Abstract:
Disclosed herein is a newly discovered problem and solution for engineering  S. cerevisiae  to ferment xylose to make ethanol utilizing xylose isomerase to convert xylose to xylulose for entry, via xylulokinase, into the pentose phosphate pathway. When grown on a media containing xylose xylitol tends to accumulate in the cell despite the absence of xylose reductase activity in  S. cerevisiae . Xylitol inhibits the activity of xylose isomerases. One solution described is to simultaneously express an exogenous xylitol dehydrogenase along with the exogenous xylose isomerase while optionally also overexpressing xylulokinase in the absence of expression of a xylose reductase. Another solution is a xylose isomerase from  Bacteroides fragilis  which is less inhibited by xylitol than other xylose isomerases, exemplified by  E. coli  xylose isomerase. Expression of the  Bacteroides fragilis  xylose isomerase may be used alone, or in combination with expression of axylitol dehydrogenase and optionally over expression of xylulokinase to improve ethanol production from xylose.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    The ability to produce ethanol from cellulosic feedstocks by fermentation on a commercial scale is a long sought goal. To be economical, the amount of ethanol produced must be sufficient to be worth the cost of preparation of the feedstock and processing of the final product. To produce sufficient amounts of ethanol requires a fermentation organism that is biologically efficient at ethanol production in comparison to production of other metabolic products. Efficiency is determined by yield and productivity, yield being expressed as a weight percentage of sugar feedstock (typically sucrose or glucose) converted into ethanol, and productivity being expressed as the maximum amount of ethanol that can be produced as a volume/volume percentage of the fermentation media before the fermentation ceases due to ethanol toxicity. In this regard, by far, the most efficient ethanol producing microorganism is the baker&#39;s yeast  S. cerevisiae    
         [0002]    Efficient ethanol producing strains of  S. cerevisiae  are capable of converting 90-93% of a sugar based carbon source into ethanol on wt/wt basis and the ethanol can typically accumulate to 16-17% of the volume of the fermentation media. The sugar for commercial scale production of ethanol is conventionally obtained by extraction of sucrose from sugar beet or cane, or by hydrolysis of corn starch to produce glucose. Sucrose and corn starch, however, represent only a small fraction of the total sugars in plant material, most of which is contained in the β glucoside polymers cellulose and hemicellulose, the later being a branched polymer of the C6 sugars glucose and mannose and the C5 sugars xylose and arabinose. 
         [0003]    There are several methods in the art for making hydrolysates of cellulose and hemicelluloses to produce feedstocks containing glucose, mannose, xylose and arabinose. For typical hydrolysates from corn stover, glucose represents 14.4%, mannose 0.9%, xylose 66.1% and arabinose 11.8% of the sugars. For typical hydrolysates of corn fiber glucose represents 48.6% xylose 25.2% and arabinose 17.6% of the sugars. Glucose and mannose are efficiently converted to ethanol during natural anerobic metabolism, however,  S. cerevisiae , lacks the enzymatic machinery to convert the dominant sugar, xylose, into ethanol. To do so requires genetic engineering of  S. cerevisiae  to express metabolic enzymes that can convert xylose into xylulose phosphate—a C5 metabolite that is part of the pentose phosphate pathway, which ultimately produces intermediates that can enter the glycolytic pathway and be converted to ethanol during anaerobic fermentation. Normally within the pentose phosphate pathway, xylulose phosphate is derived from ribulose phosphate by the action of an epimerase, but in addition,  S. cerevisiae  contains the enzyme xylulokinase which can directly phosphorylate xylulose. Xylulose however, is a rare metabolite, and the level of expression of xylulokinase in  S. cerevisiae  is low. But more importantly, xylulokinase does not phosphorylate xylose and  S. cerevisiae  lacks the necessary enzymes to convert xylose to xyululose so is unable to utilize xylose as a carbon source without metabolic engineering. 
         [0004]    There are two approaches to engineer  S. cerevisiae  to produce xylulose from xylose. The first represented by U.S. Pat. No 5,789,210 to Ho et al, is the XR-XD-XK three gene route, which is to overexpress xylulokinase (XK) simultaneously with an exogenous xylose reductase (XR) which reduces xylose to xylitol, and a xylitol dehydrogenase (XD), which oxidizes xylitol to xylulose. This approach, however, creates a redox imbalance in  S. cerevisiae  because xylose reductase utilizes NAD(H) as the reducing cofactor while xylitol dehydrogenase uses NADP +  as the oxidizing cofactor. This imbalance negatively affects the growth and productivity of  S. cerevisiae  shutting down efficient production of ethanol from xylose. One option to overcome this problem is to use a mutant xylose reductase that has a lower K m  for NAD +  than NADP +  thereby restoring the redox balance as has been described for example by Petschacher B, et. al. (Biochem J2005, 385:75-83). 
