Improved lipase for defoaming

Abstract

Disclosed are compositions and methods involving improved hybrid lipases for reducing foaming, for example, in carbohydrate fermentation processes.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 819,029, filed on March 15, 2019, the disclosure of which is incorporated by reference in its entirety.

[0002] Disclosed are compositions and methods directed to improved hybrid lipases useful for reducing foaming, for example, in carbohydrate fermentation processes. Background Art

[0003] Many commercial products are produced in fermentation processes that utilize "cell factories" (usually microorganisms). In such processes, large amounts of foam can be generated, which reduces the effective capacity per unit volume of the fermentor and can cause the fermentation broth to overflow from the fermentor through vents.

[0004] Foaming can be a particular problem in fuel ethanol production using carbohydrate substrates and yeast as the fermenting organism. Foaming appears to be exacerbated when proteases are added during or upstream of fermentation.

[0005] The use of lipases for reducing foaming in fuel ethanol production has been previously described, for example, in WO 2004029193, WO 2008135547 and WO 201875430. Nevertheless, there is still a need for superior foam suppressing enzymes at lower costs. Summary of the Invention

[0006] The compositions and methods of the present invention relate to improved variant lipase polypeptides and methods of use thereof. Aspects and embodiments of the compositions and methods of the present invention are summarized in the following, respectively numbered paragraphs:

[0007] 1. In a first aspect, a variant Thermomyces lanuginosus lipase is provided, the lipase having at least 95%, optionally at least 98%, and optionally at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, and having improved defoaming activity in a fermentation process compared to a reference lipase having the amino acid sequence of SEQ ID NO: 5, wherein the variant lipase comprises: substantially the entire contiguous amino acid sequence of the Thermomyces lanuginosus lipase, the amino acid sequence having one or more substitutions selected from the group consisting of G91A, D96W, and E99K, with reference to SEQ ID NO: 4 including the N-terminus; and substantially the entire contiguous amino acid sequence of the Thermomyces lanuginosus lipase present as a fusion protein having a contiguous amino acid sequence from Fusarium oxysporum having the amino acid sequence of SEQ ID NO: 2. oxysporum) lipase, wherein the C-terminus of the variant lipase has at least 12 but less than 55 amino acid residues derived from the C-terminus of the Fusarium oxysporum lipase, and wherein the variant lipase does not have the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0008] 2. In some embodiments, the C-terminus of the variant lipase described in paragraph 1 has at least 12 but fewer than 15 amino acid residues derived from the C-terminus of Fusarium oxysporum.

[0009] 3. In some embodiments, the C-terminus of the variant lipase described in paragraph 1 or 2 has 12 amino acid residues derived from the C-terminus of Fusarium oxysporum.

[0010] 4. In some embodiments, the variant lipase of any of paragraphs 1-3 has substitutions G91A, D96W, and E99K.

[0011] 5. In some embodiments, the variant lipase of any of paragraphs 1-4 has a small number of fewer or additional residues at the C-terminus of the contiguous amino acid sequence of the Thermomyces lanuginosus lipase.

[0012] 6. In some embodiments, the variant lipase of any of paragraphs 1-4 has a truncation of residues at the C-terminus of the contiguous amino acid sequence of the Thermomyces lanuginosus lipase.

[0013] 7. In some embodiments, the variant lipase of any one of paragraphs 1-6 has the amino acid sequence of SEQ ID NO: 4.

[0014] 8. In some embodiments of the variant lipase of any of paragraphs 1-7, the variant lipase has improved antifoaming activity in simultaneous saccharification and fermentation in the fermentation process.

[0015] 9. In another aspect, provided is an improved method for reducing foaming in an ethanol production process using a carbohydrate substrate as a feedstock, the method comprising adding a variant lipase as described in any one of paragraphs 1-7 before or during the fermentation step, wherein the variant lipase has improved antifoaming activity in the fermentation process compared to a reference lipase having the amino acid sequence of SEQ ID NO: 5.

[0016] 10. In some embodiments of the improved method of paragraph 9, the fermentation process is saccharification and / or fermentation.

[0017] 11. In some embodiments of the improved method of paragraph 9 or 10, the fermentation process is simultaneous saccharification and fermentation.

[0018] 12. In another aspect, a variant Thermomyces lanuginosus lipase is provided, the lipase having at least 95%, optionally at least 98%, and optionally at least 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 4, and having improved expression in a Trichoderma host compared to a reference lipase having the amino acid sequence of SEQ ID NO: 5, wherein the variant lipase comprises: substantially the entire contiguous amino acid sequence of Thermomyces lanuginosus lipase, the amino acid sequence having one or more substitutions selected from the group consisting of G91A, D96W, and E99K with reference to SEQ ID NO: 4 including the N-terminus; substantially the entire contiguous amino acid sequence of Thermomyces lanuginosus lipase present as a fusion protein having a contiguous amino acid sequence from a Fusarium oxysporum lipase having the amino acid sequence of SEQ ID NO: 2, wherein the C-terminus of the variant lipase has at least 12 but less than 55 amino acid residues derived from the C-terminus of the Fusarium oxysporum lipase, and wherein the variant lipase does not have the amino acid sequence of SEQ ID NO: 3. NO:3 or the amino acid sequence of SEQ ID NO:5.

