WO2015026660A2

WO2015026660A2 - Methods and compositions for making biofuels

Info

Publication number: WO2015026660A2
Application number: PCT/US2014/051298
Authority: WO
Inventors: Charles RUNCKEL
Original assignee: Runckel & Associates
Priority date: 2013-08-17
Filing date: 2014-08-15
Publication date: 2015-02-26
Also published as: WO2015026660A3

Abstract

Disclosed herein are polysaccharide, oligosaccharide, and monosaccharide processing polypeptides and polynucleotides encoding them. Also disclosed are cells that heterologously express the disclosed polypeptides or polynucleotides and methods of degrading or transforming polysaccharides into oligosaccharides or monosaccharides by contacting a feedstock with one or more of the cells or isolated polypeptides under conditions sufficient for degrading or transforming polysaccharides.

Description

METHODS AND COMPOSITIONS FOR MAKING BIOFUELS

CROSS REFERENCE TO RELATED APPLICATIONS

This claims the benefit of U.S. Provisional Application No. 61/867,029, filed August 17, 2013 and U.S. Provisional Application No. 62/011,767, filed June 13, 2014, both of which are incorporated herein by reference in their entirety.

FIELD

This disclosure relates to compositions and methods for processing biomolecules, particularly using recombinant microorganisms.

BACKGROUND

Biofuels are vehicle or power plant fuels produced from renewable, biological sources. These have traditionally involved sugary, starchy, or oily food crops, which are either fermented into ethanol in a similar process to the production of alcoholic beverages, or in the case of oils, are extracted by pressing and chemical conversion into fuels like biodiesel. These fuels were initially embraced as a means of reducing foreign fuel imports and as a means of curbing anthropogenic global warming. The use of edible food crops to produce fuels has raised controversy by raising food prices and using scarce water to grow water-inefficient crops like corn (maize). The consumables cost and quantity of fossil fuel used for producing biofuels from food crops is high, and is often not profitable without government incentives. The People's Republic of China and other countries have banned the practice of using edible food crops for fuel production due to its potential social costs.

Many national governments and international organizations, including the government of the United States (US Energy Independence and Security Act) and the European Union

(Renewable Energy Directive), encourage the production of biofuels from lignocellulosic matter. Lignocellulosic matter is rich in tough plant fibers (polysaccharides including cellulose and hemicellulose), which may be converted into simple sugars and ultimately into fuel. This class of material includes agricultural wastes such as husks, stalks and waste leaves; food processing wastes such as fruit and vegetable rinds, peels, and pulps; and purpose-grown grasses, trees, or algae, which can grow on land unsuitable for food crops and at lower expense. These wastes would otherwise create negative environmental and social impacts, but by using them as a fuel source these negative externalities may be minimized while producing an income- stream from a waste- stream. Ethanol has to date been produced primarily from sugar cane (sucrose) or starch crops using the brewer's yeast, Saccharomyces cerevisiae. This yeast is capable of breaking the sugar sucrose (a disaccharide) down into its subcomponents, glucose and fructose (monosaccharides), because it produces the necessary enzyme sucrase, along with proteins for the transport and use of those sugar products. Brewer's yeast does not produce significant quantities of amylases, or enzymes that break down starch, and these enzymes must be supplied either in direct form or through amylase-rich co-substrates, such as the traditional technique of adding barley to other grains to produce beer. The addition of these enzymes degrades the starch to monosaccharides, or to di- and tri- saccharide sugars such as maltose that the yeast cell is capable of importing and digesting.

The use of conventional brewer's yeast to produce ethanol from lignocellulosic feedstocks is a natural extension of this logic. Dozens of different enzymes must be applied to break down cellulose and hemicellulose, which the yeast does not possess. Critically, some of the final monosaccharide sugars produced from the breakdown of some hemicelluloses, such as xylose, cannot be metabolized by S. cerevisiae.

This schema is markedly different than the natural processes of breaking down lignocellulosic matter, for example in a compost heap or a cow's rumen. In these environments, microbes release enzymes that break down cellulose and hemicellulose, but these enzymes are not intended to degrade the fibrous matter all the way down to simple sugars. Rather, they degrade the cellulose and hemicellulose into sugars of 2-10 subunits, termed oligosaccharides. These are then imported into the microbial cell by proteins called "transporters," which are selective and highly efficient at locating and moving target oligosaccharides. The

oligosaccharides are fully broken down to monosaccharides inside the microbial cell and "reformatting enzymes," including isomerases, epimerases, and phosphorylases, re-arrange or modify some foreign sugars until they are in a form the microbial cell may recognize and use. Any particular fibrous feedstock thus requires the products of many genes acting in tandem, and requires many different assemblies of these genes to deal with each of the many different classes of polysaccharide matter. In a natural setting, microbes often specialize in particular

polysaccharide targets and thus a community of different microbes may colonize and consume the same feedstock in parallel.

Microbes that produce biofuels but lack the above gene sets have been engineered with foreign genes that confer the ability to produce enzymes that break down particular

polysaccharides and consume the breakdown products. This field is termed Consolidated Bio- Processing (CBP) and numerous yeast and bacteria species have been engineered for this purpose. In particular, the bacterium Escherichia coli is one suitable platform for this modification. E. coli is the workhorse of genetic engineering techniques, with virtually every gene modification technique validated in this species and modifications may be made at thousands of times the efficiency of modifying other microbes. E. coli further grows quickly in aerobic and anaerobic conditions and is capable of digesting all major monosaccharide classes found in hemicelluloses. While E. coli does not natively produce large quantities of any biofuel, it has been engineered to produce ethanol, isobutanol, butanol, pinene, fatty acids, biodiesel and a variety of other fuels. Ethanol producing CBP microbes based on E. coli have been developed to digest the polysaccharides cellulose, xylan, pectin and alginate through a combination of lignocellulose degrading enzymes and transporters (for example US20110189743 and

US20120094347, which are hereby incorporated by reference in their entirety).

SUMMARY

Disclosed herein is the isolation and identification of novel microbial genes and their polypeptide products and their use, for example to expand the ability of biofuel-producing microbes to consume lignocellulosic matter, including fruit and vegetable waste from food processing. Genes were isolated from diverse microbe communities, including compost heaps, and screened using computer and laboratory techniques to isolate useful genes not previously described. Candidate genes were engineered onto platforms amenable to incorporation in biofuel-producing microbes and their ability to break down plant waste {e.g., to degrade polysaccharides), import degradation products {e.g., oligosaccharides) into the microbe, and/or convert monosaccharides into sugar types the microbe can utilize was confirmed. Combinations of these genetic constructs were shown to provide complementary abilities and efficiencies in terms of feedstock utilization. Finally, these novel genetic constructs were shown to increase fermentation yields of ethanol, a basic biofuel, in many fruit waste types including citrus rind, pineapple peel, and apple peel and pulp.

Thus disclosed herein are polypeptides with one or more activities of degrading polysaccharides, transporting oligosaccharides into a cell, and/or modifying monosaccharides. These polypeptides include the amino acid sequences of SEQ ID NOs: 1-30 and polypeptides with at least 90% sequence identity to SEQ ID NOs: 1-30. Also disclosed are polynucleotides encoding polypeptides with polysaccharide degrading, oligosaccharide transporting, and/or monosaccharide modifying activities. These polynucleotides include nucleic acids with the sequence of SEQ ID NOs: 31-60 and nucleic acids with at least 70% identity to SEQ ID NOs: 31-60. Also disclosed herein are cells (such as recombinant microorganisms, for example, bacterial or fungal cells, also referred to herein as microbes) that include one or more of the disclosed polypeptides or polynucleotides.

Further disclosed herein are methods of processing biomolecules (such as

polysaccharides, oligosaccharides, and/or monosaccharides), utilizing the disclosed polypeptides and/or microbes expressing the disclosed polypeptides. In particular examples, the methods include producing industrially useful compounds, including biofuels, such as ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of plasmid vectors used in Examples 1-4.

FIGS. 2A-2E are a series of graphs showing activity of glycoside hydrolase and polysaccharide lyase genes. Prospective polynucleotides for use in degrading polysaccharides were assayed by means of incubating their polypeptide products with a feedstock and testing the viscosity of the solution after incubation. Less viscosity indicates more degrading activity. A total of 192 genes were assayed - all genes that displayed activity are included in this figure as well as six genes that did not (H001-003 and HI 90- 192) for comparison. Feedstocks included pineapple hemicellulose (FIG. 2A), apple hemicellulose (FIG. 2B), orange hemicellulose (FIG. 2C), commercial arabic acid (FIG. 2D), and commercial polygalacturonic acid (FIG. 2E).

Viscosity within each feedstock set was normalized to a negative control with a vector with no inserted gene. A positive control consisted of 0.2 mg per dry g of commercially prepared pectinase/hemicellulase.

FIGS. 3A-3E are a series of graphs showing reformatting enzyme growth activity.

Prospective polynucleotides with monosaccharide reformatting activity were assayed by microbe growth on acid hydrolyzed fruit and commercial hemicellulose substrates. Growth was assayed by Optical Density at 600 nm after 12 hours. Higher growth indicates improved monosaccharide digestion. Feedstocks included pineapple hemicellulose (FIG. 3A), apple hemicellulose (FIG.3B), orange hemicellulose (FIG. 3C), commercial arabic acid (FIG. 3D), and commercial polygalacturonic acid (FIG. 3E). A negative control consisted of E. coli engineered with an empty vector. A positive control consisted of an equal concentration of purified arabinose. Significantly improved growth is marked with an asterisk (* p<0.05).

FIGS. 4A-4E are a series of graphs showing transporter growth activity. Prospective polynucleotides with oligosaccharide transporter activity were assayed by microbe growth on partially hydrolyzed fruit and commercial hemicellulose substrates. Growth was assayed by Optical Density at 600 nm after 12 hours. Higher growth indicates improved oligosaccharide transport and digestion. Feedstocks included pineapple hemicellulose (FIG. 4A), apple hemicellulose (FIG. 4B), orange hemicellulose (FIG. 4C), commercial arabic acid (FIG. 4D), and commercial polygalacturonic acid (FIG. 4E). A negative control consisted of E. coli engineered with an empty vector. A positive control consisted of an equal concentration of purified arabinose. Significantly improved growth is marked with an asterisk (* p<0.05).

FIGS. 5A-5E are a series of graph showing fermentations to ethanol with pools of the identified genes. 200 mL of 4% fruit hemicellulose solutions were inoculated with 20 mL of pools of "Combined Constructs" consisting of one transporter construct and two re-formatting enzyme constructs. Solutions were further subjected to the addition of hemicellulases, as described in the text and in the figure. Feedstocks included pineapple hemicellulose (FIG. 5A), apple hemicellulose (FIG. 5B), orange hemicellulose (FIG. 5C), commercial arabic acid (FIG. 5D), and commercial polygalacturonic acid (FIG. 5E). Ethanol yield refers to mass of pure ethanol recovered by thermal distillation as determined by refractometry of the distilled material.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and amino acids, as defined in 37 C.F.R. § 1.822. In at least some cases, only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NOs: 1 and 2 are exemplary amino acid sequences of polypeptides for degrading arabinose-rich polysaccharides.

SEQ ID NOs: 3-5 are exemplary amino acid sequences of polypeptides for transporting arabinose-rich oligosaccharides.

SEQ ID NOs: 6-13 are exemplary amino acid sequences of polypeptides for modifying arabinose-rich monosaccharides.

SEQ ID NOs: 14-18 are exemplary amino acid sequences of polypeptides for degrading galactose- or galacturonic acid-rich polysaccharides.

SEQ ID NOs: 19-23 are exemplary amino acid sequences of polypeptides for

transporting galactose- or galacturonic acid-rich oligosaccharides.

SEQ ID NOs: 24-30 are exemplary amino acid sequences of polypeptides for modifying galactose- or galacturonic acid-rich monosaccharides.

SEQ ID NOs: 31 and 32 are exemplary nucleic acid sequences encoding the polypeptides of SEQ ID NOs: 1 and 2, respectively.

SEQ ID NOs: 33-35 are exemplary nucleic acid sequences encoding the polypeptides of SEQ ID NOs: 3-5, respectively.

SEQ ID NOs: 36-43 are exemplary nucleic acid sequences encoding the polypeptides of SEQ ID NOs: 6-13, respectively.

SEQ ID NOs: 44-48 are exemplary nucleic acid sequences encoding the polypeptides of SEQ ID NOs: 14-18, respectively.

SEQ ID NOs: 49-53 are exemplary nucleic acid sequences encoding the polypeptides of SEQ ID NOs: 19-23, respectively.

SEQ ID NOs: 54-60 are exemplary nucleic acid sequences encoding the polypeptides of SEQ ID NOs: 24-30, respectively.

DETAILED DESCRIPTION

I. Terms

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. "Comprising" means "including." Hence "comprising A or B" means "including A" or "including B" or "including A and B."

Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which a disclosed invention pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in

Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided: Arabinose-rich: In some examples, "arabinose-rich" refers to polysaccharides or oligosaccharides where at least 5% of the subunits are the monosaccharide arabinose or arabinose-like monosaccharides, including isomers of arabinose and arabinose monosaccharides modified with methyl, acetyl or phosphate groups. In other examples, "arabinose-rich" refers to a mixture of monosaccharides where at least 5% of the monosaccharides are arabinose or arabinose-like monosaccharides, including isomers of arabinose and arabinose monosaccharides.

