were 15 generated against each gene product to permit further moni Metabolism toring of expression at the protein level using Western blot As demonstrated in the examples herein, individual trans analysis. Proteins corresponding to the expected size band genes encoding Xylose metabolism enzymes were success were observed in extracts made from transgenic strains con fully inserted into Prototheca moriformis. To investigate the taining the corresponding inserted transgenes, but not the effect of combining these various transgenes in a single cell, untransformed parental cells. strains of P. moriformis containing certain combinations (see Although the expression of enzymes responsible for con the Genotypes in Table 6) of the transgenes discussed in version of Xylose to Xylulose-5-phosphate did not appear to Examples 4-6 were prepared and screened for Xylose metabo initially confer the ability to metabolize xylose in the P. mori lism. formis cells, the ability to grow on xylose was conferred by 25 increasing the copy number of the transgenes. The copy num Ten independent transformants of each unique genotype ber of the genes encoding these enzymes was increased using were clonally purified and assessed for growth on Solid media a selectable marker. The original parent cells had a copy containing only 2.5 percent Xylose as the Sole carbon Source. number of one for all three transgenes (suc2, XylA and Potential positive clones were further tested in liquid culture XYL3) and did not appear to grow on xylose, but after growth 30 containing Xylose as the Sole carbon Source. Growth of the under selection wherein the ability to grow on Sucrose was cells was monitored by measuring the optical density of the imparted by a gene (suc2) adjacent to the genes encoding the culture. At the end of the experiment, the culture Supernatant Xylose metabolism enzymes, three independent isolates that was analyzed for xylose content. Table 6 shows the final were characterized had increased the copy number of all three growth and Xylose consumption of the ten best strains (Strains transgenes by three to fifty-fold. The isolates with increased 35 D to M) compared with negative controls (Strains A, B and copy number were assessed for growth on Xylose. Isolate A, C). Strains D to Mare clearly capable of consumingXylose, as which displayed the greatest increase in copy number (-50 all of the xylose in the culture media had disappeared by the fold), had acquired the ability to grow on Xylose. The original end of the experiment. In contrast, the same amount of xylose parent was unable to grow on Xylose, as were the two isolates that was present at the beginning of the experiment was still (B and C) that displayed only moderate increases in copy 40 detected at the end of the experiment in culture medium number (-3-4 fold). The ability of Isolate A to grow on xylose hosting the negative control strains A, B and C. The disap demonstrates that the transgenes were expressed, retain their pearance of Xylose was accompanied by increases in cell enzymatic activity in a heterologous organism, and can con density, indicating that carbon from Xylose was being used to fer on that organism the ability to utilize xylose. Support cell growth. In contrast, the negative controls did not 45 increase in cell density throughout the course of the experi Example 6 ment, and at the end of the experiment, the culture media contained about the same amount of xylose that had been in Generation of Engineered Microalgal Strains the media at the beginning of the experiment, indicating that Expressing Translocators for Delivery of Xylose was not consumed at all, or that the amount of xylose Xylulose-5-Phosphate to the Pentose Phosphate 50 consumed was very Small. Metabolic Pathway TABLE 6 Prototheca moriformis was engineered to express one of Xylose Utilization and Growth of Engineered Strains three chimeric plant translocators that specifically transport Compared with Negative Controls. Xylulose-5-phosphate. The chimeric genes were produced by 55 Percent of Xylose replacing the transit peptide encoded by the plant translocator highest OD remaining in gene (Arabadopsis thaliana XPT) with one of three transit Strain Genotype reached the media, g/L peptides identified in endogenous translocators found in P moriformis or the SAD gene of C. protothecoides in order to A wildtype P. moriformis S.O 27.1 UTEX1435 improve export of the xylulose-5-phosphate to the final cel 60 B XylA,XYL3 5.7 24.4 lular destination/compartment. The endogenous Prototheca C XYL2, XYL3 5.7 26.4 transit peptides were identified by selecting partial DNA D XylA,XYL3, SUT1, GPT-F-XPT 80.1 O.O sequences of translocator orthologs in P. moriformis from E XylA,XYL3, SUT1, GPT-F-XPT 68.6 O.O F XylA,XYL2, XYL3, XLT1, 1OOO O.O contig assemblies and amplifying the selected sequences. S106SAD-XPT Products arising from the amplification were cloned and 65 G XylA, XYL1, XYL3, XLT1, 819 O.O sequenced, and the signal peptides were derived from the sequences obtained. The chimeric gene products were local US 8,846,352 B2 67 