# Patent

## Title
Genes and enzymes for degradation of ferulic acid crosslinks

## Abstract

Novel genes that code for a family of feruloyl esterases that break down ferulic acid crosslinks between polysaccharide chains and between polysaccharides and lignins in plant cell walls are described herein as well as a method of rapid gene discovery.

## Background
RELATED APPLICATIONS
This application is claims priority to U.S. Provisional Patent Application Ser. No. 61/247,499, filed Sep. 30, 2009 the contents of which are incorporated herein by reference in their entirety.


FIELD OF THE INVENTION
The invention relates to novel genes that code for a family of feruloyl esterases that break down ferulic acid crosslinks between polysaccharide chains and between polysaccharides and lignins in plant cell walls and a rapid method of gene discovery.
BACKGROUND OF THE INVENTION
Ferulic acid is an important component of plant material, forming crosslinks between polysaccharide chains and between polysaccharides and lignin, thus providing structural rigidity to cell walls. Ferulic acid is a cinnamic acid with the chemical name (3-methoxy-4-hydroxy)-3-phenyl-2-propenoic acid, or 3-methoxy-4-hydroxy-cinnamic acid. In the plant cell wall, ferulic acid is bonded via an ester linkage to hydroxyls on sugars, usually arabinose moieties in arabinoxylans or galactose residues in pectins. Ferulic acid dimers and trimers formed by linkages between the phenolic groups provide covalent crosslinks among cellulose, arabinoxylan, xyloglucan, pectin, lignin, as well as protein. The amount of dimers account for 0.14% and 2.5% w/w of the enzyme digest of sugar-beet pulp and corn bran, respectively, suggesting a high degree of crosslinking in the bran cell wall of corn. It has been calculated that each heteroxylan macromolecule contains  ̃75 esterified ferulic acid groups and  ̃30 diferulic bridges. These crosslinks limit carbohydrate bioavailability, resulting in lower conversion of the plant material into useful products or nutrients. To overcome this limitation, the ester bonds are hydrolyzed by feruloyl esterases, which are produced by numerous microorganisms that utilize complex plant material as nutrients. Feruloly esterases (FAE, E.C. 3.1.1.73) (ferulic acid esterases, cinnamoyl esterases, cinnamic acid hydrolases, p-coumaroyl esterases, hydroxycinnamoyl esterases, etc.) belong to a subclass of carboxylic esterases (E.C. 3.1.1). The enzyme cleaves ester bonds between hydroxycinnamic acids esterified to arabinoxylans and certain pectins present in plant cell walls.
Feruloly esterase forms a part of the enzyme complexes that are elaborated by fungi or bacteria that metabolize plant materials. FAE plays a key role in enhancing the accessibility of other biomass-degrading enzymes, and subsequent hydrolysis of plant fibers by removing the ferulic acid side chains and crosslinks. This enzyme reaction may well be an important controlling factor for increasing the extent of degradation of lignocellulosic biomass for bioenergy conversion, and pulp and paper manufacture. In biomass degradation, FAE is an integral part of an enzyme system that acts collectively and synergistically with a variety of other cellulolytic and xylanolytic enzymes to enhance biomass degradation. This in turn increases the yield of hexose and pentose sugars in the bioconversion as feedstock for yeast fermentation to biofuel or other value-added chemicals. The enzyme also aids in solubilizing lignin-polysaccharide complexes in paper pulp processing. The enzyme together with a number of glycanases and oxidases, have been implicated in the improvement of bread-making quality and related cereal processing. The importance of FAE also relates to the enzyme product, ferulic acid and feruloylated oligosaccharides, which have potential applications for food and medicinal uses. Ferulic acid and its derivatives are strong antioxidants, and have gel-forming properties. The biotransformation of ferulic acid to vanillin has been extensively investigated. The antioxidative and gelling effects have been utilized to form potential protective agents against photooxidative skin damage and for wound management.
The use of feruloyl esterases for biomass degradation and conversion has been severely hampered by the fact that there are very few known existing gene sequences and enzymes, due to the lack of workable, effective, high-throughput method for direct gene discovery of this group of enzymes. An expanded diverse pool of feruloyl esterase genes/enzymes would enable fine control over processing of complex and variable biomass materials. The immediate impact of the development is to increase the extent of degradation of lignocellulosic biomass for bioenergy conversion as well as for food and medicinal uses.
Technologies enabling rapid discovery of new genes, new enzymes, new reactions and processes are key to continuous growth in the use of biocatalytic processes in many industries. Increasing the number of enzymatic candidates feasible for increasing the commercial viability of biomass conversion is key for the fuel ethanol industry and would equally benefit other industries, such as pharmaceuticals, diagnostics, cosmetic, food and beverages and other sectors employing biocatalysis as a technology platform. A method enabling rapid discovery and isolation of more efficient biocatalytic genes is therefore desired.
SUMMARY OF THE INVENTION
An embodiment of the invention is novel ferulic acid esterase genes. Another embodiment is the ferulic acid esteases coded for by these genes. Cloning of these new genes in recombinant hosts provides a wide selection of feruloyl esterases with biochemical properties uniquely tailored for target applications.
A further embodiment is the use of these esterases in the degradation of biomass or plant matter from various waste or processing streams, including but not limited to agricultural food and animal feed processing, paper processing; wherein the phenolic by products of this processing finds further application as products in food, medicine and biofuel.
A further embodiment is a novel method for rapid direct gene discovery that includes the formulation and use of a substrate gel assay plate, and an enrichment process by serial dilutions progressive to isolate the target gene from DNA libraries constructed from individual organisms or collections of organisms.


