Biochemical upgrading of high-protein biomass and grain products

Abstract

The present invention relates to methods of upgrading biomass to provide useful chemical intermediates, fuels, amino acids, nutrients, etc. In particular examples, the biomass is a by-product of ethanol production and is mainly used as high-protein feed. Described herein are methods for upgrading such biomass, such as by implementing pre-treatment conditions and by employing fermentation conditions including modified organisms.

Claims

1. An isolated nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 14-25; wherein the isolated nucleic acid encodes a polypeptide sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-5; wherein in SEQ ID NO: 4, the Xaa at position 39 is Ile, Tyr, Val, Leu, Phe, or any other conservative amino acid substitution; and the Xaa at position 40 is Pro, Arg, His, Lys, Trp or any other conservative amino acid substitution; and wherein the isolated nucleic acid encodes a modified alcohol dehydrogenase having increased reactivity with nicotinamide adenine dinucleotide (NADH), as compared to a wild-type alcohol dehydrogenase.

2. A vector comprising the nucleic acid sequence of claim 1.

3. A vector comprising: a first nucleic acid sequence encoding a modified alcohol dehydrogenase, wherein the first nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 14-25; and a second nucleic acid sequence encoding a modified ketol-acid reductoisomerase wherein the second nucleic acid has at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 26-31.

4. The vector of claim 3, wherein the second nucleic acid has at least 95% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 26-31.

5. The vector of claim 3, wherein the second nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 26-31.

6. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a polypeptide sequence comprising the amino acid sequence of any one of SEQ ID NOs: 2-5.

7. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a polypeptide sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2.

8. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a polypeptide sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 3.

9. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a polypeptide that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 5.

10. The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a polypeptide sequence that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 4, wherein in SEQ ID NO: 4 the Xaa at position 39 is Ile, Tyr, Val, Leu, or Phe; and the Xaa at position 40 is Pro, Arg, His, Lys, or Trp.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1C shows exemplary process flow diagrams for upgrading biomass. Provided are (A) an exemplary process 10 including processing pathways and (B) another exemplary process 100 including a pre-treatment step 121,122 prior to each fermentation step 131,132. The unit operations of the process are depicted as designated boxes, where inputs and outputs are designated by arrows leading either into or away from a box, respectively. Also provided is (C) an exemplary process 1000 with an optional second pre-treatment step 1022.

(2) FIG. 2 shows another exemplary process flow diagram. Provided is an exemplary process 200 including inputs and outputs designated by arrows leading either into or away from a box, respectively.

(3) FIGS. 3A-3D shows exemplary processes with various sub-steps. Provided are (A) an exemplary process including sub-steps for the pre-treatment 310 and fermentation 320 steps; (B) an exemplary process including a distillation/extraction step 3005 between the pre-treatment 3003 and fermentation 3004 steps; (C) another exemplary process including a first pre-treatment step 3103 and a subsequent distillation/extraction step 3105 to separate a biomass solid (e.g., a bioresidue); and (D) yet another exemplary process including distillation/extraction steps 3505,3605 subsequent to each fermentation step 3504,3604.

(4) FIGS. 4A-4B shows comparative technoeconomic potential of various high-protein feedstocks, including dried distillers grains with solubles (DDGS). Provided are (A) a comparison of net theoretical amino acid yields for various biomass and (B) a comparison of unutilized high value amino acids for various biomass. The asterisk in (A) indicates that remineralized ammonia (as NH.sub.4MgPO.sub.4) was included in the mass balance.

(5) FIG. 5 shows the effect of redox cofactor engineering to produce various genetically engineered organism strains. Provided is a graph showing the concentration of fusel butanol produced by various redox mutant strains in the presence of an amino acid mixture. Redox mutant strains include AY1, AY2, AY3, AY5, VY2, VY3, VY5, IA, and IV, as described herein, which were compared to the wild-type (WT) strain.

(6) FIG. 6 shows an exemplary process flow diagram for upgrading biomass, such as DDGs.

(7) FIG. 7 shows another exemplary process flow diagram for further optimization.

(8) FIG. 8 shows carbohydrate hydrolysis and ethanol yields, in which the process included dilute acid pre-treatment and fermentation.

(9) FIG. 9 shows protein hydrolysis and fusel alcohol yields, in which the process included dilute acid pre-treatment, enzymatic pre-treatment (e.g., protease digestion), and fermentation.

(10) FIGS. 10A-10G shows the effects of cofactor engineering for E. coli alcohol dehydrogenase (YqhD). Provided are (A) an image showing structural alignment of the NADP-binding pocket for YqhD and (B) a graph showing mutant enzyme activity employing cofactor NADH, as compared to WT YqhD. Also provided are polypeptide sequences for E. coli YqhD, including sequences for (C) wild-type (SEQ ID NO:1) having Gly at positions 37-39 and Ser at position 40; (D) YqhD mutant 1 including a mutation at position 39 (SEQ ID NO:2), in which Xaa at position 39 can be any useful amino acid substitution (e.g., Ile, Tyr, Val, Leu, Phe, or any other conservative amino acid substitution described herein); (E) YqhD mutant 2 including a mutation at position 40 (SEQ ID NO:3), in which Xaa at position 40 can be any useful amino acid substitution (e.g., Pro, Arg, His, Lys, Trp, or any other conservative amino acid substitution described herein); (F) YqhD mutant 3 including a mutation at both positions 39 and 40 (SEQ ID NO:4), in which Xaa at position 39 and 40 can be any useful amino acid substitution (e.g., Ile at position 39 and Arg at position 40; Tyr at position 39 and His at position 40; as well as any other conservative amino acid substitution described herein); and (G) YqhD mutant 3 including a mutation at positions 39 and 40 (SEQ ID NO:5) with an optional substitution at positions 37 and 38, in which Xaa at position 39 and 40 can be any useful amino acid substitution (e.g., Ile at position 39 and Arg at position 40; Tyr at position 39 and His at position 40; as well as any other conservative amino acid substitution described herein). In some instances, the mutant (e.g., of SEQ ID NO:2-5) includes Gly, Ile, Tyr, Val, Leu, Phe, or any other conservative amino acid substitution described herein, for positions 37, 38, and 39; and Ser, Pro, Arg, His, Lys, Trp, or any other conservative amino acid substitution described herein for position 40 (e.g., in which position 39 and/or 40 includes an amino acid substitution as compared to the wild-type sequence, e.g., SEQ ID NO:1).

(11) FIGS. 11A-11B provides polypeptide sequences for E. coli ketol-acid reductoisomerase IlvC, including sequences for (A) wild-type (SEQ ID NO:6) having Ala at position 71, Arg at position 76, Ser at position 78, Gln at position 110, Asp at position 146, Gly at position 185, and Lys at position 433; and (B) IlvC mutant including a mutation at positions 71, 76, 78, and/or 110 (SEQ ID NO:7). Exemplary substitutions include Ser or Thr at position 71, Asp or Glu at position 76, Asp or Glu at position 78, and/or Val or Ala or Leu or Ile at position 110, as well as any other conservative amino acid substitution described herein. Optionally, SEQ ID NO:7 can include other substitutions, including Gly or Ala at position 146, Arg or Lys at position 185, and/or Glu or Asp at position 433.

(12) FIGS. 12A-12B shows the effect of adding native cofactor NADPH on yield of butanol for protein bioconversion. Provided are (A) a graph showing the effects of NADPH addition on the produced concentration fusel butanol under anaerobic fermentation conditions and (B) a graph showing the improvement of fusel butanol yield after 24 hours and 120 hours.

(13) FIGS. 13A-13C shows bioconversion of algal protein hydrolysates into fusel butanol by engineered mutant strains. Provided are (A) a graph showing fermentation performance of engineered mutant strains on an amino acid mixture; (B) a graph showing bioconversion of two algal protein hydrolysates (ATP3 or ODU) into fusel butanol employing either an engineered mutant strain AY3 or a wild-type strain YH83 (“WT”); and (C) a graph showing consumption of two algal protein hydrolysates (ATP3 or ODU) by engineered mutant strain AY3 after 72 hours.

DETAILED DESCRIPTION OF THE INVENTION

(14) The present invention relates to the upgrading biomass (e.g., high-protein biomass) into ethanol and other useful intermediates, such as amino acids, bioresidue, etc. Such intermediates, in turn, can be suitable for any useful industrial process, such as downstream refining, e.g., using known petrochemical facilities and processes. In particular embodiments, an exemplary process of the invention combines pre-treatment of the biomass to solubilize and hydrolyze the carbohydrate and protein fractions; followed by fermentation and optional distillation/extraction to recover useful alcohols, amino acids, etc. In some embodiments, the process employs biochemical steps to effectively solubilize, hydrolyze, and/or degrade components of the biomass (e.g., by employing a genetically engineered organism, such as any herein).

(15) An exemplary process 10 is shown in FIG. 1A. A biomass 11 is provided, typically including fractions of proteins, carbohydrates, and/or lipids (collectively, biocomponents). Further treatment steps can be employed to breakdown these biocomponents into useful residuals. Exemplary steps include pre-treatment 12 (e.g., employing dilute acid and/or enzymes in sequential or simultaneous steps) to hydrolyze and/or solubilize the biocomponents (e.g., such as to provide one or more sugars, including glucose, xylose, arabinose, etc.); as well as fermentation and optional distillation 13 to degrade the biocomponents into one or more alcohols (e.g., ethanol, 1-propanol, isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-phenylethanol, isopentanol, etc.), amino acids, and amines.