Expression: The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

Galactose- or galacturonic acid-rich: In some examples, "galactose- or galacturonic acid-rich" refers to polysaccharides or oligosaccharides where at least 5% of the subunits are the monosaccharide galactose, galacturonic acid or galactose-like monosaccharides, such as isomers of galactose or galacturonic acid or galactose or galacturonic acid monosaccharides modified with methyl, acetyl or phosphate groups. In other examples, "galactose- or galacturonic acid- rich" refers to a mixture of monosaccharides where at least 5% of the monosaccharides are galactose, galacturonic acid or galactose-like monosaccharides.

Hemicellulose: A polysaccharide present in the cell walls of most plants.

Hemicelluloses have a non-uniform structure and include monosaccharides other than glucose. For example, hemicellulose may include xylose, galactose, mannose, galacturonic acid, rhamnose, arabinose, and/or other monosaccharides, in addition to glucose. In contrast, cellulose and starch, for example, are polysaccharides consisting purely of linked glucose monosaccharides.

Heterologous: Originating from a different genetic sources or species. For example, a nucleic acid that is heterologous to a cell originates from an organism or species other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid includes an bacterial nucleic acid that is present or expressed in a different bacterial cell (such as an E. coli cell) or in a fungal (such as a S. cerevisiae cell), plant, or mammalian cell. Methods for introducing a heterologous nucleic acid into bacterial, fungal, plant, and mammalian cells are well known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, and particle gun acceleration.

In another example of use of the term heterologous, a nucleic acid operably linked to a heterologous promoter is from an organism or species other than that of the promoter. For example, a bacterial nucleic acid may be linked to a promoter from a different bacterium, or to a viral, fungal, plant, or mammalian promoter. In other examples of the use of the term

heterologous, a nucleic acid encoding a polypeptide or portion thereof is operably linked to a heterologous nucleic acid encoding a second polypeptide or portion thereof, for example to form a non-naturally occurring fusion protein.

Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified away from other biological components in a cell, or an organism, in which the component occurs, such as other chromosomal and extra- chromosomal DNA and RNA, proteins and/or cells. Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods or prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acid molecules and proteins.

Microbe: A single-celled organism, such as a prokaryotic cell, including bacteria and archaea, or a eukaryotic cell, including fungi (such as yeast), or algae. In particular examples, a microbe is a member of the species Escherichia coli. Other microbes include members of the bacterial genera Escherichia, Klebsiella, Zymomonas, Bacillus, and Clostridia. In other examples, a microbe is a fungus, such as Saccharomyces cerevisiae or Aspergillus niger.

Monosaccharide: Simple sugars such as glucose, fructose, xylose, galactose, galacturonic acid, arabinose, mannose, and rhamnose, among others. Monosaccharides may also be modified with various additional molecules, including methylation, acetylation, and phosphorylation, among others. In particular examples, monosaccharides may diffuse into a cell through pores in the cell membrane at a high and biologically useful rate.

Oligosaccharide: Polymers composed of two to ten monosaccharide subunits. It is commonly observed that disaccharides (molecules with two simple sugar subunits) and larger sugar polymers have substantially reduced or no detectable diffusion through the outer membrane. These molecules must either be broken down outside the cell or imported into the cell with specialized biological machinery. Although disaccharides are not commonly considered oligosaccharides in some technical fields, the definition herein of oligosaccharides includes disaccharides from a purely functional and practical standpoint, as sugars requiring active transport. Similarly, the upper limit of the definition of oligosaccharide is arbitrary. It is observed that sugars over five subunits are rarely imported into the microbe intact even with active transporters. A delineation of 10 subunits is thus appropriate as occupying a clear space between the classes of polysaccharide and oligosaccharide, while not being near a functional boundary and representing an arbitrary point in a continuum of molecule sizes.

Polynucleotide: A polymeric form of nucleotides of any length including either ribonucleotides (RNA) or deoxyribonucleotides (DNA). A polynucleotide can be circular or linear in structural arrangement. A polynucleotide may be obtained from a natural source or can be synthesized through chemical techniques, or otherwise isolated and replicated using enzymatic means. The polynucleotides described herein may be a component of a vector, an independent fragment, or integrated into the chromosome of a cell (such as a genetically engineered microbe). A polynucleotide may be present adjacent to (such as operably linked to) other polynucleotides controlling the expression of the disclosed polynucleotides from DNA to RNA, and the subsequent translation of the disclosed RNA to the disclosed polypeptides (such as a promoter).

An "exogenous polynucleotide" refers to a polynucleotide not naturally or normally found in a cell (such as a microbe). The term "heterologous polynucleotide" is also used herein, and refers to a polynucleotide originating from a different genetic source or species than the cell in which it is present. For example, a polynucleotide that is heterologous to a cell originates from an organism or species other than the cell in which it is expressed. Methods for introducing a heterologous polynucleotide into bacterial, fungal, algal, plant, and mammalian cells are well known in the art.

Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). An "exogenous polypeptide" in some examples, refers to a polypeptide not naturally or normally found in a cell, for example a microbe. The term "heterologous polypeptide" is also used herein, and refers to a polypeptide originating from a different genetic source or species than the cell in which it is present. For example, a polypeptide that is heterologous to a cell originates from an organism or species other than the cell in which it is expressed. Methods for introducing a heterologous polypeptide into bacterial, fungal, algal, plant, and mammalian cells are well known in the art, including transformation with a nucleic acid encoding the polypeptide, for example by electroporation, lipofection, and particle gun acceleration.

The disclosed polypeptides also include polypeptides that are post-translationally processed, for example upon expression in bacterial cells. In some examples, a disclosed polypeptide lacks the N-terminal methionine, and therefore in some of the disclosed embodiments, the polypeptide (for example an active polypeptide) starts at amino acid number 2 of any one of SEQ ID NOs: 1-30.

Polysaccharide: A polymer of more than 10 simple sugars (monosaccharides) such as glucose, fructose, xylose, galactose, galacturonic acid, arabinose, mannose, and rhamnose, among others. Polysaccharides include well-known polymers such as cellulose, hemicellulose, pectin, or pectic-type sugars. Monosaccharides within a polysaccharide may also be modified with various additional molecules, including methylation, acetylation, and phosphorylation, among others. Some polysaccharides are composed of long linear chains, where each monomer is typically linked to two other monomers. Other polysaccharides are highly branched polymers, where single monomers are often linked to three other subunits, or a continuum of structures between linear and highly branched structures. In some instances, polysaccharides are chemically linked to other molecules in plant matter, including lignin.

Polysaccharides also include molecules comprised of simple sugar molecules which precipitate and sediment when subjected to a 3: 1 dilution in ethanol and lOOOxg of centrifugal force for 5 minutes. Polysaccharide concentration is also functionally defined and assayed herein as conferring additional viscosity to a solution compared with a chemically similar but less polymerized solution of monosaccharides or oligosaccharides.

Recombinant: In some examples, a cell (such as a microbe) into which has been incorporated an exogenous or heterologous polynucleotide, which leads to the production of an exogenous or heterologous polypeptide. In some instances, the terms "recombinant" and

"genetically-modified" are used interchangeably in the present disclosure. In some examples, a recombinant cell is produced by incorporating a polynucleotide into a vector sequence (for example, wherein the polynucleotide is operably linked to a promoter) and introducing it to the microbe by heat shock, by electroporation, by application of a detergent, or other methods known to one of skill in the art. This may also be accomplished by incorporating the

polynucleotide directly into the microbe's chromosome using homologous recombination, a transposase or a phage-mediated integration, among many similar techniques.

In other examples, recombinant refers to a nucleic acid or protein that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of nucleotides or amino acids. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook et al.

Molecular Cloning: A Laboratory Manual, 3^rd ed., Cold Spring Harbor Laboratory Press, NY,

2001. The term recombinant also includes nucleic acids or proteins that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid sequence or amino acid sequence, respectively.

Sequence identity/similarity: The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in the art and include techniques such as the Clustal algorithm and accompanying software suite (clustal.org) or the Blastn and Blastp programs of the BLAST algorithm, available through the World Wide Web and provided by the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov). In general, the Blastn and Blastp programs are used with default parameters.

Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307- 31, 1994. Altschul et al, J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment

Search Tool (BLAST) (Altschul et al, J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biotechnology (ncbi.nlm.nih.gov), for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

One of skill in the art will appreciate that the particular sequence identity ranges provided herein are for guidance only; it is possible that strongly significant homologs or orthologs could be obtained that fall outside the ranges provided.

Transduced and Transformed: A virus or vector "transduces" a cell when it transfers nucleic acid into the cell. A cell is "transformed" by a nucleic acid transduced into the cell when the DNA becomes replicated by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal replication. As used herein, the term transformation

encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid or artificial chromosome vectors, and introduction of naked DNA by electroporation, lipofection, or particle gun acceleration. Vector: A nucleic acid molecule that can be introduced into a host cell, thereby producing a transformed or transduced host cell. Recombinant DNA vectors are vectors including recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes, a cloning site for introduction of heterologous nucleic acids, a promoter (for example for expression of an operably linked nucleic acid), and/or other genetic elements known in the art. Vectors include plasmid vectors, including plasmids for expression in gram negative and gram positive bacterial cell. Exemplary vectors include those for use in E. coli. Vectors also include viral vectors, such as, but not limited to, retrovirus, orthopox, avipox, fowlpox, capripox, suipox, adenovirus, herpes virus, alpha virus, baculovirus, Sindbis virus, vaccinia virus, and poliovirus vectors. Vectors also include vectors for expression in yeast cells.

II. Polypeptides and Polynucleotides

Disclosed herein are polypeptides with polysaccharide degrading, oligosaccharide transporting, or monosaccharide modifying ("reformatting") activity. Also disclosed are polynucleotides encoding the polypeptides. The disclosed polypeptides have functionality in breaking down complex sugar polymers (such as polysaccharides) into smaller pieces (such as to oligosaccharides or monosaccharides), in importing (transporting) those pieces into a microbe, and in altering (modifying or reformatting) monosaccharides into forms which are more easily digested by the microbe.

In some embodiments, these polypeptides or polynucleotides, when engineered into cells (such as fuel-producing microbes), enable them to better process some hemicelluloses or polysaccharides found in plant matter, for example digesting, transporting, and/or modifying the polysaccharides or their components. Different plants can produce polysaccharides having different sugar subunits. In some examples, the polysaccharides contain a substantial proportion (such as 5% or more) of the simple sugars arabinose {e.g., arabinose-rich polysaccharides) or galactose and/or galacturonic acid {e.g., galactose- or galacturonic acid-rich polysaccharides).

The structural degradation of polysaccharides may take the form of various chemical reactions wherein the chemical bonds linking atoms of sugar subunits, usually between two carbons, are replaced with two separate bonds to other molecules and have the functional consequence of separating the sugar subunits and reducing the size of the resulting

polysaccharide molecules. This may be rephrased to describe the structural degradation of polysaccharides as the reduction in the mean (average) number of monosaccharide subunits per polysaccharide molecule. In some examples, the structural degradation of polysaccharides includes a reduction in the mean (average) number of monosaccharide subunits. This state may be observed through a decrease in viscosity or through various experimental methods, such as gas chromatography.

The classes of polypeptides that degrade these bonds include glycoside hydrolases and polysaccharide lyases, among others. These bonds may also link polysaccharide subunits to other molecules in plant matter, such as lignin, a tough structural molecule. Similar effects and functional consequences are achieved for the degradation of saccharide-lignin bonds. This degradation of large polysaccharides has several desirable consequences from the perspective of industrial fermentation and production of a metabolic product, such as fuel, including 1) the degradation of polysaccharides to single sugar subunits, which are often acceptable as a food source to microbes and may be converted to fuel or other desirable products, 2) the degradation of polysaccharides into oligosaccharide subunits, which may be the subject of further processing by the microbe until ultimately degraded into simple sugars, 3) the reduction in viscosity of the fermentation solution, which allows increased mixing and superior subsequent fermentation, and 4) the increased accessibility or exposure of the partially degraded polysaccharide or

neighboring polysaccharides to further degradation (thus degradation accelerates further degradation).

In some embodiments, disclosed herein are polypeptides with the ability and utility of degrading long polysaccharide molecules into smaller polysaccharide molecules and ultimately into oligosaccharides, such as sugar molecules with 2-10 subunits. In some examples, polysaccharide degrading polypeptides include polypeptides for degrading arabinose-rich polysaccharides, such as SEQ ID NOs: 1 or 2 or a polypeptide with an amino acid sequence with at least 90% identity (such as at least 95%, 98%, 99%, or more identity) to SEQ ID NOs: 1 or 2. In other examples, polysaccharide degrading polypeptides include polypeptides for degrading galactose- or galacturonic acid-rich polysaccharides, such as SEQ ID NOs: 14-18 or a polypeptide with an amino acid sequence with at least 90% identity (such as at least 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 14-18.

In some examples, polypeptides for degrading arabinose-rich polysaccharides are encoded by polynucleotides with the nucleic acid sequence of SEQ ID NOs: 31 or 32 or with a nucleic acid sequence with at least 70% identity (such as at least 75%, 80%, 85%, 90%, 95%,

98%, 99%, or more identity) to SEQ ID NOs: 31 or 32. In additional examples, polypeptides for degrading galactose- or galacturonic acid-rich polysaccharides are encoded by polynucleotides with the nucleic acid sequence of any one of SEQ ID NOs: 44-48 or with a nucleic acid sequence with at least 70% identity (such as at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 44-48.

