# Patent

## Title
Microorganisms having enhanced tolerance to inhibitors and stress

## Abstract

The present invention provides genetically modified strains of microorganisms that display enhanced tolerance to stress and/or inhibitors such as sodium acetate and vanillin. The enhanced tolerance can be achieved by increasing the expression of a protein of the Sm-like superfamily such as a bacterial Hfq protein and a fungal Sm or Lsm protein. Further, the present invention provides methods of producing alcohol from biomass materials by using the genetically modified microorganisms of the present invention.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 12/795,882, filed on Jun. 8, 2010, now abandoned, which claims the benefit of U.S. Provisional Application No. 61/184,961, filed on Jun. 8, 2009, the contents of each of which in its entirety is incorporated herein by reference.


This invention was made with government support under Contract Number DE-AC05-00OR22725 between the United States Department of Energy and UT-Battelle, LLC. The U.S. Government has certain rights in this invention.


FIELD OF THE INVENTION
This invention generally relates to the field of microorganism and genetic modification thereof. In particular, the invention relates to microorganisms that display enhanced tolerance to stress and inhibitors as a result of increased expression of a protein of the Sm-like superfamily such as bacterial Hfq and yeast Sm or Lsm proteins. Such microorganisms are advantageous for use in fermentation of biomass materials to produce biofuels such as ethanol.
BACKGROUND OF THE INVENTION
Biomass-based bioenergy is crucial to meet the goal of making cellulosic biofuels cost-competitive with gasoline. Lignocellulosic materials represent an abundant feedstock for cellulosic-biofuel production. A core challenge in converting cellulosic material to biofuels such as ethanol and butanol is the recalcitrance of biomass to breakdown. Because of the complex structure of lignocellulosic biomass, pretreatment is necessary to make it accessible for enzymatic attack. Severe biomass pretreatments are required to release the sugars, which along with by-products of fermentation can create inhibitors in the production of ethanol or butanol, for example. During the pretreatment processes, a range of inhibitory chemicals are formed that include sugar degradation products such as furfural and hydroxymethyl furfural (HMF); weak acids such as acetic, formic, and levulinic acids; lignin degradation products such as the substituted phenolics vanillin and lignin monomers. In addition, the metabolic byproducts such as ethanol, lactate, and acetate also impact the fermentation by slowing and potentially stopping the fermentation prematurely. The increased lag phase and slower growth increases the ethanol cost due to both ethanol production rate and total ethanol yield decreases (Takahashi et al. 1999; Kadar et al. 2007).
Efficient conversion of lignocellulosic hydrolysates to biofuel requires high-yield production and resistance to industrially relevant stresses and inhibitors. To overcome the issue of inhibition caused by pretreatment processes, there are two approaches, one is to remove the inhibitor after pretreatment from the biomass physically or chemically, which requires extra equipment and time leading to increased costs. A second approach utilizes inhibitor tolerant microorganisms for efficient fermentation of lignocellulosic material to ethanol and their utility is considered an industrial requirement (Almeida et al. 2007).
Zymomonas mobilis are gram-negative facultative anaerobic bacteria with a number of desirable industrial characteristics, such as high-specific productivity and ethanol yield, unique anaerobic use of the Entner-Doudoroff pathway that results in low cell mass formation, high ethanol tolerance (12%), pH 3.5-7.5 range for ethanol production and has been generally regarded as safe (GRAS) status (Swings and De Ley 1977; Rogers et al. 1984; Gunasekaran and Raj 1999; Dien et al. 2003; Panesar et al. 2006; Rogers et al. 2007). One drawback to using wild-type Z. mobilis is its narrow substrate utilization range. However, recombinant Z. mobilis strains have been developed to ferment pentose sugars such as xylose and arabinose (Zhang et al. 1995; Deanda et al. 1996; Mohagheghi et al. 2002). On the other hand, low tolerance to acetic acid and decreased ethanol tolerance have been reported in recombinant strains (Ranatunga et al. 1997; Lawford and Rousseau 1998; Lawford et al. 2001; Dien et al. 2003).
Acetic acid is an inhibitor produced by the de-acetylation of hemicelluloses during biomass pretreatment. At pH 5.0, 36% of acetic acid is in the uncharged and undissociated form (HAc) and is able to permeate the Z. mobilis plasma membrane (Lawford and Rousseau 1993). The inhibition mechanism has been ascribed to the ability of the undissociated (protonated) form to cross the cell membrane leading to uncoupling and anion accumulation causing cytoplasmic acidification. Its importance comes from the significant concentrations of acetate that are produced relative to fermentable sugars (McMillan 1994) and the ratio of acetate to fermentable sugars is particularly high in material from hardwoods (Lawford and Rousseau 1993). Acetate may reach inhibitory levels when pretreated biomass hydrolysates are concentrated to generate high final ethanol concentrations or where process water is recycled. Acetate removal processes have been described but they are energy or chemical-intensive and their impact on processing costs have yet to be determined (McMillan 1994).
An acetate tolerant Z. mobilis mutant (AcR) has been generated by a random mutagenesis and selection strategy (Joachimstahl and Rogers 1998). The AcR mutant was capable of efficient ethanol production in the presence of 20 g/L sodium acetate while the parent ZM4 was inhibited significantly above 12 g/L sodium acetate under the same conditions. A number of studies have characterized the performance of recombinant Z. mobilis strains able to utilize both C-5 and C-6 sugars, including under acetate stress conditions (Lawford et al. 1999; Joachimsthal and Rogers 2000; Lawford and Rousseau 2001). Acetic acid was shown to be strongly inhibitory to wild-type derived strain ZM4(pZB5) on xylose medium and nuclear magnetic resonance studies indicated intracellular deenergization and acidification appeared to be the major inhibition mechanisms (Kim et al. 2000). A recombinant strain able to utilize both xylose (a C-5 sugar) and glucose (a C-6 sugar) with increased acetate resistance was generated by transforming plasmid pZBS into the AcR background (Jeon et al. 2002). Mohagheghi et al. (2004) reported a recombinant Zymomonas mobilis 8b tolerated up to 16 g/L acetic acid and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions.
Acetic acid bacteria are used for the industrial production of vinegar and are intrinsically resistant to acetic acid. Although the resistance mechanism is not completely understood, progress toward this goal has been made in recent years. Spontaneous acetic acid bacteria mutants for Acetobacter aceti (Okumura et al. 1985) and several Acetobacter pasteurianus strains (Takemura et al. 1991; Chinnawirotpisan et al. 2003) showed growth defects in the presence of acetic acid, which was associated with loss of alcohol dehydrogenase activity. Fukaya et al (1990) identified the aarA, aarB, and aarC gene cluster as being important for conferring acetic acid resistance using a genetic approach (Fukaya et al. 1990). aarA encodes citrate synthase and aarC encodes a protein that is involved in acetate assimilation (Fukaya et al. 1993), and the three aar genes have been suggested to support increased flux through a complete but unusual citric acid cycle to lower cytoplasmic acetate levels (Mullins et al. 2008). The presence of a proton motive force-dependent efflux system for acetic acid has been demonstrated as being important in A. aceti acetic acid resistance, although the genetic determinant(s) remain to be identified (Matsushita et al. 2005). In E. coli, over-expression of the ATP-dependent helicase RecG has been reported to improve resistance to weak organic acids including acetate (Steiner and Sauer 2003). Baumler et al. (2006) describe the enhancement of acid tolerance in Z. mobilis by the expression of a proton-buffering peptide in acidified TSB (HCl (pH 3.0) or acetic acid (pH 3.5)), glycine-HCl buffer (pH 3.0) and sodium acetate-acetic acid buffer (pH 3.5) (Baumler et al. 2006). Baumler et al. (2006) also note that the presence of the antibiotic also significantly increased acid tolerance by an unknown mechanism.
Aerobic, stationary phase conditions were found to produce a number of inhibitory secondary metabolites from Z. mobilis when compared to anaerobic conditions at the same time point. The Z. mobilis global regulator gene hfq has been identified as associated with stress responses generated under aerobic stationary phase conditions (Yang et al., 2009). Hfq is a bacterial member of the Sm family of RNA-binding proteins, which acts by base-pairing with target mRNAs and functions as a chaperone for non-coding small RNA (sRNA) in E. coli (Valentin-Hansen et al. 2004; Zhang et al. 2002; Zhang et al. 2003). E. coli Hfq is involved in regulating various processes and deletion of hfq has pleiotropic phenotypes, including slow growth, osmosensitivity, increased oxidation of carbon sources, and altered patterns of protein synthesis in E. coli (Valentin-Hansen et al. 2004; Tsui et al. 1994). E. coli Hfq has also been reported to affect genes involved in amino acid biosynthesis, sugar uptake, metabolism and energetics (Guisbert et al. 2007). The expression of thirteen ribosomal genes was down-regulated in hfq mutant background in E. coli (Guisbert et al. 2007). Hfq also up-regulated sugar uptake transporters and enzymes involved in glycolysis and fermentation such as pgk and pykA, and adhE (Guisbert et al. 2007). E. coli Hfq is also involved in regulation of general stress responses that are mediated by alternative sigma factors such as RpoS, RpoE and RpoH. Cells lacking Hfq induce the RpoE-mediated envelope stress response and rpoH is also induced in cells lacking Hfq (Guisbert et al. 2007), which is consistent with our results that Z. mobilis hfq was less abundant in aerobic fermentation condition in ZM4 at 26 h post-inoculation and was rpoH induced (Yang et al. 2009).