(16) Fermentation is typically employed to degrade sugars, carbohydrates, and proteins into further, smaller chemical components, such as alcohols, amino acids, and amines. In use, fermentation employs one or more organisms, such as bacteria or yeast, to degrade these biocomponents. Exemplary organisms are described herein (e.g., bacteria such as E. coli, as well as mutant forms that are selected to degrade sugars, carbohydrates, and/or proteins in a selective, specific, and/or useful manner). Typically, such organisms do not degrade lipids. Thus, conventional fermentation is usually conducted in aqueous, non-lipid samples. In some embodiments of the invention herein, organisms can be genetically modified to convert lipids. In other embodiments of the invention herein, the fermentation step includes use of one or more lipids, lipid vesicles, and/or lipid micelles within the fermentation broth.

(17) After fermentation, one or more by-products (e.g., minerals, nutrients, water, etc.) can be optionally removed from any of the fractions. Nutrients, by-products, and water can be extracted within any point of the processing stream and with any useful mixture obtained within the processing stream. Such extraction steps can include removal of by-products from the fermentation broth or a portion thereof, delivery of nitrogen (N) and/or phosphorous (P) sources (e.g., as a salt, a mineral, etc.), and/or delivery of water. Any fractions obtained from these biocomponents can be processed to recovery nutrients (e.g., N and/or P) in any useful form, such as a protonated form (e.g., ammonia for capturing N), an oxide form (e.g., phosphate for capturing P), a salt form, and/or a mineral (e.g., struvite for capturing N and P).

(18) In other examples, after the fermentation step, the aqueous and non-aqueous (e.g., lipid) fractions are phase-separated and processed in parallel steps. For instance, the non-aqueous fraction, including a bioresidue (e.g., a low nitrogen organic residue) composed of one or more lipids, can be treated by way of hydrothermal liquefaction (HTL) 14 to provide a biocrude oil. Any solid residuals, such as ash or char, can be removed after liquefaction. Any liquid residuals can be further processed to recover 15 any useful nutrients.

(19) In another example, the aqueous fraction (e.g., including water-soluble components) can be distilled to remove alcohols (e.g., along with neutral lipids). Optionally, such fractions or extracted fractions can be further processed to recover any useful nutrients 15 (e.g., for recovering nitrogen and/or phosphorus) or usable water 16 (e.g., for use in multi-pass recycle operations) in the aqueous phase, as well as to extract any lipids present in the aqueous phase.

(20) Any useful thermochemical process can be employed to process a bioresidue into a biocrude oil. Exemplary thermochemical processes include liquefaction, pyrolysis, gasification, and/or combustion in the optional presence of one or more catalysts. Experimental conditions (e.g., temperature, pressure, air composition, reactants, reagents, etc.) can be optimized in any useful manner to achieve the desired biocrude oil with appropriate viscosity, color, oxygen content, nitrogen content, etc. In addition, the biocrude oil can be further upgraded into biofuels, such as by use of hydrotreatment (e.g., as described herein).

(21) Yet another upgrading process 100 is shown in FIG. 1B, which provides a pre-treatment step 121,122 prior to each fermentation step 131,132. Overall, the exemplary non-limiting process 100 includes providing 111 a biomass (e.g., distillers grain); pre-treating 121 the biomass (e.g., with a dilute acid pre-treatment) and optionally separating a biomass solid; fermenting 131 the carbohydrate fraction (e.g., including one or more sugars) of the biocomponents with a genetically engineered organism useful for degrading carbohydrates (e.g., thereby producing ethanol) with optional distillation (e.g., of the produced ethanol); pre-treating 122 the biomass (e.g., with one or more enzymes); fermenting 132 the protein fraction (e.g., including one or more proteins, peptides, and/or amino acids) of the biocomponents with a genetically engineered organism useful for degrading amino acids (e.g., thereby producing one or more mixed alcohols, lipids, amino acids, and/or amines) with optional distillation (e.g., of the produced mixed alcohols); liquefying 140 any residual or fraction of a fermentation product (e.g., by way of hydrothermal liquefaction, thereby producing a biocrude); and recovering 150 any residual or fraction of a fermentation product (e.g., thereby producing a nutrient, such as nitrogen, phosphorous, magnesium etc.).

(22) Pre-treatment can be employed to release various biocomponents from biomass that can be difficult to process. In addition, the process can include separated fermentation steps, in which each step can be optimized or selected to degrade a particular type of biocomponent. As seen in FIG. 1B, the process 100 includes a first fermentation step 131 useful for degrading sugar and carbohydrate components, thereby producing ethanol; and a second fermentation step 132 useful for degrading protein and amino acid components, thereby producing mixed alcohols, amines, and released amino acids.

(23) Each pre-treatment step can be optionally followed by a separation step, such as by separating one or more solid components from a liquid portion. The liquid portion can be further processed (e.g., by fermentation), and the separated solid components can be re-introduced into the process at a later step in the processing pathway (e.g., enzymatic pre-treatment).

(24) A second pre-treatment step can be optional. FIG. 1C shows another exemplary upgrading process 1000 that includes providing 1011 a biomass (e.g., distillers grain); pre-treating 1021 the biomass (e.g., with a dilute acid pre-treatment); fermenting 1031 the carbohydrate fraction (e.g., including one or more sugars) with optional distillation (e.g., of the produced ethanol); optionally pre-treating 1022 the biomass (e.g., with one or more enzymes); fermenting 1032 the protein fraction (e.g., including one or more proteins, peptides, and/or amino acids) with optional distillation (e.g., of the produced mixed alcohols); liquefying 1040 any residual or fraction of a fermentation product (e.g., by way of hydrothermal liquefaction, thereby producing a biocrude); and recovering 1050 any residual or fraction of a fermentation product (e.g., thereby producing a nutrient, such as nitrogen, phosphorous, magnesium etc.)

(25) Another exemplary conversion process 200 is shown in FIG. 2, which provides various inputs and outputs during the process. As can be seen, the process 200 includes a first step of providing 211 a biomass (e.g., distillers grain), which can then be pre-treated 221. This pre-treatment step 221 can include any useful input, e.g., water, one or more acids (e.g., dilute or strong H.sub.2SO.sub.4), a neutralizer (e.g., a base, such as NaOH), and/or one or more enzymes (e.g., a protease or a protease cocktail). The fermentation step 231 results in conversion of carbohydrates into one or more smaller or simpler components (e.g., alcohols) by employing an organism strain useful for sugar fermentation. A second pretreatment step 222 generally includes one or more enzymes, and a second fermentation step 232 results in conversion of proteins into one or more smaller or simpler components (e.g., alcohols, amino acids, ketoacids, and/or amines) by employing an organism strain useful for protein fermentation. Such smaller or simpler components can be further purified (e.g., extracted, distilled, precipitated, etc.) to provide pharmaceutical intermediates, chemicals, chemical/biochemical precursors, building blocks, reagents, and/or intermediates.

(26) A distillation step (e.g., conducted after one fermentation step or after each fermentation step) can result in the separation of volatile fraction(s) from the less volatile fraction(s), resulting in, e.g., a fraction including one or more alcohols or mixtures thereof, and another fraction including a predominantly non-aqueous, lipid phase (e.g., a bioresidue). The fraction including alcohol(s) can be further purified to provide bioethanol.

(27) Optionally, the bioresidue can be thermally treated at a temperature sufficient to separate volatile lipids from solid residuals, such as by way of hydrothermal liquefaction 240, to produce a liquefied mixture. This liquefied mixture can include biocrude oil, ash, biochar, and other components. The biocrude oil, in turn, can be further processed, e.g., by way of hydrotreatment with an input of hydrogen, to produce any useful biofuel, such as biodiesel, naphtha, or light hydrocarbons. Other components from the liquefied mixture can be phase separated to extract the solid residuals. This liquid phase can be further processed for nutrient and water recovery 250.

(28) Any useful biomass can be employed. Exemplary biomass include distillers grains or co-products (e.g., wet distillers grains (WDGs), dried distillers grains (DDGs), dried distillers grains with solubles (DDGS), fatty acids from oil hydrolysis, lipids from evaporation of thin stillage, syrup, distillers grains, distillers grains with or without solubles, solids from a mash before fermentation, solids from a whole stillage after fermentation, biodiesel, and acyl glycerides), oilseed meals (e.g., soybean meal or canola meal), feeds (e.g., alfalfa meal, cottonseed meal, DDGS, rice bran, or wheat bran), yeast (e.g., extracts), algae (e.g., Nannochloropsis, wastewater algae, or any described herein), cereal by-products (e.g., whey), etc.