In additional embodiments, disclosed herein are polypeptides with the ability to actively transport external oligosaccharides into a cell (such as a microbe) through the outer membrane. In some examples, these polypeptides are referred to herein as "transporters." The

oligosaccharide transporters facilitate the digestion of hemicellulosic material by concentrating oligosaccharides inside the cell where additional enzymes may finish digestion into

monosaccharides. The volume of the cell's interior is thousands of times less than that of the external environment or total fermentation broth, providing a substantial improvement in efficiency through higher concentration of enzymes used for additional digestion. In some examples, the transporter itself may provide some oligosaccharide-degrading function with the steps of translocation and degradation occurring simultaneously or nearly simultaneously. In other examples, the cell has enzymes capable of degrading oligosaccharides into

monosaccharides. Enzyme functionality is generally highly specific to a substrate and highly efficient, or broadly specific with low efficiency. In some examples, the latter class of enzymes natively produced by the microbe processes the materials the transporters translocate.

In some examples, oligosaccharide transporter polypeptides include polypeptides for transporting arabinose-rich oligosaccharides, such as SEQ ID NOs: 3-5 or a polypeptide with an amino acid sequence with at least 90% identity (such as at least 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 3-5. In other examples, oligosaccharide transporter polypeptides include polypeptides for transporting galactose- or galacturonic acid-rich oligosaccharides, such as SEQ ID NOs: 19-23 or a polypeptide with an amino acid sequence with at least 90% identity (such as at least 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 19-23.

In some examples, polypeptides for transporting arabinose-rich oligosaccharides are encoded by polynucleotides with the nucleic acid sequence of any one of SEQ ID NOs: 33-35 or with a nucleic acid sequence with at least 70% identity (such as at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 33-35. In additional examples, polypeptides for transporting galactose- or galacturonic acid-rich oligosaccharides are encoded by polynucleotides with the nucleic acid sequence of any one of SEQ ID NOs: 49-53 or with a nucleic acid sequence with at least 70% identity (such as at least 75%, 80%, 85%, 90%, 95%,

98%, 99%, or more identity) to any one of SEQ ID NOs: 49-53.

In further embodiments, disclosed herein are polypeptides with the ability to chemically modify arabinose-like, galactose-like, or galacturonic acid-like sugars to facilitate metabolism by a microbe. These polypeptides can improve overall efficiency of polysaccharide digestion by chemically converting the variations of sugar forms used by different plants to a sugar variant usable by a microbe. As used herein, monosaccharides can include simple, single subunit sugars that are directly consumed by microbes. Monosaccharides are chemically converted through a series of steps, which may release or require energy, until the sugar resembles a component of one of several core metabolic pathways, such as the Citric Acid Cycle (TCA, or Kreb' s Cycle), the pentose phosphate pathway or the Entner-Douderoff pathway. These chemical conversions may involve the addition or removal of small molecules, including methylation (the presence of a methyl group) or acetylation (the presence of an acetyl group), and frequently the addition or removal of a phosphate group (e.g., phosphorylation). The structural arrangement of a sugar may be modified without the addition or loss of small molecules (isomerization) so that the sugar exists in a form more amenable to the microbe' s digestive pathway. For example, many yeast cannot digest the monosaccharide xylose. Some can digest the molecule xylulose, which is chemically identical to xylose but has a different structural arrangement. The addition of the enzyme xylose isomerase can allow digestion of xylose in some yeast. These monosaccharide modifying polypeptides are generally referred to as isomerases, but also as epimerases or racemases. Monosaccharides with identical molecular components but different structural arrangements may be referred to as isoforms or isomers.

In some examples, monosaccharide modifying polypeptides include polypeptides for modifying arabinose-rich monosaccharides, such as SEQ ID NOs: 6-13 or a polypeptide with an amino acid sequence with at least 90% identity (such as at least 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 6- 13. In other examples, oligosaccharide transporter polypeptides include polypeptides for modifying galactose- or galacturonic acid-rich

monosaccharides, such as SEQ ID NOs: 24-30 or a polypeptide with an amino acid sequence with at least 90% identity (such as at least 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 24-30.

In some examples, polypeptides for modifying arabinose-rich monosaccharides are encoded by polynucleotides with the nucleic acid sequence of any one of SEQ ID NOs: 36-43 or with a nucleic acid sequence with at least 70% identity (such as at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more identity) to any one of SEQ ID NOs: 36-43. In additional examples, polypeptides for modifying galactose- or galacturonic acid-rich monosaccharides are encoded by polynucleotides with the nucleic acid sequence of any one of SEQ ID NOs: 54-60 or with a nucleic acid sequence with at least 70% identity (such as at least 75%, 80%, 85%, 90%, 95%,

98%, 99%, or more identity) to any one of SEQ ID NOs: 54-60. In some embodiments, the present disclosure may include isolated polypeptides (such as SEQ ID NOs: 1-30 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 1-30). Also disclosed are isolated or purified polynucleotides (such as SEQ ID NOs: 31-60, or polynucleotides with at least 70% sequence identity to SEQ ID NOs: 31-60). The polypeptides disclosed herein can be chemically synthesized by standard methods, or can be produced recombinantly. An exemplary process for polypeptide production is described in Lu et ah, FEBS Lett. 429:31-35, 1998. Polypeptides can also be isolated by methods including preparative chromatography and immunological separations. In some examples, polypeptides are produced using molecular genetic techniques, such as by inserting a nucleic acid encoding the polypeptide (such as SEQ ID NOs: 31-60 or polynucleotides with at least 70% sequence identity to SEQ ID NOs: 31-60) into an expression vector, introducing the expression vector into a host cell, and isolating the polypeptide.

In some embodiments, one or more of the disclosed polypeptides are produced by culturing microbes expressing one or more of the polypeptides in a rich media {e.g., with simple sugar(s), salt(s), and protein or amino acid supplementation) and subsequently concentrating or purifying the polypeptide(s). The polypeptide(s) are applied to polysaccharides such as arabinose-rich polysaccharides, or galactose- or galacturonic acid-rich polysaccharides, for example to degrade, transport, or modify polysaccharides. One example of rich media is Lysogeny Broth (10 g tryptone, 5 g yeast extract, and 10 g NaCl per L water) supplemented with 5 g/L glucose. Another example of rich media is Terrific Broth (20 g tryptone, 5 g yeast extract, 0.5 g NaCl, 0.2 g KC1, and 1 g MgCl₂) supplemented with 5 g/L glucose. One of ordinary skill in the art can identify appropriate media for culturing microbes and producing polypeptides disclosed herein. III. Recombinant Cells

Disclosed herein are cells (for example, microbes, such as E. coli or S. cerevisiae) that express one or more of the disclosed polypeptides (for example, recombinant or genetically modified cells). For example, cells can be transduced or transformed with one or more of the disclosed polynucleotides to produce cells expressing the polypeptide(s) encoded by the polynucleotide(s). In one embodiment, fuel-producing cells (such as E. coli or S. cerevisiae) are genetically engineered to incorporate and express one or more of the disclosed polynucleotides to produce one or more of the disclosed polypeptides conferring the ability to degrade, import, and/or modify polysaccharides or degraded polysaccharides. This engineering may take the form of transformation with a plasmid or integration of the genes into a cell's chromosome. Methods of producing recombinant or genetically modified cells are known to one of skill in the art. In particular examples, one or more of the disclosed polynucleotides (such as one or more nucleic acids having at least 70% identity to any one of SEQ ID NOs: 31-60) is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a cell, such as a prokaryotic or eukaryotic cell. A nucleic acid encoding a disclosed polypeptide (such as a polypeptide having at least 90% identity with any one of SEQ ID NOs: 1-30) is in some examples operably linked to heterologous expression control sequences. An expression control sequence operably linked to a coding sequence is linked such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (e.g., ATG) in front of a protein-encoding nucleic acid, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The expression control sequence(s) in some examples are heterologous expression control sequence(s), for example from an organism or species other than the protein-encoding nucleic acid. Thus, the protein-encoding nucleic acid operably linked to a heterologous expression control sequence (such as a promoter) comprises a nucleic acid that is not naturally occurring.

Vectors for cloning and replication of the disclosed nucleic acid molecules include bacterial plasmids, such as bacterial cloning or expression plasmids. Exemplary bacterial plasmids into which the nucleic acids can be cloned include E. coli plasmids, such as pBR322, pUC plasmids (such as pUC18 or pUC19), pBluescript, pACYC184, pCDl, pGEM® plasmids (such as pGEM®-3, pGEM®-4, pGEM-T® plasmids; Pomega, Madison, WI), TA-cloning vectors, such as pCR® plasmids (for example, pCR® II, pCR® 2.1, or pCR® 4 plasmids; Life Technologies, Grand Island, NY) pcDNA plasmids (for example pcDNA™3.1 or pcDNA™3.3 plasmids; Life Technologies), or pCClFOS or pCC2FOS. In some examples, the vector includes a heterologous promoter which allows protein expression in bacteria. Exemplary vectors include pET vectors (for example, pET-21b), pDEST™ vectors (Life Technologies), pRSET vectors (Life Technologies), pBAD vectors, and pQE vectors (Qiagen). The disclosed nucleic acids can be also be cloned into B. subtilis plasmids, for example, pTA1060 and pHT plasmids (such as pHTOl, pHT43, or pHT315 plasmids). One of skill in the art can select additional vectors suitable for cloning and/or bacterial expression of proteins such as those disclosed herein.

In other embodiments, vectors are used for expression in yeast such as S. cerevisiae,

Pichia pastoris, or Kluyveromyces lactis. The plasmids can include nutritional markers (such as URA3, ADE3, HIS1, and others) for selection in yeast and antibiotic resistance (such as AMP) for propagation in bacteria. Exemplary yeast plasmids into which the nucleic acids can be cloned include pYES2, pYES-DEST52, pTEFl/Zeo (Invitrogen), pD1201, pD1204, pD1211 (DNA2.0), and so on. One of skill in the art can select additional vectors suitable for cloning and/or expression of proteins in yeast, such as those disclosed herein. Thus, in one example, after amplification in bacteria, plasmids can be introduced into the corresponding yeast auxotrophs by methods similar to bacterial transformation.

Nucleic acids encoding a disclosed polypeptide can be exogenously expressed by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication.

Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

Host cells can include microbial, fungal, algal, insect, plant, and mammalian host cells. Non-limiting examples of suitable host cells include bacteria (for example, E. coli), archea, insect, fungi (for example, S. cerevisiae), mycobacterium (such as M. smegmatis), algal (such as Chlamydomonas or Botryococcus), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include E. coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, Jakoby and Pastan (eds), 1979, Cell Culture. Meth.

Enzymol., volume 58, Academic Press, Inc., Harcourt Brace Jovanovich, N.Y.). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although other cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features.

Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCl₂ or RbCl can be used.

Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation, lipofection, and particle gun acceleration. Techniques for the transformation of yeast cells, such as polyethylene glycol transformation, protoplast transformation and gene guns are also known in the art (see Gietz and Woods Meth. Enzymol. 350: 87-96, 2002). In some embodiments, a recombinant cell is transformed with a single disclosed polynucleotide or expresses a single disclosed polypeptide. In other embodiments, a

recombinant cell is transformed with two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, or more) different polynucleotides or expresses two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, or more) different polypeptides. In some examples, a recombinant cell includes or expresses one or more polysaccharide degrading polynucleotide or polypeptide. In other examples, a recombinant cell includes or expresses one or more oligosaccharide transporting polynucleotide or polypeptide. In still further examples, a recombinant cell includes or expresses one or more monosaccharide modifying (or "reformatting") polynucleotide or polypeptide.

In some examples, polynucleotides encoding at least one of each of a transporter and a reformatting enzyme are expressed in a microbe, for example, combinations of two, of three, of four, of five, of six, of seven, of eight, of nine, or of ten, or of less than fifteen, or of less than twenty different polynucleotides engineered into the same strain. In other examples,

polynucleotides encoding at least one of each of a degradation enzyme and a transporter are expressed in a microbe, for example, combinations of two, of three, of four, of five, of six, of seven, of eight, of nine, or of ten, or of less than fifteen, or of less than twenty different polynucleotides engineered into the same strain. In further examples, polynucleotides encoding at least one of each of a degradation and a reformatting enzyme are expressed in a microbe, for example, combinations of two, of three, of four, of five, of six, of seven, of eight, of nine, or of ten, or of less than fifteen, or of less than twenty different polynucleotides engineered into the same strain. In additional examples, polynucleotides encoding at least one each of a degradation enzyme, a transporter, and a reformatting enzyme are expressed in a microbe, for example, combinations of two, of three, of four, of five, of six, of seven, of eight, of nine, or of ten, or of less than fifteen, or of less than twenty different polynucleotides engineered into the same strain.

In particular embodiments, modifications may be made to a single microbe strain, and groups of polynucleotides encoding polypeptides may be engineered together to confer complementary abilities. In particular non-limiting examples, cells (e.g., microbes) are transformed with or express groups of polynucleotides, for example polynucleotides encoding any of the following groups of polypeptides or polypeptides at least 90% identical to the polypeptides listed in each group: Group A (SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, and 13), Group B (SEQ ID NOs: 2, 3, 4, 5, 10, 11, and 12), Group C (SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, and 16) and Group D (SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, and 30). In another example, cells (e.g., microbes) are transformed with or express polynucleotides encoding each of SEQ ID NOs: 1-30 or polynucleotides encoding polypeptides with at least 90% sequence identity to each of SEQ ID NOs: 1-30.

In some embodiments one or more microbes engineered to produce the polypeptide(s) are contacted with an arabinose-rich polysaccharide solution or feedstock or a galactose- or galacturonic acid-rich polysaccharide solution or feedstock directly. These methods are described in more detail in Section IV, below.