SUMMARY OF THE INVENTION
It has been identified in accordance with the present invention that increased expression of a protein of the Sm-like superfamily in a microorganism confers enhanced tolerance to stress and inhibitors such as sodium acetate, ammonium acetate, potassium acetate, vanillin, furfural, hydroxymethylfurfural (HMF) and H2O2. In accordance with the present invention, microorganisms can be genetically modified to increase the expression of a protein of the Sm-like superfamily to achieve enhanced tolerance to stress and inhibitors. Such genetically modified microorganisms are particularly useful for production of biofuels based on fermentation of biomass materials.
In one aspect, the invention is directed to genetically modified microorganisms that display enhanced tolerance to stress and/or inhibitors as a result of increased expression of a protein of the Sm-like superfamily in the microorganisms.
In one embodiment, the microorganism is a genetically engineered bacterial strain, and the protein being expressed at an elevated level is a bacterial Hfq protein.
Bacteria contemplated by the present invention include both Gram-negative and Gram positive bacteria. Examples of bacteria of particular interest include Acetobacterium, Bacillus, Streptococcus, Clostridium (e.g., C. thermocellum), Zymomonas sp. (e.g., Z. mobilis), Anaerocellum (e.g., Anaerocellum thermophilum), Caldicellulosiruptor (e.g., C. saccharolyticus), Thermoanaerobacter (e.g., Thermoanaerobacter sp. X514), Gluconobacter, and E. coli. 
Bacterial strains that display enhanced tolerance to stress and/or inhibitors can be generated, e.g., by introducing to a bacterial strain an expression vector which includes the coding sequence of a bacterial Hfq protein. The expression vector directs the expression of the Hfq protein as a replicative plasmid, or mediates the integration of the coding sequence into the host genome to achieve chromosomal expression. Preferably, the bacterial Hfq protein in the vector is identical with or substantially homologous with an endogenous Hfq protein of the recipient bacterial strain.
In specific embodiments, the expression vector includes the coding sequence of a bacterial Hfq protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 2 (Z. mobilis ZM4), SEQ ID NO: 4 (E. coli), SEQ ID NO: 6 (Clostridium thermocellum), SEQ ID NO: 8 (Anaerocellum thermophilum), SEQ ID NO: 10 (Caldicellulosiruptor saccharolyticus), SEQ ID NO: 12 (Thermoanaerobacter sp. X514), and functional derivatives thereof.
In a further embodiment, the invention is directed to genetically engineered fungal strains that display enhanced tolerance to stress and/or inhibitors. Examples of fungi include Saccharomyces sp. (e.g., S. cerevisiae), Kluyveromyces sp., Pichia sp. (e.g., Pichia pastoris), Candida sp., and Schizosaccharomycetes sp.
Such fungal strains can be generated, e.g., by introducing to a fungal strain an expression vector which includes the coding sequence of a fungal protein of the Sm-like superfamily. Similarly, the expression vector can be a replicative vector or integrative vector. Preferably, the fungal protein of the Sm-like superfamily in the expression vector is identical with or substantially homologous with an endogenous Sm-like protein of the fungal strain.
In specific embodiments, the expression vector includes the coding sequence of a fungal protein of the Sm-like superfamily having an amino acid sequence selected from the group consisting of SEQ ID NOS: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 (representing 19 S. cerevisiae Sm and Lsm proteins) and functional derivatives thereof.
The genetically modified microorganisms that display enhanced tolerance to stress and inhibitors can be additionally modified as appropriate, for example, by transformation with additional recombinant genes or sequences suitable for fermentation and production of ethanol. For example, the bacterial and fungal strains can be additionally modified so as to have the ability to utilize C5 sugars such as xylose and arabinose in addition to C6 sugars.
In a further aspect, the present invent provides a method of producing biofuels from cellulosic biomass based on use of the microbial strains that are able to grow at elevated concentrations of inhibitors and/or under stress conditions.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1E. Domain and motif sites of Z. mobilis Hfq (A), E. coli Hfq (B), S. cerevisiae Sm B (C), and S. cerevisiae Lsm1 (D) proteins. Bacterial Hfq alignment and Clustal W (E). Sequences in (E), Anaerocellum thermophilum, SEQ ID NO: 8; Caldicellulosiruptor saccharol., SEQ ID NO: 10; Clostridium thermocellum, SEQ ID NO: 6; Thermoanaerobacter sp. X514, SEQ ID NO: 12; Zymomonas mobilis ZM4, SEQ ID NO: 2; Escherichia coli K12, SEQ ID NO: 4. Residues that are identical across the species are indicated by “*”, and residues that are not identical but conserved in function across the species are indicated by “:”.
FIG. 2A. Graphic map of the low copy number Gateway® compatible plasmid pBBR3-DEST42. Tc(R): Tetracycline resistance gene tet; Cm: chloramphenicol resistance gene cat. attR1 and attR2 are recombination sites allowing recombinational cloning of the gene of interest from an entry clone; ccdB is ccdB gene allowing negative selection of expression clones.
FIG. 2B. The insertion position and complementation region of ZMO0347 as well as the primers and mutation position. ZMO0346, ZMO0347, and ZMO0348 are Z. mobilis ZM4 genes. hfq_MF and hfq_MR are primers used for insertional mutant construction using pKNOCK mutagenesis system. Hfq_CF and Hfq_CR are primers used to clone the hfq gene into pBBR3-DEST42 for complementation, which resulted in a plasmid called as p42-0347. The primer sequences are: hfq_MF: cggagagatggtcagtcaca (SEQ ID NO: 51); hfq_MR: ttcttgctgctgcataatcg (SEQ ID NO: 52); Hfq_CF: atggccgaaaaggtcaacaatc (SEQ ID NO: 53); Hfq_CR: atcctcgtctcgcctttctgtc (SEQ ID NO: 54).
FIGS. 3A-3C. Hfq is responsible for sodium acetate tolerance of Z. mobilis. Z. mobilis strains were grown in RM (pH5.0) overnight, 20-μL culture were then transferred into 250-μL, RM media in the Bioscreen plate. The growth differences of different strains were monitored by Bioscreen (GrowthCurve, MA) under anaerobic conditions in RM (pH5.0) containing 0, 12, and 16 g/L NaAc (A, B, C respectively). Strains included in this study are: ZM4: Zymomonas mobilis ZM4 wild-type; AcR: ZM4 acetate tolerant mutant (Joachimstahl 1998); ZM4 (p42-0347): ZM4 containing a gateway plasmid p42-0347 over-expressing ZM4 gene ZMO0347; AcRIM0347: AcR insertional mutant of ZMO0347; AcRIM0347 (p42-0347): AcRIM0347 containing gateway plasmid p42-0347 over-expressing ZM4 gene ZMO0347. This experiment has been repeated at least three times with similar result. Triplicates were used for each condition.
FIGS. 4A-4E. Hfq contributes to Z. mobilis acetate tolerance. Z. mobilis strains were grown in RM (pH5.0) overnight, 5-μL culture were then transferred into 250-μL RM media in the Bioscreen plate. The growth differences of different strains were monitored by Bioscreen (Growth Curves USA, N.J.) under anaerobic conditions; in RM, pH 5.0 (A), RM with 195 mM NaCl, pH 5.0 (B), 195 mM NaAc, pH 5.0 (C), 195 mM NH4OAc, pH 5.0 (D), or 195 mM KAc, pH 5.0 (E). Strains included in this study are: ZM4: Zymomonas mobilis ZM4 wild-type; AcR: ZM4 acetate tolerant mutant; ZM4 (p42-0347): ZM4 containing a gateway plasmid p42-0347 to express ZM4 gene ZMO0347; AcRIM0347: AcR insertional mutant of ZMO0347; AcRIM0347 (p42-0347): AcRIM0347 containing gateway plasmid p42-0347. This experiment has been repeated at least three times with similar result. Duplicate biological replicates were used for each condition.
FIGS. 5A-5E. Z. mobilis Hfq conferred tolerance to different classes of pretreatment inhibitors. Z. mobilis strains were grown in RM (pH 5.0) overnight, 5-μL culture were then transferred into 250-μL RM media in the Bioscreen plate. The growth differences of different strains were monitored by Bioscreen (Growth Curves USA, NJ) under anaerobic conditions in RM, pH 5.0 (A), RM with 1 g/L vanillin, pH 5.0 (B), 1 g/L furfural, pH 5.0 (C), 1 g/L HMF, pH 5.0 (D) and 0.001% H2O2 (E). Strains included in this study are: ZM4: Zymomonas mobilis ZM4 wild-type; AcR: ZM4 acetate tolerant mutant; AcRIM0347: AcR insertional mutant of ZMO0347; AcRIM0347 (p42-0347): AcRIM0347 containing gateway plasmid p42-0347 over-expressing ZM4 gene ZMO0347. This experiment has been repeated at least three times with similar result for hydrogen peroxide growth and in duplicate for the vanillin growth.
FIGS. 6A-6B. Lsm-like proteins in S. cerevisiae are responsible for sodium acetate tolerance. S. cerevisiae strains were grown in CM with 2% glucose for wild-type BY4741 and the deletion mutants, CM with 2% glucose minus uracil for GST over-expression strains. Five-μL culture was then transferred into 300-μL CM broth in the Bioscreen plate. The growth differences of different strains were monitored by Bioscreen (Growth Curve USA, NJ) containing 40 g/L sodium acetate for yeast deletion mutants (A) and GST over-expression strains (B). This experiment has been repeated at least three times with similar result.
FIGS. 7A-7P. Lsm proteins in S. cerevisiae are involved in multiple inhibitor tolerance. S. cerevisiae strains were grown in CM with 2% glucose (CM+glucose) for wild-type BY4741 and the deletion mutants, CM with 2% glucose and 2% galactose minus uracil (CM+glucose+2% galactose) for GST overexpression strains. A 5-μL culture was then transferred into 250-μL CM broth in the Bioscreen plate. The growth differences of different deletion mutant strains were monitored by Bioscreen (Growth Curves USA, NJ) in CM+glucose at pH 5.5 (A), CM+glucose with 305 mM NaCl, pH 5.5 (B), 305 mM NaAc, pH 5.5 (C), 305 mM NH4OAc, pH 5.5 (D), and 305 mM KAc, pH 5.5 (E), 0.75 g/L vanillin, pH 5.5 (F), 1.5 g/L furfural, pH 5.5 (G), and 1.5 g/L HMF, pH 5.5 (H). The growth differences of different GST-over-expressing strains were monitored by Bioscreen (Growth Curves USA, NJ) in CM+glucose+2% galactose at pH 5.5 (I), CM+glucose+2% galactose with 305 mM NaCl, pH 5.5 (J), 305 mM NaAc, pH 5.5 (K), 305 mM NH4OAc, pH 5.5 (L), 305 mM KAc, pH 5.5 (M), 0.75 g/L vanillin, pH 5.5 (N), 1.5 g/L furfural, pH 5.5 (O), and 1.5 g/L HMF, pH 5.5 (P). Strains included in this study are listed in table 1. This experiment has been repeated at least three times with similar result.