(29) Pre-Treatment of the Biomass

(30) Pre-treatment can be used to convert constituents within the biomass into various biocomponents (e.g., proteins, carbohydrates, fatty acids, and/or lipids). Such biocomponents can be pre-treated to obtain more solubilized or hydrolyzed constituents, such as amino acids or sugars (e.g., glucose). For instance, carbohydrates within the biomass can be pre-treated and, thereby, be converted into a sugar and/or an alcohol, such as glucose, fucose, galactose, xylose, mannose, mannitol, ethanol, butanol, and/or pentanol. In another instance, proteins within the biomass can be treated and, thereby, hydrolyzed and converted into amino acids. Such amino acids, in turn, can be fermented to produce one or more mixed alcohols and amines. In addition, one or more extraction techniques can be applied to separate the protein/carbohydrate fraction from other constituents. Such extraction techniques can include, e.g., use of one or more ionic liquids to selectively extract a particular fraction.

(31) Pre-treatment can include the use of one or more acids, bases, oxidizers, reducers, and/or enzymes. Exemplary pre-treatment conditions include strong and/or dilute acid hydrolysis (e.g., with H.sub.2SO.sub.4 and/or HCl), base hydrolysis or neutralization (e.g., with NaOH), heat treatment, sonication, and/or enzyme degradation (e.g., with one or more proteases, such as endoproteases, exoproteases, serine proteases (e.g., subtilisin, also known as alcalase), aminopeptidases, carboxypeptidases, endoglucanases, cellobiohydrolases, glycoside hydrolases (e.g., lysozyme), endoglucanases, glucanases, endoxyalanases, pectinases, sulfatases (e.g., arylsulfatases), cellulases, xylanases, as well as mixtures thereof, such that available as commercially available Pronase®, a mixture of proteolytic enzymes that are produced in the culture supernatant of Streptomyces griseus K-1).

(32) Fermentation

(33) Fermentation conditions generally include the use of one or more organisms to convert starting reactants (e.g., biocomponents, such as carbohydrates, proteins, sugars, amino acids, etc.) into alcohol and other co-products. Fermentation can include degradation of carbohydrates into alcohol in the presence of one or more organisms. Such conditions can also release mixed alcohols and nitrogen from degradation of protein, which can contain up to about 90% of the nitrogen in a biomass. In this manner, fermentation provides useful biofuels and intermediates (e.g., alcohols). Furthermore, released nitrogen can be recovered and recycled.

(34) For fermentation, any useful organisms can be employed, such as one or more bacteria (e.g., Escherichia, such as E. coli; Zymobacter, such as Z. palmae; or Zymomonas, such as Z. mobilis) and one or more yeast (e.g., Saccharomyces, such as S. cerevisiae), including mutant forms thereof, including those that deaminate protein hydrolysates (e.g., into ketoacids, tricarboxylic acid cycle intermediates, etc.), that convert proteins to alcohols (e.g., to C4 or C5 alcohols), and/or that lack one or more quorum-sensing genes (e.g., genes luxS or lsrA), such as those described in Atsumi S et al., “Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels,” Nature 2008; 451:86-90; and Huo Y X et al., “Conversion of proteins into biofuels by engineering nitrogen flux,” Nat. Biotechnol. 2011; 29(4):346-51, which is incorporated herein by reference in its entirety; yeast (e.g., Saccharomyces, such as S. cerevisiae and S. uvarum); and fungi (e.g., Aspergillus, such as A. niger, A. terreus, and A. fumigatus).

(35) In one instance, fermentation is conducted in the presence of one or more organisms useful for sugar or carbohydrate fermentation. Such organisms can include those selected by directed evolution to employ any useful sugar substrate, to have enhanced alcohol tolerance, and/or to have increased activity. Exemplary organisms include E. coli KO11, E. coli LY01, E. coli SZ110, E. coli LY168, Z. mobilis mobilis AX101, S. cerevisiae 424A (LNH-ST), and S. cerevisiae ATCC 96581.

(36) In another instance, fermentation is conducted in the presence of one or more organisms useful for protein fermentation. Such organism can include those selected by directed evolution to switch cofactor specificity, to deaminate protein hydrolysates, and/or to reduce competing pathways. Exemplary organisms include those having mutants forms of one or more enzymes, such as YqhD and/or IlvC mutants to switch cofactor specificity, transhydrogenase overexpression in E. coli PntAB, alcohol dehydrogenase mutants (e.g., mutants of AdhE, AdhP, EutG, YiaY, YqhD, and/or YjgB), and/or ketol-acid isomerase mutants (e.g., mutants of IlvC, IlvD, IlvH, and/or IlvA), as well as any described in Brinkmann-Chen S et al., “General approach to reversing ketol-acid reductoisomerase cofactor dependence from NADPH to NADH,” Proc. Nat'l Acad. Sci. USA 2013; 110(27):10946-51; and Bastian S et al., “Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli,” Metab. Eng. 2011; 13(3):345-52, each of which is incorporated herein by reference in its entirety.

(37) Exemplary mutants include an YqhD mutant (e.g., a mutant having a polypeptide sequence with at least one amino acid substitution, as compared to wild-type YqhD (e.g., such as SEQ ID NO:1 in FIG. 10C). In one non-limiting example, the YqhD mutant has a polypeptide sequence of SEQ ID NO:2 (FIG. 10D), or a fragment thereof, in which Xaa at position 39 is Ile, Tyr, Val, Leu, Phe, or any other conservative amino acid substitution described herein. In another non-limiting example, the YqhD mutant has a polypeptide sequence of SEQ ID NO:3 (FIG. 10E), or a fragment thereof, in which Xaa at position 40 is Pro, Arg, His, Lys, Trp or any other conservative amino acid substitution described herein.

(38) In yet another non-limiting example, the YqhD mutant has a polypeptide sequence of SEQ ID NO:4 (FIG. 10F), or a fragment thereof, in which Xaa at position 39 is Ile, Tyr, Val, Leu, Phe, or any other conservative amino acid substitution described herein; and in which Xaa at position 40 is Pro, Arg, His, Lys, Trp or any other conservative amino acid substitution described herein. In another embodiment, the mutant includes Ile at position 39 and Arg at position 40; Ile at position 39 and Lys at position 40; Ile at position 39 and His at position 40; Val at position 39 and Arg at position 40; Val at position 39 and Lys at position 40; Val at position 39 and His at position 40; Leu at position 39 and Arg at position 40; Leu at position 39 and Lys at position 40; Leu at position 39 and His at position 40; Tyr at position 39 and His at position 40; Phe at position 39 and His at position 40; Phe at position 39 and Lys at position 40; Phe at position 39 and Arg at position 40; Trp at position 39 and Arg at position 40; Trp at position 39 and Lys at position 40; Trp at position 39 and His at position 40; Ser at position 39 and His at position 40; or Ser at position 39 and Thr at position 40.

(39) In another non-limiting example, the YqhD mutant has a polypeptide sequence of SEQ ID NO:5 (FIG. 10G), or a fragment thereof, in which one or more amino acid substitutions are present at positions 37, 38, 39, and/or 40. In one embodiment, the mutant includes one of the following amino acids, independently, at position 37, 38, and 39: Gly, Ile, Tyr, Val, Leu, Phe, or any other conservative amino acid substitution described herein. In another embodiment, the mutant includes one of the following amino acids at position 40: Ser, Pro, Arg, His, Lys, Trp, or any other conservative amino acid substitution described herein.

(40) Other exemplary mutants include an IlvC mutant (e.g., a mutant having a polypeptide sequence with at least one amino acid substitution, as compared to wild-type IlvC (e.g., such as SEQ ID NO:6 in FIG. 11A). In one non-limiting embodiment, the IlvC mutant has a polypeptide sequence of SEQ ID NO:7 (FIG. 11B), or a fragment thereof, in which one or more amino acid substitutions are present at positions 71, 76, 78, 110, 146, 185, and/or 433.

(41) The mutants can have any useful characteristic provided during selective evolution. In one non-limiting instance, the mutant can have increased reactivity with nicotinamide adenine dinucleotide (NADH), as compared to a wild-type reference protein. In another instance, the mutant can have increased reactivity NADH over nicotinamide adenine dinucleotide phosphate (NADPH), as compared to a wild-type reference protein. In yet another instance, the mutant can have increased specificity for NADH over NADPH, as compared to a wild-type reference protein.

(42) FIG. 3A shows a portion of an exemplary process including a pre-treatment step 310, a fermentation step 320, a distillation/extraction step 330, and a lyophilization step 340. Each of these steps, in turn, can include one or more other sub-steps. For instance, pre-treatment 310 can include acid treatment 311 in order to degrade proteins and carbohydrates into soluble and/or hydrolyzed protein and sugar; as well as enzyme treatment 312 in order to degrade proteins into amino acids. Each of these components (e.g., proteins, carbohydrates, sugars, amino acids, etc.) obtained from the pre-treatment step is considered a biocomponent. In some instances, pre-treatment 310 results in solubilization of useful biocomponents, as well as separation of biomass solids.

(43) Fermentation can include use of one or more organisms configured to facilitate degradation (e.g., specific or non-specific degradation) of one or more biocomponents. As can be seen, an exemplary fermentation step 320 includes use of at least two organisms, in which a first organism 321 is useful for degradation of sugar into alcohol and in which a second organism 322 is useful for degradation of amino acid into an alcohol (e.g., R.sup.AOH, in which R.sup.A is an optionally substituted alkyl, such as an optionally substituted C.sub.2-10 alkyl) and an amine (e.g., N.sup.+R.sup.1R.sup.2R.sup.3R.sup.4 or NR.sup.1R.sup.2R.sup.3, in which each of R.sup.2, R.sup.3, and R.sup.4 is, independently, H or an optionally substituted alkyl). The fermentation step 320 results in a fermentation product, which can include a mixture of alcohols, amino acids, amines, and/or lipids.