The disclosed microbes (such as microbes expressing the polynucleotides or

polypeptides disclosed herein) may be additionally genetically modified to increase production of particular metabolic products. In some examples, a microbe (such as E. coli) is modified to produce higher than natural {e.g., industrially useful) concentrations of ethanol, methanol, isopropanol, butanol, isobutanol, fatty acids, fatty acid methyl esters, fatty acid ethyl esters, kerosene, hydrogen, methane, nitrates and/or nitrites. In one specific example, E. coli is genetically modified to over-express the alcohol dehydrogenase (adh) gene, which results in an increase in the production of ethanol from simple sugars. In some examples, the modification is accomplished by altering the promoter of E. coli's natural adh gene or by adding non-native adh genes to the microbe.

IV. Methods of Processing Biomolecules

Disclosed herein are methods of processing polysaccharides and related compounds, for example, degrading polysaccharides, transporting oligosaccharides, and/or modifying monosaccharides. In some embodiments, the methods include contacting a mixture, composition, or solution including polysaccharides, oligosaccharides, and/or monosaccharides with one or more of the disclosed polypeptides (such as one or more isolated polypeptides and/or microbe(s) expressing one or more of the disclosed polypeptides) under conditions sufficient for polypeptide activity in processing the polysaccharides, oligosaccharides, and/or monosaccharides.

In some embodiments, the present disclosure involves the improved digestion of polysaccharides rich in arabinose or arabinose-like sugars, and/or those rich in galactose or galacturonic acid or galactose-like or galacturonic acid-like sugars. This distinction refers to the observation that polysaccharides employed by plants may involve monosaccharides that are similar but not identical to commonly considered monosaccharides. It may be difficult to detect the difference in these variations of common monosaccharides, for example typical gas chromatography experiments may fail to detect different isomers of the same sugar. These variations are important when engineering microbes to consume particular polysaccharides, as the microbe may not natively possess the biological machinery to transform one sugar variant into a variant it can use. In this case, the sugar is wasted or may serve as an easy food source for a contaminating microbe that does possess the machinery to consume that sugar, but does not produce fuel or other useful metabolic products.

In some embodiments, the disclosure includes methods to improve the ability of microbes to digest polysaccharides. Once digested, the energy produced from digestion may be directed to the execution of many metabolic processes. Different embodiments may generate biofuels from this energy or chemical products of digestion, including ethanol, isopropanol, other large alcohols, pinene, fatty acids, fatty acid esters, methane or hydrogen gas. Other embodiments may couple the improved polysaccharide digestion abilities with the production of industrial chemicals or chemical precursors, such as acetic acid, succinate, or bio-polymers for the production of plastics. Other embodiments may use the increased energy from improved polysaccharide digestion to produce specific enzymes additionally engineered into the microbe for production.

For example, a single microbe strain with a single genetic modification may be employed, a single microbe strain with multiple genetic modifications may be employed, or multiple microbe strains each with a single different genetic modification may be employed, for example, by adding them to a fermentation broth. If multiple microbe strains are used, the multiple strains may be added to a fermentation broth simultaneously or sequentially. The microbes are incubated with a fermentation broth under conditions that permit the degradation, importation and/or metabolism of polysaccharides or degraded polysaccharides (such as oligosaccharides or monosaccharides). In this embodiment, the fermentation broth into which the engineered microbes are inoculated is subsequently purified to concentrate the fuel or other metabolic product(s) of the engineered microbes.

In some embodiments, a fermentation broth is a liquid mixture including an energy- containing material (a "feedstock," such as a material including polysaccharides), salts and nutrients (such as nitrogen, phosphate, and magnesium) beneficial or even necessary for the survival and function of microbes and their polypeptide products, a pH adjusted to a state that allows fermentation and, in some examples, with little or no exposure to oxygen. Microbes and/or polypeptides (such as one or more of the microbes and/or polypeptides described herein) are added to the fermentation broth and it is then maintained at a suitable temperature (e.g., about 20-40°C, about 25-40°C, about 20-30°C, or about 30-40°C) with agitation, to support microbial growth and processing of the feedstock (such as the polysaccharides) for about 1-14 days (such as about 1- 10 days, about 5-7 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14). In some embodiments, the fermentation broth and microbes (and/or polypeptides) are incubated at ambient temperature, particularly in regions where the ambient temperature is about 20-40°C over the course of a day. In general, the pH of the fermentation broth is about 4- 10 (for example, about 5-7 or about 6-8). Compounds that can be used for adjusting pH include sodium hydroxide and dipotassium phosphate, the latter of which also serves a pH buffering function and provides other nutrients. Total salt concentration is generally less than 5% w/w, with sodium from about 0.05-3% w/w and potassium from about 0.1-3% w/w. The fermentation broth also includes nitrogen containing compounds from about 0.01-0.5% w/w (such as ammonia, nitrates, urea or amino acids) and phosphates from about 0.1%-2% w/w. Magnesium is also typically included in the fermentation broth at about 5-25 ppm (such as about 5, 10, 15, 20, or 25 ppm).

In some examples, the feedstock may provide sufficient water, salts or nutrients, and/or proper pH for microbial growth and fermentation, but in other examples, one or more components are added to the feedstock to produce a suitable fermentation broth. For example, pineapple peel generally has insufficient phosphates, and insufficient water for sufficient agitation, and so phosphates and/or water are added to produce the fermentation broth.

Pineapple peel is also generally too acidic (pH=3.8) and so the pH is adjusted. In contrast, guava cores typically have suitable (nearly ideal) levels of moisture and generally do not require dilution with water, but require supplementation with nitrogen, such as ammonia. One of skill in the art can prepare suitable fermentation broth, based on the feedstock(s) used as a base for the broth.

In some embodiments, microbes expressing one or more of the disclosed polypeptides or polynucleotides are added directly to a pre- sterilized mixture of water and chopped plant matter, or pure chopped plant matter. Chemicals are added to achieve a desirable pH level (e.g., pH 4- 10), salt concentration (e.g., 0.05-5%) and/or magnesium concentration (e.g., 5-25 ppm). The microbes are allowed to multiply and ferment at 30-40°C for a period of 1- 10 days, at the conclusion of which a metabolic product, for example ethanol, is purified by means known to one of skill in the art, including (but not limited to) thermal distillation.

In other embodiments, microbes expressing one or more of the disclosed polypeptides or polynucleotides are added to a thermo-chemically pre-treated mixture of water and chopped plant matter. Thermo-chemical pre-treatments include heating (e.g., to 120°C, to 200°C, or to

800°C), increased atmospheric pressure and/or explosive drops in pressure, addition of various chemicals (including ammonia, sulfuric acid, hydrochloric acid, other acids, or strong bases such as sodium hydroxide), oxidizing agents (including ozone, chlorine, or other bleach-like solutions), or radiation exposure (including UV, infrared, or microwave radiation). Chemicals are added to achieve a desirable pH level (pH 4-10), salt concentration (0.05-5%) and/or magnesium concentration (e.g., 5-25 ppm). The microbes are allowed to multiply and ferment at 30-40°C for a period of 1-10 days, at the conclusion of which the metabolic product, for example ethanol, is purified by means known to one of skill in the art.

In additional embodiments, microbes expressing one or more of the disclosed polypeptides or polynucleotides are added to an enzymatically pre-treated mix of water and chopped plant matter. The enzymes used to break down linkages in the plant fiber include the disclosed polypeptides (such as one or more of the disclosed polysaccharide degrading polypeptides) commercially prepared enzyme mixes (such as Cellic® CTec3 or Cellic® HTec3 (Novozymes, Franklinton, NC) or Accelerase® 1500 or Accelerase® TRIO (DuPont Industrial Biosciences, Rochester, NY)), or the sterilized broth of cultures of various bacterial and fungal strains (e.g., conditioned media from Aspergillus niger, Aspergillus Niger, Fusarium solani, Trichoderma reesei, Trichoderma koningii, Trichoderma viride, Clostridium thermocellum, Ruminococcus albus, and/or Erwinia chrysanthemi). Chemicals are added to achieve a desirable pH level (e.g., pH 4-10), salt concentration (e.g., 0.05-5%) and/or magnesium concentration (e.g., 5-25 ppm). The microbes are allowed to multiply and ferment at 30-40°C for a period of 1-10 days, at the conclusion of which the metabolic product, for example ethanol, is purified by one of any means known to one of skill in the art.

In still further embodiments, microbes expressing one or more of the disclosed polypeptides or polynucleotides are added to both enzymatically and thermo-chemically pre- treated mix of water and chopped plant matter, as described above. Chemicals are added to achieve a desirable pH level (e.g., pH 4-10), salt concentration (e.g., 0.05-5%) and/or magnesium concentration (e.g., 5-25 ppm). The microbes are allowed to multiply and ferment at 30-40°C for a period of 1-10 days, at the conclusion of which the metabolic product, for example ethanol, is purified by one of many means known to one of skill in the art.

A. Degrading Polysaccharides

Disclosed herein are methods for degrading polysaccharides, such as polysaccharides found in lignocellulosic matter. The action of polysaccharide degrading enzymes may produce oligosaccharides or monosaccharides, depending on the mechanism of the enzyme and the relative quantities of enzyme and polysaccharide substrate. Conventional techniques for treating lignocellulosic matter for fermentation rely on degrading the polysaccharide material down to monosaccharides, or in rare cases to disaccharides, prior to import into a microbe and digestion.

CBP techniques, in contrast, rely more heavily on producing oligosaccharides, including disaccharides, and adding microbial machinery to deal with the resulting products. An advantage of this is that reduced quantities of enzymes are added, and thus consumables cost is lower. CBP techniques, including those disclosed herein, thus may enjoy some benefit from polysaccharides that are degraded completely to monosaccharides, but primarily are focused on only partial, not total, degradation of the sugar polymer in the fermentation solution.

In some examples, the methods disclosed herein include contacting one or more of the disclosed polypeptides (such as an isolated polypeptide) or microbes expressing one or more of the disclosed polypeptides with polysaccharides (such as a solution including polysaccharides). For example, the methods include contacting polysaccharides with one or more isolated polypeptides for degrading arabinose-rich polysaccharides (such as SEQ ID NOs: 1 or 2) or for degrading galactose- or galacturonic acid-rich polysaccharides (such as SEQ ID NOs: 14-18) under conditions sufficient for degradation of the polysaccharides. In other examples, the methods include contacting polysaccharides with one or more microbes expressing one or more different polypeptides for degrading arabinose-rich polysaccharides (such as SEQ ID NOs: 1 or 2, for example encoded by SEQ ID NOs: 31 or 32) or for degrading galactose- or galacturonic acid-rich polysaccharides (such as SEQ ID NOs: 14-18, for example encoded by SEQ ID NOs: 44-48) under conditions sufficient for degradation of the polysaccharides. In some examples, the methods result in degradation of at least a portion of the polysaccharides to oligosaccharides.

In some embodiments, polysaccharides are contacted with a single polysaccharide degrading polypeptide or a microbe (or population of microbes) expressing a single

polysaccharide degrading polypeptide. In additional examples, polysaccharides are contacted with two or more different polysaccharide degrading polypeptides (such as two or more different arabinose-rich polysaccharide degrading polypeptides, two or more different galactose- or galacturonic acid-rich polysaccharide degrading polypeptides, or one or more arabinose-rich polysaccharide degrading polypeptides and one or more galactose- or galacturonic acid-rich polysaccharide degrading polypeptides). In further examples, the polysaccharides are contacted with two or more different microbes (or populations of microbes) each expressing different polysaccharide degrading polypeptides (such as different arabinose-rich polysaccharide degrading polypeptides, different galactose- or galacturonic acid-rich polysaccharide degrading polypeptides, or different arabinose-rich polysaccharide degrading polypeptides and galactose- or galacturonic acid-rich polysaccharide degrading polypeptides). In other examples, the polysaccharides are contacted with a microbe (or population of microbes) expressing two or more different polysaccharide degrading polypeptides (such as two or more different arabinose- rich polysaccharide degrading polypeptides, two or more different galactose- or galacturonic acid-rich polysaccharide degrading polypeptides, or one or more arabinose-rich polysaccharide degrading polypeptides and one or more galactose- or galacturonic acid-rich polysaccharide degrading polypeptides).

In other examples, microbes expressing one or more polysaccharide degrading polynucleotide or polypeptide are produced separately from a fermentation broth and lysed to destabilize the cell membrane and free the polypeptides into the growth solution. This solution may then be added to a fermentation broth, or added to a feedstock prior to fermentation in a pre-treatment step. The cells may be lysed by repeated freeze thaw cycles or the addition of chemicals capable of destabilizing the cell membrane, such as detergents. The solution may be filtered or centrifuged to concentrate or sterilize the solution.

In other examples, microbes expressing one or more polysaccharide degrading polynucleotides and polypeptides are produced during fermentation within the fermentation broth. In particular examples, the microbes have a genetic mutation conferring a "leaky" phenotype, or a state where the cell membrane is unusually unstable and allows release into the solution of polypeptides produced inside the cell. The microbe may also employ a

polynucleotide adjacent to (for example, operably linked to) the disclosed polynucleotides that produces a combined polypeptide product (such as a fusion protein), wherein the additional part of the polypeptide has the ability of translocating the disclosed polypeptides to the cell surface or for export from the cell into the fermentation broth. In some examples, the polysaccharide degrading polypeptide is fused to ompA (Yamabhai et al, J Biotech. 133:50-57, 2008), osmY

(Bokinsky et al, PNAS 108:19949-19954, 2011), or N455 (Wargacki et al, Science 335:308-313, 2012).