DESCRIPTION OF THE INVENTION
The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated, the numerical properties set forth in the following specification and claims are approximations that may vary depending on the desired properties sought to be obtained in embodiments of the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from error found in their respective measurement.
It will be understood by those skilled in the art that the terms “ferulic acid esterase” or “feruloyl esterase” as described herein refers to those esterases containing nucleic acid sequences synonymous with the exemplified coding sequences of SEQ ID NO's 1-12. Nucleic acid sequences are synonymous if the amino acid sequences encoded by those nucleic acid sequences are the same. The degeneracy of the genetic code is well known to the art. For many amino acids, there is more than one nucleotide triplet which serves as the codon for a particular amino acid, and one of ordinary skill in the art understands nucleotide or codon substitutions which do not affect the amino acid(s) encoded. It is further understood in the art that codon substitutions to conform to common codon usage in a particular recombinant host cell is sometimes desirable.
A total of 12 FAE genes have been isolated, sequenced, and cloned, represented by SEQ ID NO.'s 1-12. The primary structures are presented in FIG. 1. A BLAST of public databanks shows highest bit score in a range from 32 to 48% identity. In the case of C1, C2, C3, C8, C9, and C10 the top hit scores refer to hydrolases of α/β family. C4 is most similar (41% identical) to the fungal Orpinomyces sp. PC-2 FAE. The rest of the clones have hit scores referring to esterases of some type. All taken together, these 12 FAE genes are novel.







TABLE 1





THE PRIMARY STRUCTURES OF Cl TO C12
















Cl (SEQ ID NO: 1)
MVIFCHGFSG TKDGPLFELV ADTLQAHGIA SIRFDFNGHG



ESEGEFKDMT VPNEIVDAKK VVEYVRDLKY VSSLAIAGHS



QGGVVAAMTA GQLSEELGEP AFQAVALMAP AAVLRDDAIR



GSTMGKQYDP FDPGEYVELW GGLKLGGKYI RTAFTLPIYE



TAAKYQGPAL IIHGTADRVV PYTYGERFHQ IWPKSQLVIQ



DYFDHGFSQN VYRTTDIVSD YLIKQLTG


 


C2 (SEQ ID NO: 2)
MALFSLFSPR PSYEVFGSHG GISFTLTLPD SFDPSKDKCP



MAILMHGFMS KKEMYPMPAI AKALAKAGIA SIRFDFDAHG



KSEGRFMDMT ISSEIADAKA VLAYARNLPF VTDIALIGHS



QGGVVAGMLA GELESRPDRP KCVVQLAPAA VLKDDAIAGR



CMHAKYDASN PPEYVNVFFH KLGRSFILEA QKLPIYEVSA



QYSGPVCLIH GDKDKIVPLK YSEHYHEAYK TSELHVLKGE



GHLLNGDKTR LIETVTTFLN RHL


 


C3 (SEQ ID NO: 3)
MVIFCHGFTG RKDGPMFELI ADTLQAHGIA SIRFDFNGHG



ESEGDFKDMT VPNEIEDAKK VVAYVRDLRY VSSLAIVGHS



QGGVVAAMTA GQLSEELGRP AFKAVALMAP AAVLRDDAIR



GNTMGKQYDP FDPGEYVELW GGLKLGGQYI RTAFSLPIYE



TAVKYQGPAL IIHGNGDRVV PYTYGERFHQ IWPMSELVIQ



EYFDHGFSQN IYRTTDIVSD YLIKQLK


 


C4 (SEQ ID NO: 4)
VNISYTAHDT EANGRTYTKK ANVYLPAGYS PDKKYNVLYL



LHGIGGNENE WGMTGNNSTV KAIMDNLSYY GDIDSFIVVT



PNGKASASGS TNSFYNFGAE LRYDLIPYID SHYSTNADRD



HRAIAGLSMG GMQTINIGIG ECVDLFGYFG AFSAAPTSNA



ASKTASLLNG NSYPIHYFYN VCGLQDGIAY SSHSQAAKNL



PSVCNQFVNG QNYMWQELNG GHDFNIWYLG FYNFAQIAFK


 


C5 (SEQ ID NO: 5)
MKKTILSVCM CCLSAVAMAQ PAGGFGGFQA PQVKLETSQE



WKDVNYAGDD QAYHTCDIYL PKQEKASYPV VIHIYGSAWF



SNNSKGMADL GTIVKSLLDA GFAVVCPNHR SSMDAKWPAQ



IHDIRAVIRF VRGEAKKYKF DTKFIATSGF SSGGHLASTA



ATTSGTKQTK VGTVDIDLEG NVGNYLNESS AVDAACDWSG



PIDLTAMDCG ESMKMGENSP EDVLLNSKLA KEPDKYLSLS



ANTYVDKNDP PIIIFHGEKD NVVPCCQGKA FFETLKAAGV



KTEATFVPEG SHGGPAMYVE ENLQKMVNFL KALL


 