[0019] 13. In some embodiments, the C-terminus of the variant lipase of paragraph 12 has at least 12 but fewer than 15 amino acid residues derived from the C-terminus of Fusarium oxysporum.

[0020] 14. In some embodiments, the C-terminus of the variant lipase of paragraph 12 or 13 has 12 amino acid residues derived from the C-terminus of Fusarium oxysporum.

[0021] 15. In some embodiments, the variant lipase of any of paragraphs 12-14 has substitutions G91A, D96W, and E99K.

[0022] 16. In some embodiments, the variant lipase of any of paragraphs 12-15 has a small number of fewer or additional residues at the C-terminus of the contiguous amino acid sequence of the Thermomyces lanuginosus lipase.

[0023] 17. In some embodiments, the variant lipase of any of paragraphs 12-16 has a truncation of residues at the C-terminus of the contiguous amino acid sequence of the Thermomyces lanuginosus lipase.

[0024] 18. In some embodiments, the variant lipase of any of paragraphs 12-17 has the amino acid sequence of SEQ ID NO: 4.

[0025] 19. In some embodiments of the variant lipase of any of paragraphs 11-18, the variant lipase has improved antifoaming activity in simultaneous saccharification and fermentation in the fermentation process.

[0026] These and other aspects and embodiments of the compositions and methods will be apparent from the description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Is a diagram depicting the simplified structure of the lipase molecule described herein. The black band is the amino acid sequence derived from Thermomyces lanuginosus lipase (TLL). The grey band is the amino acid sequence derived from Fusarium oxysporum lipase (FOX).

[0028] Figure 2 is a Coomassie stained SDS-PAGE gel loaded with samples of the lipase molecules described herein.

[0029] Figure 3 is a graph showing the total protein concentration in culture supernatants from cells expressing LIP3 (squares), LIP4 (diamonds), and LIP5 (triangles).

[0030] Figure 4 It is a graph showing the culture broth lipase activities of LIP3 (squares), LIP4 (diamonds), and LIP5 (triangles).

[0031] Figure 5 is a graph showing the stability of LIP4 (diamonds), LIP5 (triangles) and an unrelated commercially available lipase and its truncated variants (shape 1 and shape 2, respectively). pH values ​​are indicated by a + sign. DETAILED DESCRIPTION

[0032] Prior to describing the various aspects and embodiments of the compositions and methods of the present application, the following definitions and abbreviations are described.

[0033] 1. Definitions and Abbreviations

[0034] In accordance with this detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "a dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0035] This document is organized into sections to facilitate reading; however, the reader will appreciate that statements made in one section can apply to other sections. In this manner, headings for different sections of the disclosure should not be construed as limiting.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following terms are provided below.

[0037] 1.1. Abbreviations and Acronyms

[0038] The following abbreviations / acronyms have the following meanings unless otherwise indicated:

[0039] BSA bovine serum albumin

[0040] °C degrees Celsius

[0041] DCW dry cell weight

[0042] DNA deoxyribonucleic acid

[0043] DS dissolved solids

[0044] FFAeq free fatty acid equivalent

[0045] g or gm gram

[0046] GA glucoamylase

[0047] GAU / g ds glucoamylase activity units per gram of dry solids

[0048] H2O water

[0049] hr hour

[0050] kDa kilodalton

[0051] kg kilogram

[0052] M Moore

[0053] mg milligrams

[0054] min

[0055] mL and ml milliliters

[0056] mm millimeter

[0057] mM millimole

[0058] MW molecular weight

[0059] ppm parts per million, e.g. μg protein / g dry solids

[0060] REMI restriction enzyme-mediated integration

[0061] SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

[0062] sec seconds

[0063] sp. species

[0064] SSF simultaneous saccharification and fermentation

[0065] TP total protein

[0066] Tris-HCl Tris(hydroxymethyl)aminomethane hydrochloride

[0067] U units

[0068] v / v volume / volume

[0069] w / v weight / volume

[0070] w / w weight / weight

[0071] wt% weight percentage

[0072] μg microgram

[0073] μL and μl microliter

[0074] μm micrometer

[0075] μM micromolar

[0076] 1.2. Definitions

[0077] The term "starch" refers to any material consisting of a complex polysaccharide carbohydrate of a plant having the formula (C6H 10 O5) xThe term "starch" refers to a starch that is composed of a plurality of chains of amylose and amylopectin (where "X" can be any number). The term includes plant-based materials such as grains, cereals, grasses, tubers and roots, and more specifically, materials obtained from wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, milo, potato, sweet potato, and tapioca starch. The term "starch" includes granular starch. The term "granular starch" refers to raw starch (i.e., uncooked starch), for example, starch that has not undergone gelatinization.