In additional examples, microbes including or expressing one or more of the

polysaccharide degrading polynucleotides or polypeptides may be produced during fermentation within the fermentation broth and a portion of the broth lysed and returned to the original fermentation chamber or to a pre-treatment chamber containing new feedstock. The lysis of the microbes in the broth may be accomplished by standard methods, such as temperature change, (e.g., freeze-thaw cycles), addition of a chemical, application of radiation (for example UV or microwave radiation), sonication or other vibration, by filtration, or application of high pressure.

In some examples, the method includes contacting a feedstock, such as a suspension of chopped or ground fruit and/or fruit waste (such as fruit peel or pulp) with one or more of the polysaccharide degrading polypeptides or one or more different microbes expressing one or more of the polypeptides. The feedstock may be a solution that is primarily water, with a low concentration of salts (for example, about 0.05-5%) and a mild pH (for example, greater than 4 and less than 10). The pH may be controlled by the addition of acids or bases, or by the addition of pH buffering chemicals. In some examples, trace minerals are added passively, such as by the use of unfiltered water. In particular examples, the magnesium content is about 5-25 ppm (such as about 10-20 ppm, about 5-15 ppm, or about 10 ppm). Magnesium containing compounds, such as magnesium chloride, magnesium sulfate, dolomitic lime, agricultural lime, or magnesium oxide, are added to achieve the desired magnesium level, if needed. A suspension of chopped or ground fruit material in water at a dilution of 1 part fruit to 20 parts water ranging to pure chopped fruit generally is sufficient to provide necessary salts and minerals, while also presenting the polysaccharides for structural degradation.

B. Transporting Oligosaccharides

Also disclosed herein are methods for transporting oligosaccharides into a cell, such as a bacteria or yeast cell, for example, utilizing one or more of the oligosaccharide transporting polypeptides disclosed herein. The polypeptide may function by facilitating the active transport of oligosaccharides into the cell (e.g., across the cell membrane, such as from a fermentation liquid into the cell), or may function by modifying oligosaccharides in such a way as to render them recognizable and transportable by the microbe' s existing machinery, or a combination of the two. The ability of a particular polypeptide, or a polynucleotide encoding a particular polypeptide inserted into a microbe, to facilitate the transport of oligosaccharides may be determined by observing a genetically modified microbe' s growth or metabolism, such as the fermentation of oligosaccharides to ethanol, when fed a solution containing predominantly oligosaccharides, wherein improved ability to transport the oligosaccharide confers improved growth or metabolic product.

The ability to transport, and subsequently digest, a particular oligosaccharide is both beneficial from the perspective of the microbes (for example, microbes producing fuel) and from the perspective of decreasing or even preventing contamination of the fermentation with non- fuel producing wild microbes. Wild microbes may possess a variety of oligosaccharide transporting abilities and oligosaccharides that are not used by the fuel-producing microbes would be expected to build up and provide an attractive environment for the growth of contaminating microbes.

Oligosaccharide-containing solutions may be produced by subjecting a polysaccharide solution to incomplete hydrolysis through thermo-chemical stress (e.g., sulfuric acid and high heat, e.g., about 200°C) or to enzymatic degradation where the particular enzymes used are capable of degrading the particular polysaccharides present in the solution. These enzymes may include the polypeptides disclosed in SEQ ID NOs: 1, 2, or 14-18 (for example, utilizing the methods described above), or commercial enzyme preparations. In general, production of oligosaccharides includes cessation of treatment prior to complete degradation of

polysaccharides to monosaccharides. The distribution of sugars in a solution between monosaccharide and oligosaccharide form may be observed by a variety of laboratory techniques, most commonly chromatography.

In some examples, the methods disclosed herein include contacting one or more microbes expressing one or more of the disclosed oligosaccharide transporting polypeptides with oligosaccharides (such as a solution including oligosaccharides). For example, the methods include contacting oligosaccharides with microbes expressing one or more polypeptides for transporting arabinose-rich oligosaccharides (such as SEQ ID NOs: 3-5, for example encoded by SEQ ID NOs: 33-35) or for transporting galactose- or galacturonic acid-rich oligosaccharides (such as SEQ ID NOs: 19-23, for example encoded by SEQ ID NOs: 49-53) under conditions sufficient for transport of oligosaccharides into the microbe. In other examples, the methods include contacting oligosaccharides with one or more different microbes expressing one or more different polypeptides for transporting arabinose-rich oligosaccharides (such as SEQ ID NOs: 3- 5) or for transporting galactose- or galacturonic acid-rich oligosaccharides (such as SEQ ID NOs: 19-23) under conditions sufficient for transport of oligosaccharides into the microbe. In some examples, the methods result in transport of at least a portion of the oligosaccharides from outside the microbes to inside the microbes.

In some embodiments, oligosaccharides are contacted with a microbe (or population of microbes) expressing a single oligosaccharide transporting polypeptide. In some examples, the oligosaccharides are produced as described above, for example utilizing one or more of the disclosed polysaccharide degrading polypeptides. In additional examples, oligosaccharides are contacted with two or more different microbes (or populations of different microbes) each expressing different oligosaccharide transporting polypeptides (such as different arabinose-rich oligosaccharide transporting polypeptides, different galactose- or galacturonic acid-rich oligosaccharide transporting polypeptides, or different arabinose-rich oligosaccharide transporting polypeptides and galactose- or galacturonic acid-rich oligosaccharide transporting polypeptides). In other examples, the polysaccharides are contacted with a microbe (or population of microbes) expressing two or more different oligosaccharide transporting polypeptides (such as two or more different arabinose-rich oligosaccharide transporting polypeptides, two or more different galactose- or galacturonic acid-rich oligosaccharide transporting polypeptides, or one or more arabinose-rich oligosaccharide transporting polypeptides and one or more galactose- or galacturonic acid-rich oligosaccharide transporting polypeptides).

In some examples, the method includes contacting a feedstock, such as a suspension of chopped or ground fruit and/or fruit waste (such as fruit peel or pulp) with microbes expressing one or more of the disclosed oligosaccharide transporting polypeptides or with two or more microbes each expressing one or more different oligosaccharide transporting polypeptides. The feedstock may be a solution that is primarily water, with a low concentration of salts (for example, about 0.05-5%) and a mild pH (for example, greater than 4 and less than 10). The pH may be controlled by the addition of acids or bases, or by the addition of pH buffering chemicals. In some examples, trace minerals are added passively, such as by the use of unfiltered water. In particular examples, the magnesium content is about 5-25 (such as about 10 ppm). Magnesium containing compounds, such as magnesium chloride, magnesium sulfate, dolomitic lime, agricultural lime, or magnesium oxide, are added to achieve the desired magnesium level, if needed. A suspension of chopped or ground fruit material in water at a dilution of 1 part fruit to 20 parts water ranging to pure chopped fruit generally is sufficient to provide necessary salts and minerals, while also presenting oligosaccharides for transport into a microbial cell.

C. Modifying Monosaccharides

Also disclosed herein are methods for modifying one or more monosaccharides, for example, utilizing one or more of the monosaccharide modifying polypeptides disclosed herein. In some embodiments, the modification of monosaccharides occurs in a cell, for example, a genetically modified microbe disclosed herein. An advantage of altering a cell' s ability to digest monosaccharides by converting them to other, similar forms of monosaccharides includes increasing the overall proportion of feedstock material that may be consumed by a metabolic process. Many modifications from one monosaccharide form to another take place

spontaneously at a slow, and not industrially useful, rate. Polypeptides converting one monosaccharide class to another can increase the rate of monosaccharide use from a not- industrially useful rate to an industrially useful rate, for example from the time scale of 30 days to 3 days.

The accumulation of unused monosaccharides which cannot be utilized by fuel- producing microbes present in a fermentation broth can lead to contamination of the

fermentation broth with wild (e.g., non-fuel producing) microbes which may have the ability to utilize a particular monosaccharide type. Further, the accumulation of unused monosaccharides can be toxic to microbes, thus the ability to utilize those monosaccharides is further beneficial to the health of the microbe population. The ability of a particular polypeptide, or a polynucleotide encoding a particular polypeptide inserted into a microbe, to facilitate the modification of monosaccharides may be determined by observing a genetically modified microbe's growth or metabolism, such as the fermentation of monosaccharides to ethanol, when fed a solution containing predominantly monosaccharides, wherein improved ability to digest the

monosaccharide confers improved growth or metabolic product, for example, compared to a microbe that does not express the monosaccharide modifying polypeptide.

In some embodiments, the methods disclosed herein include contacting monosaccharides (such as a solution including monosaccharides) with one or more of the disclosed

monosaccharide modifying polypeptides. For example, the methods include contacting monosaccharides with one or more isolated polypeptides for modifying arabinose-rich monosaccharides (such as SEQ ID NOs: 6-13) or for modifying galactose- or galacturonic acid- rich monosaccharides (such as SEQ ID NOs: 24-30) under conditions sufficient for modifying the monosaccharides. In other examples, the methods include contacting monosaccharides with one or more microbes expressing one or more different polypeptides for modifying arabinose- rich monosaccharides (such as SEQ ID NOs: 6-13, for example encoded by SEQ ID NOs: 36- 43) or for modifying galactose- or galacturonic acid-rich monosaccharides (such as SEQ ID NOs: 24-30, for example encoded by SEQ ID NOs: 54-60) under conditions sufficient for modifying the monosaccharides.

In some embodiments, the methods disclosed herein include contacting monosaccharides

(such as a solution including monosaccharides) with one or more microbes expressing one or more of the disclosed monosaccharide modifying polypeptides (such as microbes expressing one or more of SEQ ID NOs: 6-13 or 24-30). In some examples, monosaccharides are contacted with a single monosaccharide modifying polypeptide or a microbe (or population of microbes) expressing a single monosaccharide modifying polypeptide. In additional examples,

monosaccharides are contacted with two or more different monosaccharide modifying polypeptides (such as two or more different arabinose-rich monosaccharide modifying polypeptides, two or more different galactose- or galacturonic acid-rich monosaccharide modifying polypeptides, or one or more arabinose-rich monosaccharide modifying polypeptides and one or more galactose- or galacturonic acid-rich monosaccharide modifying polypeptides).

In further examples, the monosaccharides are contacted with two or more different microbes (or different populations of microbes) each expressing different monosaccharide modifying polypeptides (such as different arabinose-rich monosaccharide modifying polypeptides, different galactose- or galacturonic acid-rich monosaccharide modifying polypeptides, or different arabinose-rich monosaccharide modifying polypeptides and galactose- or galacturonic acid-rich monosaccharide modifying polypeptides). In other examples, the monosaccharides are contacted with a microbe (or population of microbes) expressing two or more different monosaccharide modifying polypeptides (such as two or more different arabinose-rich monosaccharide modifying polypeptides, two or more different galactose- or galacturonic acid- rich monosaccharide modifying polypeptides, or one or more arabinose-rich monosaccharide modifying polypeptides and one or more galactose- or galacturonic acid-rich monosaccharide modifying polypeptides).

In other examples, microbes expressing one or more monosaccharide modifying polynucleotide or polypeptide are produced separately from a fermentation broth and lysed to destabilize the cell membrane and free the polypeptides into the growth solution. This solution may then be added to a fermentation broth, or added to a feedstock prior to fermentation in a pre-treatment step. The cells may be lysed by repeated freeze thaw cycles or the addition of chemicals capable of destabilizing the cell membrane, such as detergents. The solution may be filtered or centrifuged to concentrate or sterilize the solution.

In other examples, microbes expressing one or more monosaccharide modifying polynucleotides or polypeptides may be produced during fermentation within the fermentation broth. In particular examples, the microbes have a genetic mutation conferring a "leaky" phenotype, or a state where the cell membrane is unusually unstable and allows the release of polypeptides produced inside the cell. The microbe may also employ a polynucleotide adjacent to (for example, operably linked to) the disclosed polynucleotides that produces a combined polypeptide product (such as a fusion protein), wherein the additional part of the polypeptide has the ability of translocating the disclosed polypeptides to the cell surface or for export from the cell into the fermentation broth.

In additional examples, microbes expressing one or more of the monosaccharide modifying polynucleotides and polypeptides may be produced during fermentation within the fermentation broth and a portion of the broth lysed and returned to the original fermentation chamber or to a pre-treatment chamber containing new feedstock. The lysis of the microbes in the broth may be accomplished by standard methods, such as temperature change, (e.g., freeze- thaw cycles), addition of a chemical, application of radiation (for example UV or microwave radiation), sonication or other vibration, by filtration, or application of high pressure.

In some examples, the method includes contacting a feedstock, such as a suspension of chopped or ground fruit and/or fruit waste (such as fruit peel or pulp) with one or more of the monosaccharide modifying polypeptides or microbes expressing one or more of the polypeptides. The feedstock may be a solution that is primarily water, with a low concentration of salts (for example, about 0.05-5%) and a mild pH (for example, greater than 4 and less than 10). The pH may be controlled by the addition of acids or bases, or by the addition of pH buffering chemicals. In some examples, trace minerals are added passively, such as by the use of unfiltered water. In particular examples, the magnesium content is about 5-25 ppm (such as about 10 ppm). Magnesium containing compounds, such as magnesium chloride, magnesium sulfate, dolomitic lime, agricultural lime, or magnesium oxide, are added to achieve the desired magnesium level, if needed. A suspension of chopped or ground fruit material in water at a dilution of 1 part fruit to 20 parts water ranging to pure chopped fruit generally is sufficient to provide necessary salts and minerals, while also presenting the monosaccharides for structural modification.