68 TABLE 6-continued cent of dry cell weight. All engineered Strains performed similarly to the parental, non-engineered control strains when Xylose Utilization and Growth of Engineered Strains glucose was fed to the cultures. The negative control strains Compared with Negative Controls. (A, B, C) produced small amounts of lipid when cultured on Percent of Xylose xylose (6-8 percent of the amount normally made by Strain A highest OD remaining in in glucose). In contrast, all ten Xylose metabolizing stains Strain Genotype reached the media, g/L were capable of producing significant amounts of lipid when H XylA, XYL1, XYL3, GXS1, 62.8 O.O cultured in xylose. The best strain, L, accumulated up to 60 GPTA-XPT percent of the amount of lipid normally made by Strain A I XYL1, XYL2, XYL3, symporter, 67.9 O.O 10 GPTF-XPT when grown on glucose. J XYL1, XYL2, XYL3, GXS1, 81.8 O.O GPTF-XPT TABLE 7 K XYL2, XYL3, symporter, 744 O.O S106SAD-XPT Lipid Productivity of Xylose-metabolizing L XYL2, XYL3, XLT1, 82.9 O.O 15 Strains Grown in Glucose or Xylose. S106SAD-XPT M XYL2, XYL3, GXS1, 92.0 O.O Percent Percent GPTA-XPT of control of control ipid lipid production production Multiple strains with unique genotypes showed clear 2O Strain Genotype in glucose in Xylose increases in biomass using Xylose as the sole carbon Source. A wildtype P. moriformis UTEX1435 OO 8.9 These results demonstrate that microalgae were engineered to B XylA,XYL3 93.8 6.6 grow on Xylose via transformation with multiple transgenes C XYL2, XYL3 93.5 6.2 or groups of transgenes that transported Xylose into the cell, D XylA,XYL3, SUT1, GPT-F-XPT 93.5 32.4 biochemically converted Xylose toxylulose-5-phosphate, and E XylA,XYL3, SUT1, GPT-F-XPT O3.8 34 25 F XylA,XYL2, XYL3, XLT1, S106SAD- O1 3O.S delivered xylulose-5-phosphate to an endogenous, central XPT metabolic pathway. G XylA, XYL1, XYL3, XLT1, S106SAD- OO.8 32.2 XPT Example 8 XylA,XYL1,XYL3, GXS1, GPT-A-XPT OO 32.4 I XYL1, XYL2, XYL3, symporter, GPTF- O4.7 45.7 30 XPT Lipid Production Capability of Xylose-Metabolizing J XYL1, XYL2, XYL3, GXS1, GPT-F-XPT 99.9 30.6 Strains K XYL2, XYL3, symporter, S106SAD-XPT O2S 35 L XYL2, XYL3, XLT1, S106SAD-XPT OO.8 6O2 Xylose-metabolizing strains A-M from Example 7 were M XYL2, XYL3, GXS1, GPT-A-XPT O2 27.8 evaluated for the ability to produce oil when grown using Xylose as the Sole carbon Source. The Xylose metabolizing 35 These results show that the inserted pathways can divert stains were subjected to a lipid productivity assay, together carbon from Xylose and utilize it to produce a lipid. with negative control strains. Cells were first grown up in Although this invention has been described in connection media containing glucose, and then Subjected to a lipid pro with specific embodiments thereof, it will be understood that ductivity phase using either glucose or Xylose as the sole it is capable of further modifications. This application is Source of carbon. As Sugar became depleted from the cultures, 40 intended to cover any variations, uses, or adaptations of the more of the corresponding Sugar was fed to the cells until invention following, in general, the principles of the invention consumption ceased. At this point, the experiment was termi and including Such departures from the present disclosure as nated and cells were analyzed for lipid content. Table 7 shows come within known or customary practice within the art to the results of the lipid analysis. All strains were normalized to which the invention pertains and as may be applied to the Strain A's performance on glucose expressed as lipid as per essential features hereinbefore set forth. SEQUENCE LISTING <16 Os NUMBER OF SEO ID NOS: 52 <21 Os SEQ ID NO 1 &211s LENGTH: 541 &212s. TYPE: DNA <213> ORGANISM: Prototheca kruegani <4 OOs SEQUENCE: 1 tgttgaagaa tagc.cggcg agittaaaaag agtggcatgg ttaaagaaaa tact ctggag 60 ccatagc gala agcaagttta gtaagct tag gt cattcttt ttagacccga aaccgagtga 12O tot acccatg at Cagggtga agtgttagta aaataac atg gaggc.ccgaa cc.gactaatg 18O ttgaaaaatt agcggatgaa ttgttggg tag gggcgaaaaa ccaatcgaac toggagittag 24 O Ctggttct C C cc.gaaatgcg tittaggcgca gcagtag cag tacaaataga ggggtaaagc 3 OO actgtttctt ttgtgggctt cqaaagttgt acct caaagt ggcaaactict gaatacticta 360 US 8,846,352 B2 79 - Continued <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer <4 OOs, SEQUENCE: 13 tgttgaagaa tagg.cggcg ac <210s, SEQ ID NO 14 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer <4 OOs, SEQUENCE: 14 cagtgagcta ttacgcactic <210s, SEQ ID NO 15 &211s LENGTH: 437 212. TYPE: PRT <213> ORGANISM: Piromyces sp. <4 OOs, SEQUENCE: 15 Met Ala Lys Glu Tyr Phe Pro Glin Ile Glin Lys Ile Llys Phe Glu Gly 1. 5 1O 15 Lys Asp Ser Lys Asn Pro Lieu Ala Phe His Tyr Tyr Asp Ala Glu Lys 2O 25 3O Glu Val Met Gly Llys Llys Met Lys Asp Trp Lieu. Arg Phe Ala Met Ala 35 4 O 45 Trp Trp His Thir Lieu. Cys Ala Glu Gly Ala Asp Glin Phe Gly Gly Gly SO 55 6 O Thr Lys Ser Phe Pro Trp Asn Glu Gly Thr Asp Ala Ile Glu Ile Ala 65 70 7s 8O Lys Glin Llys Val Asp Ala Gly Phe Glu Ile Met Glin Llys Lieu. Gly Ile 85 90 95 Pro Tyr Tyr Cys Phe His Asp Val Asp Leu Val Ser Glu Gly Asn Ser 1OO 105 11 O Ile Glu Glu Tyr Glu Ser Asn Lieu Lys Ala Val Val Ala Tyr Lieu Lys 115 12 O 125 Glu Lys Gln Lys Glu Thr Gly Ile Llys Lieu. Lieu. Trp Ser Thr Ala Asn 13 O 135 14 O Val Phe Gly His Lys Arg Tyr Met Asn Gly Ala Ser Thr Asn Pro Asp 145 150 155 160 Phe Asp Val Val Ala Arg Ala Ile Val Glin Ile Lys Asn Ala Ile Asp 1.65 17O 17s Ala Gly Ile Glu Lieu. Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg 18O 185 19 O Glu Gly Tyr Met Ser Lieu. Lieu. Asn. Thir Asp Glin Lys Arg Glu Lys Glu 195 2OO 2O5 His Met Ala Thr Met Lieu. Thir Met Ala Arg Asp Tyr Ala Arg Ser Lys 21 O 215 22O Gly Phe Lys Gly Thr Phe Lieu. Ile Glu Pro Llys Pro Met Glu Pro Thr 225 23 O 235 24 O Llys His Glin Tyr Asp Val Asp Thr Glu Thir Ala Ile Gly Phe Lieu Lys 245 250 255 Ala His Asn Lieu. Asp Lys Asp Phe Llys Val Asn. Ile Glu Val Asn His US 8,846,352 B2 91 92 - Continued Ctgggcgc.cg agaact acgt gttctggggc ggcc.gc.gagg gctacatgtc. cctdctgaac acggaccaga agcgc.gagaa ggagcacatg gcgac catgc tgacgatggc cc.gcgacitac 660 gcc.cgcticga agggct tcaa gggcacct tc Ctgat cago cCaag.ccgat ggagc.ccacc 72 O alagcaccagt acgacgt.cga Caccgaga.cc gcgat.cggct t cctgaaggc gcaca acctg gacaaggact tca aggtgaa catcgaggtg aaccacgc.ca CCCtggcggg CCaCaccitt C 84 O gagc acgagc tigg.cgt.gc.gc C9tggacgcc ggCatgctgg gcago atcga cgc.galaccgc 9 OO ggcgact acc agaacggctg gga caccgac Cagttc.ccca tcqaccagta cgagctggtg 96.O Caggcctgga tiggagatcat ccdc.ggcggc ggctt.cgt.ca ccggcggcac gaactitcgac gccalagaccc gcc.gcaactic Caccgacctg gagga catca t catcgc.gca 108 O atggacgc.ca toccgc.gc cctggagaac gcc.gc.gaagc tgctgcagga gagcc cctac 114 O accalagatga agaaggagcg ctacgc.ct co titcgact cqg gcatcggcaa. ggactitcgag 12 OO gacggcaa.gc tigaccctgga gcaggtotac gagtacggca agaagaacgg cgagcc.caag 126 O Cagacctic cq gcaa.gcagga gctgtacgag gcc atcgtgg cgatgtacca gtag 1314 <210s, SEQ ID NO 24 &211s LENGTH: 398 212. TYPE: PRT <213> ORGANISM: Arabidopsis thaliana <4 OOs, SEQUENCE: 24 Met Arg Val Glu Ile Trp Arg Thr Gly Ser Pro Ala Wall Pro Glu 1. 5 1O 15 Gly Leu Tyr Trp Val Glu Ser Asp Lieu. Gly Ala Ala Thir His Arg Glu 2O 25 Ser Glu Pro Ser Arg Gly Gly Thir Lieu. Lieu. Arg Gly Pro Ala Lieu. Thir 35 4 O 45 Pro Arg Pro Pro Ala Cys Ile Arg Asp Lieu. Arg Arg Gly Arg Ala Ala SO 55 6 O Wall Gly Ser Ser Asp Ser Asn Pro Asp Glu Lys Ser Asp Luell Gly Glu 65 8O Ala Glu Lys Lys Glu Lys Llys Ala Lys Thir Lieu Glin Lell Gly Ile Wall 85 90 95 Phe Gly Leu Trp Tyr Phe Glin Asn. Ile Wall Phe Asn Ile Phe Asn Lys 1OO 105 11 O Ala Lieu. Asn. Wall Phe Pro Tyr Pro Trp Leu Lell Ala Ser Phe Glin 115 12 O 125 Lell Phe Ala Gly Ser Ile Trp Met Lieu. Wall Lieu. Trp Ser Phe Llys Lieu. 13 O 135 14 O Tyr Pro Cys Pro Lys Ile Ser Llys Pro Phe Ile Ile Ala Luell Lieu. Gly 145 150 55 160 Pro Ala Lieu. Phe His Thir Ile Gly His Ile Ser Ala Wall Ser Phe 1.65 17O 17s Ser Llys Val Ala Val Ser Phe Thir His Wall Ile Ser Ala Glu Pro 18O 185 19 O Wall Phe Ser Wall Ile Phe Ser Ser Lieu. Lieu. Gly Asp Ser Pro Leu 195 2OO 2O5 Ala Val Trp Lieu. Ser Ile Lieu. Pro Ile Wal Met Gly Ser Lieu Ala 21 O 215 22O Ala Wall Thr Glu Wal Ser Phe Asn Lieu. Gly Gly Lell Ser Gly Ala Met 225 23 O 235 24 O US 8,846,352 B2 93 94 - Continued Ile Ser Asn Val Gly Phe Wall Luell Arg Asn Ile Tyr Ser Lys Arg Ser 245 250 255 Lell Glin Ser Phe Glu Ile Asp Gly Luell Asn Lieu. Tyr Gly Cys Ile 26 O 265 27 O Ser Ile Lieu. Ser Lieu Lell Tyr Luell Phe Pro Wall Ala Ile Phe Wall Glu 28O 285 Gly Ser His Trp Val Pro Gly His Ala Ile Ala Ser Wall Gly 29 O 295 3 OO Thir Pro Ser Thir Phe Tyr Phe Wall Trp Luell Ser Gly Val Phe Tyr 3. OS 310 315 His Leu Tyr ASn Glin Ser Ser Glin Ala Lieu. Asp Glu Ile Ser Pro 3.25 330 335 Lell Thir Phe Ser Wall Gly Asn Thir Met Lys Arg Wall Wal Wall Ile Ile 34 O 345 35. O Ser Thir Wall Lieu Wall Phe Arg Asn Pro Wall Arg Pro Lieu. Asn Ala Luell 355 360 365 Gly Ser Ala Ile Ala Ile Cys Gly Thir Phe Lieu. Tyr Ser Glin Ala Thir 37 O 375 38O Ala Lys Ile Glu Wall Gly Gly Asp Asn 385 390 395 SEO ID NO 25 LENGTH: 60 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide <4 OOs, SEQUENCE: 25 Met Arg Val Glu Ile Trp Arg Thr Gly Ser Pro Tyr Ala Wall Pro Glu 1. 5 1O 15 Gly Lieu. Tyr Trp Val Glu Ser Asp Luell Gly Ala Ala Thr His Arg Glu 2O 25 Ser Glu Pro Ser Arg Gly Gly Thr Luell Luell Arg Gly Pro Ala Lieu. Thir 35 4 O 45 Pro Arg Pro Pro Ala Cys Ile Arg Asp Luell Arg Arg SO 55 6 O SEQ ID NO 26 LENGTH: 60 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide <4 OOs, SEQUENCE: 26 Met Arg Val Glu Ile Trp Arg Thr Gly Ser Pro His Ala Ala Glin Gly 1. 5 1O 15 Gly Lieu. Cys Trp His Val Ser Asp Luell Gly Ala Ala Thr His Arg Glu 2O 25 Ser Glu Pro Ser Arg Gly Gly Thr Luell Phe Arg Gly Pro Ala Lieu. Thir 35 4 O 45 Pro Arg Pro Pro Ala Cys Ile Arg Asp Luell Arg Arg SO 55 6 O <210s, SEQ ID NO 27 &211s LENGTH: 713 US 8,846,352 B2 103 104 - Continued gtc.ccgatcc tggaga cctg gaaggcc.ctg gagaa.gctgg t caaggcggg caagat.ccgc 48O tccatcggcg tgagcaactt CCC cqgcgc.c Ctgctgctgg acctgctg.cg cggcgcgacc 54 O atcaag.ccgt. cc.gtcc tigca ggtcgagcac CaCCCCtaCC tgcagcagcc cc.gc.ctgat c gagttcgc.cc agt ccc.gcgg catcgc.cgtg accgc.gtaca gct cott cq9 cc.cccagt cc 660 titcgtggagc tgalaccaggg cc.gc.gc.cctg aac acct cqc c cctdtt cqa gaacgagacg 72 O atcaaggc.ca tcqc.cgc.caa gcacggcaag agc.ccc.gc.cc aggtoctgct gcgctggtcC tcgcagcgcg gcatcgcgat catc.cccaag agcaa.caccg gctggagaac 84 O aaggacgtga act cott.cga Cctggacgag Caggactitcg ccgacat cqc Caagctggac 9 OO atcaacct gc gct tcaacga CCC ctgggac tgggacaaga t cc coat citt cgtgtag 95.7 <210s, SEQ ID NO 35 &211s LENGTH: 318 212. TYPE : PRT &213s ORGANISM: Pichia stipitis <4 OOs, SEQUENCE: 35 Met Pro Ser Ile Llys Lieu. Asn Ser Gly Tyr Asp Met Pro Ala Val Gly 1. 