         [0005]    The second approach is the XI-XK two gene route, which is to overexpress xylulokinase along with an exogenous xylose isomerase (XI) which directly converts xylose to xylulose without reduction and subsequent oxidation. This approach is represented by: U.S. Pat. No. 7,622,284 and US Pat. Pub Nos: US20060216804, US20080261287. Genes from a variety of bacterial and fungal source of xylose isomerase share the common name xylA. Several species of xylA genes have been identified from bacterial and fungal sources and some, but not all, have been shown to be useful in producing ethanol from xylose simultaneously overexpressed with xylulokinase. Proposed bacterial sources for such xylA genes include  Thermus thermophilus  (U.S. Pat. No. 7,622,284),  Bacteroides thetaiotaomnicron , (US20060216804, US20080261287) and  Xanthamonus . Several fungal sources of xylA genes have also been proposed, including from  Neocallimastix, Caecomyces, Piromyces, Orpinomyces , or  Rumnomyces . (US20080261287). See also Curr Op Biotech 17:320 (2006). Of these, only the xylA genes from  Piromyces  (20080014620)  Orpinomyces  (Madhavan A, Tamalampudi S, Ushida K, Kanai D, Katahira S, Srivastava A, Fukuda H, Bisaria VS, Kondo A. Appl Microbiol Biotechnol. 2009 82(6):1067-78.) and  Bacteroides thetaiotaomnicron  (US20080261287) have been shown to improve ethanol production in  S. cerevisiae  when co-overexpressed with xylulokinase. See also FEMS Yeast Res 4:69, FEMS Yeast Res 5:399, FEMS Yeast Res 4:655, FEMS Yeast Res 5:925. 
         [0006]    Although ethanol production from both the three gene approach and the two gene approach has been demonstrated, the ethanol yield form xylose remains lower than expected for strains only containing those features. To improve productivity requires further genetic manipulation, either by way of mutation, evolutionary selection or by further genetic engineering. For example, US20070082386 proposes that ethanol production from xylitol by either the two gene or three gene route could be improved by increasing expression of a xylose transporting enzyme and/or by overexpression of genes encoding enzymes of the pentose phosphate pathway. US20060234364 discloses that mutants having a deletion in an endogenous gre3 gene encoding a non specific aldol reductase could improve ethanol production from xylitol using the two gene approach. US20070155000, from a different perspective, teaches than ethanol production from xylose containing hydrolysates utilizing the two gene route could be improved by further selection for resistance against growth inhibitors such as furfural and hydroxymethyl furfural that are typically found in hydrolysates of lignocellulosic biomass. 
         [0007]    There remains a need in the art to discover other xylose isomerase genes and other multi gene combinations to improve the efficiency of xylose utilization in  S. cerevisiae  for ethanol production. The disclosure that follows presents such alternatives in the form of a particular xylose isomerase xylA gene from  Bacteroides fragilis  and alternative three gene route that includes simultaneous overexpression of an xylose isomerase, xylitol dehydrogenase and xylulokinase activity, without the need for overexpressing xylose reductase. 
       SUMMARY OF THE INVENTION 
       [0008]    Described herein are recombinant yeast strains and methods of producing ethanol therefrom where xylose is a carbon source for growth and ethanol production. One broad aspect includes  S. cerevisiae  strains having a recombinant nucleic acid comprising a sequence encoding an exogenous xylose isomerase and an exogenous xylitol dehydrogenase, each operably linked to a promoter to over express the exogenous genes; with the proviso the strain does not also overexpress and exogenous xylose reductase activity in the  S. cerevisiae  strain. One feature of an exemplary exogenous xylose isomerase is that it is less inhibited by xylitol than is a homologue xylose isomerase from  E. coli . One particular example is a xylose isomerase derived from a xylA gene from a strain of  Bacteroides fragilis . Exemplary sequences and vectors encoding such genes are also described. These aspects may optionally be included with yeast strains that also over-express a xylulokinase activity. 