D. Combinations

In additional embodiments, disclosed herein are methods for processing oligosaccharides that include contacting microbes expressing one or more oligosaccharide transporting polypeptides or polynucleotides and one or more monosaccharide modifying polypeptides with an arabinose-rich, galactose-rich, and/or galacturonic acid-rich oligosaccharide solution under conditions sufficient for the microbes to transport the oligosaccharides into the cell and process the oligosaccharides and resulting monosaccharides (for example to produce an industrially relevant compound such as a ethanol).

In some embodiments, oligosaccharides are contacted with a microbe (or population of microbes) expressing a single oligosaccharide transporting polypeptide and a single

monosaccharide modifying polypeptide. In other examples, the oligosaccharides are contacted with a microbe (or population of microbes) expressing two or more different oligosaccharide transporting polypeptides and one or more monosaccharide modifying polypeptides or one or more oligosaccharide transporting polypeptides and two or more different monosaccharide modifying polypeptides. In still further examples, the oligosaccharides are contacted with at least one microbe (or a population of microbes) expressing one or more oligosaccharide transporting polypeptides and at least one microbe (or a population of microbes) expressing one or more monosaccharide modifying polypeptides.

In some examples, the microbes are contacted with a solution containing

oligosaccharides, such as oligosaccharides produced using one or more of the disclosed polysaccharide degrading polypeptides disclosed herein, as described above. In other examples, the microbes are contacted with a solution containing a feedstock, such as a suspension of chopped or ground fruit and/or fruit waste (such as fruit peel or pulp). In some examples, the feedstock is pre-treated (for example, thermally, chemically, or a combination thereof, for example, as described above). The feedstock may be a solution that is primarily water, with a low concentration of salts (for example, about 0.05-5%) and a mild pH (for example, greater than 4 and less than 10). The pH may be controlled by the addition of acids or bases, or by the addition of pH buffering chemicals. In some examples, trace minerals are added passively, such as by the use of unfiltered water. In particular examples, the magnesium content is about 5-25 ppm (such as about 10 ppm). Magnesium containing compounds, such as magnesium chloride, magnesium sulfate, dolomitic lime, agricultural lime, or magnesium oxide, are added to achieve the desired magnesium level, if needed. A suspension of chopped or ground fruit material in water at a dilution of 1 part fruit to 20 parts water ranging to pure chopped fruit generally is sufficient to provide necessary salts and minerals.

In another embodiment, one or more of the disclosed polypeptides are produced separately and added to a fermentation broth in the presence of fuel-producing microbes. In some examples, fuel-producing microbes include naturally occurring organisms such as the yeast Saccharomyces cerevisiae or the bacterium Zymomonas mobilis, both of which consume sugar and produce ethanol. Fuel-producing microbes also include organisms that have been modified to produce fuel or to produce more fuel than they naturally would, such as the bacterium Escherichia coli, which has been modified to produce ethanol (for example Chen et al, Biotechnol Lett 32:87-96, 2010 or Qureshi et al, Food and Bioproducts Processing 84: 114- 122, 2006) or other advanced biofuels such as Butanol, Pinene or Fatty- Acid Ethyl esters, a precursor to diesel fuel (Bokinsky et al, PNAS 108: 19949-19954, 2011). In some examples, one or more polynucleotides encoding the disclosed polypeptides may be genetically engineered into the fuel-producing microbes. In other examples, several polynucleotides encoding polypeptides with different but complementary functions are genetically engineered into the same fuel- producing microbe strain and the microbe is cultured under conditions sufficient to produce fuel (such as ethanol). This allows synergistic functions, such as one or more polypeptides degrading a polysaccharide to an oligosaccharide, one or more transporters importing the oligosaccharide product of that degradation, and one or more monosaccharide-modifying polypeptides re-formatting the monosaccharide breakdown products into usable sugars. The fuel-producing microbes then digest the sugars and produce fuel (such as ethanol). Other engineered microbes can be used to produce other metabolic products, including commodity chemicals. In some examples, the microbes produce fuels such as methanol, ethanol, isopropanol, butanol and isobutanol, long-chain alcohols, fatty acid ethyl esters, methane, ethane, pinene, or hydrogen. These fuels may also be useful as industrial chemicals, for example as solvents, or as chemical feedstocks for the synthesis of other chemicals. Other commodity chemicals that may be produced with the disclosed polypeptides include acetic acid, plastic precursors such as polylactic acid, glycerol, and furfural.

In particular examples, a polysaccharide containing solution (such as a feedstock or fermentation broth described herein) is contacted with a microbe (or population of microbes) expressing all of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, and 13 (Group A), a microbe (or population of microbes) expressing all of SEQ ID NOs: 2, 3, 4, 5, 10, 11, and 12 (Group B), a microbe (or population of microbes) expressing all of SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, and 16 (Group C), and/or a microbe (or population of microbes) expressing all of SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, and 30 (Group D). In some examples, a polysaccharide containing solution (such as a feedstock or fermentation broth described herein) is contacted with each of the microbes of Group A, Group B, Group C, and Group D. In additional examples, a polysaccharide solution is contacted with a microbe (or population of microbes) expressing all of the polypeptides of SEQ ID NOs: 1-30.

In other examples, a polysaccharide containing solution is contacted with a microbe (or population of microbes) expressing SEQ ID NO: 1 and a microbe (or population of microbes) expressing all of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, and 13. In further examples, a polysaccharide containing solution is contacted with a microbe (or population of microbes) expressing SEQ ID NO: 2 and a microbe (or population of microbes) expressing all of SEQ ID NOs: 3, 4, 5, 10, 11, and 12. In additional examples, a polysaccharide containing solution is contacted with a microbe (or population of microbes) expressing all of SEQ ID NOs: 14, 15, and 16 and a microbe (or population of microbes) expressing all of SEQ ID NOs: 19, 20, 24, 25, and 26. In yet additional examples, a polysaccharide containing solution is contacted with a microbe (or population of microbes) expressing SEQ ID NOs: 17 and 18 and a microbe (or population of microbes) expressing all of SEQ ID NOs: 21, 22, 23, 27, 28, 29, and 30.

E. Exemplary Feedstocks

In various embodiments, the plant matter used as a source of polysaccharides include pineapple waste (e.g., peel and/or fiber), apple waste (e.g., peel and/or fiber), orange waste (e.g., rind and/or pulp), oil palm fruit waste (e.g., crushed fruit pulp, crushed kernel, and/or empty fruit bunch), beet waste (e.g., crushed beet pulp and/or peel), as well as other waste fruit and vegetable types. The plant matter also includes non-traditional plant and plant- like materials, such as algaes and kelps. Many agricultural wastes or agricultural crops contain arabinose-, galactose-, or galacturonic acid-rich polysaccharides and are potential feedstocks for the methods disclosed herein. The waste products of fruit processing, such as juicing, canning or drying, are typically rich in these types of polysaccharides and may be used for the methods disclosed herein, for example as feedstock for the engineered microbes described herein. In some examples, specific types of feedstocks include the peels, pulps or whole fruit of pineapples, oranges, tangerines, grapefruits, pomelos, other citrus fruits, apples, pears, peaches, plums, grapes, bananas, plantains, mangos, papayas, or guavas. Agricultural wastes or crops that are not commonly considered fruits may also be used as feedstocks, including sugar-beet peel, pulp or whole root.

Peels used as feedstock include outer layers of fibrous material as well as layers of edible flesh that are removed in the peeling process. Peels are typically removed from the whole fruit using automatic or manually operated blades and removal may be assisted by the use of chemicals to soften the peel, such as the application of lye (for example, to soften papaya peels). Peels may also be produced as a waste product of juicing when a whole or partial fruit is squeezed or otherwise mechanically stressed to extract fruit juice without any specific removal of the peel.

Pulps include fruit material that is not liquefied during juice extraction as well as material that is removed from juice by filtration or sedimentation for various reasons including adjusting juice texture, lowering viscosity, improving mouthfeel, for sanitary reasons, or for ease of subsequent liquids handling. Peels and pulps may be separated from each other or left connected to each other.

Whole fruit may also be used as a feedstock. Whole fruit may be grown and used directly as feedstock, may be redirected into feedstock when produced or delivered in surplus of processing capacity, or may be redirected into feedstock when fruit is rejected from sale or processing due to disease, age, physical damage, appearance, or state of decay.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. Example 1

Identification and Characterization of Target Genes

Deep Sequencing Library Preparation and Analysis

A residential compost pile was used to inoculate separate piles of plant waste, including corn cob and husk, rice husk, pineapple peel, apple peel, citrus rinds and beet pulp, as well as commercially supplied refined hemicelluloses, such as xylan, arabinogalactan, pectin and other polysaccharides. After one week, DNA was extracted from each sub-pile and subjected to deep sequencing using the technique described in Runckel et al. (PLoS One 6:e20656, 2011).

Metagenomes, or the genomes of multiple life-forms present in a sample, were assembled from the deep sequencing data and putative genes were predicted from this data, as well as possible gene functions, using the methods described (Runckel et al, 2011) and employing the Geneious DNA analysis software suite.

DNA sequences that shared motifs with genes whose protein products are known to break down polysaccharides, such as glycoside hydrolases or polysaccharide lyases, were selected as were genes whose protein products produced known transporters of oligosaccharides or whose products were known to perform re-formatting activities on mono- and di-saccharides. In total, over 99.5% of all predicted genes were discarded at this stage. 6592 genes were selected for further analysis from the datasets derived from fruit wastes.

Target genes were prioritized based on length of coding region (ideally 1000-2500 nt due to superior PCR efficiency and cloning efficiency within this size range) and diversity as determined by all-against-all translated amino acid similarity alignments. Target genes which had previously been described in NCBI's GenBank DNA and amino acid sequence repository, with 90% or greater amino acid similarity, were removed from the candidate list. The highest priority 192 glycoside hydrolases or polysaccharide lyases, 1000 transporters and 1000 reformatting enzymes were selected for recovery and testing.

Strain Creation

Each of 192 target glycoside hydrolase or lyase genes was retrieved by specific PCR. Briefly, the terminal 20-25 DNA nucleotides of each target gene were chemically synthesized as DNA oligonucleotide primers (IDT, Coralville, IA) and used in a PCR reaction performed with Phusion® DNA polymerase (Thermo Scientific, Pittsburgh, PA) with HF buffer as per manufacturer's instructions. Specifically, samples were denatured at 98°C for 2 minutes and cycled 30 times from 98°C for 15 seconds, annealing at either 58°C or 62°C for 30 seconds, and a 3 minute extension step at 68°C. A final extension step for 10 minutes at 68°C was followed by hold at 4°C. DNA extracted from compost piles was used as the template with 10 ng DNA per reaction.

After PCR, successful reactions were confirmed by gel electrophoresis and the DNA was purified by column-spin purification (Zymo Research, Irvine, CA). Amplified DNA was then phosphorylated by reaction with T4 Polynucleotide Kinase (ThermoScientific, Pittsburgh, PA) per manufacturer's instructions, again cleaned by spin column, and ligated to a tetracycline- resistant derivative of the pBR322 vector using T4 DNA Ligase (ThermoScientific) per manufacturer's instructions for blunt-end ligation. Recovered DNA constructs were transformed into DHlOb E. coli by electroporation as per manufacturer's instructions (Cole-Parmer, Vernon Hills, IL) and successful transformants selected with tetracycline.

Target genes were cloned into derivatives of the pBR322 plasmid, a classic low-copy vector with two constitutively active antibiotic resistance genes. One gene was replaced with the gene of interest (FIG. 1) and the second gene was replaced with each of four different antibiotic resistance markers. The target gene and antibiotic were thus both always expressed with strong ribosome binding sites.

Microarray PCR primers for the 1000 transporter targets and 1000 reformatting enzyme targets were designed using similar criteria as above, with an additional 20 nt "Common sequence A" added to all forward primers and a 20 nt "Common sequence B" added to all reverse primers. The sequence of Common sequence A includes a ribosome-binding site at a suitable location to the coding start site to facilitate translation of the gene target. 500 ng of template DNA was incubated with Kapa 2x Library Amplification Kit (Kapa Biosystems, Wilmington, MA) and 0.8 ng of oligonucleotide mix (consisting of all 4000 microarray produced primers suspended in water, prepared under contract by LC Biosciences, Houston, TX). The solution was denatured at 98°C for 2 minutes and cycled 10 times from 98°C for 15 seconds, annealing at 55°C for 30 seconds, and a 3 minute extension step at 68°C. 10 pmols of 20 nt oligonucleotides of Common sequence A and B (IDT, Coralville, IA) were added and PCR continued by cycling 25 times from 98°C for 15 seconds, annealing at 62°C for 30 seconds, and a 3 minute extension step at 68°C. A final extension step for 10 minutes at 68°C was followed by hold at 4°C. Both primers were ordered with a 5' phosphorylation modification to facilitate later engineering steps. At the conclusion of the reaction, the amplified DNA was incubated at 72°C for 2 hours to facilitate re-annealing of the complex DNA mixture. The amplified DNA was purified by spin-column, ligated and transformed into E. coli as described above. Transporters were ligated into vectors containing ampicillin resistance and reformatting enzymes were transformed into vectors containing either kanamycin or chloramphenicol resistance. Feedstock Sample Preparation

Two commercially- supplied purified hemicelluloses (Sigma- Aldrich, St. Louis, MO), polygalacturonic acid (a polymer of galacturonic acid) and arabic acid (a polymer of arabinose), were used as substrates to determine the various polypeptide activities, as were the purified hemicellulose fractions of orange rind, pineapple peel, and apple peel and pulp. In the case of orange rind, oranges were first peeled to remove the oily outer layer (often colloquially referred to as the zest and technically as the flavedo) and the rind (or pith or albedo) was subsequently used for further analysis. In the case of pineapple, the outer peel from a pineapple canning operation was used. This peel was mechanically removed from the fruit, lightly pressed to remove juice and de- sugared with 65°C water for 20 minutes. In the case of apple, spent cider pressings (peel and pulp) were further de-sugared in 65°C water for 20 minutes and re -pressed to partially dry the apple pressings. All fruit feedstocks were coarsely chopped to <2 cm pieces, suspended 1: 1 in 0.5% NaCl by weight and passed through a macerator pump to reduce size to less than 2 mm particles. Pure arabinose was used as a positive control monosaccharide solution, and was similarly treated. All hemicellulose, fruit hemicellulose and monosaccharide solutions were used at 0.5% calculated final weight after various dilutions during treatment.