5 1O 15 Phe Gly Cys Trp Llys Val Asp Wall Asp Thr Cys Ser Glu Glin Ile Tyr 25 Arg Ala Ile Lys Thr Gly Tyr Arg Lieu. Phe Asp Gly Ala Glu Asp Tyr 35 4 O 45 Ala Asn Glu Llys Lieu Val Gly Ala Gly Val Lys Lys Ala Ile Asp Glu. SO 55 6 O Gly Ile Wall Lys Arg Glu Asp Lieu Phe Lieu. Thir Ser Luell Trp Asn 65 70 8O Asn His His Pro Asp Asn Val Glu Lys Ala Lell Asn Arg Thir Lieu. 85 90 95 Ser Asp Luell Glin Val Asp Tyr Val Asp Lieu. Phe Lell Ile His Phe Pro 105 11 O Wall Thir Phe Llys Phe Val Pro Leu Glu Glu Lys Pro Pro Gly Phe 115 12 O 125 Cys Gly Lys Gly Asp Asn. Phe Asp Tyr Glu Asp Wall Pro Ile Lieu. 13 O 135 14 O Glu Thir Trp Lys Ala Lieu. Glu Lys Lieu Val Lys Ala Gly Ile Arg 145 150 155 160 Ser Ile Gly Wall Ser Asn. Phe Pro Gly Ala Lieu. Lell Lell Asp Lieu. Luell 1.65 17O 17s Arg Gly Ala Thir Ile Llys Pro Ser Wall Lieu. Glin Wall Glu His His Pro 18O 185 19 O Luell Glin Glin Pro Arg Lieu. Ile Glu Phe Ala Glin Ser Arg Gly Ile 195 Ala Wall Thir Ala Tyr Ser Ser Phe Gly Pro Gln Ser Phe Wall Glu Lieu. 21 O 215 22O Asn Glin Gly Arg Ala Lieu. Asn Thr Ser Pro Leu Phe Glu Asn Glu Thir 225 23 O 235 24 O Ile Ala Ile Ala Ala Lys His Gly Lys Ser Pro Ala Glin Wall Lieu 245 250 255 Lell Arg Trp Ser Ser Glin Arg Gly Ile Ala Ile Ile Pro Lys Ser Asn 26 O 265 27 O Thir Wall Pro Arg Lieu. Lieu. Glu Asn Lys Asp Wall Asn Ser Phe Asp Lieu. US 8,846,352 B2 107 108 - Continued Met Ser Ser Glin Asp Ile Pro Ser Gly Wall Glin Thir Pro Ser Asn Ala 15 Ser Phe Luell Glu Lys Asp Glu Asp Lys Ile Glu Glu Wall Pro Glin Asn 2O 25 His Asp Ala Thir Lell Wall Ala Luell Glu Ser Lys Gly Ile Ser Glu 35 4 O 45 Lell Luell Ile Phe Phe Cys Luell Luell Wall Ala Phe Gly Gly Phe Wall SO 55 6 O Phe Gly Phe Asp Thir Gly Thir Ile Ser Gly Phe Wall Asn Met Ser Asp 65 70 8O Phe Luell Glu Arg Phe Gly Glin Thir Arg Ala Asp Gly Thir His Tyr Luell 85 90 95 Ser Asn Wall Arg Wall Gly Lell Luell Wall Ser Ile Phe Asn Ile Gly 105 11 O Ala Ile Gly Gly Ile Phe Lell Ser Ile Gly Asp Wall Gly Arg 115 12 O 125 Arg Wall Gly Ile Met Ala Ser Met Wall Ile Wall Wall Gly Ile Ile 13 O 135 14 O Wall Glin Ile Ala Ser Glin His Ala Trp Glin Wall Met Ile Gly Arg 145 150 155 160 Ala Ile Thir Gly Lell Ala Wall Gly Thir Wall Ser Wall Lell Ser Pro Luell 1.65 17O 17s Phe Ile Gly Glu Ser Ser Pro His Luell Arg Gly Thir Luell Wall 18O 185 19 O Phe Glin Luell Cys Ile Thir Luell Gly Ile Phe Ile Gly Wall 195 2OO Thir Tyr Gly Thir Lys Arg Lell Ser Asp Ser Arg Glin Trp Arg Wall Pro 21 O 215 Lell Gly Luell Phe Lell Trp Ala Ile Phe Luell Wall Wall Gly Met Luell 225 23 O 235 24 O Ala Met Pro Glu Ser Pro Arg Luell Wall Glu Arg Ile Glu 245 250 255 Asp Ala Lys Ser Wall Ala Arg Ser Asn Lell Ser Pro Glu Asp 26 O 265 27 O Pro Ser Wall Thir Glu Ile Glin Luell Ile Glin Ala Gly Ile Asp Arg 28O 285 Glu Ala Ile Ala Gly Ser Ala Ser Trp Thir Glu Lell Ile Thir Gly 29 O 295 3 OO Pro Ala Ile Phe Arg Wall Wall Met Gly Ile Ile Met Glin Ser Luell 3. OS 310 315 32O Glin Glin Luell Thir Gly Wall Asn Phe Phe Gly Thir Thir Ile 3.25 330 335 Phe Glin Ala Wall Gly Lell Asp Ser Phe Glin Thir Ser Ile Ile Luell 34 O 345 35. O Gly Wall Wall Asn Phe Ala Ala Thir Phe Ile Gly Ile Trp Ala Ile Glu 355 360 365 Arg Phe Gly Arg Arg Ser Cys Luell Luell Wall Gly Ser Ala Gly Met Phe 37 O 375 Wall Phe Ile Ile Tyr Ser Thir Ile Gly Ser Phe His Luell Lys 385 390 395 4 OO Asp Gly Glu Tyr Asn Asn Asp Asn Thir Tyr Pro Ser Gly Asn Ala 4 OS 41O 415 US 8,846,352 B2 111 112 - Continued ggcaac Ctgg gct coaacgt. Ctt CttcatC tiggcggct tca acctggc ctg.cgtgttc 1380 titcgc.ctggit actitcatcta Cagaccalag ggcctgtc.cc tigagcaggt cacgagctg 144 O tacgag cacg tdt CCaaggc ctggalagt cc aagggct tcg tocctic cala gcact cott C 15OO cgcgagcagg taccagca gatggacticc aagaccgagg C catcatgtc. c.gaggaggcg 1560 tcggtgttgat acgtac 1576 <210s, SEQ ID NO 39 &211s LENGTH: 522 212. TYPE: PRT <213> ORGANISM: Candida intermedia <4 OOs, SEQUENCE: 39 Met Gly Lieu. Glu Asp Asn Arg Met Val Lys Arg Phe Val Asin Val Gly 1. 