(44) After distillation/extraction 330, various fractions of the mixture can be separated into different components, including a first fraction including mostly lipids and lipid products (e.g., a bioresidue); a second fraction including mixed alcohols and, optionally, neutral lipids; and a third fraction including amines and/or amino acids. The first fraction can be further processed (e.g., by way of liquefaction and/or pyrolysis) to produce a biocrude oil, which can be treated to form a biofuel. The second fraction can be further purified into, e.g., bioethanol. The third fraction can be further processed to isolate high-value amino acids. In some embodiments, the pre-treatment and fermentation conditions herein can provide enhanced alcohol yield (see, e.g., FIG. 8), as well as enhanced amino acid degradation and enhanced fusel alcohol yield (FIG. 9).

(45) The pre-treatment, distillation/extraction, and fermentation steps can be conducted in any useful order. For instance, the fermentation step can be conducted prior to distillation/extraction, meaning that lipids, proteins, and carbohydrates, as well as derived components thereof, are present during fermentation.

(46) Alternatively, the distillation/extraction step can be conducted prior to fermentation, meaning that the lipid fraction will be omitted from the fermentation step. FIG. 3B shows a portion of an exemplary process including a pre-treatment step 3003, a distillation/extraction step 3005, and a fermentation step 3004. Pre-treatment 3003 can include the sub-steps of acid treatment 3013 in order to degrade proteins and carbohydrates into soluble and/or hydrolyzed protein and sugar; as well as enzyme treatment 3023 in order to degrade proteins into amino acids, thereby providing one or more biocomponents.

(47) Next, a distillation/extraction 3005 step is conducted to provide a first fraction including mostly lipids and lipid products (e.g., a bioresidue) and a second fraction including soluble biocomponents. The first fraction can be further processed (e.g., by way of liquefaction and/or pyrolysis) to produce a biocrude oil, which can be treated to form a biofuel. The second fraction can be fermented and further purified into, e.g., bioethanol. As can be seen, an exemplary fermentation step 3004 includes use of at least two organisms, in which a first organism 3014 is useful for degradation of sugar into alcohol and in which a second organism 3024 is useful for degradation of amino acid into alcohol and an amine (e.g., including ammonium).

(48) The process can include a pre-treatment step that precedes each fermentation step. FIG. 3C shows a portion of an exemplary process including a first pre-treatment step 3103, a distillation/extraction step 3105, and a first fermentation step 3104. Pre-treatment 3103 can include acid treatment 3113 in order to degrade proteins and carbohydrates into soluble and/or hydrolyzed protein and sugar, followed by fermentation 3104 with a first organism 3114 that is useful for degradation of sugar into alcohol.

(49) The process can be followed by a second pre-treatment step 3203 and a second fermentation step 3214. Pre-treatment 3203 can include enzyme treatment 3213 in order to degrade proteins into amino acids, thereby providing one or more biocomponent, followed by fermentation 3204 with a second organism 3214 that is useful for degradation of amino acid into alcohol and an amine (e.g., an ammonium).

(50) Distillation/extraction steps can be conducted at any useful time during the process. FIG. 3D shows a portion of an exemplary process including a first pre-treatment step 3503, a first fermentation step 3504, a second pre-treatment step 3603, and a second fermentation step 3604.

(51) Pre-treatment steps 3503,3603 can include acid treatment 3513 in order to degrade proteins and carbohydrates into soluble and/or hydrolyzed protein and sugar; or enzyme treatment 3613 in order to degrade proteins into amino acids. Fermentation steps 3504,3604 can include use of a first organism 3514 that is useful for degradation of sugar into alcohol and a second organism 3614 that is useful for degradation of amino acid into alcohol and an amine (e.g., an ammonium). Optionally, a first distillation/extraction step 3505 can be conducted after the first fermentation step 3504, thereby isolating bioethanol and removing this potentially inhibitory product from the fermentation product. In another example, a second distillation/extraction step 3605 can be conducted after the second fermentation step 3604, thereby isolating mixed alcohols from the fermentation product. In yet another example, a further separation step (e.g., a lyophilization step 3606) can be employed to isolate amino acids from the fermentation product.

(52) Distillation/Extraction

(53) The alcohol fermentation products, lipids, and amino acids from the biomass can be captured by distillation and solvent co-extraction. Retaining the lipids through the protein fermentation has been demonstrated to increase yield by reducing product inhibition by phase segregation into lipid microparticles, which can be extracted by lipophilic solvents, such as hexane and ethyl acetate, avoiding high energy fractional distillation of the more than C.sub.2 alcohol (e.g., C.sub.2-10 alcohol) and lipid products.

(54) Any useful distillation and extraction techniques can be employed, including flash extraction, ionic liquid extraction, etc., to isolate one or more biocrude oil, aqueous phases, aqueous co-products, nutrients, etc.

(55) Thermal Conversion, Liquefaction or Pyrolysis

(56) High-temperature treatment (e.g., liquefaction or pyrolysis) can be used to separate or convert particular components of the biomass solids, bioresidue, etc. Exemplary thermal conversion conditions include use of catalysts, use of hydrogen (e.g., in hydrotreatment), use of water (e.g., in liquefaction, including sub-critical or super-critical water), use of aerobic conditions, use of anaerobic conditions (e.g., in pyrolysis), use of high pressure (e.g., of from about 2,000 psi to about 3,000 psi), and/or use of high temperatures (e.g., of from about 200° C. to about 800° C.) to decompose the bioresidue into small molecules, which in turn can react and repolymerize to form oily compounds within a biocrude oil.

(57) In one instance, the thermal conversion condition includes liquefaction, which is generally conducted in the presence of water. By using high temperature and/or high pressure conditions, water becomes a reactive compound that converts the bioresidue into a biocrude oil. Exemplary liquefaction conditions include a wet biomass (e.g., more about 70% moisture), a temperature of from about 200° C. to about 500° C., and a pressure of from about 4 to about 25 MPa.

(58) In another instance, the thermal conversion condition includes pyrolysis, which is generally conducted in the absence of water and in anaerobic conditions. Exemplary pyrolysis conditions include a dry biomass (e.g., less than about 5% moisture), a temperature of from about 200° C. to about 750° C., and a pressure of from about 0.1 to about 0.5 MPa.

(59) Exemplary thermal conversion conditions are described in Ma F et al., “Biodiesel production: a review,” Bioresourc. Technol. 1999; 70:1-15; Naik S N et al., “Production of first and second generation biofuels: a comprehensive review,” Renew. Sustain. Energy Rev. 2010; 14:578-97; Raheem A et al., “Thermochemical conversion of microalgal biomass for biofuel production,” Renew. Sustain. Energy Rev. 2015; 49:990-9; Ringer M et al., “Large-scale pyrolysis oil production: a technology assessment and economic analysis,” National Renewable Energy Laboratory Technical Report NREL/TP-510-37779, November 2006, 93 pp.; and Schneider R C S et al., “Potential production of biofuel from microalgae biomass produced in wastewater,” in Biodiesel—Feedstocks, Production and Applications, Prof. Zhen Fang (ed.), InTech, 2012, 22 pp., each of which is incorporated herein by reference in its entirety.

(60) Any of the liquefaction steps herein can be replaced by any other thermal conversion step (e.g., pyrolysis) in which high temperature conditions are employed to thermally degrade a bioresidue.

(61) Hydrotreatment

(62) Hydrotreatment is generally used to convert compositions into useful intermediate products or end-use products. Such hydrotreatment generally includes use of high temperatures to institute any useful chemical change, e.g., to break apart triglycerides; to form low molecular weight carbon species, such as optionally substituted alkanes, cycloalkanes, or aryls; to saturate carbon chains with hydrogen; to denitrogenate species; and/or to deoxygenate species to form alkanes, such as n-alkanes. For instance, hydrotreatment can be used to upgrade biocrude oil into biofuels, biochar, or ash; as well as to convert aqueous co-products into biogas. Biocrude oil produced from the post-fermentation residuals by HTL is indicated to have ˜50% reduction in nitrogen (primary and secondary amines), thus making it acceptable for hydrotreatment using the existing petrochemical infrastructure.

(63) Hydrotreatment can include isomerization, hydrocracking, distillation, hydrodeoxygenation, catalytic processing (e.g., such as use of one or more catalysts to remove nitrogen, oxygen, and/or sulfur from the biocrude oil under any useful condition, such as a pressure of from about 5 MPa to about 15 MPa and a temperature of from about 200° C. to about 450° C.), liquefaction (e.g., such as hydrothermal liquefaction (HTL) or catalytic liquefaction of a biocrude oil into a biofuel or a biofuel intermediate by use of an operating temperature of from about 100° C. to about 500° C.), transesterification (e.g., treatment of biocrude oil with an alcohol and an optional catalyst to produce methyl ester biodiesel), and/or catalytic hydrothermal gasification (CHG) (e.g., of an aqueous co-product into biogas).