C6 (SEQ ID NO: 6)
MKKLAMISMT ALLAGCTAAP DLEKQIDELY QKMPQEERIA



QLRSMYMDEL FDEAGNLDTA KCRELIPYGI GHFSQFALQK



PRDPNDIRDK VVAVQDWLMH NTPNGIPALF HEEVLSGINT



KGSTIYPQQI GQAGSFNTAL AELKTRQTST AMRKMGGVLA



LSPMVDVCRT PSFNRLEESY GEDAYLSAAM GVAFVKGLQQ



GDLKKGVGAC TKHYLGYGGG GDAEEKELME EILLPHETMI



RKTGSVAVMP GYHDVHGTRC VCNSEILQDI LRDYVGFDGM



VVSDYTAIDQ IPGLDSVVQK AAAAINNGND VDFPHGANYK



FLQDAIDQEL VKPEVLERAV KNVLRIKFRA GLFDKDAYLY



STENITLDTP EERQTAYDIA TQSVVLLENK GVLPLKEAKN



ILLTGPNANT MWAMLGDYSF PAMSYFWKRV QDDLDHPHTI



TLLEGMKAKA PEGVNLMYER GCDWTEEIET KYGELGDARA



WEYELLHRKV DSGEKADKAN ALKLAKLADV IVAAVGENVM



LCGENRDRKG LRLPGKQEQF VKELLATGKP VVLVMFGGRA



QVVSGLAEQC AAVIQAWYPG EEGGNAVADI LYGKVSPSAK



LSVSYPNTEV YEPLCYNCQA EKDPRVQWPF GYGLSYTTFE



YQNLKVDSAA TTADQSINLS FEVKNTGQVA ADEIAQIYLS



PTADDQQIRP IQLQGFARVS LNPGETKTVK VKLYTEQFGF



YTNDGKRLWI VRPGSFIVKV GASSQDIRLQ QQVTLSGNLV



SNPLKEFYFS KTSIE


 


C7 (SEQ ID NO: 7)
MAFITVNFMS EALMRTVTVH VVLPADKIAE PGMPEPKHTD



FPALYLLHGV FGNQTDWALR TRVQRMAENS DLALIMPAGE



NAFYLDQEAT HANYGDFVGR ELPEIMRRMF PLSPRREDCF



IAGLSMGGYG ALRNGLKYHE TFSRIGAFSA ALVLDGIENR



TNDSPLFIER RDYAEAIFGP LDKVAESDIN PLWIARRLVE



SGTELPGLYL ACGTEDFLFE PNVRFRDEVR KLGCELTWDE



GPYGHEWDFW NLQVEKFIDW LPLSESGTGI DSGNVGI


 


C8 (SEQ ID NO: 8)
MKRKNHISLA MAFLAIGLMG TTVAKAQSAQ PDFDDKYATE



MVKAGTTAPD FKMKTPDGKT IQLSKYIKAR PKDKGKTVVL



DFWASWCPDC RKDAPEVVRL YEKYRPYGIE FIGISMDTDV



EAWKKAIEQY GITYPQVSEL KKFKETDIAK AYGVKWIPSM



VVVGPDGEVK LSTVLTYKVD KYLKELTTGK YAGPGKGEAV



FIDGDHGRLK AIIQKPELQQ GEKCPMVIFC HGFSGRKDGP



MFELIADTLQ AHGIASIRFD FNGHGESEGE FKDMTVPNEI



EDAKKVVEYV RDLRYVSSLA IVGHSQGGVV AAMTAGQLSE



ALGEPAFKAV ALMAPAAVLR DDAIRGNTMG KQYDPFDPGE



YVELWGGLKL GGKYIRTAFS LPIYETAAKY QGPALVIHGN



ADRVVPYTYG ERFHQIWPNS ELVIQEYFDH GFSQNLYRTT



DIVSEYLIKQ LKK


 


C9 (SEQ ID NO: 9)
GKGETVFIDG DLGRLKALIQ KPALQQGEKC PMVIFCHGFS



GTKDGPLFEL VADTLQAHGI ASIRFDFNGH GESEGEFKDM



TVPNEIEDAK KVVEYVSDLR YVSSLAIVGH SQGGVVAAMT



AGQLSEELGE SPFKAVVLMA PAAVLRDDAI RGSTMGKQYD



PFDPGEYVEL WGGLKLGGQY IRTAFSLPIY ETAAKYQGPA



LVIHGNADRV VPYTYGERFH QIWPKSELVI QEYFDHGFSQ



NIYRTTDIVS EYLIKQLKSK


 


C10 (SEQ ID NO: 10)
MGARVNAVMD EAVSGNKIVG AELIVYRHGD LVLRRTAGHF



DREAGTPMIE NAIYRLASVT KPIVAATALA MIDKGLLRLD



DLVSDHLDYF APLLDDGSPA PITIHHLLTH TAGLAYGYDD



EAISTGLGPT DNDFRSNFTR IAKAPLLFAP GSGWNYSVAI



DVLGAVLAAV HGGSLQDAVH AHITGPLGMD ETGFFVADLA



RLAKPYADGA PEPTAMTDPQ SVIGEDGGSV VFSPSRIFSD



KAFQSGGAGM AGTPENIAKF LETLRQGGGS VLRPDTVALA



FSNRIGDLYR QDQGQRFGYF GAVIDDPVAA ESPSGAGTVN



WGGVYGHSWL VDPTNAITIV SMSNTALEGC TGRYPKDLIR



AVYDDLS


 