DETAILED DESCRIPTION OF THE INVENTION
It has been identified in accordance with the present invention that increased expression of a protein of the Sm-like superfamily in a microorganism confers enhanced tolerance to stress and inhibitors. Based on this discovery, the present invention provides strains of microorganisms displaying enhanced tolerance to stress and/or inhibitors, which are particularly advantageous for use in fermentation of biomass materials to produce biofuels.
In one aspect, the invention is directed to genetically modified strains of microorganisms that display enhanced tolerance to stress and/or growth inhibitor as a result of increased expression of a protein of the Sm-like superfamily in the microorganisms.
Sm-like superfamily proteins are a highly conserved family of proteins found in eukaryotes, archaea and bacteria, and are characterized by an Sm-like superfamily domain having two conserved motifs referred to as Sm1 motif and Sm2 motif. The Sm1 and Sm2 motifs were first defined for human Sm snRNP proteins (Hermann et al. 1995), and were subsequently found to be highly conserved in other Sm and Lsm (Sm-like) proteins in eukaryotes including plant, drosophila, C. elegans, and S. cerevisiae. Eukaryotic Sm and Lsm proteins are integral to RNA processing and mRNA degradation complexes. Subsequently, the E. coli global response regulator Hfq was reported to be a homolog of the Sm and Lsm proteins (Zhang et al. 2002). The bacterial Hfq proteins contain a first region that shares significant similarity with the Sm1 motif found in eukaryotes, and a second region of particularly high conservation among the bacterial proteins which contains a number of conserved hydrophobic residues that align with hydrophobic residues found in the Sm2 motif of eukaryotic cells (Zhang et al. 2002). Similar to the eukaryotic Sm and Lsm proteins, the E. coli Hfq protein also forms a multisubunit ring and is believed to also function to enhance RNA-RNA pairing.
As used herein, the term “Sm-like superfamily” includes both Sm and Lsm proteins of eukaryotes and archaea, and Hfq proteins of bacteria.
A eukaryotic protein is considered to be a protein of the Sm-like superfamily in the context of the present invention if the protein contains an Sm-like superfamily domain characterized by the Sm1 motif and Sm2 motif defined by Hermann et al. (1995). Specifically, the Sm1 motif typically spans 32 amino acids, with positions 13 and 23 being Gly and Asn, respectively, positions 1, 3, 11, 15, 18 and 26 being a hydrophobic residue, and positions 19 and 31 being an acidic amino acid (Asp or Glu). The Sm2 motif typically spans only 14 amino acids, and has the consensus sequence (I or L) (R or K) (G or C) at positions 6-8, with positions 1, 4, 11, 13 and 14 being a hydrophobic residue, and positions 9-10 being a hydrophilic residue. Examples of eukaryotic proteins of the Sm-like superfamily include S. cerevisiae Sm B, Sm D1, Sm D2, Sm D3, Sm E, Sm F, Sm G, Lsm1, Lsm2, Lsm3, Lsm4, Lsm5, Lsm6, Lsm7, Lsm8, Lsm9, Lsm 13, and Lsm16 (SEQ ID NOS: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50, respectively). The locations of Sm1 and Sm2 motifs are illustrated for Sm B and Lsm1 proteins in FIGS. 1C-1D.
In the context of the present invention, a bacterial protein is considered to be an Hfq protein and therefore a protein of the Sm-like superfamily if the bacterial protein contains an Sm-like superfamily domain characterized by a first motif similar to the Sm1 motif of eukaryotic proteins defined above, and a second highly conserved region. Generally, the bacterial Sm1 motif spans 26 amino acids, and like the eukaryotic Sm1 motif, has Gly at position 13, an acidic amino acid at position 19 (typically Asp), a hydrophobic residue at positions 1 (preferably V), 3, 11 (preferably L), 15 and 18 (preferably F). Additionally, the second highly conserved region (“the bacterial Sm2 motif”) generally spans 12 amino acids, and has a “KHA” sequence at positions 6-8; and preferably, with Y, I and S at positions 5, 9, and 10, respectively, and with a hydrophobic residue at positions 3-4 and 11. Examples of bacterial Hfq proteins include SEQ ID NO: 2 (Z. mobilis ZM4), SEQ ID NO: 4 (E. coli), SEQ ID NO: 6 (Clostridium thermocellum), SEQ ID NO: 8 (Anaerocellum thermophilum), SEQ ID NO: 10 (Caldicellulosiruptor saccharolyticus), and SEQ ID NO: 12 (Thermoanaerobacter sp. X514). Alignment of these bacterial Hfq proteins is provided in FIG. 1B, and the locations of the Sm-like superfamily domain including the Sm1 and Sm2 motifs of Z. mobilis and E. coli proteins are illustrated in FIG. 1A. It is clear that these six bacterial Hfq proteins share significant homologies, having conserved V, L, G, L, G, F, D and F at positions 1, 5, 8, 11, 13, 18, 19 and 21 of the Sma1 motif, and Y, K, H, A, I, and S at positions 5-10 of the Sm2 motif.
Functional derivatives and homologs of a given protein of the Sm-like superfamily are also suitable for use in the present invention. As used herein, “functional derivatives” and “homologs” of a protein of the Sm-like superfamily refer to proteins that share at least 45% identity or similarity, or preferably at least 50%, 60%, 75%, or 85% identity or similarity, or more preferably 90%, 95%, 98%, or 99% identity or similarity, with the protein of the Sm-like superfamily. Similarity between two protein sequences can be determined, for example, using the well known Lipman-Pearson Protein Alignment program with the following choice of parameters: Ktuple=2, Gap Penalty=4, and Gap Length Penalty=12. Preferably, the derivatives and homologs share consensus motifs of the Sm-like superfamily, which are believed to be critical to the function of the proteins.
A functional derivative of a given protein includes derives where modifications are made to non-conserved residues, as well as a functional or enzymatically active fragment of the protein. The term “functional fragment” or “enzymatically active fragment” means a polypeptide fragment of a full length protein, which substantially retains the activity of the full-length protein. By “substantially” it is meant at least about 50%, or preferably at least 70%, or even 80% or more of the activity of the full-length protein is retained.
The genetically engineered microbial strains of the present invention display enhanced tolerance to stress and/or one or more inhibitors as a result of increased expression of a protein of the Sm-like superfamily.
The term “stress”, as used herein, refers generally to environmental stress, i.e., stress received from the environment, such as high temperatures, low temperatures, low pH, oxidation (i.e., the presence of reactive oxidative species such as H2O2), osmotic, drought, the presence of inhibitors, or nutrient limit such as starvation, among others. For example, “cold stress” is stress on microorganism due to exposure to environments below the minimum optimal growth temperature of the microorganism. “Drought stress” is stress due to exposure of the microorganism to environments under the minimum optimal growth moisture concentration. “Osmotic stress” is stress on microorganisms due to exposure of the microorganisms to environments over or under the maximum or minimum optimal growth osmotic of the microorganisms.
The term “inhibitors” as used herein refer particularly to inhibitory chemical compounds that are formed during biomass pretreatments, including sugar degradation products such as furfural and hydroxymethyl furfural (HMF), weak acids such as acetic, formic, and levulinic acids, lignin degradation products such as the substituted phenolics vanillin and lignin monomers, reactive oxidative species generating hydrogen peroxide (H2O2) and vanillin, as well as metabolic byproducts such as ethanol, lactate, and acetate. A particularly desirable trait of microorganisms is an enhanced tolerance to sodium and acetate ions, e.g., in the form of sodium acetate, ammonium acetate, and potassium acetate.
In the present invention, microorganisms with enhanced tolerance to stress and/or one or more inhibitors refer to microorganisms which, as a result of genetic modification to increase the level of proteins of the Sm-like superfamily in the microorganisms, demonstrate improved tolerance as compared to microorganisms without the genetic modification. Improved tolerance can be determined by an improved growth profile (either as a shorter lag phase, a shorter doubling time, or a higher maximum density) under a given stress condition or inhibitor concentration. Alternatively, improved tolerance can be determined by an increase in the concentration of an inhibitory molecule which the microorganisms can tolerate.
For example, microorganisms having an elevated expression of Sm-like superfamily proteins exhibit enhanced tolerance to acetate. “Tolerance to acetate” is meant herein to include resistance to acetate salts including, for example, sodium acetate, ammonium acetate and potassium acetate, and/or to acetic acid. Tolerance of a strain to acetate can be determined by assessing the growth of the strain in media containing various concentrations of acetate (e.g., sodium acetate). The microbial strains containing a desirable genetic modification of the present invention are able to grow in media containing a higher concentration of acetate (e.g., sodium acetate) than the unmodified strains. For example, the concentration of sodium acetate that can be tolerated by a strain can be increased by 15%, 20%, 30%, or 50% or higher, as a result of a genetic modification. As demonstrated herein below, wild type Z. mobilis strain ZM4 is unable to grow in media containing 16 g/L (195 mM) sodium acetate, while ZM4-p42-0347 (expressing additional ZM4 Hfq proteins) is able to grow at this concentration. Alternatively, “enhanced tolerance” can be measured by a shorter lag time (e.g., shortened by 10%, 20%, 30% or 50% or greater), a shorter doubling time (e.g., shortened by 10%, 20%, 30% or 50% or greater) or a higher cell density reached at the end of the exponential growth phase (e.g., 25%, 50%, 75%, 100%, 150%, 200%, 500%, or even 1000% or higher cell density). See FIGS. 3A-3C.
Microorganisms encompassed within the scope of the present invention include both bacteria and fungi.
In accordance with the present invention, bacterial strains having enhanced tolerance to stress and inhibitors as a result of increased expression of Sm-superfamily proteins include both Gram-positive and Gram-negative bacteria. Examples of Gram-positive bacteria include those from the genus of phylum Firmicutes, particularly strains of Acetobacterium, Bacillus, Streptococcus, Clostridium (e.g., C. thermocellum), Anaerocellum (e.g., Anaerocellum thermophilum), Caldicellulosiruptor (e.g., C. saccharolyticus), and Thermoanaerobacter (e.g., Thermoanaerobacter sp. X514). Examples of Gram-negative bacteria of particular interest include those generally considered medically safe, such as Zymomonas sp. (e.g., Z. mobilis), E. coli, Gluconobacter sp. (e.g., Gluconobacter oxydans, previously known as Acetobacter suboxydans), Cyanobacteria, Green sulfur and Green non-sulfur bacteria.
Fungal strains contemplated by the present invention include filamentous and unicellular fungal species, particularly the species from the class of Ascomycota, for example, Saccharomyces sp., Kluyveromyces sp., Pichia sp., Candida sp., and Schizosaccharomycetes sp. Preferred fungal strains contemplated by the present invention are S. cerevisiae, S. pombe, and Pichia pastoris. Where the fungal strains are S. cerevisiae, additional genetic modifications are preferred besides the genetic modification that results in an increased expression of a Sm-like superfamily protein. For example, S. cerevisiae is also modified such that the strain is able to utilize C5 sugars.