5 1O 15 Glu Lys Lys Ala Gly Ser Thr Ala Met Ala Ile Ile Val Gly Lieu. Phe 2O 25 3O Ala Ala Ser Gly Gly Val Lieu Phe Gly Tyr Asp Thr Gly Thr Ile Ser 35 4 O 45 Gly Val Met Thr Met Asp Tyr Val Lieu Ala Arg Tyr Pro Ser Asn Lys SO 55 6 O His Ser Phe Thr Ala Asp Glu Ser Ser Lieu. Ile Val Ser Ile Leu Ser 65 70 7s 8O Val Gly Thr Phe Phe Gly Ala Lieu. Cys Ala Pro Phe Lieu. Asn Asp Thr 85 90 95 Lieu. Gly Arg Arg Trp Cys Lieu. Ile Lieu Ser Ala Lieu. Ile Val Phe ASn 1OO 105 11 O Ile Gly Ala Ile Lieu. Glin Val Ile Ser Thr Ala Ile Pro Lieu. Lieu. Cys 115 12 O 125 Ala Gly Arg Val Ile Ala Gly Phe Gly Val Gly Lieu. Ile Ser Ala Thr 13 O 135 14 O Ile Pro Leu Tyr Glin Ser Glu Thir Ala Pro Llys Trp Ile Arg Gly Ala 145 150 155 160 Ile Val Ser Cys Tyr Gln Trp Ala Ile Thir Ile Gly Lieu. Phe Leu Ala 1.65 17O 17s Ser Cys Val Asn Lys Gly Thr Glu. His Met Thr Asn Ser Gly Ser Tyr 18O 185 19 O Arg Ile Pro Lieu Ala Ile Glin Cys Lieu. Trp Gly Lieu. Ile Lieu. Gly Ile 195 2OO 2O5 Gly Met Ile Phe Leu Pro Glu Thr Pro Arg Phe Trp Ile Ser Lys Gly 21 O 215 22O Asn Glin Glu Lys Ala Ala Glu Ser Lieu Ala Arg Lieu. Arg Llys Lieu Pro 225 23 O 235 24 O Ile Asp His Pro Asp Ser Lieu. Glu Glu Lieu. Arg Asp Ile Thir Ala Ala 245 250 255 Tyr Glu Phe Glu Thr Val Tyr Gly Lys Ser Ser Trp Ser Glin Val Phe 26 O 265 27 O Ser His Lys Asn His Glin Lieu Lys Arg Lieu. Phe Thr Gly Val Ala Ile 27s 28O 285 Glin Ala Phe Glin Gln Lieu. Thr Gly Val Asin Phe Ile Phe Tyr Tyr Gly 29 O 295 3 OO Thir Thr Phe Phe Lys Arg Ala Gly Val Asin Gly Phe Thr Ile Ser Lieu. 3. OS 310 315 32O Ala Thr Asn Ile Val Asn Val Gly Ser Thr Ile Pro Gly Ile Lieu. Leu US 8,846,352 B2 115 116 - Continued gacgagaagt ccggcggcaa. Ctt Cotggag gtgttcCagg gcc cca acct gaaggcc ctg accatcggcg ggggcctggt cctgttccag Cagat Caccg gcc agcc ct c cgtgctgtac 14 O tacgc.cggct c catcc tigca gaccgc.cggc ttcticggcgg Caccc.gc.gtg 2OO tcc.gtgat ca Caagctgctg atgacctggg tcgcggit cqc Caaggtggac 26 O gacctgggcc gcc.gc.ccc ct gctgatcggc gcatcgc.gct gtc.gctgttc 32O CCtact acaa. gttcCtcggc ggct tcc ccc ggg.cgcCCtg Ctgctgtacg tgggctgcta ccagatct co titcggc.ccca t ct cotggct gatggtgtcC 44 O gagatct tcc c cct cogcac ggcatct coc gacca acttic SOO ggct coaacg c catcgtgac citt cqcct tc tcc cc cctoga aggagttcct ggg.cgc.cgag 560 aacctgttcc t cctgttctgg cggcatcgc.c tcc tigttcgt. catcCtggtg gtgc.ccgaga cCaagggc ct Ctcgctggag gagat.cgagt c caagat cot gaagtga 677 <210s, SEQ ID NO 41 &211s LENGTH: 558 212. TYPE : PRT &213s ORGANISM: Arabidopsis thaliana <4 OOs, SEQUENCE: 41 Met Ala Phe Ala Wall Ser Wall Glin Ser His Phe Ala Ile Arg Ala Lieu 1. 5 15 Lys Arg Asp His Phe Asn Pro Ser Pro Arg Thir Phe Cys Ser Cys 2O 25 Phe Ser Arg Pro Asp Ser Ser Luell Ser Lell Lys Glu Arg Thr 35 4 O 45 Phe Wall Ser Lys Pro Gly Luell Wall Thir Thir Arg Arg His Ile SO 55 6 O Phe Glin Wall Gly Ala Glu Thir Gly Gly Asp Phe Ala Asp Ser Gly Glu 65 70 8O Wall Ala Asp Ser Luell Ala Ser Asp Ala Pro Glu Ser Phe Ser Trp Ser 85 90 95 Ser Wall Ile Leul Pro Phe Ile Phe Pro Ala Luell Gly Gly Luell Lieu. Phe 1OO 105 11 O Gly Asp Ile Gly Ala Thir Ser Gly Ala Thir Lell Ser Luell Glin Ser 115 12 O 125 Pro Ala Luell Ser Gly Thir Thir Trp Phe Asn Phe Ser Pro Wall Gln Lieu. 13 O 135 14 O Gly Luell Wall Wall Ser Gly Ser Luell Gly Ala Lell Lell Gly Ser Ile 145 150 155 160 Ser Wall Gly Val Ala Asp Phe Luell Gly Arg Arg Arg Glu Lieu. Ile 1.65 17O 17s Ile Ala Ala Wall Lieu. Lell Luell Gly Ser Luell Ile Thir Gly Cys Ala 18O 185 19 O Pro Asp Luell ASn Ile Lell Lell Wall Gly Arg Luell Lell Tyr Gly Phe Gly 195 Ile Gly Luell Ala Met His Gly Ala Pro Luell Ile Ala Glu Thr Cys 21 O 215 22O Pro Ser Glin Ile Arg Gly Thir Luell Ile Ser Luell Glu Luell Phe Ile 225 23 O 235 24 O Wall Luell Gly Ile Lieu. Lell Gly Phe Ser Wall Gly Ser Phe Glin Ile Asp 245 250 255 US 8,846,352 B2 117 118 - Continued Val Val Gly Gly Trp Arg Tyr Met Tyr Gly Phe Gly Thir Pro Wall Ala 26 O 265 27 O Lieu. Luell Met Gly Lieu. Gly Met Trp Ser Lieu. Pro Ala Ser Pro Arg Trp 27s 285 Lieu. Luell Luell Arg Ala Val Glin Gly Lys Gly Glin Lell Glin Glu 29 O 295 3 OO Glu Lys Ala Met Lieu Ala Lieu. Ser Llys Lieu. Arg Gly Arg Pro Pro Gly 3. OS 310 315 32O Asp Llys Ile Ser Glu Lys Lieu Val Asp Asp Ala Lell Ser Val Lys 3.25 330 335 Thr Ala Tyr Glu Asp Glu Lys Ser Gly Gly Asn Phe Lell Glu Wall Phe 34 O 345 35. O Gln Gly Pro Asn Lieu Lys Ala Lieu. Thir Ile Gly Gly Gly Luell Wall Lieu 355 360 365 Phe Glin Glin Ile Thr Gly Glin Pro Ser Wall Lieu Tyr Ala Gly Ser 37 O 375 Ile Lieu. Glin Thr Ala Gly Phe Ser Ala Ala Ala Asp Ala Thir Arg Val 385 390 395 4 OO Ser Wall Ile Ile Gly Val Phe Lys Lieu. Luell Met Thir Trp Wall Ala Wall 4 OS 41O 415 Ala Lys Val Asp Asp Lieu. Gly Arg Arg Pro Lieu. Lell Ile Gly Gly Val 425 43 O Ser Gly Ile Ala Lieu. Ser Lieu. Phe Lieu. Luell Ser Ala Tyr Llys Phe 435 44 O 445 Lieu. Gly Gly Phe Pro Leu Wall Ala Val Gly Ala Lieu Lieu Luell Tyr Val 450 45.5 460 Gly Cys Tyr Glin Ile Ser Phe Gly Pro Ile Ser Trp Lell Met Wall Ser 465 470 47s 48O Glu Ile Phe Pro Lieu. Arg Thr Arg Gly Arg Gly Ile Ser Luell Ala Wall 485 490 495 Lieu. Thir Asn Phe Gly Ser Asn Ala Ile Wall. Thir Phe Ala Phe Ser Pro SOO 505 Lieu Lys Glu Phe Lieu. Gly Ala Glu Asn Lieu. Phe Lell Lell Phe Gly Gly 515 525 Ile Ala Lieu Wall Ser Lieu Lleu Phe Wall Ile Lieu. Wall Wall Pro Glu Thir 53 O 535 54 O Lys Gly Lieu. Ser Lieu. Glu Glu Ile Glu Ser Lys Ile Lell 5.45 550 555 <210s, SEQ I D NO 42 &211s LENGT H: 1552 212. TYPE : DNA <213> ORGANISM: Trichoderma rees ii <4 OOs, SEQUENCE: 42 atgtaccgca tctggaacat citacgtcc td gcggcgttcg gcaccatcgg cgg catgat C 6 O tittggctt.cg agatct cqtc gatgtcggcg tggat.cggct cc.gagcagta Cctggagtac 12 O ttcaac Cacc ccgacticcac cgagc agggc ggcatcaccg cc.gc.catgtc cgc.cggct cc 18O Ctggtgggct CCCtgctggc gcggaccgc.c cctggc.catc 24 O cagat.cgc.ct cc.gtggactg gatcgtgggc gcggit Cotgc agtgctcgt.c gCagaacgtg 3OO gcccacctgg