C11 (SEQ ID NO: 11)
MTTLLLILLL ALALIAAAAW LFTVRTVRKV EAFLPPQGRF



VEVPGARLHI VEKGEGRPLL LIHGLAGVLN HFTYGMVDEL



AKHYRVIAVD RPGSGYSVRG AGASANLFDQ ADVMAALIDN



LKLDRPVVVG HSLGGAVSLA LAQRHPQKVA ALALIAPLTH



KPARISPAFD GLKIPSNWLR HALAWTLAVP VGLIKRDEIL



AIVFGPEPVL EDFGTQGGGL LGVRPSHFIA ACADMNSELA



QLLDMEKRYP AMQLPVGVFF GRGDLILDYR EQGEALASKV



PGAELLLVDG GHMLPLTMVQ KSADFVRGVV ARA


 


C12 (SEQ ID NO: 12)
MPIPDMAEFF TLAHFSDVHL PPVFGSGWRH WNAKRALGYM



NWLRKRRRVH HGEVADKLLA DAAALRVDHI AITGDLINLG



LPSEYEAAHA WLQSIGSPED VTVVPGNHDI YSSLHGDPGV



ARWAEYMGGE NDTLAFPFVR RVGPIAVVGL NSAVETPPFI



ASGRLGAHQL EIAGEQLEAL GEKGIARVVM IHHPPLPDLA



PPRRALSDAA HFAHLLERGN AELVIYGHNH QSRVDWLPSR



TKPIPVVGVA SASAGVTHGD EPLASYNLFT FFKSDSGLRI



RHVVRGIDAP NSPVRKISEA VLTPPP









Some of the genes possess notable features. The C1, C3, C8, and C9 genes have a TlpA-like ORF (thioredoxin-like open reading frame) at the 5′ end of the sequence. Theoredoxin-like proteins are known to function in various cellular processes to reduce, oxidize, or isomerize thiol:disulfides. A partial sequence of a pullulanase is found in the opposite orientation of the C5 gene sequence. A sequence coding for ketopantoate reductase is located in opposite direction of the C9 gene.







TABLE 2







FAE genes and proteins characterization

















Number







%
of







Identity
Residues





Gene Location

to Ref
(Includes
SEQ ID
Recombinant


Clone Name
(Insert Size)
Reference Sequence
Seq
tag)
NO
Size (kDa)
