Strains of microorganisms that display enhanced tolerance to stress and/or inhibitors as a result of increased expression of a Sm-like superfamily protein can be made using any of the known genetic engineering techniques. For example, the 5′ upstream regulatory region of an endogenous Sm-like superfamily gene can be modified to achieve enhanced expression of the encoded endogenous Sm-like superfamily protein.
In one embodiment, a microbial strain having enhanced tolerance is created by introducing an exogenous expression vector into the strain which contains the coding sequence of a protein of the Sm-like superfamily.
In a preferred embodiment, the protein encoded by the expression vector is identical with an endogenous protein of the Sm-like superfamily or a functional derivative thereof, even though homologs from other related species can also be utilized.
Generally, the nucleotide sequence coding for a protein of the Sm-like superfamily is placed in an operably linkage to a promoter and a 3′ termination sequence that are functional in a recipient microbial host. The promoter can be a constitutive promoter or an inducible promoter. The promoter can be the native promoter of the Sm-like superfamily gene being expressed, or a heterologous promoter from a different gene. Promoters suitable for use in expression in a bacterial host include, for example, lac promoter, T7, T3 and SP6 phage RNA polymerase promoters. Specific examples of promoters suitable for use in expression in Zymomonas species include Z. mobilis pdc promoter and adhB promoter. Specific examples of promoters suitable for use in expression in yeast including S. cerevisiae include adh1+(constitutive high expression), fbp1+(carbon source responsive), a tetracycline-repressible system based on the CaMV promoter, and the nmt1+(no message in thiamine) promoter. These and other examples of promoters are well documented in the art.
A variety of vector backbones can be used for purpose of the present invention. Choices of vectors suitable for transformation and expression in bacteria and fungi have been well documented in the art. For example, numerous plasmids have been reported for transformation and expression in Zymomonas, including, e.g., pZB serial plasmids developed based on Zymomonas cryptic plasmid, as described in U.S. Pat. Nos. 5,712,133, 5,726,053, and 5,843,760, and a cloning-compatible broad-host-range destination vector described by Pelletier et al. (2008), among many others.
In addition to the Sm-like superfamily protein expression unit, the expression vector can include other sequences where appropriate, such as sequences for maintenance and selection of the vector, e.g., a selection marker gene and a replication origin. The selection marker gene can be a gene that confers resistance to antibiotics such as ampicillin resistance (Ampr), tetracycline resistance (Tetr), neomycin resistance, hygromycin resistance, and zeocin resistance (Zeor) genes, or a gene that provides selection based on media supplement and nutrition.
The vector can be a replicative vector (such as a replicating circular plasmid), or an integrative vector which mediates the introduction of the vector into a recipient cell and subsequent integration of the vector into the host genome for chromosomal expression.
For industrial applications, the inhibitors generated from the biomass pretreatments will select for plasmid maintenance where hfq expression confers an advantage to the strain (i.e., enhanced tolerance to inhibitors) in the absence of additional marker or antibiotic selection. The vectors can also be modified to include the parDE genes to enhance plasmid stability in bacteria in the absence of selection using standard molecular biology approaches, as described in the art (Brown et al., 2002; Pecota et al., 1997). Alternatively and preferably, the desired expression unit (such as an hfq coding sequence operably linked to a promoter) is integrated into the chromosome of the microorganism for expression and enhanced stability. Methods for chromosomal integration in bacteria include modified homologous Campbell-type recombination (Kalogeraki et al. 1997) or transposition (Koch et al. 2001). Methods for chromosomal integration in yeast are well known and are described in Amberg et al. (2005).
An expression vector can be introduced into a microbial host by various approaches known in the art, including transformation (e.g., chemical reagent based transformation), electroporation and conjugation.
The genetic modification to a microbial strain results in an increased expression of a Sm-like superfamily protein. Where the exogenously introduced expression unit codes for a protein identical with an endogenous protein, the level of such protein (expressed from both the native sequence and the exogenous sequence) is increased. Where the exogenously introduced expression unit codes for a protein that is not identical with any endogenous protein but is a functional derivative of or most homologous to an endogenous protein, the collective level of the endogenous protein and the exogenous protein is increased as compared to the unmodified strain. The extent of increase in expression contemplated by the present invention is at least 40%, 50%, 75%, 100% (i.e., twice the level of parental strain), or more preferably at least four or five times, or even more preferably at least ten to fifteen times, the level of parental strain. As a practical matter, the level of expression can be assessed both at the mRNA level and at the protein level.
Pretreatment of biomass by chemical or enzymatic methods yields a mixture of hexose sugars (C6 sugars, primarily glucose and mannose) and pentose sugars (C5 sugars, primarily xylose and arabinose). The fermentation of almost all the available C6 and C5 sugars to ethanol or other liquid biofuel is critical to the overall economics of these processes. Most microorganisms are able to ferment glucose but few have been reported to utilize xylose efficiently and even fewer ferment this pentose to ethanol.
The genetically modified strains of microorganisms of the present invention, which display enhanced tolerance to stress and/or one or more inhibitors as a result of increased expression of a Sm-like superfamily protein, can be additionally modified as appropriate. For example, Z. mobilis strains overexpressing Z. mobilis Hfq can be additionally modified in order to expand the range of substrates that can be utilized by the strains for efficient ethanol production. For instance, Z. mobilis strains over-expressing Hfq can also be introduced with additional genes so that the strains can ferment xylose, arabinose or other pentose sugars as the sole carbon source to produce ethanol. See, e.g., U.S. Pat. No. 5,514,583. Additionally, yeast strains over-expressing a Sm or Lsm protein, particularly S. cerevisiae strains, can be additionally modified to have an enhanced ability to ferment xylose, arabinose or other pentose sugars to produce ethanol. For example, yeast cells can be modified to overexpress (via transformation with additional expression unit) xylose reductase, xylulokinase, or xylose isomerase; or modified to have reduced expression of xylitol dehydrogenase, PHO13 or a PHO13 ortholog. See, e.g., U.S. Pat. No. 7,285,403, US 20060234364 A1, and US 20080254524 A1, the teachings of which are incorporated herein by reference.
The isolated or genetically modified microbial strains of the present invention are particularly useful for production of biofuels based on fermentation of biomass materials. Therefore, in a further aspect, the present invent provides a method of producing biofuels from cellulosic biomass based on use of the microbial strains of the present invention that are able to grow at elevated concentrations of acetate.
Biofuels contemplated by the present invention include particular the types of biologically produced fuels, such as bioalcohols, based on the action of microorganisms and enzymes through fermentation of biomass materials. Examples of bioalcohols include ethanol, butanol, and propanol.
In a typical cellulosic biomass to alcohol process, raw cellulosic biomass material is pretreated in order to convert, or partially convert, cellulosic and hemicellulosic components into enzymatically hydrolyzable components (e.g., poly- and oligo-saccharides). The pretreatment process also serves to separate the cellulosic and hemicellulosic components from solid lignin components also present in the raw cellulosic material. The pretreatment process typically involves reacting the raw cellulosic biomass material, often as a finely divided mixture or slurry in water, with an acid, such as sulfuric acid. Other common pretreatment processes include, for example, hot water treatment, wet oxidation, steam explosion, elevated temperature (e.g., boiling), alkali treatment and/or ammonia fiber explosion. The pretreated biomass is then treated by a saccharification step in which poly- and oligo-saccharides are enzymatically hydrolyzed into simple sugars. The free sugars and/or oligosaccharides produced in the saccharification step are then subjected to fermentation conditions for the production of ethanol or butanol, for example. Fermentation can be accomplished by combining one or more fermenting microorganisms with the produced sugars under conditions suitable for fermentation.
One can also add enzyme to the fermentor to aid in the degradation of substrates or to enhance alcohol production. For example, cellulase can be added to degrade cellulose to glucose simultaneously with the fermentation of glucose to ethanol by microorganisms in the same fermentor. Similarly, a hemicellulase can be added to degrade hemicellulose.
Because the pretreatment processes and by-products of fermentation can create a range of inhibitors including acetate, it is especially advantageous to utilize the genetically modified microbial strains described herein which display enhanced resistance to acetate and are able to continue fermentation despite acetate present in the fermentation broth, either in the fermentation substrate carried over from pretreatment of biomass material, or built up as a byproduct of fermentation.
For purpose of fermentation, one strain or a mixture of several strains, some or all of which display enhanced tolerance to stress and/or inhibitors, can be used.
Specific fermentation conditions can be determined by those skilled in the art, and may depend on the particular feedstock or substrates, the microorganisms chosen and the type of biofuel desired. For example, when Zymomonas mobilis is employed, the optimum pH conditions range from about 3.5 to about 7.5; substrate concentrations of up to about 25% (based on glucose), and even higher under certain conditions, may be used; and no oxygen is needed at any stage for microorganism survival. Agitation is not necessary but may enhance availability of substrate and diffusion of ethanol.
After fermentation, alcohol is separated from the fermentation broth by any of the many conventional techniques known to separate alcohol from aqueous solutions, including evaporation, distillation, solvent extraction and membrane separation. Particles of substrate or microorganisms may be removed before separation to enhance separation efficiency.
Table 1. List all the sequence identifiers for the nucleotide and protein sequences of the Sm-like superfamily molecules exemplified in the present application.
The present invention is further illustrated and by no means limited by the following examples.