(64) The hydrotreatment process can employ any useful catalyst (e.g., a metal catalyst, such a copper-based catalyst (e.g., CuCr, CuO), a nickel-based catalyst (e.g., NiMo), a ruthenium-based catalyst, a palladium-based catalyst (e.g., Pd/C), a platinum-based catalyst, a rhenium-based catalyst, or a cobalt-based catalyst (e.g., CoMo)) in the presence of any carrier (e.g., a zeolite, an alumina, etc.); any useful reagent, such as hydrogen (e.g., H.sub.2) or water (e.g., supercritical water); any useful pressure, e.g., such as from about 3 MPa to about 30 MPa (e.g., from about 5 MPa to about 20 MPa); and/or any useful temperature, e.g., such as from about 100° C. to about 500° C. (e.g., from about 250° C. to about 350° C.). Further exemplary hydrotreatment conditions are described in Ma F et al., “Biodiesel production: a review,” Bioresourc. Technol. 1999; 70:1-15; Tran N H et al., “Catalytic upgrading of biorefinery oil from micro-algae,” Fuels 2010; 89:265-74; and Wildschut J et al., “Catalyst studies on the hydrotreatment of fast pyrolysis oil,” Appl. Catalysis B 2010; 99:298-306, each of which is incorporated herein by reference in its entirety.

(65) Exemplary biofuels formed by hydrotreatment include naphtha, biodiesel (e.g., including one or more unsaturated fatty acids or fatty acid esters, such as of from about 10% to about 35% of a long chain fatty acid having a C.sub.13-C.sub.21 tail, such as a palmitic fatty acid (C.sub.16 tail), linoleic fatty acid (C.sub.18 tail), oleic fatty acid (C.sub.18 tail), and/or stearic fatty acid (C.sub.18 tail)), green diesel, renewable aviation fuel, hydrocarbons (e.g., light hydrocarbons), alcohol (e.g., ethanol; propanol, such as 1-propanol; butanol, such as n-butanol, isobutanol, 2-butanol, 3-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, etc.), and/or a biogas (e.g., hydrogen or methane). Other products formed by hydrotreatment include solid residuals (e.g., biochar and ash), aqueous co-products (e.g., ketoacids, amines, nutrients, etc.), as well as other useful co-products (e.g., animal feed, fertilizer, glycerine, biopolymers, etc.).

(66) Phase Separation

(67) Phase separation steps can be employed to separate components of a liquefied mixture, fermentation broth, aqueous fraction, a non-aqueous fraction, alcohol fraction, etc. Such steps include any that separate liquid from solid phases, as well as separate two or more phases that can be differentiated based on solubility, miscibility, etc. (e.g., as those present in non-aqueous phases, aqueous phases, lipophilic phases, etc.) in any useful solvent (e.g., an organic solvent, an aqueous solvent, water, buffer, etc.). Phase separation techniques include flash separation (e.g., separation of liquefied mixture into biocrude oil, solid residuals, aqueous phase, and/or aqueous co-products), acid absorption (e.g., absorption of acid in a matrix to provide recovered nutrients and water for recycled use), filtration, distillation, solvent extraction, ion liquid extraction, etc. The resultant products and co-products can include one or more intermediate products that can optionally be processed to form useful end-use products.

EXAMPLES

Example 1: Biochemical Upgrading of Dried Distillers Grains

(68) Dried distillers grains are a high-protein biomass that is recalcitrant to further processing. The methods described herein provide a process to upgrade this biomass into useful intermediates and by-products. In one embodiment, the method includes an integrated sugar and protein fermentation process, with pre-treatment steps to facilitate fermentation. The method can include use of a dilute acid (e.g., use of 2-10% H.sub.2SO.sub.4 at a temperature of from about 90° C. to 145° C. for any useful incubation period, such as about 30 minutes to 6 hours) prior to use of a sugar fermentation strain (e.g., E. coli K011) to convert sugars into ethanol. The method can also include use of an enzyme (e.g., about 0.5 g/L to 2 g/L of a protease or protease cocktail at a temperature of from about 37° C. to 55° C. for any useful incubation period, such as from about 12 to 48 hours) prior to use of a protein fermentation strain (e.g., E. coli YH83 with one or more additional mutated variations) to convert amino acids to alcohols (e.g., more than C.sub.2 alcohols, such as C.sub.3-10 alcohols) and amines (e.g., NH.sub.4).

(69) Any useful biomass can be processed. In particular, high-protein biomass can be particularly beneficial. Exemplary biomass include dried distillers grains with solubles (DDGS), soy products (e.g., soy meal), yeast products (e.g., yeast extract), whey, algae (e.g., microalgae, macroalgae, diatoms, green algae, yellow algae, phytoplankton, plankton, protists, haptophytes, chlorophyta, and/or cyanobacteria), etc. FIGS. 4A-4B provides the potential conversion yield of various biomass, as well as the potential yield of unutilized high value amino acids. As an initial step, processing methods in this Example were employed with DDGS samples.

(70) Fermentation strains were developed and identified. As seen in FIG. 5, genetic engineering of E. coli resulted in organisms with high activity under anaerobic or microaerobic conditions. Our approach focused on, in part, altering the cofactor specificity of two enzymes in the pathway that provides isobutanol. In particular, two enzymes in the keto-acid pathway were modified: ketol-acid reductoisomerase IlvC and alcohol dehydrogenase YqhD. Generally, the wild-type strain employs a NADPH cofactor having a 2′-phosphate group. Modifications for the mutants were optimized to provide increased activity in the presence of a non-native NADH cofactor, as compared to the wild-type strain.

(71) Mutant strains included AY1 (replacing IlvC with mutant A combined with replacing YqhD with mutant Y1), AY2 (replacing IlvC with mutant A combined with replacing YqhD with mutant Y2), AY3 (replacing IlvC with mutant A combined with replacing YqhD with mutant Y3), AY5 (replacing IlvC with mutant A combined with replacing YqhD with mutant Y5), VY2 (replacing IlvC with mutant V combined with replacing YqhD with mutant Y2), VY3 (replacing IlvC with mutant V combined with replacing YqhD with mutant Y3), VY5 (replacing IlvC with mutant V combined with replacing YqhD with mutant Y5), IA (replacing IlvC with mutant A), and IV (replacing IlvC with mutant V). Additional details for these mutant strains are described herein. In particular, five new E. coli strains showed increased conversion yield, as compared to the wild-type YH83 strain.

(72) FIG. 6 shows an exemplary process flow for use with modified fermentation stains. This exemplary process can be optimized by varying the dilute acid pre-treatment conditions, by employing enzymatic pre-treatment at particular points of the process, and by separating the two fermentation steps, in which each step uses a different strain (see, e.g., FIG. 7).

(73) Dilute acid pre-treatment and fermentation achieved enhanced bioconversion of carbohydrates (e.g., sugars) into ethanol. As seen in FIG. 8, dilute acid hydrolysis pre-treatment resulted in more than 90% hydrolysis of various carbohydrates, including glucose, xylose, and arabinose. After treatment with 4% hot acid for 72 hours, the concentrations of these three representative sugars were decreased, and the concentration of ethanol increased at a theoretical conversion yield of about 60.7%. Improved xylose degradation can be promoted in any useful manner, e.g., by increasing the incubation temperature, increasing the concentration of the acid during pre-treatment, including a pre-treatment step with basic conditions (e.g., in the presence of NaOH or KOH), and/or by including one or more enzymes to cleave xylose, such as an isomerase, a reductase, etc.

(74) Enzymatic pre-treatment and fermentation achieved enhanced bioconversion of proteins into fusel alcohol (e.g., more than C.sub.2 alcohols, such as isobutanol, isopentanol, and/or phenylethanol) and amines. Enzymatic pre-treatment combined with dilute acid pre-treatment resulted in more than 90% hydrolysis of various carbohydrates. Furthermore, as seen in FIG. 9, after treatment for 72 hours, the concentration of amino acids was decreased, and the concentration of various fusel alcohols increased at a theoretical conversion yield of about 40.8%. Improved resistance to product inhibition can be promoted in any useful manner, e.g., by employing one or more separation or extraction steps; or by employing one or more lipids to promote vesicle formation.

Example 2: Cofactor Engineering to Improve the Fusel Alcohol Yield

(75) Algal protein, as well as other high-protein feedstock, can be feasibly converted into fusel butanol with an engineered E. coli strain. In particular, algal protein has been used for producing fuel compounds, but the titer of fusel butanol is generally relatively low. One possible reason is cofactor imbalance during the algal protein fermentation.

(76) To resolve this problem, a direct evolution approach was applied to switch the cofactor specificity of two enzymes (IlvC and YqhD) in the isobutanol pathway. These two enzymes rely on native cofactor NADPH in this pathway. Cofactor switching can allow for reactivity in the presence of a non-native cofactor (NADH), even if the native cofactor (NADPH) is lacking (e.g., such as in microaerobic or anaerobic conditions). Through high throughput screening, more than 20 YqhD mutants showed the activity with NADH.