Fruit hemicelluloses were recovered by chemical treatment and centrifugation. Briefly, homogenized fruit slurry was digested with Proteinase K (0.5 mg per dry g fruit) to hydrolyze protein. The slurry was then suspended in alternating rounds of ethanol and acetone and gently centrifuged to remove free sugars, cellular debris and amino acids. The pellet was resuspended in 17.5% NaOH and again centrifuged. The pellet, containing cellulose and very large cellular debris, was discarded. The liquid fraction was neutralized with hydrochloric acid to pH 7.0. This fraction is considered to contain hemicellulose and polysaccharides often referred to as pectins; as these compounds are structurally similar and may be attached to one another, they are dealt with as "hemicelluloses." One twentieth of the material was oven dried at 120°C to determine the proportion dry weight, and this dry- weight was used to calculate the dilution necessary for the remaining hemicellulose.

All test feedstocks were split into three different treatment conditions. One stream was used directly. This stream is referred to as the polysaccharide feedstock. A second stream was partially digested with commercially prepared pectinase (Sigma, from Aspergillus niger) at 5 units per g dry weight for 24 hours at 45°C. This formulation is the filtered supernatant of

Aspergillus (a fruit digesting fungus) culture and contains a wide variety of hemicellulases, not just pectinases. The added enzymes were inactivated by incubation at 80°C for 20 minutes.

This stream is referred to as the oligosaccharide feedstock. The third stream is similarly digested with pectinase, followed by the addition of hydrochloric acid to pH of 2 and heating at 90°C for four hours and acid neutralization with sodium hydroxide. This stream is referred to as the monosaccharide feedstock. All feedstocks were combined with M9 mineral salts (Sigma) to lx concentration as per manufacturer's instructions and adjusted to pH 7.0. Phosphate buffer was added to maintain neutral pH.

Glycoside Hydrolase and Polysaccharide Lyase Selection

Single plasmid microbe strains containing a glycoside hydrolase or polysaccharide lyase gene were grown up overnight in 10 mL of LB and antibiotic. Mature cultures were subjected to three cycles of freeze-thaw at -20°C and 37°C to lyse cells and liberate enzymes trapped within the cell. 5 mL of polysaccharide solutions were incubated with 1 mL of lysed cells at 37°C for 6 hours with shaking at 200 rpm. Viscosity was measured by the timed cup method for each sample at the conclusion of the incubation (FIG. 2). Seven polypeptides were chosen for further analysis.

Initial Transporter and Reformatting Gene Selections

Double plasmid strains containing one reformatting gene and one transporter were grown to OD600 = 1.0 and diluted 1: 100 in 2 mL of feedstock with the appropriate antibiotic. Each oligosaccharide and monosaccharide feedstock described above was used. Cultures were maintained at 200 rpm shaking under aerobic conditions at 37°C for 2 days. Cultures were passaged by diluting each culture by 1: 100 into the same feedstock. In total, each selection involved three passages. Cultures were then diluted 1: 10 into 10 mL of LB media plus antibiotic. Cultures were then extracted for plasmid DNA. Plasmid DNA was prepared for deep sequencing as described (Runckel et ah, 2011) and sequenced on an Illumina HiSeq 2000 (under contract by Elim BioPharmaceuticals, Hay ward, CA). Each feedstock selection was assigned a different DNA barcode for subsequent subdivision of the data as described in (Stenglein et ah, mBio 3:e00180-12, 2012). All samples were prepared in biological duplicate, including the selection process.

Deep sequencing produced 182 million short DNA sequences. The sequences were aligned to the set of known gene targets with a 95% nucleotide similarity cutoff; 12% of sequences matched target genes with the majority of the remainder mapping to vector or the E. coli genome. The proportion of sequences aligning to each target gene compared to the total number of sequences aligned was calculated separately for each sample. Each was then compared to the arabinose positive control. Genes that were over-represented by at least 5-fold compared to a control sample in both replicates and possessed at least 50 mapped sequences were deemed to provide a selective advantage under those conditions and chosen for further analysis.

Approximately 5-20 million reads were recovered for each experimental condition. Of 2000 target genes where recovery was attempted by microarray PCR, 27% were present with greater than 1: 10,000 proportion of mapped reads and 19% were present at greater than 1: 1000 proportion, indicating that at least 380-540 targets were recovered depending on the threshold. It was observed that the rarest gene recovered that indicated enrichment and was subsequently confirmed by other techniques was initially present at a proportion of 1 : 11 ,428, thus the higher estimate of target genes is likely applicable. Given the requirement for 50 mapped reads to confirm a positive result, the range of genes observed is estimated to be over a 10,000-fold variation.

Target genes performing transporter or reformatting activities were recovered by specific inverse PCR. Briefly, for each target two DNA oligonucleotide primers were designed to be adjacent in the gene's DNA sequence. These primers are positioned on opposite DNA strands such that in the presence of a polymerase they would prime a Polymerase Chain Reaction in opposite directions, (away from each other). PCR was performed with Phusion® DNA polymerase with HF buffer as per manufacturer's instructions and a 5 minute extension step at 68°C by default. Primers were all designed to employ a 62°C annealing temperature, and this temperature was used for all specimens. After PCR, successful reactions were confirmed by gel electrophoresis and the DNA was purified by column- spin purification (Zymo Research).

Amplified DNA was then phosphorylated by reaction with T4 Polynucleotide Kinase

(ThermoScientific) per manufacturer's instructions, again cleaned by spin column, and ligated using T4 DNA Ligase (ThermoScientific) per manufacturer's instructions for blunt-end ligation. Recovered DNA constructs were transformed into DHlOb E. coli and selected with the appropriate antibiotic.

Verification of Polypeptide Function

The activity of 24 reformatting enzymes was confirmed by observing growth rate on fully hydrolyzed monosaccharide feedstocks. Recovered gene constructs were grown to OD600 = 1.0 and diluted 1: 1000 into 2 mL of each monosaccharide feedstock. Cultures were incubated at 37°C and 200 rpm shaking for 12 hours, at which time their optical density was again measured (FIG. 3). A positive control consisted of purified arabinose. Experiments were performed in triplicate and averaged. Fifteen constructs were selected for further use.

The activity of 17 transporter enzymes was confirmed by observing growth rate on partially hydrolyzed oligosaccharide feedstocks. Recovered gene constructs were grown to OD600 = 1.0 and diluted 1: 1000 into 2 mL of each oligosaccharide feedstock. Cultures were incubated at 37°C and 200 rpm shaking for 12 hours, at which time their optical density was again measured (FIG. 4). Experiments were performed in triplicate and averaged. A positive control consisted of the purified arabinose. Eight constructs were selected for further use.

Combinations of constructs were created by mixing equal quantities of plasmid DNA of the chosen reformatting or transporter gene constructs. DHlOb E. coli were then sequentially transformed with one transporter and two reformatting gene constructs. Given equal proportions of transformants, there are 1800 possible combinations. Efficiencies of 7 x 10⁶, 2 x 10⁶ and 4 x 10⁵ transformants were achieved in serial transformations, suggesting that virtually all possible combinations would have been created. These pools are termed the "Construct Combinations." Construct combinations were assayed on the oligosaccharide feedstocks and are included in FIG. 4. Combination pools grown on partially hydrolyzed pineapple, apple and orange feedstocks achieved 72%, 79% and 90% of cell growth, respectively, compared to growth on pure arabinose, a non-ideal but still easily digested pentose monosaccharide.

Example 2

Fermentations to produce Ethanol

To validate the effectiveness of these gene constructs in producing ethanol, the

"Construct Combination" microbe pools were transformed with plasmid pETH7, redirecting some sugar digestion into ethanol production under anaerobic conditions and using tetracycline as a selectable marker. In addition, a control strain with three empty vectors and the pETH7 plasmid was prepared and fermented in parallel to provide a baseline. Each strain or set of strains was grown up overnight in 20 mL of LB media plus antibiotic under aerobic conditions, centrifuged at 1000 x g to pellet microbes and resuspended in 200 mL of 4% pineapple, citrus or apple hemicellulose feedstock or one of the two purified hemicelluloses. Feedstocks were prepared as described in Example 1 for full polysaccharide feedstocks, including the addition of M9 mineral salts and phosphate solution to buffer pH. Feedstocks were heat inactivated at 80°C for 1 hour to prevent gelling associated with high temperature sterilization.

Each fermentation contained either commercial pectinase (0.1 mg per g dry weight feedstock), 10 mL of an equal mix of the previously engineered glycoside hydrolase and polysaccharide lyase enzyme producing strains, both the engineered microbes and commercial pectinase, or 10 mL DHlOb E. coli with an empty tetracycline-based vector as a control, with e coli cultures freeze thawed three times to release enzymes. The unmodified E. coli was also added into the pectinase only sample to maintain consistency between sample conditions. With only one antibiotic resistance (tetracycline), any surviving microbes from this step cannot survive in the fermentations, which contained ampicillin, kanamycin, chloramphenicol and tetracycline at half standard concentrations.

Samples were incubated at 37°C with 100 rpm shaking and a sealed rubber covering with a pinhole for overpressure relief for five days and analyzed for ethanol production by thermal distillation (FIG. 5). Ethanol concentrations were determined by refractometry.

In all feedstocks, microbes engineered with transporters and reformatting enzymes outperformed the negative control microbes in ethanol production, and the presence of strains engineered with transporters and reformatting enzymes had more impact on ethanol production than the addition of a commercial enzyme preparation. Commercial pectinase was found to outperform the engineered polysaccharide degrading enzyme mix in orange, apple and purified polygalacturonase feedstocks and the engineered enzyme mix was found to be slightly superior for pineapple and Arabic acid feedstocks. While commercial pectinase mixes have many off- target effects, this is a reasonable result as the breakdown of a relatively low pectin feedstock like pineapple is a use the product was not designed for.

The polysaccharide degrading enzyme mix was outperformed by commercial pectinase in several substrates but showed utility in improving ethanol yields in all tested feedstocks. Further, a combination of the two outperformed either alone in all feedstocks. When these capabilities are combined the use of microbes engineered with transporters and reformatting enzymes and the ability to ferment ethanol, strains capable of producing fuel from fruit wastes approach industrial viability.

Example 3

Ethanol Fermentation with Microbes Expressing Multiple Target Genes The "Construct Combinations" described and tested in Examples 1 and 2 were further refined. Four microbe strains were engineered, each with the polynucleotides encoding one of the following groups of polypeptides: Group A (SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 13), Group B (SEQ ID NOs: 2, 3, 4, 5, 10, 11, 12), Group C (SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, 16) and Group D (SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, 30). To choose and assemble these groups, the fermentations containing the "Commercial Pectinase," the "Enzyme Pool" and the

"Construct Combinations" from Example 2 and Figure 5, were streaked on LB-Agar plates using a toothpick with selection by ampicillin, chloramphenicol and kanamycin (ACK) under the same conditions as in Example 2. The fermentation of each of the five feedstocks

(Pineapple, Apple and Orange Hemicellulose, and Commercial Arabic acid and Polygalacturonic Acid) was streaked separately. Eight colonies from each plate were grown up in 2 mL of LB+ACK under aerobic conditions.

The plasmid DNA of each "Construct Combination" was extracted using the Zyppy™ miniprep kit, as per manufacturer' s instructions (Zymo Research, Irvine, CA) and the identities of each of the three genes confirmed by either Sanger sequencing (Sequetech, Mountain View, CA) or by PCR. PCR was performed with the corresponding primers from the initial microarray mix, but ordered and synthesized individually (IDT, Coralville, IA). The PCR was performed with the Phusion® polymerase in the default buffer with the following thermocycling program: 98°C for 2 min, (98°C for 15 sec, 62°C for 30 sec, 68°C for 3 min) x 20 cycles, 68°C for 10 min, 4°C hold. PCR products were run on a 1.5% agarose gel stained with Sybr Gold for 30 minutes at 100V for size analysis. The "Overlap Extension PCR Cloning" technique (for example in Bryskin and Matsumura, Biotechniques 48:463-465, 2010) was then used to assemble the three genes from each "Construct Combination" together in each of the pBR322 plasmid backbones used previously (each has only either Amp, Chi or Kan resistance).

The one-hundred and twenty new constructs (forty constructs in each of three

backbones) were transformed into DHlOb E. coli as in Example 1, generating virtually all possible combinations as before. Each microbe possessed three randomly installed plasmids, each with three genes derived from a single "Construct Combination" strain. These strains were grown in the five feedstocks as above, streaked onto LB-Agar plates and eight colonies were selected from each. The nine genes present in each strain were identified by PCR exactly as above with the same primers. Four groupings were selected based on their frequency among the streaked colonies. Each grouping could have up to nine unique genes, but it was observed that groupings sometimes possessed the same gene multiple times on different plasmids. The four groupings (Groups A, B, C and D) were assembled, each on its own plasmid, with between seven and nine unique genes each using the same methods as described above. These single- plasmid constructs were used for further testing. It is noted that the function of each Group may be achieved with all genes on one plasmid or with the genes distributed among several plasmids, or otherwise present in the microbe such as with a chromosomal insertion.