TABLE 1





SEQ ID NO
Description
















1
Zymomonas mobilis hfq nucleotide


2
Zymomonas mobilis Hfq amino acid


3
E. coli hfq nucleotide


4
E. coli Hfq amino acid


5
Clostridium thermocellum hfq nucleotide


6
Clostridium thermocellum Hfq amino acid


7
Anaerocellum thermophilum hfq nucleotide


8
Anaerocellum thermophilum Hfq amino acid


9
Caldicellulosiruptor saccharolyticus hfq nucleotide


10
Caldicellulosiruptor saccharolyticus Hfq amino acid


11
Thermoanaerobacter sp. X514 hfq nucleotide


12
Thermoanaerobacter sp. X514 Hfq amino acid


13
S. cerevisiae SMB1 nucleotide


14
S. cerevisiae Sm B amino acid


15
S. cerevisiae SMD1 nucleotide


16
S. cerevisiae Sm D1 amino acid


17
S. cerevisiae SMD2 nucleotide


18
S. cerevisiae Sm D2 amino acid


19
S. cerevisiae SMD3 nucleotide


20
S. cerevisiae Sm D3 amino acid


21
S. cerevisiae SME1 nucleotide


22
S. cerevisiae Sm E amino acid


23
S. cerevisiae SMX3 nucleotide


24
S. cerevisiae Sm F amino acid


25
S. cerevisiae SMX2 nucleotide


26
S. cerevisiae Sm G amino acid


27
S. cerevisiae LSM1 nucleotide


28
S. cerevisiae Lsm1 amino acid


29
S. cerevisiae LSM2 nucleotide


30
S. cerevisiae Lsm2 amino acid


31
S. cerevisiae LSM3 nucleotide


32
S. cerevisiae Lsm3 amino acid


33
S. cerevisiae LSM4 nucleotide


34
S. cerevisiae Lsm4 amino acid


35
S. cerevisiae LSM5 nucleotide


36
S. cerevisiae Lsm5 amino acid


37
S. cerevisiae LSM6 nucleotide


38
S. cerevisiae Lsm6 amino acid


39
S. cerevisiae LSM7 nucleotide


40
S. cerevisiae Lsm7 amino acid


41
S. cerevisiae LSM8 nucleotide


42
S. cerevisiae Lsm8 amino acid


43
S. cerevisiae LSM9 nucleotide


44
S. cerevisiae Lsm9 amino acid


45
S. cerevisiae LSM12 nucleotide


46
S. cerevisiae Lsm12 amino acid


47
S. cerevisiae LSM13 nucleotide


48
S. cerevisiae Lsm13 amino acid


49
S. cerevisiae LSM16 nucleotide


50
S. cerevisiae Lsm16 amino acid









Example 1
This example describes the materials and methods used in the experiments described in the subsequent examples.
Strains and Culture Conditions
Bacterial strains and plasmids used in this study are listed in Table 2. E. coli strains were cultured using Luria-Bertani (LB) broth or plates. E. coli WM3064 was supplemented with 100 μg/mL diaminopimelic acid (DAP). Z. mobilis ZM4 was obtained from the
American Type Culture Collection (ATCC31821) and the Z. mobilis acetate tolerant strain AcR has been described previously (Joachimsthal et al. 2000). ZM4 and AcR were cultured in RM medium at 30° C. S. cerevisiae wild-type, deletion mutant and GST-fusion ORF over-expression strains were obtained through Open Biosystems (Huntsville, Ala.). S. cerevisiae strains were cultured in rich YPD media. CM media with 2% glucose was used for S. cerevisiae wild-type and S. cerevisiae deletion mutants, CM media with 2% glucose minus uracil was used for S. cerevisiae GST-over expressing strains, 2% galactose was used to induce the GST-fusion strains. Plasmid-containing strains were routinely grown with antibiotics at the following concentrations (μg/mL): kanamycin of 50 for E. coli and 200 for ZM4; tetracycline, 10 for E. coli and 20 for ZM4; and gentamicin, 10 for E. coli. G418 of 200 for S. cerevisiae YKO deletion mutants. Growth was monitored turbidometrically by measuring optical density at 600nm periodically with the Bioscreen C automated microbiology growth curve analysis system (Growth Curve USA, Piscataway, N.J.).
PCR and DNA Manipulations
Genomic DNA from Z. mobilis was isolated using a Wizard Genomic DNA purification kit, following the manufacturer's instructions (Promega, Madison, Wis.). The QIAprep Spin Miniprep and HiSpeed Plasmid Midi kits (Qiagen, Valencia, Calif.) were used for plasmid isolation, respectively. PCR, restriction enzyme digestion, DNA ligation, DNA cloning, and DNA manipulations were done following standard molecular biology approaches (Sambrook 2000).
Construction of the Novel Tetracycline Resistant Gateway Entry Vector and ZMO0347 Over-Expression Plasmid
The construction of the broad-host-range, tetracycline resistant Gateway® compatible destination plasmid vector pBBR3DEST42 (FIG. 2A) was carried out essentially as described previously (Pelletier et al. 2008), except that pBBRMCS-3 tetracycline resistance cassette was used in this study instead of pBBRMCS-5 gentamicin resistance cassette used to construct pBBR3DEST42. Briefly, pBBR1MCS3 plasmid DNA was restricted with the KpnI and Pvul enzymes, treated with calf intestine alkaline phosphatase and purified using a Qiagen gel purification kit according to the manufacturer's instructions (Qiagen, Valencia, Calif.). The recombination region on pET-DEST42 vector DNA (Invitrogen, Carlsbad, Calif.) was PCR-amplified using the primers 42F and 42R that include KpnI and Pvul restriction sites as described previously (Pelletier et al. 2008). The gel-purified PCR product was ligated with pBBR1MCS3 KpnI/PvuI fragment with Fast-LinkTM DNA Ligation Kit (Epicentre, Madison, Wis.). Ligation products were transformed into E. coli DB3.1 chemically competent cells (Invitrogen, Carlsbad, Calif.) and the transformants were selected by plating on LB agar plates containing tetracycline. Individual colonies were grown overnight in LB containing 30 μg/mL chloramphenicol and 10 μg/mL tetracycline, and plasmid DNA was prepared using QIAprep spin miniprep or HiSpeed Plasmid Midi Kit following the manufacturer's protocol (Qiagen, Valencia, Calif.). Plasmid DNA was digested with KpnI and PvuI and digestion products were analyzed on an agarose gel to confirm the presence of products of the expected sizes.