(77) Five YqhD mutants were selected and then combined with one of two IlvC mutants to reengineer the production strain. Upon combining the beneficial mutations of IlvC and YqhD, the engineered E. coli strain AY3 provided an optimized performance, in which fusel butanol yield increased by about 60%, as compared to wild-type E. coli, under anaerobic fermentation with an amino acid mixture. When applied to algal protein hydrolysates, the engineered AY3 strain produced from about 38% to 100% more fusel butanol in the fermentation broth, as compared to wild-type. This study provides a promising approach to improve bioconversion of algal protein into fusel butanol (e.g., as advanced fuel compounds) and amino acids (e.g., for further processing as nutrients or chemical intermediates). Additional details for this study are provided in Example 3. The following materials and methods were employed for this study.

(78) Strains and plasmids: The mutant E. coli strain YH83 (BW25113/F′ [traD36, proAB+, lacI.sup.q ZAM15]ΔglnAΔgdhAΔluxSΔlsrA) containing plasmids pYX68, pYX90, and pYX97 was generously provided by Professor James C Liao from University of California, Los Angeles (UCLA) (see, e.g., Huo Y X et al., Nat. Biotechnol. 2011; 29(4):346-51). The strain was engineered for bioconversion of protein hydrolysates into isobutanol. Plasmids pYX90 and pYX97 contained IlvC and YqhD, which use NADPH as the cofactor. Plasmid pBbE1a and the E. coli strain DH1 were used for the expression and screening of mutant libraries of enzyme YqhD, as well as for the creation of IlvC mutants.

(79) Selection of cofactor binding sites: The amino acids for mutation in YqhD were selected by inspecting the cofactor NADPH binding site. Autodock 4 was used to investigate the cofactor-enzyme interaction. The protein X-ray structure of E. coli K-12 YqhD (Entry No. 1OJ7) containing cofactor NADPH was extracted from RCSB Protein Data Bank.

(80) Mutant library construction, expression, and high-throughput screening: Plasmid pBbE1a was generously provided by Dr. Taek Soon Lee from Joint BioEnergy Institute. Genes IlvC and YqhD were amplified from plasmids (pYX90 and pYX97), and then sub-cloned into vector pBbE1a under restriction cutting sites (EcoRI and BamHI) to achieve vectors pBbE1a-IlvC and pBbE1a-YqhD, respectively. Saturation mutagenesis was applied to create a recombinant mutant library of enzyme YqhD, following the description in the previous study (see, e.g., Wu W et al., “Site-saturation mutagenesis of formate dehydrogenase from Candida bodinii creating effective NADP.sup.+-dependent FDH enzymes,” J. Molec. Catal. B 2009; 61(3-4):157-61). For the recombinant library of YqhD, primers with degenerate codons were used to create mutations at the selected amino acid sites. High fidelity DNA polymerase fusion Q5 (New England BioLabs, Inc. (NEB), Ipswich, Mass.) was used to generate the library and to express library members in E. coli DH1. Strains containing YqhD mutants were diluted properly and spread onto Luria-Bertani (LB) plates supplemented with ampicillin (100 μg/mL) for the following library screening.

(81) According to the previous study (see, e.g., Bastian S et al., Metab. Eng. 2011; 13(3):345-52), the two mutant IlvCs (A71S, R76D, S78D Q110V/Q110A) switched the cofactor specificity from NADPH to NADH with the relatively high activity with NADH. Site mutagenesis (point mutation) was used to create two mutants of enzyme IlvC at the four target amino sites mentioned above. An iterative strategy was used to create all four sites of mutations. High fidelity DNA polymerase fusion Q5 was again used to create point mutations of IlvC in the vector pBbE1a-IlvC, as described before (see, e.g., Wu W et al., J. Molec. Catal. B 2009; 61(3-4):157-61). Mutations were confirmed through DNA sequencing. All the primers used herein are listed in Table 1, below.

(82) TABLE-US-00001 TABLE 1 Primers and mutants for YqhD and IlvC SEQ ID Name Sequence NO: Notes YqhD-pBbE1a-F 5-CTC AGC GAA TTC ATG AAC AAC TTT AAT CTG CAC 10 Clone YqhD ACC CCA AC-3 into pBbE1a YqhD-pBbE1a-R 5-TGACCTGGATCCTTA GCG GGC GGC TTC GTA TAT AC-3 11 IlvC-pBbE1a-F 5-CTC AGC GAA TTC ATG GCT AAC TAC TTC AAT ACA 12 Clone IlvC CTG AAT CTG C-3 into pBbE1a IlvC-pBbE1a-R 5-TGACCTGGATCC TTA ACC CGC AAC AGC AAT ACG TTT 13 C-3 YqhD-S40-F 5-GTATTGATTACCTACGGCGGC GGC NNN GTG AAA AAA 14 Create mutant ACC GGC GTT CTC-3 library S40 YqhdS40-R 5-GAG AAC GCC GGT TTT TTT CAC NNN GCC GCC GCC 15 GTA GGT AAT CAA TAC-3 YqhD-G39S40-F 5-GTATTGATTACCTACGGCGGC NNN NNN GTG AAA AAA 16 Create mutant ACC GGC GTT CTC-3 library Yqhd-G39S40-R 5-GAG AAC GCC GGT TTT TTT CAC NNN NNN GCC GCC 17 G39S40 GTA GGT AAT CAA TAC-3 YqhD-S40P-F 5-GTATTGATTACCTACGGCGGC CCG GTG AAA AAA ACC 18 Mutant Y1 GGC GTT CTC-3 (S40P) Yqhd-S40P-R 5-GAG AAC GCC GGT TTT TTT CAC CGG GCC GCC GTA 19 GGT AAT CAA TAC-3 YqhD-S40R-F 5-GTATTGATTACCTACGGCGGC CGT GTG AAA AAA ACC 20 Mutant Y2 GGC GTT CTC-3 (S40P) Yqhd-S40R-R 5-GAG AAC GCC GGT TTT TTT CAC CGT GCC GCC GTA 21 GGT AAT CAA TAC-3 YqhD- 5-GTATTGATTACCTACGGCGGC ATCCGT GTG AAA AAA 22 Mutant Y3 G39I/S40R-F ACC GGC GTT CTC-3 (G39I/S40R) YqhD- 5-GAG AAC GCC GGT TTT TTT CAC ACG GAT GCC GCC 23 G39I/S40R-R GTA GGT AAT CAA TAC-3 YqhD- 5-GTATTGATTACCTACGGCGGC TATCAT GTG AAA AAA 24 Mutant Y5 G39Y/S40H-F ACC GGC GTT CTC-3 (G39Y/S40H) Yqhd- 5-GAG AAC GCC GGT TTT TTT CAC ATG ATA GCC GCC 25 G39Y/S40H-R GTA GGT AAT CAA TAC-3 IlvCA71SR76DS78D- 5-CGT AAA GAA  CG ATT GCC GAG AAG G  GCG  26 F TGG-3 IlvCA71SR76DS78D- 5-CCA ATC CGC ATC CTT CTC GGC AAT CGA TTC TTT 27 R ACG-3 ilvCQ110A-F 5-CGG ACA AGG CGC ACT CTG ATG TAG-3 28 Mutant A ilvCQ110A-R 5-CTA CAT CAG AGT GCG CCT TGT CCG-3 29 (A71S/R76/ S78D/Q110) ilvCQ110V-F 5-CGG ACA AGG TGC ACT CTG ATG TAG-3 30 Mutant V ilvCQ110V-R 5-CTA CAT CAG AGT GCA CCT TGT CCG-3 31 (A71S/R76D/ S78D/Q110) ILVC-Pyx90- 5-GAA AGC TCT CTA GGT CGA CGA GGA ATC ACC ATG 32 Replace IlvC Gib-F1 GCT AAC TAC TTC AAT ACA CTG AAT CTG-3 with mutant ILVC-Pyx90- 5-GTA CTT AGG CAT GGT ATA TCT CCT TCC GGG TTA 33 A or V Gib-R1 ACC CGC AAC AGC AAT ACG TTT CAT ATC-3 ILVD-AvTA- 5-GAT ATG AAA CGT ATT GCT GTT GCG GGT TAA CCC 34 pYX90-Gib-F2 GGA AGG AGA TAT ACC ATG CCT AAG TAC-3 ILVD-AvTA- 5-GGA TTT GTC CTA CTC AGG AGA GCG TTC ACC GAC 35 pYX90-Gib-R2 AAA CAA CAG ATA AAA CGA AAG GCC CAG-3 Spec-alsS- 5-CTG GGC CTT TCG TTT TAT CTG TTG TTT GTC GGT GAA 36 pYX90-Gib-F3 CGC TCT CCT GAG TAG GAC AAA TCC-3 Spec-alsS- 5-CAG ATT CAG TGT ATT GAA GTA GTT AGC CAT GGT 37 pYX90-Gib-R3 GAT TCC TCG TCG ACC TAG AGA GCT TTC-3 YqhD-Pyx97- 5-GGA GAA AGG TCA CAT GAA CAA CTT TAA TCT GCA 38 Replace YqhD Gib-F1 CAC CCC AAC CCG CAT TC-3 with mutant YqhD-Pyx97- 5-CTC TAG CAC GCG TAC CAT GGG ATC CTT AGC GGG 39 Y1, Y2, Y3, or Gib-R1 CGG CTT CGT ATA TAC-3 Y5 ColE-Amp- 5-GTA TAT ACG AAG CCG CCC GCT AAG GAT CCC ATG 40 pYX97-Gib-F2 GTA CGC GTG CTA GAG-3 ColE-Amp- 5-CAT GAT AAT AAT GGT TTC TTA GAC GTC AGG TGG 41 pYX97-Gib-R2 CAC TTT TCG GGG AAA TGT GCG CGG AAC-3 LeuDH-KivD- 5-GTT CCG CGC ACA TTT CCC CGA AAA GTG CCA CCT 42 pYX97-F3 GAC GTC TAA GAA ACC ATT ATT ATC ATG-3 LeuDH-KivD- 5-GAA TGC GGG TTG GGG TGT GCA GAT TAA AGT TGT 43 pYX97-R3 TCA TGT GAC CTT TCT CC-3