Rum-C1
1811-2497 
ZP_01061020.1 
45
229
1
25.2



(687 bp)
Hydrolase of ~ Family






Rum-C2
 587-1396 
ZP_01061020.1 
45
269
2
29.7



(810 bp)
Hydrolase of ~ Family






Rum-C3
 663-1343 
ZP_01061020.1 
46
381
3
41.9



(681 bp)
Hydrolase of ~ Family






Rum6-C4
112-831 
AAF70241 
41
246
4
27.1



(720 bp)
Feruloyl Esterase A






Rum-C5
  60-1001 
ZP_01122099.1 
32
416
5
45



(942 bp)
probable lipase/esterase






Rum-C6
2871-3827 
YP_169760.1 
34
422
6
45.6



(957 bp)
hypothetical protein 








lipase/esterase






Rum-C7
 447-1277 
ZP_01777732 
48
277
7
32.3



(831 bp)
putative esterase






Rum-C8
1849-3207 
ZP_01061020.1 
47
382
8
41.9



(1359 bp)
Hydrolase of ~ Family






Rum-C9
  66-1403 
ZP_01061020.1 
47
383
9
41.9



(1338 bp)
Hydrolase of ~ Family






Mul-C10
 181-1281 
ZP_01421329 
44
367
10
39.7



(1101 bp)
Beta-lactamase






Mul-C11
 116-1057 
YP_583166 
54
322
11
34.7



(942 bp)
alpha/beta hydrolase






Mul-C12
1495-2412 
YP_578455 
43
319
12
34.8



(918 bp)
metallophosphoesterase









The gene sequences range from 681 bp (C3) to 1359 bp (C8). The upper range is the result of the additional Tlp-like protein sequence at the 5′ end, which was not used for cloning. The structural gene sequences vary from 681 (C3) to 1101 (C10) bp. In the gene-vector construction, the signal sequence was deleted before ligation to the Trx-tag sequence in the pET32b vector.
Phylogenetic tree analysis of relatedness with known FAE's suggests that the novel FAE's fall within three major clusters. C5 and C6 in cluster 1 have a common node, so do C7 and C11, C9 and C10 in cluster 2, as well as C3 and C8 in cluster 3. This relationship is also reflected in the sequence homology. For example, C5 and C6 sequences share 84% identity and 90% similarity. In terms of calculated distance values, C2 and C5 show lesser degree of divergence than the others. C1, C3, C7, C8, C9, C11, and C12 have greater divergence, while C1, C2, and C10 are in the intermediate range. All these results provide further support on the diverse and novel nature of the genes.
The C1, C2, C4, and C7 genes containing the signal sequence cloned into pET29b, were expressed in BL21 as soluble proteins. The C3, C5, C6, C8, C9, C10, C11, C12, were constructed in fusion with the 342 bp TrxA tag into pET32b resulting in the production of soluble protein. The sizes of the recombinant enzymes range from 25.2 (C1) to 45.6 kD (C6). The enzyme preparations obtained by Ni-Sepharose affinity chromatography of the cell extracts were >90% pure by SDS-PAGE. The final stocks of the enzyme preps had concentrations ranging from 1.7 mg to 7.3 mg per ml.
The recombinant FAE's have varying pH optima in neutral and alkaline range, and retained substantial activities from pH 7.0 to 9.0, using ground corn fiber as the substrate. C4, C5, C6, and C7 remained quite active at pH 6, while C6 showed considerable activity at pH 5. The FAE's operated at various temperature optima. C4, C5, C7, and C10 showed optimum activity at 40° C. C6 was most active at 50° C. C6 and C7 maintained considerable activity at 60° C. C8 and C9 were cold-active enzymes, showing optimum activity below ambient temperature. C4 had the highest activity in releasing FA from corn fiber, followed by C6 and C5, at pH 7.0, 37° C., 2 hr incubation. The observed effects of pH and temperature may not be exclusively on the enzyme, as alkaline pH and high temperature could affect the fiber and effectively increase or decrease substrate accessibility to enzymatic degradation.
The choice of vector in which the DNA of interest is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., replication, protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing recombinant DNA molecules. The vector desirably includes a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally when introduced into a prokaryotic host cell, such as a bacterial host cell. Such replicons are well known in the art. In addition, preferred embodiments that include a prokaryotic replicon also include a gene whose expression confers a selective advantage, such as a drug resistance, to the bacterial host cell when introduced into those transformed cells. Typical bacterial drug resistance genes are those that confer resistance to ampicillin or tetracycline, among other selective agents. The neomycin phosphotransferase gene has the advantage that it is expressed in eukaryotic as well as prokaryotic cells.
Those vectors that include a prokaryotic replicon also typically include convenient restriction sites for insertion of a recombinant DNA molecule of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories (Richmond, Calif.) and pPL, pK and K223 available from Pharmacia (Piscataway, N.J.), and pBLUESCRIPT and pBS available from Stratagene (La Jolla, Calif.). A vector of the present invention may also be a Lambda phage vector including those Lambda vectors described in Molecular Cloning: A Laboratory Manual, Second Edition, Maniatis et al., Eds., Cold Spring Harbor Press (1989) and the Lambda ZAP vectors available from Stratagene (La Jolla, Calif.). Other exemplary vectors include pCMU [Nilsson et al. (1989) Cell 58:707]. Other appropriate vectors may also be synthesized, according to known methods; for example, vectors pCMU/Kb and pCMUII used in various applications herein are modifications of pCMUIV (Nilson et al., supra). Typical expression vectors capable of expressing a recombinant nucleic acid sequence in plant cells and capable of directing stable integration within the host plant cell include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al. (1987) Meth. in Enzymol. 153:253-277, and several other expression vector systems known to fraction in plants. See for example, Verma et al., No. WO87/0055 1; Cocking and Davey (1987) Science 236:1259-1262.
A transgenic plant can be produced by any means known to the art, including but not limited to Agrobacterium tumefaciens-mediated DNA transfer, preferably with a disarmed T-DNA vector, electroporation, direct DNA transfer, and particle bombardment (See Davey et al. (1989) Plant Mol. Biol. 13:275; Walden and Schell (1990) Eur J. Biochem. 192:563; Joersbo and Burnstedt (1991) Physiol. Plant. 81:256; Potrykus (1991) Annu. Rev. Plant Physiol. Plant Mol Biol. 42:205; Gasser and Fraley (1989) Science 244:1293; Leemans (1993) Bio/Technology. 11:522; Beck et al. (1993) Bio/Technology. 11:1524; Koziel et al. (1993) Bio/Technology. 11:194; and Vasil et al. (1993) Bio/Technology. 11:1533). Techniques are well-known to the art for the introduction of DNA into monocots as well as dicots, as are the techniques for culturing such plant tissues and regenerating those tissues.