The construction of entry vector and expression clone of target gene hfq (ZMO0347) was carried out as described previously (Pelletier et al. 2008). Briefly, target gene hfq (ZMO0347) was PCR amplified using AcR genomic DNA as template and primer hfq_CF and hfq_CR as primers. PCR products were then cloned into Gateway® entry clone pDONR221 using BP Clonase II enzyme mix following the manufacturer's protocol (Invitrogen, Carlsbad, Calif.), and then transformed into chemically competent DH5a cells (Invitrogen, Carlsbad, Calif.) and plated onto LB with appropriate antibiotic selection. The inserts were confirmed by sequencing using M13 forward and reverse primers (Integrated DNA Technologies, Inc., Coralville, Iowa). The confirmed entry clone vector was then recombined with the destination vector pBBR3DEST42 using LR Clonase II enzyme mix (Invitrogen Carlsbad, Calif.) to create the expression vector as described previously (Pelletier et al. 2008). The resulting expression vector construct was designed as p42-0347. The plasmid construct p42-0347 was confirmed by sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, Calif.).
Mutant Plasmid Construction
Briefly, a 262-bp hfq internal part PCR product was purified and cloned into pKnock-Km suicide vector (Alexeyev 1999) digested with XbaI and HindIII restriction enzymes followed by de-phosphorylation. The plasmid construct named as pKm-0347 was then sequenced to confirm the presence of the target gene fragment, which was then electroporated into E. coli WM3064 strain. The transformant E. coli WM3064 (pKm-0347) was verified by PCR and sequencing for the presence of correct plasmid construct pKm-0347. E. coli WM3064 (pKm-0347) was then conjugated with AcR. The conjugant of potential hfq mutant grown on RM plate with kanamycin concentration of 200 μg/mL and no DAP was selected based on PCR size shift by comparing the PCR size of wild-type AcR and conjugants using primer hfq_OCF and hfq_OCR (Table 2). Wild-type AcR has a 1050-bp PCR product and hfq mutant candidates have a 2.9-kb PCR product. The PCR product was sequenced for mutant confirmation.
The internal part of the Z. mobilis hfq gene (ZMO0347) was amplified by PCR using primers hfq_MF and hfq_MR supplied by MWG-Biotech (Huntsville, Ala.). The hfq gene and the primer positions used for mutant construction and an hfq gene-expressing vector are shown in FIG. 2B. The 262-bp hfq internal part PCR product was then purified and cloned into pCR2.1-TOPO and then transformed into E. coli TOPO one competent cell (Invitrogen, Carlsbad, Calif.). Transformants containing the correct construct were confirmed by PCR and sequencing. The plasmid was then extracted using Qiagen Midiprep and digested XbaI and HindIII restriction enzyme, the 262-bp hfq internal part was then purified by Qiagen Gel purification kit. Similarly, pKnock-Km suicide vector was also digested with XbaI and HindIII restriction enzyme followed by de-phosphorylation, and then ligated with 262-bp purified hfq internal part using Fast-LinkTM DNA Ligation Kit (Epicentre, Madison, Wis.). The ligation product (pKm-0347) was then transformed into TransforMax EC100D pir-116 Electrocompetent E. coli competent cells (Epicentre, Madison, Wis.) by electroporation. Transformants containing plasmid pKm-0347 were selected on LB agar plate with 50 μg/mL kanamycin. The plasmid was then extracted from the transformants, sequenced to confirm the presence of the target gene fragment, and was then electroporated into E. coli WM3064 strain. Transformants were verified by PCR and sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, Calif.) for the presence of the correct plasmid construct pKm-0347.
Plasmid Transformation of Z. mobilis 
Z. mobilis wild-type ZM4 and acetate tolerant strain AcR cultures were grown aerobically at 30° C. in RM, and E. coli WM3064 containing plasmid pKm-0347 or p42-0347 cultures were grown at 37° C. in LB containing 100 μg/mL DAP and 10 μg/mL tetracycline to exponential phase. E. coli WM3064 cells containing plasmid pKm-0347 or p42-0347 were washed with RM for three times by centrifugation at 13,000 rpm for 1 min and resuspended in RM. AcR cells were mixed with E. coli WM3064 (pKm-0347) cells in different ratios (1:3, 1:1, and 3:1). Similarly, ZM4 or AcR cells were mixed with E. coli WM3064 (p42-0347) cells in different ratios (1:3, 1:1, and 3:1). The mixtures of cells were plated onto RM agar plates with 100 μg/mL DAP and 10 μg/mL tetracycline for plasmid p42-0347 conjugation or 50 μg/mL kanamycin for plasmid pKm-0347 conjugation. The cells were incubated at 30° C. overnight. Conjugants were selected by plating on RM agar plates containing 20 μg/mL tetracycline for p42-0347 plasmid conjugants or 200 μg/mL kanamycin for pKm-0347plasmid conjugants at 30° C. The conjugants were confirmed for the presence of correct plasmid constructs by PCR and sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems Inc., Foster City, Calif.).







TABLE 2







Bacterial strains, plasmids and primers used in this application.









Strain,




plasmid, or
Genotype, phenotype, or



primer
sequence of primer (5′ to 3′)
Reference





E. coli




K-12
K-12 MG1655 Wild-type strain
Joachimstahl




et al. (1998)


 


DH5α
F− φ80dlacZΔM15 Δ(lacZYA-argF)
Novagen



U169 recA1 endA1 hsdR17(rk−, mk+)




phoA supE44 λ− thi-1 gyrA96 relA1



 


DB3.1
F− gyrA462 endA1Δ(sr1-recA) mcrB
Invitrogen



mrr hsdS20(rB−, mB−) supE44 ara-14




galK2 lacY1 proA2 rpsL20(SmR)




xyl-5λ- leu mtl1



 


WM3064

Denef et al.