         [0009]    Another aspect is yeast strains obtained by evolutionary selection of the forgoing types of parent strains. The evolutionary selection includes expressing in a parent yeast strain, a nucleic acid encoding an exogenous xylose isomerase operably linked to a promoter to express the exogenous xylose isomerase in the parent yeast strain. Performing a first growing of the parent yeast strain in a liquid media containing xylose as a principle carbon source to produce a population of offspring yeast strains. performing a second growing of the population of the offspring yeast strains on a solid media containing xylose as a principle carbon source. The selection is to obtain a fast growing daughter strain from the offspring yeast strains, characterized in the fast growing daughter yeast strains grow faster than other yeast in the population of offspring yeast strains on the solid media. The steps of first and second growing and selection are repeated to finally obtain a final selected yeast strain that produces more ethanol from xylose than the parent yeast strain. The method is exemplified with a yeast strain herein the xylose isomerase is encoded by a xylA gene of  B. fragilis . The method may further be implemented when the parent strain further expresses a nucleic acid encoding an exogenous xylitol dehydrogenase operably linked to a promoter to express the exogenous xylitol dehydrogenase in the parent yeast strain. An exemplary strain made by such a process is described in the Figures as Y500+bsd+XIbf “e” and deposited as strain number NRRL Y-50424. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0010]      FIG. 1  illustrates growth (OD600) of  S. cerevisiae  flask cultures growing on YEP media+2% xylose over time (hours). All strains were derived from parent ethanol producing strain Y500 and each carries a blasticidin resistance marker (bsd). 421 i is a transformant carrying the XI (xylA) from  Bacteroides fragilis  driven by the HXT7 promoter from  S. cerevisiae  and has been deposited under terms of the Budapest Treaty at the Agricultural Research Services Northern Regional Research Laboratories as deposit number NRRL Y-50423 on Oct. 12, 2010. 437i carries the same XI and XD (xyl2) from  Pichia stipitis  driven by the  P. stipitis  XD promoter also deposited with number NRRL Y-50424. 437:xks carries the same XI and XD plus carries an extra copy of the XKS (xks) from  S. cerevisiae  also deposited with number NRRL Y-50425. 
           [0011]      FIG. 2  shows a comparison of  E. coli  xylose isomerase and  B. fragilis  xylose isomerase activity in the presence of varying concentrations of xylitol. Activity is reported as the percent of activity obtained in the absence of xylitol. 
           [0012]      FIG. 3  depicts a plasmid map of bsdYEMhxt-XIbf-cycl, which is a yeast replicating plasmid carrying xylA from  B. fragilis  under control of the  S. cereiviase  hxt7 promoter. 
           [0013]      FIG. 4 . depicts a plasmid map of bsdYIMrDNA Pxd-XD Phxt-XIbf, which is an integrative vector used to cross xylA from  B. fragilis  under control of the  S. cerevisae  hxt7 promoter and XD from  Pichia stipitis  (see U.S. Pat. No 5,789,210) under the control of the  P. stipitis  xd promoter into the chromosome of  S. cerevisiae.    
           [0014]      FIG. 5 . depicts a plasmid map of plasmid 421, which is a yeast integrative vector used to cross xylA from  B. fragilis  under control of the yeast hxt7 promoter and blasticidin resistance into the chromosome of  S. cerevisiae.    
           [0015]      FIG. 6 . depicts a plasmid map of plasmid 426, a yeast integrative vector used to cross xylA from  B. fragilis  under control of the yeast hxt7 promoter into the chromosome of  S. cerevisiae.    
           [0016]      FIG. 7  shows the wild type protein sequence for XI from the xylA gene  B. fragilis  (SEQ. ID NO: 2). 
           [0017]      FIG. 8  shows the mutant protein sequence for XI from the xylA gene of  B. fragilis  where a change in the nucleotide sequence to enhance transcription results in a mutant protein where a serine substitutes for the alanine at position 2 (SEQ. ID NO: 4). 
           [0018]      FIG. 9  shows the wild type nucleotide sequence for xylA from  B. fragilis . (SEQ. ID NO: 1) 
           [0019]      FIG. 10  shows the mutant nucleotide sequence (SEQ. ID NO: 3) encoding the mutant xylA according to SEQ. ID NO: 4. 
           [0020]      FIG. 11  shows another the mutant nucleotide sequence (SEQ. ID NO: 5) also encoding the mutant xylA according to SEQ. ID NO: 4 optimized for expression in  S. cerevisiae.    
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0021]    The following description and forgoing Background make citations to certain references that may aid one of ordinary skill in the art understand the present invention and that may provide materials, information, techniques, proteins, vectors and nucleotide sequences that may assist one of ordinary skill in the art to make and use aspects of the present invention in its fullest scope. Accordingly, each cited reference is incorporated into this application as if originally filed herewith to the extent the teaching of the cited references do not conflict with the teaching of the present application, in which case the teaching of this application shall be deemed to control over the conflicting teaching of art incorporated herein by reference. 