Feruloyl esterase of the invention can be used to improve the ferulic acid availability in a food or feed. Ferulic acid has antioxidant activity, and can be made available through feruloyl esterase treatment of a foodstuff. Agricultural grains may be treated with feruloyl esterase, advantageously in combination with xylanase, and be consumed to serve as a ferulic acid based antioxidant supplement, especially in humans. In addition to plant-derived solid food or feed treated with feruloyl esterase or the combination of feruloyl esterase and xylanase, liquids (beverages, e.g.) can also comprise feruloyl esterase (or feruloyl esterase and xylanase) treated material or soluble products thereof. If the beverage contains the solid foodstuff or feedstuff, enzymes are added at a ratio of from about 1 to 200 units of enzyme per kg, desirably from about 10 to about 50 U/kg of esterase and for xylanase, from about 100 to about 10,000 U/kg dry weight of plant-derived material in the liquid foodstuff or beverage. A feruloyl esterase described herein, desirably in combination with a cellulase and/or xylanase, for example that from Orpinomyces PC-2, can also be used in the pulping and paper recycling industries. The ratio of the esterase to solids is from about 0.1 to about 200 U/kg dry weight, desirably from about 1 to about 100 U/kg, and advantageously from about 10 to about 50 U/kg. The feruloyl esterases and mixtures thereof or combination of feruloyl esterase and xylanase can be formulated as dry materials or as liquid concentrates for subsequent use in combination with a source of plant-derived non-starch polysaccharide or poorly digestible plant fiber material to be treated. Such a formulation can be freeze-dried in the case of a dry material or it can be a liquid concentrate.feruloyl esterase and xylanase to improve nutrition.
The feruloyl esterases of the invention may be used to improve the digestibility or energy source availability for humans, poultry (e.g., chickens, turkeys, ducks, geese, and other fowl), swine, sheep, cattle, horse, goats, fish (including but not limited to salmon, catfish, tilapia and trout) and shellfish, especially shrimp, and other farmed animals. Food or feed ingredients which are improved by treatment with feruloyl esterase include, without limitation, wheat, rye, barley, oats, corn, rice, soybean, millet, sorghum, grasses, legumes and other pasture and forage plants. Fresh or dry feed or food components can be treated with a liquid comprising the ferulic acid esterase so that the particles of the food or feed are coated with the enzymes. Similarly, wet or dry enzyme compositions can be added to a liquid food or feed composition so that the ratio of enzymes to dry weight or plant material is as taught herein.
Feruloyl esterase proteins are also characterized by at least a portion having from at least about 50% amino acid sequence identity with an amino acid sequence as given in SEQ ID NO's:1-12. Sequences included in this invention are also those amino acid sequences which are 50, 60, 70, 75, 80, 85, 90, 95 to 100%, identical to the amino acid sequences encoded by the ferulic acid esterases coding sequence of SEQ ID NO's: 1-12 and corresponding to or identifying encoded proteins which exhibit feruloyl esterase activity. In comparisons of protein or nucleic acid sequences, gaps go introduced into either query or reference sequence to optimize alignment are treated as mismatches. It is well-known in the biological arts that certain amino acid substitutions may be made in protein sequences without affecting the function of the protein. Generally, conservative amino acid substitutions or substitutions of similar amino acids are tolerated without affecting protein function. Similar amino acids can be those that are similar in size and/or charge properties, for example, aspartate and glutamate, and isoleucine and valine, are both pairs of similar amino acids. Similarity between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas of protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22, pp. 345-352, which is incorporated by reference herein provides frequency tables for amino acid substitutions which can be employed as a measure of amino acid similarity. Dayhoff et al.'s frequency tables are based on comparisons of amino acid sequences for proteins having the same fraction from a variety of evolutionarily different sources. A further embodiment of the invention is the use of the FAE's for crosslink degradation releasing fibers from lignocellulosic biomass without further action of other enzymes to obtain intact oligosaccharides and polysaccharides for feed, food, and industrial applications. This process also finds utility in soluble fiber release from brewer's grain, beet pulp, corn steeping, residual food processing, agricultural residue and waste. Oligosaccharides and controlled conversion of polysaccharides to oligosaccharides can perform as prebiotics for both human and animals, and serve to enhance gut and intestinal health.
Additional applications for the feruloyl esterase enzymes of the present invention, include producing ferulic acid from wheat bran or agricultural byproducts, pretreating biomass to for downstream waste or sewer treatment, using the enzyme to treat grasses, grains, maize bran, generation of fiber in situ, waste residue of rice bran oil or other plant materials which are used in the pulp and paper industry, wet milling, feed processing—including improving the digestibility of animal feed, as a food additive. The Biotransformation of the ferulic acid to vanillin may also be accomplished by microbial transformation using Generally Recognized as Safe Microorganisms. The present invention further provides methods for improved pulping of plant material or recycled fiber materials, wherein the improvement comprises the step of adding a feruloyl acid esterases of the invention to the pulping mixture and incubating under conditions allowing enzymatic action of the esterase on the non-starch polysaccharides in the mixture. If further hydrolysis of polysaccharides is required, the substrate specificity and other catalytic properties imparted by each esterase allows for greater control and processing in the products formed.
Cloning of the genes of the invention into recombinant hosts provides a selection of feruloyl esterases that may be uniquely tailored for targeted applications including the applications set forth above. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art, standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (Eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (Eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles, of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Haines and Higgins (Eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York.