[0078] The term "lipase" refers to an enzyme that catalyzes the hydrolysis of fats (i.e., lipids). Lipase is a subclass of esterase. As used herein, the term lipase is intended to be broadly interpreted to encompass enzymes classified as EC.3.1.1.X, especially EC.3.1.1.1 and EC.3.1.1.2.

[0079] The term "titratable phospholipase unit (TIPU)" refers to the amount of enzyme that releases 1 μmol free fatty acid equivalents (FFAeq) per minute at 30°C and pH 7.0.

[0080] The terms "protease" and "proteinase" refer to enzyme proteins that have the ability to perform "proteolysis" or "proteolytic cleavage," which refers to the hydrolysis of the peptide bonds that link the amino acids in the peptide or polypeptide chain that form the protein. This activity of a protease as a protein-digesting enzyme is referred to as "proteolytic activity." As used herein, the term lipase is intended to be broadly interpreted to encompass enzymes classified as EC.3.4.X.

[0081] The term "serine protease" refers to an enzyme that cleaves peptide bonds in proteins, wherein the enzyme serine acts as a nucleophilic amino acid at the enzyme active site. Serine proteases are divided into two major categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like. These enzymes are classified as EC.3.4.16.

[0082] The term "glucoamylase" refers to enzymes classified under EC.3.2.1.3 (glucoamylase, α-1,4-D-glucan glucohydrolase) that remove consecutive glucose units from the non-reducing ends of starch. These enzymes can also hydrolyze α-1,6 and α-1,3 linkages, but at a much slower rate than α-1,4 linkages.

[0083] The term "α-amylase" refers to an enzyme classified under EC 3.2.1.1 (α-D-(1→4)-glucan glucanohydrolase), which cleaves α-D-(1→4) O-glycosidic bonds in starch.

[0084] The terms "thermostable" and "thermostability" with respect to enzymes refer to the ability of an enzyme to maintain activity after exposure to elevated temperatures. The thermostability of an enzyme (e.g., an amylase) is measured by its half-life (t1 / 2) given in minutes, hours, or days, during which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring the residual α-amylase activity after exposure to (i.e., challenge with) elevated temperatures.

[0085] The terms "wild-type," "parent," or "reference" with respect to a polypeptide or polynucleotide refer to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid or nucleotide positions.

[0086] References to a wild-type polypeptide are understood to include the mature form of the polypeptide.A "mature" polypeptide or variant thereof is one in which the signal sequence is not present, eg, cleaved from the immature form of the polypeptide during or after expression of the polypeptide.

[0087] The term "variant" with respect to a polypeptide refers to a polypeptide that differs from a specified wild-type, parent, or reference polypeptide because it includes one or more naturally occurring or artificial amino acid substitutions, insertions, or deletions. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parent, or reference polynucleotide. The characteristics of the wild-type, parent, or reference polypeptide or polynucleotide will be apparent from the context.

[0088] The term "recombinant" when used in reference to a subject cell, nucleic acid, protein, or vector indicates that the subject has been modified from its native state.

[0089] The terms "recovered," "isolated," and "separated" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other designated material or component that is removed from at least one other material or component with which it is naturally associated as found in nature.

[0090] "pH range" with respect to an enzyme refers to the range of pH values ​​at which the enzyme exhibits catalytic activity.

[0091] The terms "pH stable" and "pH stability" with respect to an enzyme relate to the ability of an enzyme to remain active for a predetermined period of time (eg, 15 min., 30 min., 1 hour) over a wide range of pH values.

[0092] The term "amino acid sequence" is synonymous with the terms "polypeptide," "protein," and "peptide," and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes." Amino acid sequences are represented using the standard amino-terminal to carboxyl-terminal orientation (i.e., N→C) using the conventional single-letter or three-letter code for amino acid residues.

[0093] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules that are capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded and can contain chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Unless otherwise indicated, nucleic acid sequences are presented in 5'- to 3' orientation.

[0094] The terms "transformed," "stably transformed," and "transgenic" as used with respect to cells mean that the cells contain a non-native (eg, heterologous) nucleic acid sequence integrated into their genome or as an episome that is maintained through multiple generations.

[0095] The term "fermenting organism" refers to any organism suitable for producing a desired fermentation product, including bacteria and fungal organisms (including yeast and filamentous fungi).

[0096] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus or other DNA construct, including a polynucleotide encoding a polypeptide of interest (eg, amylase), has been introduced. The term "host cell" includes protoplasts generated from a cell.

[0097] The term "filamentous fungi" refers to all filamentous forms of species of the subdivision Eumycotina, in particular of the subdivision Pezizomycotina.

[0098] The term "heterologous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in the host cell.

[0099] The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0100] The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes both transcription and translation.

[0101] "Selectable marker" or "selectable marker" refers to a gene that can be expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include, but are not limited to, antimicrobial agents (e.g., hygromycin, bleomycin, or chloramphenicol) and / or genes that confer a metabolic advantage (e.g., a nutritional advantage) on the host cell.