         [0022]    One aspect of the invention is discovery that the xylose isomerase from  Bacteroides fragilis  is less sensitive to xylitol inhibition than are other xylose isomerase. Examples of other xylose isomerases include, but are not limited, those from the fungi  Piromyces  or  Orpinomyces  or from the sibling bacterial isomerases from  E. coli, L. lactis  and  Bacteroides thetaiotaomnicron  that have been previously described in the art. Two  B. fragilis  xylose isomerases of the present invention with reduced sensitivity to xyitol inhibition have the peptide sequences according to SEQ. ID NOS:2 and 4 shown in  FIGS. 7 and 8  respectively. SEQ. ID NO: 2 is a wild type sequence for the xylA gene from  B. fragilis  which was isolated by the present inventors and which is encoded by nucleotide sequence according to SEQ. ID NO: 1 shown in  FIG. 9 . SEQ. ID NO: 4 is a mutant xylA made by the present inventors to enhance transcription in  S. cerevisiae  and which is encoded by the nucleotide sequence according to SEQ. ID NO: 3 shown in  FIG. 10 . The XI proteins according to SEQ. ID NO: 2 and 4 are identical except that the mutation to enhance transcription also results in a change of an alanine to a serine at position 2 of the protein according to SEQ ID NO:4. SEQ. ID NO: 4 is also encoded by SEQ. ID NO: 5 shown in  FIG. 11  which preserves the change in nucleotide sequence for enhancement of transcription, but which also has changes in codon usage to optimize for protein synthesis in  S. cerevisiae . The  B. fragilis  XI proteins according to SEQ. ID NO: 2 and 4 are only 78.5% identical to the protein sequences for XI from  Piromyces,  77.9% identical to the sequence from  Orpinomyces,  46.2% identical to the sequence from  L. lactic,  52.5% identical to the sequence from  C. difficile,  47.3% identical to the sequence from  E. coli , and 90% identical to the sequence from  Bacteroides thetaiotamnicron . Accordingly, a  B. fragilis  XI useful for the present invention has greater than 90%, greater than 92.5%, greater than 95%, or greater than 98% amino acid sequence identity to SEQ.ID NO: 2 or SEQ.ID NO: 4. 
         [0023]    A distinguishing feature of the  B. fragilis  isomerases described herein is that enzymes are less inhibited by xylitol than orthologue XI sequences from other organism.  FIG. 3  shows a comparison of the xylitol inhibition of XI from  B. fragilis  according to SEQ. ID NO: 4 in comparison to its  E. coli  orthologue. At 12.5 mM xylitol, the  E. coli  XI activity is &gt;97% inhibited, in contrast to the  B. fragilis  XI, which retains at least 30% of its activity at the same xylitol concentration. 
         [0024]    While the precise differences in protein structure that accounts for the lower inhibition by xylitol for the  B. fragilis  enzyme is not presently known, the relationship between the structural and more important functional differences can be characterized in various ways. One characterization of the scope of the  B. fragilis  XI provided herein, is that it is a protein encoding a xylose isomerase activity to convert xylose to xylulose and having at least 90% identity to SEQ. ID NO: 2 or 4. Another is that it is a protein encoding a xylose isomerase activity to convert xylose to xylulose and that is more identical to SEQ. ID NO: 2 or 4 than to a XI protein sequence from  Piromyces, Orpinomyces, E. coli , or  Bacteroides thetaiotaomnicron . Yet another characterization is that is a protein encoding a xylose isomerase activity to convert xylose to xylulose that is less inhibited by xylitol than a XI protein sequence from  Piromyces, Orpinomyces, E. coli , or  B. thetaiotaomnicron . The polynucleotides provided herein track the same functional definitions as being nucleotide sequences that encode such functional proteins as characterized above, and/or that would hybridize to SEQ. ID NO: 1, 3 or 5 under stringent hybridization conditions under which they would not hybridize to a nucleotide sequences encoding a protein with XI activity from  Piromyces, Orpinomyces  or  Bacteroides thetaiotaomnicron.    
         [0025]    Another aspect of the present teaching is use of the forgoing  B. fragilis  XI genes and proteins expressed thereby to engineer  S. cerevisiae  for the production of ethanol from xylose. In a first embodiment, a xylA polynucleotide according to SEQ. ID NO: 5 encoding the XI protein according to SEQ. ID NO:4 was operably linked to the  S. cerevisiae  hxt7 promoter and cyc terminator in the multicopy autonomously replicating yeast plasmid bsdYEMhxt-XIbf-cycl shown in  FIG. 3 . In other embodiments, the same transcriptional unit for expression of the  B. fragils  XI protein was also engineered into integrating plasmids 421 bsd rDNA Phxt-Xibf (the 421 construct) and 423 rDNA Phxt-XIbf (the 423 construct) shown in  FIGS. 5 and 6 , respectively. Each of these integrative plasmids contains nucleotide sequences for yeast ribosomal RNA genes for targeted recombination into the chromosome in a region encoding a ribosomal RNA, which are multicopy genes in  S. cerevisiae . The 423 plasmid carries the marker gene for kanomycin resistance, while the 421 plasmid additionally carries the marker gene bsd, conferring resistance to the fungicide blasticidin. 