An additional embodiment of the invention is a novel method for rapid screening and isolation of genes of interest from complex genomes of any bioresources including microbial, plant or animal. The method includes the formulation and use of a substrate gel assay plate, and an enrichment process by serial dilutions progressive to isolate the target gene from a DNA library, wherein the DNA library may be genomic or cDNA. Initial extraction of the genomic or cDNA libraries proceed by methods known to one of skill in the art. The substrate gel contains a combination of a detecting/expression solution and a gelling growth medium. The base components of the detection/expression solution contain (1) a buffer at a suitable pH; (2) a non-ionic detergent at concentration 1-3% (v/v), preferably 1-2% (v/v); (3) a suitable substrate for detecting expression of the target gene. The growth medium contains a low melt agarose at 1-3% (w/v), preferably 1.5% (w/v) melted and mixed into a suitable nutrient broth such as Luria Bertani (LB) or other suitable growth medium. Types of substrates may include chromogenic, fluorogenic, soluble or insoluble dye-crosslinked substrates, which enable the detection of the targeted enzyme reaction product to be quickly visualized and/or measured spectroscopically. The inclusion of antibiotics and expression inducers depends on the type of libraries, which can be constructed based on Escherichia coli, bacteriophages, phagemid, and other expression vectors.
The approach departs from conventional methods used for screening genomic or cDNA libraries conferring the advantages of: (1) screening library of clones in split pools; (2) localizing and intensifying positive signals on a substrate gel plate; and (3) progressive enrichment and isolation of positive clones using serial dilutions with high-throughput robotic capabilities. Rapid isolation of a diverse repertoire of candidate genes for tailor-making enzymes and routes leading to new bioprocesses and bioproducts are achieved. This invention may be packaged into commercial kits to provide a convenient and universal tool for gene discovery.
Methods
Genomic Library Construction and Activity Screening
Genomic DNA was extracted and purified from the microflora of cow's rumen or other environmental samples, followed by partial digestion separately with EcoRI and ApoI. The digests were combined and selected for fragments of sizes from 1 to 8 kb by agarose gel electrophoresis. The fragments were purified and ligated into X-ZAP-II predigested EcoRI/CIAP-treated vector. The recombinant vector mixture was packaged using Gigapack III packaging extract, and the resulting primary library was amplified to a titer of 1010 pfu/ml based on blue-white color selection. This amplified library was subjected to enzyme activity screening as described in [0027] to [0039].
Subcloning of FAE Genes
The identified gene sequence was amplified from the genomic DNA by PCR and cloned in pET29b and pET32b, both containing a C-terminal His-tag. The pET32b vector contains additionally a Trx-tag fusion at the N-terminal end. The N-terminal end His-tag between the Trx-tag and the C-terminal His-tag in the vector was deleted during the gene-vector construction. The final construction was verified by DNA sequencing before used for transformation.
Expression and Purification
The recombinant gene-vector construct was used to transform E. coli BL21(DE3). Positive transformants were selected by halo formation growing on ethyl ferulate agar plates. Positive clones were further confirmed by liquid assay of FAE activity using p-nitrophenyl ferulate as the substrate. A single positive transformant was used to inoculate 2 ml LB and incubated overnight at 37° C. at 225 rpm. A 2 ml overnight culture was transferred to 200 ml fresh LB, and incubated for 3 hr to an OD600 nm of 0.7. Induction of protein expression was initiated by the addition of 0.1 mM of IPTG, and incubation was allowed to proceed for 4 more hr at 30° C. Thereafter, the cells were pelleted and lyzed using 8 ml of primary amine-free Bug-Buster reagent. The extracted protein was purified by Ni Sepharose affinity chromatography, using 20 mM sodium phosphate, 0.5M NaCl, 20 mM imidazole, pH 7.4 binding buffer, and the same buffer containing 500 mM imidazole as the elution buffer. Collected 1-ml fractions were analyzed for purity by SDS-PAGE. Combined active fractions were buffered exchanged into 20 mM sodium phosphate buffer, pH 7.4 with 10% glycerol, and concentrated by ultrafiltration.
Electrophoresis
The purified and concentrated enzyme was run on bis-tris gels using 50 mM MOPS running buffer, pH 7.7 at 200 V constant for 50 min, and stained with Coomassie G-250. For molecular weight determination, the bands were analyzed against protein standards by image analysis software (Alpha Innotech, CA).
Enzyme Activity Measurements
Enzyme activity in the transformants was detected by halo formation by plating the colonies on ethyl ferulate agar plates. The enzyme activity was further confirmed by liquid assay using p-nitrophenyl ferulate as the substrate. The purified FAE's were characterized for biochemical properties using corn fiber as the substrate. Enzyme activity was expressed as μg ferulic acid released per 100 mg corn fiber at pH 7.0 and 37° C.
Determination of pH and Temperature Optima
For pH optimum, the reaction mixture was incubated for 2 h at 37° C., using corn fiber as the substrate at varying pH using a citric acid/sodium phosphate universal buffer. Parallel controls were conducted for all pH points without the enzyme. For temperature optimum, the reaction was incubated for 2 hr in universal buffer, pH 7.0. Parallel controls were conducted for all temperature points without the enzyme.
HPLC Analysis of Ferulic Acid
Ferulic acid released from enzymatic hydrolysis of corn fiber was measured by HPLC. The analysis was performed using a SphereClone 5μ ODS column with H2O/HCOOH/CH3CN (7:1:2) as the solvent, at a flow rate of 0.2 ml/min at ambient temperature. The effluent was monitored with a UV detector at 300 nm. Ferulic acid was applied as external standard for calculation.
Method of Gene Discovery
I. Making Substrate Gel Assay Plate
A substrate gel is formulated and set in a 96 well microtiter plate as an assay plate for screening and detecting the target gene expression. The DNA library is split into small pools (sub-libraries) for growth and amplification of the cell populations in each of the wells in the assay plate. Cells grown in each well are in direct contact with the substrate gel with enhanced permeability and diffusion to maximize cell-substrate interactions and reactions. The substrate gel set in each well enables the localization of the reaction products confined within the well area with enhanced detection sensitivity.