catcgtgtc.c ggCCtgg.cga tcqgcatcac gtcct cocag 360 tgcatcgtgt acctgtc.cga gctggcc.ccc tcc.cgcatcc ggtgggcatc US 8,846,352 B2 121 122 - Continued Ala Wall Pro Gly Ala Wall Lell Phe Phe Ser Luell Phe Phe Phe Pro Glu 18O 185 19 O Ser Pro Arg Trp Lell Ala Thir Lys Asp Arg Trp Glu Glu Cys His Glu 195 2O5 Wall Luell Ala Asn Lell His Ala Gly Asp Arg Asn Asn Ile Glu Wall 21 O 215 22O Lell Ala Glu Luell Glu Glu Wall Arg Glu Ala Ala Arg Ile Ala Ala Glu 225 23 O 235 24 O Ser Glu Ile Gly Lell Gly Luell Phe Ala Pro Met Trp Lys 245 250 255 Arg Thir Luell Wall Gly Wall Ser Ala Glin Ile Trp Glin Glin Luell Luell Gly 26 O 265 27 O Gly Asn Wall Met Lell Luell Wall Phe Asn Met Ala Gly 285 Met Ser Gly Asn Thir Ala Lell Thir Ser Ser Ile Glin Wall Ile 29 O 295 3 OO Phe Luell Wall Thir Thir Gly Gly Wall Luell Phe Wall Asp Arg Ile Gly 3. OS 310 Arg Arg Trp Luell Lell Ile Wall Gly Ala Ile Gly Wall Ile His 3.25 330 335 Phe Ile Wall Gly Ala Wall Met Ala Wall His His Wall Asp Ser 34 O 345 35. O Wall Asp Gly Asn Asp Ile Lell Arg Trp Glin Gly Gly Pro Pro Ala 355 360 365 Ala Ile Ile Ala Lieu Cys Ile Phe Wall Gly Wall Gly Wall 37 O 375 Thir Trp Ala His Gly Ala Trp Ile Gly Glu Wall Phe Pro Luell 385 390 395 4 OO Arg Ala Lys Gly Wall Gly Luell Ala Ala Ala Gly Asn Trp Ala 4 OS 415 Phe Asn Luell Ala Lell Ala Phe Phe Wall Pro Pro Ala Phe Thir Asn Ile 425 43 O Glin Trp Lys Ala Tyr Met Ile Phe Gly Thir Phe Ile Ala Met Wall 435 44 O 445 Phe His Ile Phe Met Tyr Pro Glu Thir Wall Lys Ser Luell Glu 450 45.5 460 Glu Ile Asp Wall Lell Phe Glu Gly Asp Ile Pro Ala Trp Arg Ser Ala 465 470 Ser Ala Wall Ser Thir Phe Asp Glu Wall Ala Arg Ala Glu Ala 485 490 495 Gly Gly Luell Glu Glu Phe Ser Glin Ala Asp Ile His Glu Glu SOO 505 51O Wall SEQ ID NO 44 LENGTH: 11.89 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide SEQUENCE: 44 atgagggtgg agatctggag aactgggit cq cct catgcc.g. c9cagggggg attgttgctgg Catgtgagtg atttgggtgc ggcgacgcac cqggaga.gcg agcc.ca.gc.cg aggcgg tact 12 O US 8,846,352 B2 125 126 - Continued 1.65 17O 17s Gly Arg Lieu. Val His Pro Arg Asp Glin Val Arg Arg Ala Arg Wall Lieu 18O 185 19 O Arg Asp Lieu. Lieu. Lieu Ala Ala Gly Arg Lieu. Lieu. Pro Pro Gly Arg Val 195 Ala Wal His Pro Ala His Arg Asp Gly Lieu. Lieu. Pro Gly Arg Arg Asp 21 O 215 22O Arg Gly Lieu. Val Glin Pro Gly Arg Pro Val Arg Arg His Asp Lieu. Glin 225 23 O 235 24 O Arg Gly Lieu. Arg Ala Ala Glin His Lieu. Lieu. Glin Ala Lell Pro Ala Wall 245 250 255 Lieu. Glin Gly Asp Arg Arg Pro Glin Pro Val Arg Lell His Luell His Pro 26 O 265 27 O Wall Pro Ala Val Pro Val Pro Arg Gly His Lieu. Arg Gly Gly Leul Pro 27s 285 Lieu. Gly Ala Arg Lieu Pro Glin Gly His Arg Lieu. Arg Gly His Pro Leu 29 O 295 3 OO His Lieu. Lieu. Lieu. Lieu. Gly Lieu Ala Val Gly Arg Wall Lell Pro Pro Wall 3. OS 310 315 32O Glin Pro Wall Leu Lleu Pro Gly Pro Gly Arg Asp Lell Pro Pro Asp Lieu. 3.25 330 335 Lieu. Gly Arg Gln His His Glu Ala Arg Gly Gly Asp His Luell His Arg 34 O 345 35. O Ala Gly Val Pro Gln Pro Arg Ala Pro Pro Glu Arg Pro Gly Lieu. Arg 355 360 365 His Arg His Lieu. Arg His Lieu Pro Wall Leu Pro Gly His Arg Glin Glu 37 O 375 Glu Glu Asp Arg Gly Gly Arg Arg Glin Glu Glu Lell 385 390 395 <210s, SEQ I D NO 46 &211s LENGT H: 11.89 212. TYPE : DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATU RE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide <4 OOs, SEQUENCE: 46 atgagggtgg agatctggag aactgggit cq cct tatgcc.g tgc.cggaggg Cttgtactgg 6 O gttgaga.gtg atttgggtgc ggcgacgcac aggcgg tact 12 O ggc.ccgcc ct cacgc.ccc.gc ccaccc.gcat gcatc.cgcga Cctgcgcagg 18O t cct cogact cgalaccc.cga cgagaagt cc gacCt999C9 aggcc.gagaa 24 O galaggagaag aaggccaaga c cctdcagct gggcatcgtg titcggcctgt gg tact tcca 3OO gaac atcqtc ttcaa.cat Ct t caacaagaa ggc cctgaac gtgttcc cct acc cctdgct 360 cctggcct co titccagctgt tcgc.cggctic Catctggatg ggtcgttcaa 42O gctgtacc cc tgc.cccalaga t ct cqaagcc gttcatcatc gcc cc.gc.cct 48O gttccacacc atcggccaca t ct cogcctg cgtgtcc titc tccalaggtgg cc.gtc.tc.gtt 54 O cacccacgtg atcaagtc.cg cc.gagc.ccgt. gttct cogtg a tott ct cott cgctgctggg 6OO cgactic ctac catcc togcc.c atcgtgatgg gctgctic cct 660 accgaggit ct cgttcaac ct tcc.ggcgc.ca tgat citccaa 72 O US 8,846,352 B2 127 128 - Continued cgtgggct tc gtgctg.cgca acatctactic caa.gc.gcticc ctgcagt cct tcaaggagat 78O cgacggcctic aacctgtacg gctgcatcto catcc tdtcc ctdctgtacc togttcc.ccgt. 84 O ggc.cat Cttic gtggagggct C cc actgggit gcc.cggctac Cacaaggcca t cqcct CC9t 9 OO gggcaccc cc ticcaccittct acttctgggit ctdgctgtcg gg.cgtgttct accacct gta 96.O caac cagt cc ticcitaccagg ccctggacga gat ct coccc ctacct tct cqgtcggcaa. 1 O2O Caccatgaag cqcgtggtgg tat catcto Caccgtgctg gtgttcc.gca accc.cgtgcg 108 O cc.ccctgaac gcc ctdggct c cqccatcgc catctg.cggc acct tcc tdt act cocaggc 114 O Caccgc.caag aagaagaaga t caggtggg C9gcgacaag aagaactga 11.89 <210s, SEQ ID NO 47 &211s LENGTH: 396 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide <4 OOs, SEQUENCE: 47 Met Arg Val Glu Ile Trp Arg Thr Gly Ser Pro Tyr Ala Val Pro Glu 1. 