[0102] "Vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like.

[0103] "Expression vector" refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, said coding sequence being operably linked to appropriate control sequences capable of effecting DNA expression in a suitable host.

[0104] The term "operatively linked" means that the specified components are in a relationship that permits them to function in the intended manner, including but not limited to juxtaposition. For example, a regulatory sequence is operatively linked to a coding sequence such that expression of the coding sequence is controlled by the regulatory sequence.

[0105] "Fusion" polypeptide sequences are linked, i.e., operatively linked, by a peptide bond between the two subject polypeptide sequences.

[0106] A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of a protein outside the cell lacks the signal sequence that is cleaved off during the secretion process.

[0107] The term "specific activity" refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specified conditions. Specific activity is typically expressed as units (U) per mg of protein.

[0108] "Percent sequence identity" means that a specified sequence has at least a certain percentage of amino acid residues identical with the amino acid residues in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are:

[0109] Gap open penalty: 10.0

[0110] Gap extension penalty: 0.05

[0111] Protein weight matrix: BLOSUM series

[0112] DNA weight matrix: IUB

[0113] Delay divergent sequences %: 40

[0114] Gap separation distance: 8

[0115] DNA transition weight: 0.50

[0116] List of hydrophilic residues: GPSNDQEKR

[0117] Use negative matrix: No

[0118] Switch special residue penalty: On

[0119] Switch hydrophilic penalty: On

[0120] Switch end gap separation penalty: No

[0121] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process for the production of a biochemical in which a microorganism, such as an ethanol-producing microorganism, and at least one enzyme, such as an amylase, are present in the same process step. SSF includes the simultaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) to sugars (including glucose) and fermentation of the sugars to an alcohol or other biochemical or biomaterial in the same reaction vessel.

[0122] The term "fermented beverage" refers to any beverage produced by a process that includes a fermentation process (e.g., microbial fermentation, such as bacterial and / or fungal fermentation). "Beer" is one example of such a fermented beverage, and the term "beer" is meant to include any fermented wort produced by fermenting / brewing starch-containing plant material.

[0123] The term "malt" refers to any malted cereal grain, such as malted barley or wheat.

[0124] The term "wort" refers to the unfermented liquid run-off after the extraction of the grain during mashing.

[0125] The term "about" refers to ± 15% of the referenced value.

[0126] 2. Variant lipase polypeptides

[0127] One aspect of the compositions and methods of the present application is a variant lipase molecule comprising a combination of mutations that improve its performance in controlling foam inhibition in a fermentation process.

[0128] The variant lipase and methods of using the same are derived from Thermomyces lanuginosus lipase (TLL; see, e.g., NCBI Accession Nos. 059952.1, AOE45082.1, 1DT3_A, and 1GT6_A), as represented by SEQ ID NO: 1 below:

[0129] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFGLIGTCL

[0130] Variant lipases comprise one or more of the substitutions G91A, D96W, and E99K with reference to SEQ ID NO: 1 (see, e.g., SEQ ID NO: 2 in WO 2003 / 099016 A2). In some embodiments, the variant lipase comprises all three substitutions G91A, D96W, and E99K.

[0131] The variant lipase is a fusion protein and further comprises a portion of the C-terminus of Fusarium oxysporum lipase (FOX; NCBI Accession No. ABR12479.1), as represented by the following SEQ ID NO: 6:

[0132] MLLLPLLSAITLAVASPVALDDYVNSLEERAVGVTTTDFGNFKFYIQHGAAAYCNSEAAAGSKITCSNNGCPTVQGNGATIVTSFGSKTGIGGYVATDSARKEIVVSFRGSINIRNWLTNLDFGQEDCSLVSGCGVHSGFQRAWNEISSQATAAVASARKANPSFKVISTGH SLGGAVAVLAAANLRVGGTPVDIYTYGSPRVGNVQLSAFVSSNQAGGEYRVTHADDPVPRLPPLIFGYRHTTPEFWLSGGGGDTVDYTISDVKVCEGAANLGCNGGTLGLDIAAHLHYFQATDACNAGGFSWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS

[0133] In the fusion polypeptide of the present invention, based on the amino acid sequence of SEQ ID NO: 6, the portion of the C-terminus of FOX fused to the TLL portion of the lipase should not exceed 50 consecutive amino acid residues of the most C-terminal portion of FOX, and should not be less than 12 consecutive amino acid residues. In some embodiments, the portion of the C-terminus of FOX should not exceed 15 consecutive amino acid residues of the most C-terminal portion of FOX, and should not be less than 12 consecutive amino acid residues. In some embodiments, the portion of the C-terminus of FOX is 12 consecutive amino acid residues of the most C-terminal portion of FOX.

[0134] In some embodiments, the C-terminal portion of the TLL portion of the variant lipase can have a small number of fewer residues or a small number of additional residues, for example, due to the use of convenient restriction sites for cloning purposes. In some embodiments, the number of fewer or additional residues is 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or even 1 or fewer. In particular embodiments, the number of fewer residues is 7 ± 3, 7 ± 2, 7 ± 1, or exactly ± 7. In one particular embodiment, the number of fewer residues is exactly -7.