Substrate Gel Assay Plate
In one tube, low-melt agarose at 1.5% (w/v) is melted into LB or any suitable growth medium and cooled to 50° C. In a separate tube, buffer, detergent, antibiotic, inducer, and substrate are added to the growth medium and equilibrated to 50° C. An example would include 50 mM sodium phosphate buffer, pH 6.5, 1.67% (v/v) detergent Triton X-100, 50 μg/ml antibiotic ampicillin, 2 mM expression inducer IPTG, and 2 mM substrate p-nitrophenyl ferulate in DMSO added to LB medium to a final volume of 25 ml and equilibrated to 50° C. After equilibrating both tubes, one containing the agarose mix and the other substrate mix, to 50° C., the two tubes were combined and poured in 10-ml aliquots into a reservoir. Using an 8-channel pipetter fitted with 1.2 ml tips, the combined mix was dispensed to 96-well, half-area microtiter plates (polystyrene, flat-bottom from Greiner) at 50 μl per well. A volume of 50 ml was sufficient for making 9 assay plates. After hardening, the assay plates are used immediately, or can be stored at 4° C. for several weeks. The gelling growth medium must contain 1.67% low-melt agarose. The substrate tube should at least contain the base components (1) a buffer at a suitable pH, (2) a non-ionic detergent, (3) a suitable substrate for detecting expression of the target gene. Types of substrates may include chromogenic, fluorogenic, soluble or insoluble dye-crosslinked substrates, which enable the detection of the targeted enzyme reaction product to be quickly visualized and/or measured spectroscopically by a plate reader. The inclusion of antibiotics and inducers depends on the type of libraries, which can be constructed based on Escherichia coli, bacteriophages, phagemid, and suitable expression vectors known to one of skill in the art.
II. Application of Substrate Gel Plates for Screening DNA Libraries
In a general scheme, 5 μl of an overnight culture of each DNA library pool (see below) is transferred from culture plates into individual wells of the premade substrate gel assay plate. The assay plates are covered with a porous membrane and incubated and monitored for the formation of reaction products due to expression of the target gene. Wells showing product formation indicate active pools, which are subjected for enrichment to isolate the positive clone.
A Protocol Example for Screening a Phagemid Library
An aliquot of a phagemid library containing  ̃1×105 cfu (cell formation units) is diluted into 80 ml of LB ampicillin (50 μg/ml), and 0.1 ml of the diluted phagemid is dispensed per well into eight 96-well, flat-bottomed microtiter plates. Each well therefore contains 1×105 cfu divided by 800, equivalent to  ̃125 cfu per well. Each individual well represents a sub-library (sub-populations) of the original DNA library. The plates are covered with a permeable membrane and incubated overnight at 850 rpm and 37° C. in a microtiter plate incubator shaker. These culture plates are used to inoculate the pre-made substrate gel assay plates, and afterwards stored at 4° C. as the master plate containing sub-libraries.
The overnight cultures from individual wells of the culture plates are transferred in 5 μl volume onto the wells of the assay plates. The assay plate was covered with a permeable membrane and incubated without shaking at 30° C. or 37° C. overnight. Typically, assay plate wells containing active pools (that is, containing a positive clone in the cell population) would produce the reaction product detectable within 8 hours at 37° C.
III. Enrichment for Isolation of an Active Clone from a Positive Pool
Positive Wells of the assay plate are identified and referred to the corresponding sub-libraries in the master plates. A serial dilution scheme is developed to enable quick enrichment and isolation of the active clone(s) from the cell population in the positive pool, as described in the following protocol.
On a 96-well microtiter plate, each column represents an individual sampling from a single well of the master sub-library growth plate. Each row is a serial dilution of that sampling starting from row A and continuing to row H (8 dilutions). Usually, 2 to 4 samplings of the master well were done and each sampling was serially diluted.
An aliquot of 2.5 μl of the sub-library is added to 122.5 μl of growth medium in row A, resulting in a 1:5 dilution. Next, 25 μl from row A is diluted in 100 μl growth medium in row B, resulting in a further 1:5 dilution. This is continued to row H to a final dilution of  ̃8×105. The plate was covered with a porous membrane and shaken overnight at 850 rpm and 37° C. The next day, 5 μl of the overnight culture in each well is assayed using the premade substrate gel assay plates as described above The serial dilution growth plates are stored at 4° C., as the master dilution culture plates. The most dilute well showing positive detection is sampled, serially diluted, and assayed again in the same manner as described above. Eventually, all the wells in the assay plate will become positive, indicating a homogeneous population of the positive clone.

## Claims

1. A feruloyl esterase composition comprising a feruloyl esterase selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and mixtures thereof; and a plant derived composition containing ferulic acid crosslinks.

2. The feruloyl esterase composition of claim 1, wherein the plant derived composition is selected from the group consisting of agricultural food, agricultural waste, animal feed and paper.

3. The feruloyl esterase composition of claim 2, wherein the agricultural food and waste is selected from the group consisting of grasses, grains, maize, bran, corn steeping and vegetable pulp.

4. A method for increasing free ferulic acid content of a plant-derived composition, said method comprising the step of contacting a plant-derived composition with a feruloyl esterase wherein said feruloyl esterase comprises a feruloyl esterase selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and mixtures thereof.

5. The method of claim 4, wherein the plant derived composition is selected from the group consisting of agricultural food, agricultural waste, animal feed, paper, municipal waste and industrial waste.

6. The method of claim 5, wherein the agricultural food and waste is selected from the group consisting of grasses, grains, maize, bran, corn steeping and vegetable pulp.