(2006)


 


BL21(DE3)
F- ompT hsdSB(rB-mB-) gal dcm (DE3)
Invitrogen


 


Zymomonas




mobilis




ZM4
ATCC31821



 


AcR
ZM4 acetate tolerant strain
Joachimstahl



generated by random mutagenesis
et al. (1998)


 


ZM4(p42-0347)
ZM4 containing plasmid p42-0347
This




application


 


AcRIM0347
Insertional mutant of AcR gene
This



ZMO0347
application


 


AcRIM0347
AcRIM0347 containing plasmid p42-0347
This


(p42-0347)

application


 


S. cerevisiae




BY4741
MATa his3Δ1 leu2Δ0 ura3Δ0 met15Δ0-
Open



s288c background
Biosystems


 


YSC1021-
Yeast: Yeast Knock Out Strain, NHA1
Open


547768
Clone Id: 14095
Biosystems



Accession: YLR138W



 


YSC1021-
Yeast: Yeast Knock Out Strain, VNX1
Open


551633
Clone Id: 1123
Biosystems



Accession: YNL321W



 


YSC1021-
Yeast: Yeast Knock Out Strain, ARR3
Open


553567
Clone Id: 5616
Biosystems



Accession: YPR201W



 


YSC1021-
Yeast: Yeast Knock Out Strain, NHX1
Open


555633
Clone Id: 4290
Biosystems



Accession: YDR456W



 


YSC1021-
Yeast: Yeast Knock Out Strain, transporter
Open


551475
Clone Id: 610
Biosystems



Accession: YMR034C



 


YSC1021-
Yeast: Yeast Knock Out Strain, PSR1
Open


551268
Clone Id: 1498
Biosystems



Accession: YLL010C



 


YSC1021-
Yeast: Yeast Knock Out Strain, PSR2
Open


551318
Clone Id: 1574
Biosystems



Accession: YLR019W



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


555189
Clone Id: 2341
Biosystems



Accession: YIR005W



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


554440
Clone Id: 1301
Biosystems



Accession: YJL124C



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


552226
Clone Id: 4214
Biosystems



Accession: YDR378C



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


556031
Clone Id: 7383
Biosystems



Accession: YNL147W



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


552677
Clone Id: 3501
Biosystems



Accession: YCR020C-A



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


552563
Clone Id: 1949
Biosystems



Accession: YHR121W



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


552280
Clone Id: 255
Biosystems



Accession: YEL015W



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


553518
Clone Id: 5544
Biosystems



Accession: YPR129W



 


YSC1021-
Yeast: Yeast Knock Out Strain
Open


553919
Clone Id: 3618
Biosystems



Accession: YDR259C




YAP6



 


YSC4515-
Yeast GST-Tagged Strain
Open


98809240
Clone Id: YLR138W
Biosystems



Accession: YLR138W



 


YSC4515-
Yeast GST-Tagged Strain
Open


98810980
Clone Id: YLL010C
Biosystems



Accession: YLL010C



 


YSC4515-
Yeast GST-Tagged Strain
Open


98807049
Clone Id: YJL124C
Biosystems



Accession: YJL124C



 


YSC4515-
Yeast GST-Tagged Strain
Open


98805426
Clone Id: YCR020C-A
Biosystems



Accession: YCR020C-A



 


YSC4515-
Yeast GST-Tagged Strain
Open


98809076
Clone Id: YEL015W
Biosystems



Accession: YEL015W



 


YSC4515-
Yeast GST-Tagged Strain
Open


98808930
Clone Id: YPR129W
Biosystems



Accession: YPR129W



 


YSC4515-
Yeast GST-Tagged Strain
Open


98806813
Clone Id: YHR121W
Biosystems



Accession: YHR121W



 


YSC4515-
Yeast GST-Tagged Strain
Open


98811389
Clone Id: YDR378C
Biosystems



Accession: YDR378C



 


YSC4515-
Yeast GST-Tagged Strain
Open


98805850
Clone Id: YDR259C
Biosystems



Accession: YDR259C



 


Plasmids




pKNOCK-Km
Kmr, mob, broad host range
Alexeyev



cloning vector, 1.8 kb
(1999)


 


pET-DEST42
Apr, Cmr, C-terminal 6xHis and
Invitrogen


pBBR1MCS-3
V5 epitope Tcr, mob, broad host




range cloning vector



 


pBBR3DEST42
Cmr Tcr, C-terminal 6xHis and
This



V5 epitope
application


 


pDONR221
Kmr, gateway entry vector Gmr,
Invitrogen



N-terminal GST



 


F42-0347
pBBR3DEST42 containing ZM4 gene
This



ZMO0347
application


 


Primers




hfq_MF
cggagagatggtcagtcaca
262-bp



(SEQ ID NO: 51)



 


hfq_MR
ttcttgctgctgcataatcg




(SEQ ID NO: 52)



 


hfq_CF
atggccgaaaaggtcaacaa
483-bp



(SEQ ID NO: 53)



 


hfq_CR
tcaatcctcgtctcgccttt




(SEQ ID NO: 54)



 


hfq_OCF
caaagcttgagctcgaattcatttttgccgtggtagttgc
1050-bp



(SEQ ID NO: 55)



 


hfq_OCR
caggtacctctagaattcaccactcaatcctcgtctcg




(SEQ ID NO: 56)









Example 2
This example describes the results of the experiments showing that overexpression of the Zymomonas mobilis global regulator gene hfq confers enhanced tolerance to sodium acetate.
The Z. mobilis hfq gene (SEQ ID NO: 1) was cloned into the vector pBBR3-DEST42 (FIG. 2A) and the resulting plasmid construct p42-0347 was transformed into the wild-type strain ZM4 and acetate mutant AcR through conjugation. In addition, an insertional mutant of Z. mobilis strain AcR hfq gene (ZMO0347) was created using the pKNOCK system (Brown 2006; Alexeyev 1999) and complemented with plasmid p42-0347. The hfq gene and the primer positions used for mutant construction and hfq gene over-expression are shown in FIG. 2B. An insertional mutant of hfq (ZMO0347) was generated in the AcR background and designated as strain “AcRIM0347”. The hfq gene was over-expressed via plasmid p42-0347 in both wild-type ZM4 and the acetate mutant AcR backgrounds, their susceptibilities to sodium acetate and other stressors were tested in growth assays along with strains ZM4 and AcR. The AcR acetate tolerant mutant is more tolerant to sodium acetate than its wild-type ZM4 parental strain (Joachimstahl et al. 1998); however, the insertional inactivation of the hfq gene in AcR reduces its sodium acetate tolerance (FIGS. 3A-3C). These strains were tested for their growth responses in four different concentrations of sodium acetate: 0, 12 g/L, 16 g/L (195 mM) and 20 g/L (FIGS. 3A-3C).
The sum of these data show that hfq expression contributed to sodium acetate tolerance. The AcRIM0347 mutant strain grew slightly more slowly in RM medium compared to the parental strain, i.e., in the absence of the sodium acetate stressor (FIG. 3A). Strains ZM4 and AcR with intact hfq genes grew faster than the hfq mutant AcRIM0347 strain in the presence of 12 g/L sodium acetate (FIG. 3B). The AcRIM0347 mutant phenotype was mostly restored by hfq expression and complementation via plasmid p42-0347. Similar, but more dramatic growth phenotypes were observed for sodium acetate of 16 g/L with the wild-type strain unable to grow at this concentration (FIG. 3C).
Example 3
This example describes the experiments performed to compare the negative effects of pretreatment inhibitors on Z. mobilis growth, and to demonstrate that Hfq overexpression confers tolerance to pretreatment inhibitors.
Pretreatment Inhibitors had Negative Effects on Z. mobilis Growth
The growth of Z. mobilis strains was reduced in the presence of acetate, vanillin, furfural, or HMF with increased lag phases and/or slower growth rates and/or final bacterial cell densities depending on the respective condition and strain (Tables 3-4; FIGS. 4A-4E and 5A-5E). Among the different forms of acetate counter-ions tested, sodium acetate had the most significant inhibitory effect on wild-type Z. mobilis growth. This was followed by potassium acetate and ammonium acetate, and sodium chloride had the least negative influence on wild-type Z. mobilis growth (Table 3; FIGS. 4A-4E). Wild-type ZM4 growth was completely inhibited when RM medium was supplemented with 195 mM sodium acetate (Table 3; FIG. 4C). Among the pretreatment inhibitors of vanillin, furfural, and HMF, vanillin had the most significant inhibitory effect on Z. mobilis, while HMF had the least effect (Table 4). It took Z. mobilis a longer period of time to complete active growth and reach the stationary phase, which was about 16, 19 or 21 h in the presence of HMF, furfural or vanillin, respectively, as compared to 11 h without any inhibitor present in the medium (FIGS. 5A-5D).







TABLE 3







Growth rate and final cell density of different Z. mobilis strains


in the absence or presence of different sodium and acetate ions.
