[0135] The characteristics of various lipase molecules are summarized in Table 2 in the Examples, and graphical representations of the molecules are provided in Figure 1 Table 1. The amino acid sequence of a particular variant lipase is set forth below as SEQ ID NO: 4:

[0136] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIANLNFWLKKINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFQATDACNAGGFS

[0137] LIP5 described herein is related to Ultra (a baking enzyme that has apparently been renamed a Defoamer for use in ethanol facilities that also use heat-stable proteases) is the same. LIP5 serves as a benchmark for the improved lipase variants described herein. The amino acid sequence of LIP5 is set forth below as SEQ ID NO: 5: EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIANLNFWLKKINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFQATDACNAGGFSWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS

[0138] In some embodiments, the lipase variants of the application have the indicated combination of mutations and a determined degree of amino acid sequence homology / identity to SEQ ID NO: 4, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence homology / identity. Preferably, the variant lipases do not have the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0139] The lipases of the application can include any number of conservative amino acid substitutions. Exemplary conservative amino acid substitutions are listed in Table 1.

[0140] Table 1. Conservative amino acid substitutions

[0141]

[0142]

[0143] The reader will appreciate that some of the foregoing conservative mutations can be produced by genetic manipulation, while others by the introduction of synthetic amino acids into the polypeptide, either genetically or otherwise.

[0144] The variant lipases of the present invention can be "precursor," "immature," or "full-length," in which case they contain a signal sequence and / or a presequence; or "mature," in which case they lack a signal sequence. The mature form of the polypeptide is generally the most useful. Unless otherwise indicated, the amino acid residue numbering used herein refers to the mature form of the corresponding variant lipase polypeptide. The lipase variant polypeptides of the present invention can also be truncated to remove the N-terminus or the C-terminus, as long as the resulting polypeptide retains lipase activity.

[0145] 3. Metal salts

[0146] Any suitable metal salt can be used in combination with the lipase variants of the present invention. Preferred metal salts include metal salts selected from the group consisting of calcium, magnesium, sodium and potassium. Preferred metal salts include divalent ions such as CaCl2, CaCO3, Ca(OH)2, although salts including monovalent metals may also be used.

[0147] 4. Use of the improved variant lipase

[0148] Improved foam suppressing lipase described herein is preferably used in fermentation process well known in the art.Fermentation process generally comprises liquefaction and saccharification of the raw material that comprises starch (for example from cereal).Any variation of liquefaction or saccharification can be used in combination with fermentation process of the present invention.For example, liquefaction and saccharification can be carried out simultaneously or in overlapping mode.Similarly, saccharification and fermentation can be carried out separately or simultaneously, as the situation of saccharification and fermentation (SSF) simultaneously.

[0149] The raw materials for the fermentation process can be obtained from tubers, roots, stems, cobs, beans, cereals or whole grains. More specifically, granular starch can be obtained from corn, cobs, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas, or potatoes.

[0150] The improved anti-foam lipase variants described herein are suitable for use in fermentation processes that include thermal gelatinization of ground grains (i.e., "conventional fermentation" processes), as well as in fermentation processes that do not include such thermal gelatinization (i.e., "raw starch hydrolysis" or "cold cooking" processes), where liquefaction is performed at or below the gelatinization temperature. Conventional fermentation processes to which the anti-foam system of the present invention can be applied are described, for example, in WO 199628567 and WO 200238787. Cold cooking processes to which the anti-foam system of the present invention can be applied are described, for example, in WO 2003 / 66816, WO 2003 / 66826, and WO 2004 / 080923.

[0151] The lipase of the present invention, and optionally other enzymes and metal salts, are preferably added before fermentation or early in the fermentation, when foaming is the biggest problem. Typically, addition is made sometime during saccharification. In the case of SSF, addition is typically made early in the SSF process. Addition can even be made during liquefaction, as long as the variant lipase is not destroyed by heat. Addition can be made simultaneously with yeast addition, and yeast products mixed with the variant lipase, or even yeast expressing the variant lipase, are contemplated.

[0152] Examples

[0153] Example 1

[0154] Construction of lipase expression vector

[0155] A series of expression vectors were constructed to express codon-optimized Thermomyces lanuginosus lipase (TLL; SEQ ID NO: 1) and variants thereof in Trichoderma reesei. Figure 1 The characteristics of the parent molecule and variants are shown, including mutations relative to the parent TLL. For numbering and naming convenience, LIP1 is wild-type TLL (i.e., SEQ ID NO: 1), and LIP2-LIP5 (SEQ ID NO: 2-5) are variants. All four variants include substitutions G91A, D96W, and E99K (see, e.g., SEQ ID NO: 2 in WO 2003 / 099016 A2). LIP3-LIP5 also include a small truncation of the C-terminus of TLL and a fusion of different lengths of the C-terminus of Fusarium oxysporum lipase (FOX; SEQ ID NO: 6).