5 1O 15 Gly Lieu. Tyr Trp Val Glu Ser Asp Lieu. Gly Ala Ala Thr His Arg Glu 2O 25 3O Ser Glu Pro Ser Arg Gly Gly. Thir Lieu. Lieu. Arg Gly Pro Ala Lieu. Thir 35 4 O 45 Pro Arg Pro Pro Ala Cys Ile Arg Asp Lieu. Arg Arg Gly Arg Gly Lieu SO 55 6 O Lieu. Arg Lieu. Glu Pro Arg Arg Glu Val Arg Pro Gly Arg Gly Arg Glu 65 70 7s 8O Glu Gly Glu Glu Gly Glin Asp Pro Ala Ala Gly His Arg Val Arg Pro 85 90 95 Val Val Lieu Pro Glu. His Arg Lieu Gln His Lieu. Glin Glin Glu Gly Pro 1OO 105 11 O Glu Arg Val Pro Lieu Pro Lieu Ala Pro Gly Lieu Lleu Pro Ala Val Arg 115 12 O 125 Arg Lieu. His Lieu. Asp Ala Gly Ala Val Val Val Glin Ala Val Pro Lieu 13 O 135 14 O Pro Glin Asp Lieu. Glu Ala Wal His His Arg Ala Ala Gly Pro Arg Pro 145 150 155 160 Val Pro His His Arg Pro His Lieu. Arg Lieu. Arg Val Lieu. Lieu. Glin Gly 1.65 17O 17s Gly Arg Lieu Val His Pro Arg Asp Glin Val Arg Arg Ala Arg Val Lieu. 18O 185 19 O Arg Asp Lieu. Lieu. Lieu Ala Ala Gly Arg Lieu. Lieu Pro Pro Gly Arg Val 195 2OO 2O5 Ala Wal His Pro Ala His Arg Asp Gly Lieu. Lieu Pro Gly Arg Arg Asp 21 O 215 22O Arg Gly Lieu Val Glin Pro Gly Arg Pro Val Arg Arg His Asp Lieu. Glin 225 23 O 235 24 O Arg Gly Lieu. Arg Ala Ala Glin His Lieu. Lieu. Glin Ala Lieu Pro Ala Val 245 250 255 Lieu. Glin Gly Asp Arg Arg Pro Glin Pro Val Arg Lieu. His Lieu. His Pro 26 O 265 27 O Val Pro Ala Val Pro Val Pro Arg Gly His Lieu. Arg Gly Gly Lieu Pro US 8,846,352 B2 131 132 - Continued polypeptide <4 OOs, SEQUENCE: 49 Met Ala Thir Ala Ser Thir Phe Ser Ala Phe ASn Ala Arg Gly Asp 1. 5 15 Lell Arg Arg Ser Ala Gly Ser Gly Pro Arg Arg Pro Ala Arg Pro Luell 25 Pro Wall Arg Gly Arg Gly Lell Luell Arg Luell Glu Pro Arg Arg Glu Wall 35 4 O 45 Arg Pro Gly Arg Gly Arg Glu Glu Gly Glu Glu Gly Glin Asp Pro Ala SO 55 6 O Ala Gly His Arg Wall Arg Pro Wall Wall Luell Pro Glu His Arg Luell Glin 65 70 His Luell Glin Glin Glu Gly Pro Glu Arg Wall Pro Lell Pro Luell Ala Pro 85 90 95 Gly Luell Luell Pro Ala Wall Arg Arg Luell His Luell Asp Ala Gly Ala Wall 105 11 O Wall Wall Glin Ala Wall Pro Lell Pro Glin Asp Luell Glu Ala Wall His His 115 12 O 125 Arg Ala Ala Gly Pro Arg Pro Wall Pro His His Arg Pro His Luell Arg 13 O 135 14 O Lell Arg Wall Luell Lell Glin Gly Gly Arg Lel Wall His Pro Arg Asp Glin 145 150 155 160 Wall Arg Arg Ala Arg Wall Lell Arg Asp Lel Luell Lell Ala Ala Gly Arg 1.65 17O 17s Lell Luell Pro Pro Gly Arg Wall Ala Wall His Pro Ala His Arg Asp Gly 18O 185 19 O Lell Luell Pro Gly Arg Arg Asp Arg Gly Lel Wall Glin Pro Gly Arg Pro 195 Wall Arg Arg His Asp Lell Glin Arg Gly Lel Arg Ala Ala Glin His Luell 21 O 215 Lell Glin Ala Luell Pro Ala Wall Luell Glin Gly Asp Arg Arg Pro Glin Pro 225 23 O 235 24 O Wall Arg Luell His Lell His Pro Wall Pro Ala Wall Pro Wall Pro Arg Gly 245 250 255 His Luell Arg Gly Gly Lell Pro Luell Gly Ala Arg Lell Pro Glin Gly His 26 O 265 27 O Arg Luell Arg Gly His Pro Lell His Luell Luell Luell Lell Gly Luell Ala Wall 28O 285 Gly Arg Wall Luell Pro Pro Wall Glin Pro Wall Luell Lell Pro Gly Pro Gly 29 O 295 3 OO Arg Asp Luell Pro Pro Asp Lell Luell Gly Arg Glin His His Glu Ala Arg 3. OS 310 315 Gly Gly Asp His Lell His Arg Ala Gly Wall Pro Glin Pro Arg Ala Pro 3.25 330 335 Pro Glu Arg Pro Gly Lell Arg His Arg His Luell Arg His Luell Pro Wall 34 O 345 35. O Lell Pro Gly His Arg Glin Glu Glu Glu Asp Arg Gly Gly Arg Arg Glin 355 360 365 Glu Glu Luell 37 O <210s, SEQ ID NO 50 &211s LENGTH: 363 US 8,846,352 B2 133 134 - Continued 212. TYPE : PRT &213s ORGANISM: Pichia stipitis <4 OOs, SEQUENCE: SO Met Thir Ala Asn Pro Ser Lell Wall Luell Asn Lys Ile Asp Asp Ile Ser 1. 5 15 Phe Glu Thir Tyr Asp Ala Pro Glu Ile Ser Glu Pro Thir Asp Wall Luell 25 Wall Glin Wall Lys Lys Thir Gly Ile Gly Ser Asp Ile His Phe Tyr 35 4 O 45 Ala His Gly Arg Ile Gly Asn Phe Wall Luell Thir Lys Pro Met Wall Luell SO 55 6 O Gly His Glu Ser Ala Gly Thir Wall Wall Glin Wall Gly Gly Wall Thir 65 70 Ser Luell Wall Gly Asp Asn Wall Ala Ile Glu Pro Gly Ile Pro Ser 85 90 95 Arg Phe Ser Asp Glu Tyr Ser Gly His Asn Lell Cys Pro His 105 11 O Met Ala Phe Ala Ala Thir Pro Asn Ser Glu Gly Glu Pro Asn Pro 115 12 O 125 Pro Gly Thir Luell Tyr Phe Ser Pro Glu Asp Phe Luell Wall 13 O 135 14 O Lys Luell Pro Asp His Wall Ser Luell Glu Luell Gly Ala Lell Wall Glu Pro 145 150 155 160 Lell Ser Wall Gly Wall His Ala Ser Luell Gly Ser Wall Ala Phe Gly 1.65 170 175 Asp Wall Ala Wall Phe Gly Ala Gly Pro Wall Gly Lell Luell Ala Ala 18O 185 19 O Ala Wall Ala Thir Phe Gly Ala Gly Wall Ile Wall Wall Asp Ile 195 Phe Asp Asn Lell Met Ala Asp Ile Gly Ala Ala Thir His 21 O 215 22O Thir Phe Asn Ser Thir Gly Gly Ser Glu Glu Lell Ile Ala Phe 225 23 O 235 24 O Gly Gly Asn Wall Pro Asn Wall Wall Luell Glu Cys Thir Gly Ala Glu Pro 245 250 255 Ile Luell Gly Wall Asp Ala Ile Ala Pro Gly Gly Arg Phe Wall 26 O 265 27 O Glin Wall Gly Asn Ala Ala Gly Pro Wall Ser Phe Pro Ile Thir Wall Phe 27s 28O 285 Ala Met Glu Lell Thir Lell Phe Gly Ser Phe Arg Gly Phe Asn 29 O 295 3 OO Asp Thir Ala Wall Gly Ile Phe Asp Thir Asn Tyr Glin Asn Gly 3. OS 310 315 Arg Glu Asn Ala Pro Ile Asp Phe Glu Glin Luell Ile Thir His Arg Tyr 3.25 330 335 Phe Asp Ala Ile Glu Ala Tyr Asp Luell Wall Arg Ala Gly 34 O 345 35. O Gly Ala Wall Cys Lell Ile Asp Gly Pro Glu 355 360 <210s, SEQ ID NO 51 &211s LENGTH: 623 212. TYPE : PRT <213> ORGANISM: Pichia stipitis US 8,846,352 B2 135 136 - Continued <4 OOs, SEQUENCE: 51 Met Thir Thir Thr Pro Phe Asp Ala Pro Asp Lys Lell Phe Luell Gly Phe 1. 