         [0026]    The 421 construct was integrated into the chromosome of a parent  S. cerevisiae  strain Y500, which is an exemplary commercial ethanol producing yeast strain. This commercial strain has characteristics typical of other commercially available strains such as Ethanol Red™, available from Lesaffre Group (Cedar Rapids Iowa) in terms of ethanol productivity. Transformants containing the 421 construct were selected for blasticidin resistance. An initial transformant referred to in  FIG. 1  as 421i was tested for its ability to grow in flasks on media containing yeast extract and peptone (YEP) with 2% wt/v xylose as the sole carbon source in comparison to a control containing the same bsd vector, but lacking the  B. fragilis  XI coding region (Y500+bsd). As shown in  FIG. 1 , the control strain ceased growth in YEP media within the first few hours, presumably after exhaustion of residual carbon sources from the YEP media. In contrast, the strain bearing the 421 construct expressing the  B. fragilis  XI continued growth on xylose for period of at least 150 hours. 
         [0027]    Another aspect of the present invention is recognizing that one of the problems with the xylose isomerase/xylulokinase two gene route for the production ethanol from xylose is unexpected accumulation of xylitol within the cell. This is a surprising finding because  S. cerevisiae  does not naturally contain a xylose reductase activity. While not be bound by theory, it is believed that the activity of one or more non specific aldol reductases, such as those encoded by the gre3 gene of  S. cerevisiae  may be converting a significant portion of the aldol sugars, including xylose, into xylitol. Because as mentioned above, xylitol is an inhibitor of xylose isomerases, the intracellular accumulation of xylitol may inhibit the isomerization of xylose to xylulose, slowing the production of ethanol from xylitol. US Pat. Pub No 20080261287 may at least partially address this problem in another system by deletion of the gre3 gene while simultaneously overexpressing a  Piromyces  xylose isomerase xylA gene in  S. cerevisiae  However, despite reporting that such strains hardly accumulate xylitol, ethanol yields from xylose remains low, which the present inventors believe is due to other factors that may cause the accumulation of xylitol. 
         [0028]    To compensate for this, one embodiment of the invention is to simultaneously express an exogenous xylitol dehydrogenase along with the exogenous xylose isomerase. This represents a novel 2 gene solution in that while xylose is indirectly being driven to xylulose by XI, simultaneously, the non specifically accumulated pool of xylitol is also being swept toward xylulose by XD preventing the inhibition of XI conversion of xylose to xylulose by xylitol. 
         [0029]    Y500 was engineered with the chromosomal integration vector, plasmid bsdYIMrDNA.Pxd-XD Phxt-Xibf (437), shown in  FIG. 4 . This plasmid carries the bsd, resistancegene, the xylA gene encoding XI from  B. fragilis  operably linked under the control of the yeast Hxt7 promoter, as in the 421 and 423 constructs mentioned above, but further carries the xyl2 gene from  P. stipitis  (see U.S. Pat. No. 5,789,210) encoding XD operably linked under control of the  P. stipitis  XD promoter. Blasticidin resistant strains having his vector integrated into the Y500 chromosome were selected and tested for the ability to utilize xylose for growth and to ferment xylose to produce ethanol. 
         [0030]      FIG. 1  shows the growth of the XI/XD construct 437 in shake flasks on media containing 2% xylose as the sole added carbon source in comparison to the control strains Y500, Y500 expressing XD alone, and Y500 expressing XIbf alone. The growth rate of the strain expressing both XI and XD was enhanced over each of the control strains. 
         [0031]    The solution of simultaneously expressing XI and XD can advantageously also be implemented with a three gene solution that further includes over-expressing xylulokinase (XK). Unlike the three gene route using the combination of xylose reductase and xylitol dehydrogenase and over-expressed xylulokinase, the present invention does not require, and indeed, preferably omits expression of a xylose reductase activity. Over-expression of XK would improve ethanol production from xylose in cases where the over-accumulation of xyulose is rate limiting. Such a condition may be the case where one or more of the XI or XD activities are also over-expressed, or when those activities have been modified to produce a higher turnover number or reduced allosteric inhibition.