[0156] Genes encoding variants were generated using standard molecular biology techniques based on the codon-optimized sequence of SEQ ID NO: 7. All genes were under the transcriptional control of (i.e., operably linked to) the native Trichoderma reesei cbhl promoter and terminator. The expression vector included the pyr2 selectable marker (encoding orotate phosphoribosyltransferase) and the TLL gene upstream of the cbhl promoter.

[0157] The characteristics of various lipase molecules are summarized in Table 2 and Figure 1 A schematic representation of the molecule is shown in FIG. Ultra (a baking enzyme, apparently renamed PROTREAT TM Defoamers, for use in ethanol applications where heat-stable proteases are also used) are the same.

[0158] Table 2. Characteristics of TLL lipase and its variants

[0159]

[0160] The amino acid sequences of LIP1-LIP5 and FOX, as well as the nucleotide sequence of the codon-optimized gene encoding TLL are shown below. It should be noted that SEQ ID NOs refer to mature polypeptide sequences (ie, without signal sequences) unless otherwise indicated.

[0161] The mature amino acid sequence of LIP1 is shown in SEQ ID NO: 1:

[0162] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVRE HPDYRVVFTGHSLGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFGLIGTCL

[0163] The mature amino acid sequence of LIP2 is shown in SEQ ID NO: 2:

[0164] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIANLNFWLKKINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVRE HPDYRVVFTGHSLGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFGLIGTCL

[0165] The mature amino acid sequence of LIP3 is shown in SEQ ID NO: 3:

[0166] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIANLNFWLKKINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFQATDACNAGGF

[0167] The mature amino acid sequence of LIP4 is shown below as SEQ ID NO: 4:

[0168] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIANLNFWLKKINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFQATDACNAGGF

[0169] The mature amino acid sequence of LIP5 is shown below as SEQ ID NO: 5:

[0170] EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDVTGFLALDNTNKLIVLSFRGSRSIENWIANLNFWLKKINDICSGCRGHDGFTSSWRSVADTLRQKVEDAVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRITHTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVTRNDIVKIEGIDATGGNNQPNIPDIPAHLWYFQATDACNAGGFSWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS

[0171] The mature amino acid sequence of FOX lipase (NCBI Accession No. ABR12479.1) (SEQ ID NO: 6):

[0172] MLLLPLLSAITLAVASPVALDDYVNSLEERAVGVTTTDFGNFKFYIQHGAAAYCNSEAAAGSKITCSNNGCPTVQGNGATIVTSFGSKTGIGGYVATDSARKEIVVSFRGSINIRNWLTNLDFGQEDCSLVSGCGVHSGFQRAWNEISSQATAAVASARKANPSFKVISTGHSLGGAVAVLAAANLRVGGTPVDIYTYGSPRVGNVQLSAFVSNQAGGEYRVTHADDPVPRLPPLIFGYRHTTPEFWLSGGGGDTVDYTISDVKVCEGAANLGCNGGTLGLDIAAHLHYFQATDACNAGGFSWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS

[0173] The nucleotide sequence of the codon-optimized gene encoding LIP1 (i.e., TLL) is shown below as SEQ ID NO: 7 (the start codon is underlined):

[0174] CACAAGTTTGTACAAAAAAGCAGGCTCCGCGCCACC ATGCGCAGCTCCCTTGTTCTGTTCTTCGTCAGCGCGTGGACGGCCTTGGCCTCCCCTATTCGTCGAGAGGTCTCGCAAGATCTGTTCAACCAGTTCAATCTCTTCGCTCAGTATTCTGCAGCCGCCTACTGCGGAAAGAACAACGACGCCCCCGCTGGTACCAACATCACGTGCACGGGCAACGCCTGCCCCGAGGTCGAGAAGGCGGACGCCACGTTTCTCTACTCGTTCGAGGACAGCGGCGTGGGCGATGTCACCGGCTTCCTGGCTCTCGACAACACGAACAAGCTCATCGTCCTCTCTTTCCGCGGCAGCCGGTCCATCGAGAACTGGATCGGCAACCTTAACTTCGACCTCAAGGAGATCAACGACATCTGCTCCGGCTGCCGCGGCCACGACGGCTTCACTTCGTCCTGGAGGAGCGTCGCCGACACGCTGCGCCAGAAGGTGGAGGACGCTGTGCGCGAGCATCCCGACTACCGCGTTGTTTTTACCGGACACAGCCTCGGTGGTGCGCTCGCTACTGTTGCCGGAGCCGACCTGCGCGGCAATGGGTACGACATCGACGTGTTCAGCTATGGCGCCCCCCGAGTCGGAAACCGCGCTTTCGCCGAGTTCCTGACCGTCCAGACCGGCGGCACTCTCTACCGCATCACCCACACCAACGATATTGTCCCTCGCCTCCCCCCGCGCGAATTCGGTTACAGCCACTCTAGCCCCGAGTACTGGATCAAGTCTGGCACCCTCGTCCCCGTCACCCGAAACGACATCGTGAAGATCGAGGGCATCGATGCCACCGGCGGCAACAACCAGCCTAACATTCCGGACATCCCTGCGCACCTGTGGTACTTCGGTCTGATCGGTACCTGTCTTTGAGCGCGCCGACCCAGCTTTCTTGTACAAAGT