15 Asp Luell Ser Thir Glin Glin Lell Ile Ile Wall Thir Asp Glu Asn Luell 25 3O Ala Ala Luell Thir Asn Wall Glu Phe Asp Ser Ile Asn Ser Ser 35 4 O 45 Wall Glin Gly Wall Ile Ala Ile Asn Asp Glu Ile Ser Gly Ala SO 55 6 O Ile Ile Ser Pro Wall Tyr Met Trp Luell Asp Ala Lell Asp His Wall Phe 65 70 Glu Asp Met Lys Asp Gly Phe Pro Phe ASn Wall Wall Gly Ile 85 90 95 Ser Gly Ser Cys Glin Glin His Gly Ser Wall Trp Ser Arg Thir Ala 1OO 105 11 O Glu Wall Luell Ser Glu Lell Asp Ala Glu Ser Ser Lell Ser Ser Glin 115 12 O 125 Met Arg Ser Ala Phe Thir Phe His Ala Pro Asn Trp Glin Asp His 13 O 135 14 O Ser Thir Gly Glu Lell Glu Glu Phe Glu Arg Wall Ile Gly Ala Asp 145 150 155 160 Ala Luell Ala Asp Ile Ser Gly Ser Arg Ala His Arg Phe Thir Gly 1.65 17O 17s Lieu Gln Ile Arg Lys Lieu Ser Thr Arg Phe Lys Pro Glu Lys Asn 18O 185 19 O Arg Thir Ala Arg Ile Ser Lell Wall Ser Ser Phe Wall Ala Ser Wall Luell 195 Lell Gly Arg Ile Thir Ser Ile Glu Glu Ala Asp Ala Gly Met Asn 21 O 215 Lell Asp Ile Glu Lys Arg Glu Phe Asn Glu Glu Lell Luell Ala Ile 225 23 O 235 24 O Ala Ala Gly Wall His Pro Glu Luell Asp Gly Wall Glu Glin Asp Gly Glu 245 250 255 Ile Arg Ala Gly Ile Asn Glu Luell Arg Lell Gly Pro Wall 26 O 265 27 O Pro Ile Thir Tyr Glu Ser Glu Gly Ile Ala Ser Phe Wall 28O 285 Thir Arg Gly Phe Asn Pro Asp Ile Tyr Ser Phe Thir Gly 29 O 295 3 OO Asp Asn Luell Ala Thir Ile Ile Ser Luell Pro Luell Ala Pro Asn Asp Ala 3. OS 310 315 Lell Ile Ser Luell Gly Thir Ser Thir Thir Wall Luell Ile Ile Thir Lys Asn 3.25 330 335 Ala Pro Ser Ser Glin Tyr His Luell Phe Lys His Pro Thir Met Pro 34 O 345 35. O Asp His Tyr Met Gly Met Ile Cys ASn Gly Ser Luell Ala Arg 355 360 365 Glu Lys Wall Arg Asp Glu Wall Asn Glu Phe Asn Wall Glu Asp 37 O 375 38O Lys Ser Trp Asp Phe Asn Glu Ile Luell Asp Ser Thir Asp Phe 385 390 395 4 OO Asn Asn Luell Gly Ile Phe Pro Luell Gly Glu Ile Wall Pro Asn US 8,846,352 B2 137 138 - Continued 4 OS 41O 415 Ala Ala Ala Glin Ile Arg Ser Wall Luell Asn. Ser Asn Glu Ile 425 43 O Wall Asp Wall Glu Lieu. Gly Asp Lys Asn Trp Glin Pro Glu Asp Asp Wall 435 44 O 445 Ser Ser Ile Wall Glu Ser Glin Thir Lieu. Ser Arg Lell Arg Thr Gly 450 45.5 460 Pro Met Luell Ser Lys Ser Gly Asp Ser Ser Ala Ser Ser Ser Ala Ser 465 470 47s Pro Glin Pro Glu Gly Asp Gly Thir Asp Lieu. His Wall Glin Asp 485 490 495 Lieu Val Llys Llys Phe Gly Asp Luell Tyr Thir Asp Lys Gn. Thir SOO 505 51O Phe Glu Ser Luell Thir Ala Arg Pro Asn Arg Tyr Wall Gly Gly 515 525 Ala Ser Asn. Asn Gly Ser Ile Ile Arg Met Gly Ser Ile Lieu Ala 53 O 535 54 O Pro Wall Asn Gly Asn Tyr Wall Asp Ile Pro Asn Ala Ala Lieu 5.45 550 555 560 Gly Gly Ala Tyr Lys Ala Ser Trp Ser Tyr Glu Glu Ala Lys Lys 565 st O sts Glu Trp Ile Gly Tyr Asp Glin Ile Asn Arg Leul Phe Glu Wall Ser 585 59 O Asp Glu Met Asn. Ser Phe Glu Wall Asp Trp Lell Glu Tyr Ala 595 605 Asn Gly Val Gly Met Lell Ala Met Glu Ser Glu Lell Lys His 610 615 SEQ ID NO 52 LENGTH: 4 TYPE PRT ORGANISM: Unknown FEATURE: OTHER INFORMATION: Description of Unknown: Retention signal peptide <4 OOs, SEQUENCE: 52 Lys Asp Glu Lieu. 1. What is claimed is: 6. The recombinant oleaginous microalga of claim 5. 1. A recombinant oleaginous microalga comprising an 50 wherein the microalga is Prototheca kruegani, Prototheca exogenous gene that encodes a xylulose-5-phosphate trans wickerhamii, Prototheca stagnora, Prototheca moriformis, locator, wherein the Xylulose-5-phosphate translocator is or Prototheca Zopfii. S106SAD-XPT having SEQID NO:49, GPT-A-XPT having SEQID NO:45, or GPT-F-XPT having SEQID NO: 47. 7. The recombinant oleaginous microalga of claim 1, 2. The recombinant oleaginous microalga of claim 1, fur wherein the xylulose-5-phosphate translocator is GPT-A- ther comprising one or more exogenous genes that encode a 55 XPT having SEQID NO: 45. Xylose transporter protein, a symporter protein, an oxi 8. The recombinant oleaginous microalga of claim 1, doreductase pathway protein, or a Xylose isomerase pathway protein. wherein the xylulose-5-phosphate translocator is GPT-F- 3. The recombinant oleaginous microalga of claim 1, XPT having SEQID NO: 47. wherein the xylulose-5-phosphate translocator is S106SAD 60 9. The recombinant oleaginous microalga of claim 2, XPT having SEQ ID NO: 49. wherein the one or more exogenous genes that encode a 4. The recombinant oleaginous microalga of claim 2, xylose isomerase pathway protein is XylA having SEQ ID wherein the one or more exogenous genes that encode an NO: 15. oxidoreductase pathway protein is XYL3 having SEQ ID 10. The recombinant oleaginous microalga of claim 2, NO: 51. 65 wherein the one or more exogenous genes that encode an 5. The recombinant oleaginous microalga of claim 2, oxido-reductase pathway protein is XYL1 having SEQ ID wherein the microalga is of the genus Prototheca. NO: 35. US 8,846,352 B2 139 140 11. The recombinant oleaginous microalga of claim 2, wherein the one or more exogenous genes that encode an oxido-reductase pathway protein is XYL2 having SEQ ID NO: 50.