The parent strain for all four new strains was a non-pathogenic E. coli strain that had previously been modified to over-produce ethanol and under-produce other metabolic products when fermenting saccharides under anaerobic conditions. The background of this strain is DHlOb with the pflB-focA and ldhA genes deleted and further modified with the addition of the pETH7 plasmid. Fresh fruit waste was removed from raw fruit. Pineapple peel was cut from the fruit with a knife, minimizing non-peel flesh removed. Pineapple top and bottom stems were discarded. Pineapple peel was then size-reduced using a blender. Small quantities of fresh water were added as required to achieve a particle size of less than 3 mm as determined by a sieve. Papaya peels were treated similarly to pineapple peels. Oranges were initially peeled with a vegetable peeler to remove the oily outer layer, which was discarded. The remaining soft, white peel was then removed by hand and size-reduced in a blender. The remaining orange fruit was then squeezed using a hand-operated press to remove liquids, soaked in warm water to dilute residual sugar, and then pressed again. Solid pressings (orange pulp) were then size -reduced in a blender. Guava cores were recovered by removing the flesh and skin of whole fruit with a knife and size-reducing the cores with a blender.

The moisture content of fruit wastes was then determined by drying at 120°C for 1.5 hours and comparing weight measurements taken before and after drying. Fruit wastes were diluted to 10% solids content with freshwater.

Size-reduced fruit wastes were all found to be acidic. Fruit wastes were neutralized using Dipotassium Phosphate (0.5 g per 100 mL of fruit waste). Fruit wastes were then divided into 1 L glass bottles with 630 mL of acid-neutralized waste in each bottle and sterilized by autoclaving (121°C at 15 psi pressure for 20 minutes). Wastes were cooled to room temperature before microbes were added.

Microbes were grown in standard Luria-Bertani media supplemented with 0.5% w/w glucose. Antibiotics, either kanamycin or chloramphenicol, were added to assist in maintaining the genetic modifications in the microbe strains. As used previously, half- standard

concentrations of antibiotics were employed, specifically 25 mg/L kanamycin and 10 mg/L chloramphenicol. Each of the four microbe strains was grown separately. 1 mL of media in an 8 mL glass tube was incubated with 20 μΐ_^ of frozen microbe and grown up overnight at 37°C with vigorous shaking in an aerobic environment. The 1 mL of media was then added to 100 mL of media in a 250 mL flask and cultured under similar conditions for an additional eight hours. 70 mL of the microbe liquid was then added to each bottle. For the test of all four microbe strains together, 17.5 mL of each of the four microbe strains was added to the bottle. A conventional distiller's yeast was used for comparison. 500 mL of water with 0.5% sucrose was warmed to 30°C and 5 g of dried yeast starter was added and agitated for one hour prior to addition to fruit waste bottles as above.

Fermentation bottles were agitated by hand for one minute three times a day and stored at 30°C. Anaerobic conditions were maintained using a rubber stopper and plastic water airlock. After seven days, fermentation liquids were filtered through a sieve. Ethanol content was determined by thermal distillation of 250 mL of sieve-filtered fermentation liquid, and refractometry of the distilled liquid. Each engineered microbe group individually produced more ethanol than conventional yeast, and the combination of all four engineered strains outperformed the conventional yeast or any one individual strain (Table 1).

Table 1. Calculated ethanol content of fermentations using 9% fruit waste solids and various microbe strains

Example 4

Ethanol Fermentation Scale-Up

A 250 L volume stainless steel tank was modified to function as a fermenter by the addition of a totally enclosed-head polypropylene pump for circulation and 1 inch cross-linked polyethylene (PEX) pipe with brass fittings for liquid connections. A Kynar Venturi injector was situated between the pump outflow and the tank inflow and supplied by an ozone generator via ethylene propylene diene monomer (EPDM) tubing. The outflow of the tank was similarly EPDM tubing, and leads to a water-airlock to prevent oxygen entering the system, then to a desiccator to remove humidity, and finally a manganese dioxide catalytic converter to safely dispose of excess ozone.

Each week, approximately 200 kg of wet fruit waste was obtained from local sources. Mixed fruit waste was composed of pineapple peel, papaya peel, guava cores, tangerine peels and pulp, and pomelo peels. Each fruit waste type was individually analyzed for moisture content by drying at 120°C for 1.5 hours and comparing weight measurements before and after drying. The solids content (non-moisture content as determined by this analysis) was used for all calculations. Fruit waste was chopped to less than 3 mm particles using a rotating cylinder grinder. Some wastes (including papaya peels) required three or more passes through the device to achieve proper grinding, while others (pineapple peel and guava cores) only ever required one pass. Liquids that were produced during grinding were preserved and mixed into the chopped fruit.

Each fermentation was composed of the following: 10% microbe inoculation by volume, 12.5% fruit waste solids by weight, 20% by volume UV-sterilized fresh water and the remainder UV-sterilized, recycled process water. All items were initially mixed together except for the microbe inoculation. The liquid was circulated and the acidity adjusted to pH 6.2 with sodium hydroxide (caustic soda). The Ozone Generator was then used to sterilize the entire

fermentation and fermentation system, including the pipes and pump through circulation. After one half hour of sterilization, the Ozone Generator was shut off and the system circulated for six hours, during which time most remaining ozone was chemically reacted and destroyed. The microbe inoculation was then added and the entire fermentation circulated twice a day for six days.

The microbe inoculation consisted of 22 strains of E. coli, each engineered with a different combination of genes for the hydrolysis, transport or utilization of saccharides. These strains included microbes containing genetic construct Groups A, B, C or D, as well as other engineered strains with similarly generated but different genetic constructs. All microbes were derived from the ethanol-producing strain described in Example 3. The inoculation was grown under aerobic conditions in rich media (including hydrolyzed yeast extract, glucose and mild salt solution in a near-neutral pH buffer) and with antibiotic selection to avoid other contaminating microbe types and to maintain genetic modifications in the absence of other selective pressure. In addition, two public-domain S. cerevisiae strains were also included in the inoculation to provide improved sucrose-digesting abilities. The strains used were Lalvin 7 IB- 1122 and LD Carlson Red Star DADY. The yeasts were grown in the same media under the same conditions to confluence and each yeast strain was inoculated at the same concentration as any individual E. coli strain, or 0.45% of the total fermentation volume, or a ratio of 1:222.

At the conclusion of the fermentation, the liquid fraction of the fermentation was separated from any remaining solids by a sieve. The liquid was then distilled to purify ethanol.

Ethanol-depleted liquid was UV-sterilized, and some liquid was recycled into the next fermentation. This fermentation scheme was deployed for six weeks, with ethanol production during the first week predictably lower than average due to a lack of recycled process water and the use of fresh water instead. The microbe system adequately processed mixes of fruit waste which were varied in composition each week. Further, all fruit wastes were combined together in a "single-pot" fermentation, which is advantageous for industrial processes.

To determine the usefulness of the engineered microbes over the conventional saccharide-to-ethanol process, which employs unmodified yeast, fermentations with equivalent chopped fruit waste at 1 L scale were carried out alongside the main fermentations with only yeast added during the microbe inoculation step, no engineered E. coli. Ethanol content was determined by thermal distillation of 250 mL of sieve-filtered fermentation liquid, and refractometry of the distilled liquid. The engineered microbe mix produced more ethanol than conventional yeast alone in all tested fruit waste mixes (Table 2)

Table 2 Input and output data for mixed fruit waste-to-ethanol fermentations

In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

What is claimed is:

1. A cell comprising one or more heterologous polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 1-30 or one or more heterologous polypeptides with at least 90% sequence identity to any one of SEQ ID NOs: 1-30.

2. The cell of claim 1, wherein the one or more heterologous polypeptides comprise each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, and 13 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, and 13.

3. The cell of claim 1, wherein the one or more heterologous polypeptides comprise each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 10, 11, and 12 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 10, 11, and 12.

4. The cell of claim 1, wherein the one or more heterologous polypeptides comprise each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, and 16 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, and 16.

5. The cell of claim 1, wherein the one or more heterologous polypeptides comprise each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, and 30 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, and 30.

6. The cell of claim 1, wherein the one or more heterologous polypeptides comprise one or more polysaccharide degrading polypeptides.

7. The cell of claim 6, wherein the one or more polysaccharide degrading polypeptides comprise the amino acid sequence of any one of SEQ ID NOs: 1, 2, or 14-18 or an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1, 2, or 14-18.

8. The cell of claim 7, wherein the one or more polysaccharide degrading polypeptides are encoded by a nucleic acid comprising the sequence of any one of SEQ ID NOs: 31, 32, or 44-48 or a nucleic acid with at least 70% sequence identity to any one of SEQ ID NOs: 31, 32, or 44- 48.

9. The cell of claim 1, wherein the one or more heterologous polypeptides comprise one or more oligosaccharide transporting polypeptides.

10. The cell of claim 9, wherein the one or more oligosaccharide transporting polypeptides comprise the amino acid sequence of any one of SEQ ID NOs: 3-5 or 19-23 or an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 3-5 or 19-23.

11. The cell of claim 10, wherein the one or more oligosaccharide transporting polypeptides are encoded by a nucleic acid comprising the sequence of any one of SEQ ID NOs: 33-35 or 49- 53 or a nucleic acid with at least 70% sequence identity to any one of SEQ ID NOs: 33-35 or 49- 53.

12. The cell of claim 1, wherein the one or more heterologous polypeptides comprise one or more monosaccharide modifying polypeptides.

13. The cell of claim 12, wherein the one or more monosaccharide modifying polypeptides comprise the amino acid sequence of any one of SEQ ID NOs: 6-13 or 24-30 or an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 6-13 or 24-30.

14. The cell of claim 13, wherein the one or more monosaccharide modifying polypeptides are encoded by a nucleic acid comprising the sequence of any one of SEQ ID NOs: 36-43 or 54- 60 or a nucleic acid with at least 70% sequence identity to any one of SEQ ID NOs: 36-43 or 54- 60.

15. The cell of any one of claims 1 to 14, wherein the cell comprises a bacterial cell, a fungal cell, an algal cell, a plant cell, or a mammalian cell.

16. The cell of claim 15, wherein the bacterial cell comprises E. coli.

17. The cell of claim 16, wherein the E. coli further comprises one or more modifications to the adh gene or promoter.

18. An isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-30, or an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-30.

19. An isolated nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NOs: 31-60 or a nucleic acid sequence with at least 70% sequence identity to any one of SEQ ID NOs: 31-60.

20. The nucleic acid of claim 19, operably linked to a promoter.

21. A vector comprising the isolated nucleic acid of claim 19 or 20.

22. An isolated cell comprising the vector of claim 21.

23. A method of processing one or more of polysaccharides, oligosaccharides, or

monosaccharides, comprising contacting a solution comprising polysaccharides,

oligosaccharides, and/or monosaccharides with one or more of the cells of any one of claims 1 to 17, one or more of the isolated polypeptides of claim 18, or a combination thereof, under conditions sufficient for processing the polysaccharides, oligosaccharides, and/or

monosaccharides.

24. The method of claim 23, wherein contacting the solution comprising the polysaccharides, oligosaccharides, and/or monosaccharides comprises contacting a feedstock with the one or more microbes, one or more isolated polypeptides, or a combination thereof.

25. The method of claim 24, wherein the feedstock comprises plant matter.

26. The method of claim 25, wherein the plant matter comprises whole fruit or vegetable, or fruit or vegetable peel or pulp.

27. The method of claim 25 or 26, wherein the feedstock comprises peel, pulp, or whole fruit of one or more of pineapples, oranges, tangerines, grapefruits, pomelos, other citrus fruits, apples, pears, peaches, plums, grapes, bananas, plantains, mangos, papayas, guavas, or sugar- beet.

28. The method of any one of claims 23 to 27, wherein the conditions sufficient for processing the polysaccharides, oligosaccharides, and/or monosaccharides comprise incubating the solution comprising the polysaccharides, oligosaccharides, and/or monosaccharides with the one or more microbes and/or one or more polypeptides at about 30-40°C for about 1-10 days.

29. The method of claim 28, further comprising isolating one or more metabolic products from the solution.

30. The method of claim 29, wherein the metabolic product comprises ethanol.

31. The method of any one of claims 23 to 30, wherein the contacting a solution comprising polysaccharides, oligosaccharides, and/or monosaccharides comprises contacting the solution with a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, and 13 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, and 13.

32. The method of any one of claims 23 to 30, wherein the contacting a solution comprising polysaccharides, oligosaccharides, and/or monosaccharides comprises contacting the solution with a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 10, 11, and 12 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 2, 3, 4, 5, 10, 11, and 12.

33. The method of any one of claims 23 to 30, wherein the contacting a solution comprising polysaccharides, oligosaccharides, and/or monosaccharides comprises contacting the solution with a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, and 16 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, and 16.

34. The method of any one of claims 23 to 30, wherein the contacting a solution comprising polysaccharides, oligosaccharides, and/or monosaccharides comprises contacting the solution with a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, and 30 or polypeptides with at least 90% sequence identity to SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, and 30.

35. The method of any one of claims 23 to 30, wherein the contacting a solution comprising polysaccharides, oligosaccharides, and/or monosaccharides comprises the solution with a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, and 13, a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 10, 11, and 12, a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 14, 19, 20, 24, 25, 26, 15, and 16, and a cell comprising each of the polypeptides comprising the amino acid sequence of SEQ ID NOs: 17, 18, 21, 22, 23, 27, 28, 29, and 30.