[0175] 实例2

[0176] 原生质体制备和转化

[0177] Spores of Trichoderma were inoculated into 50 mL of YEG medium (5 g / L yeast extract, 20 g / L glucose) and grown overnight in a shaking incubator at 28 °C at 180 rpm in 250 mL shake flasks with a throw of 50 mm. Germinated spores were collected by 10-min centrifugation (3,000 g) and washed twice with 10 mL of 1.2 M gSO4, 10 mM Na-phosphate (pH 5.8). The pellet was resuspended in 40 mL of the same buffer supplemented with 1.2 g lyticase (Sigma, St. Louis, MO) and incubated in a shaking incubator at 100-200 rpm at 28 °C until protoplasts were formed. The suspension was filtered through MIRACLOTH TM (Millipore-Sigma) to remove mycelium and an equal volume of 0.6 M sorbitol, 0.1 M Tris-HCl (pH 7.0) was gently added on top of the protoplast solution, centrifuged at 4,000 rpm for 15 min. Protoplasts were collected from the interphase region and transferred to a new tube. An equal volume of 1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl (pH 7.5) was added and protoplasts were pelleted and washed with 1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl (pH 7.5) at 4,000 rpm for 15 min (4 °C). Finally, protoplasts were resuspended in the same buffer to a concentration of 1 x 10 8 protoplasts / mL and 50 pL of 25% PEG 6000, 50 mM CaCl2, 10 mM Tris-HCl (pH 7.5) was added per 200 pL of protoplasts and the resulting suspension was stored at -80 °C.

[0178] PCR products containing the genes described in Example 1 were used to transform protoplasts. If REMI was used, 5-20 units of a restriction endonuclease were added with the DNA. 5-20 pg of DNA was added to 200 pL of protoplasts and incubated on ice for 20 min. After that, the transformation mixture was transferred to room temperature and 2 mL of 25% PEG 6,000, CaCl2, 10 mM Tris-HCl (pH 7.5) and 4 mL of 1.2 M sorbitol, 10 mM CaCl2, 10 mM Tris-HCl (pH 7.5) was added.

[0179] Transformants were selected for uridine prototrophy on AmdS medium supplemented with 10 mM NH3Cl. To prepare this medium, 2X AmdS solution (30 g / L KH2PO4, 20 mM acetamide, 1.2 g / L MgSO4 7H2O, 1.2 g / L CaCl2 2H2O, 0.48 g / L citric acid H2O, 0.5 g / L FeSO4 7H2O, 40 mg / L ZnSO4 7H2O, 8 mg / L CuSO4 5H2O, 3.5 mg / L MnSO4 H2O, 2 mg / L H3BO3 (boric acid), 40 g / L glucose (pH 4.5) was mixed with an equal volume of 4% agar containing 2 M sorbitol. Other minimal media without uridine are also suitable.

[0180] Example 3

[0181] Expression and characterization of TLL molecules

[0182] The expression of proteins in suspension Trichoderma cultures has been described. Transformants expressing various TLL molecules from Example 1 were inoculated into conventional Trichoderma fermentation medium and subjected to standard fermentations.

[0183] The fermentation samples were analyzed for lipase expression levels by SDS-PAGE analysis and using a lipase activity assay. Images of Coomassie-stained SDS-PAGE gels are shown in Figure 2 The horizontal lines under LIP3-LIP5 indicate that two transformants require two gel lanes for growth. The expression levels of LIP1, LIP3, and LIP4 are slightly higher than that of LIP5.

[0184] Total protein (TP) production, phospholipase activity, and specific activity were measured in submerged fermentation cultures grown at 28°C, pH 5.75-6.0 with a sugar feed rate of 0.06 g glucose / g DCW / hr.

[0185] The total protein concentration in the culture supernatant from cells expressing LIP3-LIP5 was measured using the Biuret method with BSA as a standard and is shown in Figure 3In the figure. Phospholipase activity was determined using Triton-X 100 as an emulsifier and L-α-phosphatidylcholine (Avanti 441601G, Avanti Polar Lipids, USA) as a substrate, which was dissolved in 50 mM HEPES buffer containing 5 mM CaCl2. The amount of free fatty acids released during the enzymatic reaction was measured using the NEFA kit (Wako Chemicals GmbH, Germany). The results are reported in titratable phospholipase units (TIPU), which refers to the amount of enzyme that releases 1 μmol free fatty acid equivalent (FFAeq) per minute at 30°C and pH 7.0. The results are shown in Figure 4 In the picture.