(83) Single colonies of YqhD mutant and wild-type were cultured into 96-well plates. Each well contained 200 μl of LB medium with corresponding antibiotics. Cultures were incubated at 300 rpm and at 37° C. overnight (16 hours) and induced by 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for another 24 hours at 250 rpm, 30° C. in a humidified shaker. Cells were harvested by centrifugation (3000 rpm, 4° C., 15 min.) and stored at −20° C. before conducting assays. For high-throughput screening assays, E. coli cells were lysed with 200 μl of 50 mM MOPS (pH 7.0) containing 1 mg/ml of lysozyme (Sigma-Aldrich Corp., St. Louis, Mo.) and 20 U/ml of DNase (NEB) at 4° C. for 6 hours under gentle shaking. Lysed cells were spun down, and 100 μl of the cell free extract was transferred into another 96 well plate. YqhD and its mutant activity assay buffer contained 50 μl of 0.25 mM NADH, 10 μl of isobutyraldehyde, and 40 μl of MOPS (pH 7.0). Consumption of NADH was monitored at 340 nm in a plate reader (Molecular Devices, LLC, Sunnyvale, Calif.).

(84) Plasmids and strain construction for anaerobic bioconversion of algal protein hydrolysates: Positive hits with high activity with NADH were selected to replace the wild-type YqhD gene in plasmid pYX97. Two mutant genes of IlvC were selected to replace the wild-type IlvC gene in plasmid pYX90. A Gibson assembly was applied to replace wild-type YqhD and IlvC with the corresponding mutant genes. Plasmids pYX97 and pYX90 with mutant genes, as well as pYX68 were co-transformed into strain YH40 for the bioconversion of algal protein hydrolysates into fusel butanol. All the primers used herein are listed in Table 1, above.

(85) Addition of NADPH into fermentation media: Wild-type strain YH83 was cultured in 20 ml of LB media with 100 μg/ml ampicillin, 34 μg/ml chloramphenicol, and 25 μg/ml spectinomycin overnight. Then, 5 ml of culture was transferred into 150 ml of 1× M9 medium (see, e.g., Huo Y X et al., Nat. Biotechnol. 2011; 29(4):346-51; and Wu W, “Fuel ethanol production using novel carbon sources and fermentation medium optimization with response surface methodology,” Int. J. Agri. Biol. Eng. 2013; 6(2):42-53) (in 160 ml serum bottle) containing 20 g/L of an amino acid mixture, 2 g/L of LB, and 150 μM NADPH with corresponding aforementioned antibiotics in a rotary shaker at 220 rpm, 37° C. The culture was induced by 1 mM IPTG at 37° C. when the OD reached 0.6 for the production of fusel butanol. Samples were tested at regular time intervals to determine the concentration of fusel butanol in each sample. Each run was performed in biological duplicate.

(86) Investigation of the fusel butanol yields with an amino acid mixture: Engineered strains of YH83 containing various combinations of mutant YqhD and IlvC were cultured in 20 ml of LB media with corresponding aforementioned antibiotics, overnight. Then, 3 ml of culture was transferred into 30 ml of 1× M9 medium containing 20 g/L of an amino acid mixture (Sigma-Aldrich, Corp.), 5 g/L of LB, and antibiotics. Fermentation was performed as described above. Each run was performed in biological duplicate. Strains containing the details of the mutants are listed in Table 1, above.

(87) Bioconversion of algal protein hydrolysates into fusel butanol: ATP algae biomass samples were pretreated with diluted acid, according to the protocol from the National Renewable Energy Laboratories (referenced hereinafter as “ATP3”) or according to the protocol described previously (see, e.g., Garcia-Moscoso J L et al., “Kinetics of peptides and arginine production from microalgae (Scenedesmus sp.) by flash hydrolysis,” Ind. Eng. Chem. Res. 2015; 54(7):2048-58) through a thermal flash hydrolysis (referenced hereinafter as “ODU”). Hydrolyzed algal carbohydrate was converted into ethanol through alcoholic fermentation using an ethanogenic strain, E. coli KO11. The cell mass was removed through centrifugation (6,000 rpm, 4° C., 10 min.) at the end of KO11 fermentation. The supernatant containing ethanol and algal protein was air-bubbled at room temperature for 10 minutes to remove ethanol. Then, the resultant supernatant was concentrated and digested with 2 mg/mL Pronase® (Promega Corp., Madison, Wis.) following the manufacturer's protocol. The protease-digested, algal protein hydrolysate was sterilized through a 0.45 μm PTFE membrane and used as the fermentation media for fusel butanol production. This hydrolysate was incubated with mutant strain AY3 in the presence of the amino acid mixture. Samples were tested at regular intervals with GC-MS analysis. Each run was performed in biological duplicate.

(88) Analytic determination of the presence of fusel butanol and amino acid: Concentrations of amino acids were analyzed using an amino acid analyzer (Hitachi Ltd., Tokyo, Japan) at the genome center of University of California, Davis, following their protocol. Fusel butanol was extracted using ethyl acetate at a ratio of 1:1 (ethyl acetate: fermentation broth), with 2-methyl-pentanol as an internal reference. The mixture was vortex at 1,200 rpm for 20 min. and centrifuged at 14,000 rpm, 5 min. The ethyl acetate layer was collected for further GC-MS analysis. Two μl of sample was inserted into the injection port (220° C.) of an Agilent 7890A Gas Chromatograph containing a 30 m×0.25 mm i.d. DB wax capillary column with a film thickness of 0.25 μm. The column was temperature programmed as follows: 40° C. for 4 min., increasing to 65° C. at 10° C./min. and holding for 10 min., then increasing to 120° C. at 10° C./min. and holding for 2 min., and then increasing to 220° C. at 20° C./min and holding for 5 min. The carrier gas was ultra-high purity helium at a constant flow rate of 1.8 ml/min. The initial column head pressure was 16.188 psi with a split ratio of 10. The gas chromatograph was coupled to a quadrupole mass selective detector (MSD), Agilent 5975C. The MSD parameters included EI at 70 eV, mass range at 10-650 Da, and scan speed at 2 scans/sec. Spectral components were searched against the NIST 2015 mass spectral library. Serial of dilutions of isobutanol and 2-methyl-butanol in ethyl acetate were analyzed to determine a standard curve. Concentrations of fusel butanol were calculated by referring samples to the standard curve.

Example 3: Cofactor Engineering of Ketol-Acid Reductoisomerase (IlvC) and Alcohol Dehydrogenase (YqhD)

(89) Increasing concerns about diminishing fossil fuels and global environmental problems have attracted interest in sustainable biofuels obtained from renewable resources (see, e.g., Peralta-Yahya P P et al., “Microbial engineering for the production of advanced biofuels,” Nature 2012; 488(7411):320-8; and Keasling D, “Sustainable production of advanced biofuels,” 241st ACS National Meeting & Exposition, held on 27-31 Mar. 2011 in Anaheim, Calif., Abstract 202). Algae-based biofuel is considered to be one sustainable alternative biofuel due to several benefits, including simplified pretreatment as compared to lignocellulosic biomass, higher biomass yields as compared to plants, possible cultivation on nonarable land, and possible reclamation of waste water (see, e.g., Razeghifard R, “Algal biofuels,” Photosynth. Res. 2013; 117(1-3):207-19; and Luque R, “Algal biofuels: the eternal promise?,” Energy Environ. Sci. 2010; 3:254-7). So far, algae biomass have been converted to versatile biofuel chemicals, such as bioethanol, biohydrogen, biogas, crude oil, and biodiesel (see, e.g., Li K et al., “An overview of algae bioethanol production,” Int. J. Energy Res. 2014; 38(8):965-77; Melis A et al., “Hydrogen production: green algae as a source of energy,” Plant Physiol. 2001; 127(3):740-8; Hernández D et al., “Biofuels from microalgae: lipid extraction and methane production from the residual biomass in a biorefinery approach,” Bioresour. Technol. 2014; 170:370-8; Lopez Barreiro D et al., “Assessing microalgae biorefinery routes for the production of biofuels via hydrothermal liquefaction,” Bioresour. Technol. 2014; 174:256-65; Sharma K K et al., “High lipid induction in microalgae for biodiesel production,” Energies 2012; 5(5):1532-53; and Scott S A et al., “Biodiesel from algae: challenges and prospects,” Curr. Opin. Biotechnol. 2010; 21(3):277-86).