[0186] The amount of lipase in the samples was quantified as the fraction of TP based on the density ratio of the bands on Coomassie Brilliant Blue-stained SDS-PAGE gels analyzed using the gel analysis module in ImageJ software. The specific activity relative to the lipase protein was calculated and summarized in Table 3.

[0187] Table 3. Examples of lipase expression and specific activity analysis

[0188]

[0189] The expression of LIP4 was better than that of LIP3 and LIP5, as were its activity and specific activity in culture medium (see, e.g., Figure 4 and Table 3 ).

[0190] Example 4

[0191] Antifoaming properties of TLL variants

[0192] The effects of lipase molecules LIP2-LIP5 on foam formation and ethanol production were tested in a laboratory-scale simultaneous saccharification and fermentation (SSF) using protease-treated and untreated liquefacts. LIP1 had previously been shown to be a poor defoaming enzyme (data not shown) and was therefore not included in the experiments. LIP5, a representative commercial product, was used as a benchmark.

[0193] Corn kernels were ground using a Retsch ZM200 mill (Arie Blok Animal Nutrition, NL-3440AA Woerden, product number 377) and set as follows: 3 mm screen, 1,0000 rpm. The resulting corn flour was used to produce a 2 kg slurry batch at 34% dry solids by adding tap water to the flour. The pH of the slurry was adjusted to pH 5.1 with H2SO4. A product containing α-amylase ( RSL, DuPont), followed by the addition of thermostable protease ME-3 (WO 2018 / 118815) to a final concentration of 4 μg / g DS. The mixture was incubated at 85°C for 2 h with overhead stirring. A control sample was generated in which no protease was added to the liquefied slurry. After incubation, the treated material (liquefact) was used in subsequent SSF experiments.

[0194] The pH of the liquefact was adjusted to pH 4.8 with H2SO4. Urea and glucoamylase product ( PRIME LC, DuPont Industrial Biosciences). 0.1% w / w dry active yeast ( PRIME ADY; DuPont Industrial Biosciences) was used for fermentation. Acid fungal protease (FERMGEN) with zero or 0.1 SAPU / g DS was used for fermentation. TM 2.5x (DuPont Industrial Biosciences, abbreviated as AFP in this article).

[0195] Add antifoaming agent lipase to SSF at a dosage of 0.5 ug / g DS. Dispense the SSF mixture into 250 mL polypropylene graded cylinders. Place the cylinders with foam plugs in a 32°C water bath and stir magnetically at 350 rpm.

[0196] After a few hours of fermentation, foam began to accumulate on top of the SSF mixture, leaving a streak on the cylinder walls that remained visible even after the foam collapsed. The level of foam produced during the SSF was recorded after 16 hours of incubation and expressed as volume. The reduction in levels for replicate samples is shown in Table 3.

[0197] Table 3. Summary of relative foam levels in different SSF mixtures after 16 h

[0198]

[0199] The results showed that the addition of lipase had a positive impact on controlling the foam level of the fermentation system, even when increasing urea was used and acid proteases were added to the SSF mixture. During SSF, LIP4 exhibited the lowest level of foaming in both the presence and absence of a thermostable protease.

[0200] Example 5

[0201] Stability of TLL variants

[0202] The stability of LIP4, LIP5 and unrelated commercially available lipases and their truncated variants was determined under SSF conditions. Briefly, a 50 mL volume of SSF substrate (representing corn liquefact obtained from corn flour and tap water at unadjusted pH 5.5 as described in Example 4) was mixed with a commercially relevant dose of glucoamylase ( XP, DuPont) and 0.1% w / w dry active yeast (ETHANOL Yeast (Lesaffre Advanced Fermentations) was incubated in the presence of 352 ppm urea at 33°C in an orbital shaker at 150 rpm. The dosage of LIP4, LIP5, and other lipases was 0.9 TIPU / g DS. Fermentation broth samples were regularly withdrawn and centrifuged at 12,000 × g to pellet insoluble material, and the supernatant was tested for residual lipase activity. Figure 5 As shown in the figure, LIP4 is clearly more stable than the other molecules tested.

Claims

1. A method for reducing foaming in an ethanol production process using a carbohydrate substrate as a feedstock, the method comprising adding a fusion protein before or during a fermentation step, wherein the fusion protein has improved antifoaming activity in the fermentation process compared to a reference lipase having the amino acid sequence of SEQ ID NO: 5, the fusion protein having the amino acid sequence of SEQ ID NO: 4, wherein the fusion protein consists of: 1) the entire contiguous amino acid sequence of a Thermomyces lanuginosus lipase variant, said amino acid sequence comprising, with reference to SEQ ID NO: 4, an N-terminus having substitutions G91A, D96W, and E99K; and 2) as its C-terminus, 13 amino acid residues derived from the C-terminus of a Fusarium oxysporum lipase.

2. The method of claim 1, wherein the fermentation process is saccharification and / or fermentation.

3. The method of claim 1 or 2, wherein the fermentation process is simultaneous saccharification and fermentation.

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