(90) Current state-of-the-art algal biofuels have primarily focused on producing biodiesel by boosting algal lipid yield under nutrient stress conditions. This strategy ignores another major component of algae: proteins. Under conditions that support robust algae growth, algal carbohydrate and proteins are two of the major components of biomass, including up to ˜80% of the ash-free dry weight (AFDW) of microalgae biomass, in which up to 60% can be proteins (see, e.g., Luque R, Energy Environ. Sci. 2010; 3:254-7; Becker E W, “Microalgae: biotechnology and microbiology,” Cambridge University Press, Cambridge, U.K., 1994 (293 pp.); and Singh J et al., “Commercialization potential of microalgae for biofuels production,” Renew. Sustain. Energy Rev. 2010; 14(9):2596-610).

(91) Recently, engineered E. coli strains have been employed to convert algal protein into fusel butanol. Modified strains can be selected to exhibit deamination of protein hydrolysates to C4 and C5 alcohols at 56% of the theoretical yield (see, e.g., Huo Y X et al., “Conversion of proteins into biofuels by engineering nitrogen flux,” Nat. Biotechnol. 2011; 29(4):346-51). By combining the Ehrlich pathway with three exogenous transamination and deamination cycles, the engineered E. coli strain produced up to 0.183 g of fusel butanol/g of amino acids under aerobic or microaerobic fermentation conditions (see, e.g., Huo Y X et al., Nat. Biotechnol. 2011; 29(4):346-51). In some instances, anaerobic conditions can be preferred for large scale fermentation due to lower operating costs and higher theoretical yield.

(92) In the Ehrlich pathway for isobutanol production, two enzymes use nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor: ketol-acid reductoisomerase (IlvC) and alcohol dehydrogenase (YqhD). Thus, bioconversion of protein hydrolysates requires at least two reducing equivalents of NADPH to convert glucose to isobutanol (see, e.g., Atsumi S et al., “Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels,” Nature 2008; 451(7174):86-9). Under anaerobic fermentation, glycolysis can only produce nicotinamide adenine dinucleotide (NADH) as an available reducing equivalent, while the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle are not functional due to the lack of oxygen. This may result in a cofactor imbalance during anaerobic fermentation or oxygen limited fermentation conditions (e.g., microaerobic conditions). Thus, although anaerobic or microaerobic fermentation is preferred to minimize processing costs and to increase theoretical yields, these very fermentation conditions can result in an inhibitory cofactor imbalance that can limit alcohol production.

(93) NADPH limitation can result in cofactor imbalance, as reported previously for isobutanol production from glucose (see, e.g., Shi A et al., “Activating transhydrogenase and NAD kinase in combination for improving isobutanol production,” Metab. Eng. 2013; 16:1-10; and Bastian S et al., “Engineered ketol-acid reductoisomerase and alcohol dehydrogenase enable anaerobic 2-methylpropan-1-ol production at theoretical yield in Escherichia coli,” Metab. Eng. 2011; 13(3):345-52). Therefore, we hypothesized that fusel butanol yield can be improved through resolving the cofactor imbalance present in anaerobic fermentation of algal protein.

(94) To achieve this, we first tested our hypothesis that lack of NADPH affects fusel alcohol production. Thus, we performed experiments by adding NADPH in the fermentation media and observing the effect of NADPH on alcohol yield. Then, we designed two isobutanol biosynthesis pathway enzymes (IlvC and YqhD) to switch cofactor specificity from NADPH to NADH through directed evolution. Upon combining the beneficial mutations of two enzymes in the isobutanol biosynthesis pathway, the resultant engineered E. coli strain improved fusel butanol yield by about 60%, as compared to wild-type, under anaerobic fermentation conditions with an amino acid mixture as the feedstock. When applied to algal protein hydrolysates, the mutant strain with best performance produced 38% to 100% more fusel butanol, as compared to wild-type.

(95) Engineering the cofactor specificity of E. coli IlvC and YqhD to switch to NADH: The x-ray crystal structure of YqhD had been reported previously (see, e.g., Sulzenbacher G et al., “Crystal structure of E. coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme,” J. Mol. Biol. 2004; 342(2):489-502). This structure contained cofactor NADP.sup.+ as a ligand. The protein possesses a GGGS (residues 37-40) motif, which binds the 2′-phosphate groups of NADP through hydrogen bonds, as shown in FIG. 10A (phosphorous and oxygen atoms of the 2′-phosphate is indicated by P, O, and dashed lines). These hydrogen bonds provide a preference for binding NADPH over NADH, making the motif GGGS a major cofactor binding site. Therefore, two amino acids (Gly39Ser40) were chosen to identify mutations that would confer cofactor switching from NADPH to NADH through site-saturation mutagenesis.

(96) Two mutant libraries (G39S40 and S40) were generated and screened for NADH activity using a cell free extract. More than 20 positive mutants showed various activities with cofactor NADH. Five mutants showed 60-80 times higher activity with NADH (as shown in FIG. 10B), as compared to wild-type YqhD (FIG. 10C). The five mutants were selected to replace wild-type YqhD in plasmid pYX97, including two single mutations of S40 (Y1, Y2) (see, e.g., FIGS. 10D-10E) and three double mutations of G39S40 (Y3, Y4, Y5) (see, e.g., FIG. 10F).

(97) E. coli IlvC has been engineered to switch cofactor from NADPH to NADH for isobutanol production under anaerobic fermentation (see, e.g., Bastian S et al., Metab. Eng. 2011; 13(3):345-52). Two mutants of IlvC (A71S, R76D, S78D, Q110V/A) with higher activity with NADH were chosen to replace wild-type IlvC in plasmid pYX90 (see, FIGS. 11A-11B).

(98) Improved fusel butanol yield under anaerobic fermentation with NADPH: We hypothesized that cofactor imbalance compromised fusel butanol yield during protein bioconversion. To test this hypothesis, the native cofactor NADPH of YqhD and IlvC was added into the fermentation medium as an external source of NADPH. FIGS. 12A-12B show that addition of NADPH significantly improved yields of isobutanol and ⅔-methyl-butanol during protein bioconversion. Within 5 days of cultivation, yields of isobutanol, ⅔-methyl-butanol, and total fusel butanol increased by about 34%, 28%, and 30%, respectively, confirming that cofactor imbalance can be one factor affecting fusel butanol production.

(99) Bioconversion of amino acid mixtures and algal protein into fusel butanol: The fusel butanol yields of the engineered YH83 strains, which contained both IlvC and YqhD mutants, were initially investigated through bioconversion of amino acid mixtures in the 1× M9 medium. As seen in FIG. 13A, the mutant strains produced various amounts of fusel butanol in the fermentation broth. Among the seven mutant strains, four strains (AY1, AY3, VY3, and VY5) yielded at least 30% higher concentrations of fusel butanol, as compared to wild-type. In particular, the AY3 strain produced more than 60% higher amount of fusel butanol than wild-type, i.e., up to 6 g/L fusel butanol from 20 g/L amino acids. The wild-type produced 3.7 g/L fusel butanol.

(100) Further studies were conducted with pretreated algal protein hydrolysates. Two hydrolysates were used as medium to investigate fermentation performance of mutant strain AY3. As shown in the FIG. 13B, mutant strain AY3 produced higher titers of isobutanol, ⅔-methyl-butanol, and total fusel butanol with both pretreated algal protein hydrolysates (ATP3 and ODU), as compared to wild-type strain YH83.

(101) Within 3 days of fermentation, the mutant strain AY3 yielded more than 100% and 38% higher total fusel butanol titers than wild-type with ATP3 and ODU, respectively, as shown in FIG. 13B. Yields using algal hydrolysates were lower than that observed with amino acid mixtures. Yields can be optimized, e.g., by providing supplemental nutrients with the algal hydrolysates, adjusting the ionic strength of the hydrolysate, neutralizing the hydrolysate, etc.

(102) As shown in FIG. 13C, only about 25% of algal protein hydrolysates were consumed after 3 days of fermentation. Based on substrate consumption, the strain AY3 yielded 0.2 to 0.18 g fusel butanol/g amino acid for converting algal hydrolysates AY3 and ODU, respectively. Yields could be optimized in any useful manner, e.g., by increasing incubation times, increasing protease digestion times, repeating and recycling separated fractions, etc.

(103) Here, we engineered two enzymes (IlvC and YqhD) in the isobutanol biosynthesis pathway to resolve cofactor imbalance during fermentation. By combining beneficial mutations of these two enzymes, the engineered AY3 strain improved fusel butanol yield with an algal hydrolysate, as compared to the wild-type strain. Fusel butanol possess the higher energy density, lower vapor pressure, lower hygroscopicity than fuel ethanol and has been considered as an advanced fuel compounds (see, e.g., Lan E I et al., “Microbial synthesis of n-butanol, isobutanol, and other higher alcohols from diverse resources,” Bioresour. Technol. 2013; 135:339-49; and Smith K M et al., “An evolutionary strategy for isobutanol production strain development in Escherichia coli,” Metab. Eng. 2011; 13(6):674-81). Recently, algal protein hydrolysates were reported into fusel butanol but at a relative low titer (see, e.g., Huo Y X et al., Nat. Biotechnol. 2011; 29(4):346-51). One of possible factors that results in the lower fusel titer could be the co-factor imbalance during the fermentation, in which strategies to address these factors are described herein.

Other Embodiments

(104) All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

(105) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

(106) Other embodiments are within the claims.