AcRIM0347
ZM4



ZM4
AcR
AcRIM0347
(p42-0347)
(p42-0347)

















Growth
RM
0.42 ± 0.01 
0.39 ± 0.01 
0.32 ± 0.003
0.33 ± 0.002
0.38 ± 0.003


rate
RM (NaCl)
0.24 ± 0.008
0.29 ± 0.005
0.21 ± 0.008
0.22 ± 0.009
0.25 ± 0.008


(hour−1)
RM (NH4OAc)
0.20 ± 0.008
0.19 ± 0.005
NA
0.22 ± 0.002
0.19 ± 0.007



RM (Kac)
0.15 ± 0.004
0.12 ± 0.000
NA
0.09 ± 0.003
0.12 ± 0.006



RM (NaAc)
NA
0.29 ± 0.04 
0.12 ± 0.004
0.16 ± 0.002
0.27 ± 0.004


Final
RM
0.95 ± 0.006
1.01 ± 0.006
0.94 ± 0.004
0.92 ± 0.002
1.02 ± 0.004


Cell
RM (NaCl)
0.73 ± 0.01 
0.96 ± 0.01 
0.73 ± 0.03 
0.72 ± 0.02 
0.84 ± 0.01 


Density
RM (NH4OAc)
0.43 ± 0.01 
0.42 ± 0.006
NA
0.32 ± 0.007
0.37 ± 0.008


(OD600 nm)
RM (Kac)
0.42 ± 0.002
0.40 ± 0.000
NA
0.28 ± 0.007
0.34 ± 0.004



RM (NaAc)
NA
0.63 ± 0.02 
0.25 ± 0.001
0.45 ± 0.002
0.59 ± 0.002





“NA” indicates that the data are not available due to the lack of growth in that condition. The concentration for all the chemicals (NaCl, NH4OAc, KAc, NaAc) supplemented into the RM is 195 mM. NaCl: sodium chloride, NH4OAc: ammonium acetate, KAc: potassium acetate, NaAc: sodium acetate. Strains included in this study are: ZM4: Zymomonas mobilis ZM4 wild-type; AcR: ZM4 acetate tolerant mutant; ZM4 (p42-0347): ZM4 containing a gateway plasmid p42-0347 to express ZM4 gene ZMO0347; AcRIM0347: AcR insertional mutant of ZMO0347; AcRIM0347 (p42-0347): AcRIM0347 containing gateway plasmid p42-0347. This experiment has been repeated at least three times with similar result. Duplicate biological replicates were used for each condition.













TABLE 4







Growth rate and final cell density of different Z. mobilis strains


in the absence or presence of different pretreatment inhibitors.












ZM4
AcR
AcRIM0347
AcRIM0347(p42-0347)
















Growth
RM
0.48 ± 0.03 
0.46 ± 0.003
0.35 ± 0.004
0.32 ± 0.003


rate
HMF
0.36 ± 0.02 
0.35 ± 0.01 
0.19 ± 0.02 
0.22 ± 0.001


(hour−1)
Furfural
0.31 ± 0.01 
0.30 ± 0.005
0.19 ± 0.03 
0.20 ± 0.01 



Vanillin
0.26 ± 0.001
0.26 ± 0.01 
0.20 ± 0.006
0.20 ± 0.003


Final
RM
0.91 ± 0.01 
0.98 ± 0.006
0.95 ± 0.003
0.92 ± 0.006


Cell
HMF
0.93 ± 0.003
0.96 ± 0.006
0.67 ± 0.03 
0.78 ± 0.02 


Density
Furfural
0.88 ± 0.006
0.89 ± 0.009
0.67 ± 0.001
0.80 ± 0.02 


(OD600 nm)
Vanillin
0.69 ± 0.006
0.71 ± 0.01 
0.66 ± 0.01 
0.70 ± 0.01 





The concentration for the inhibitor supplemented into the RM is: HMF: 0.75 g/L, furfural, or vanillin: 1 g/L. Strains included in this study are: ZM4: Zymomonas mobilis ZM4 wild-type; AcR: ZM4 acetate tolerant mutant; AcRIM0347: AcR insertional mutant of ZMO0347; AcRIM0347 (p42-0347): AcRIM0347 containing gateway plasmid p42-0347. This experiment has been repeated at least three times with similar result. Duplicate biological replicates were used for each condition.






Hfq Contributes to Sodium and Acetate Ion Tolerances
Although the final cell density of hfq mutant AcRIM0347 was similar to that of AcR parental strain (Table 3; FIG. 5A), the growth rate of AcRIM0347 was reduced by about one-fifth even without any inhibitor in the RM, indicating that hfq plays a central role in normal Z. mobilis physiology. Wild-type ZM4 that contained p42-0347 was able to grow in the presence of 195 mM sodium acetate and had a similar growth rate and final cell density to that of acetate tolerant strain AcR (Table 3; FIG. 4C). The wild-type ZM4 was unable to grow under this condition.
The inactivation of the hfq gene in AcR decreased this acetate tolerant strain's resistance to both sodium ion (sodium chloride) and acetate ion (ammonium acetate and potassium acetate) (Table 3; FIGS. 4A-4E). hfq mutant AcRIM0347 was unable to grow in the presence of 195 mM ammonium acetate or potassium acetate (Table 3; FIGS. 4D-4E). Both the growth rate and final cell density of hfq mutant AcRIM0347 were reduced by at least a quarter in the presence of 195 mM sodium chloride, and about 60% in the presence of 195 mM sodium acetate compared to that of the parental strain AcR (Table 3; FIGS. 4B-4C). The AcRIM0347 hfq mutation was complemented by the introduction of an hfq-expres sing plasmid (p42-0347) into the strain. The complemented mutant strain recovered at least half of the parental strains growth rate and 70% of its final cell density in the presence of 195 mM acetate ion (whether as sodium, ammonium or potassium acetate) (Table 3; FIGS. 4A-4E).
Hfq Contributes to Vanillin, Furfural, HMF and H2O2 Tolerances
AcRIM0347 growth rates were lower than that of ZM4 and AcR under all conditions tests, except for growth in RM broth (Table 4; FIGS. 5A-5D). AcRIM0347 also achieved lower final cell densities compared to ZM4 and AcR (Table 4; FIGS. 5A-5D). When AcRIM0347 was provided functional Z. mobilis Hfq via p42-0347, growth rates under all conditions were largely unchanged (Table 4). However, shorter lag phases were observed for AcRIM0347 (p42-0347) grown with vanillin, furfural or HMF and increases in final cell densities were also observed under these conditions (Table 4; FIGS. 5A-5D). These data indicate that hfq is important for optimal Z. mobilis growth and its ability to resist furfural, HMF and vanillin toxicity.
Hfq also contributed to tolerance of other stress such as the reactive oxidative species generating hydrogen peroxide (H2O2). hfq mutant AcRIM0347 was sensitive to hydrogen peroxide H2O2 and no observable growth was detected in RM medium with 0.001% H2O2 (FIG. 5E). The wild-type strain ZM4 and acetate tolerant strain AcR grew well at this concentration. Complementation of the hfq mutant strain allowed strain AcRIM0347(p42-0347) to grow in RM medium with 0.001% H2O2.
Example 4
This Example describes experiments to show that Yeast Lsm proteins contribute to pretreatment inhibitor tolerance.
Lsm Protein and Yeast Tolerance to Sodium and Acetate Ions
S. cerevisiae Sm and Sm-like (Lsm) proteins are similar to Z. mobilis Hfq at the level of protein sequence. Growth of yeast Lsm deletion mutants and Lsm over-expressing strains in 305 mM ammonium acetate, potassium acetate, or sodium acetate was assessed to test whether S. cerevisiae Lsm proteins and ZM4 Hfq had functionally similar roles.
Deletion of seven Lsm genes affecting three Lsm heteroheptameric ring components (Lsm1, Lsm6, Lsm7) and four other Lsm proteins containing an Sm domain (Lsm9, Lsm12, Lsm13, Lsm16), was shown to have negative effects on the growth of S. cerevisiae in the presence of sodium acetate 40 g/L (FIG. 6A). On the other hand, six Lsm protein over-expressing S. cerevisiae strains (Lsm1, Lsm6, Lsm9, Lsm12, Lsm13, Lsm16) displayed enhanced growth in the presence of sodium acetate 40 g/L (FIG. 6B).
Growth differences between the Lsm mutants and yeast wild-type BY4741 in the CM broth without the addition of acetate or with 305 mM NaCl were not observed (FIGS. 7A-7B, respectively). S. cerevisiae Lsm proteins involved in RNA processing ring complex formation (Lsm1, 6, 7), especially Lsm6, played a role in acetate tolerance (FIGS. 7C-7E, 7K-7M). Lsm protein deletion mutants Lsm1, 6, and 7 showed decreased acetate tolerance compared to the wild-type control strain, especially in early growth stages for acetate with sodium, ammonium and potassium counter-ions (FIGS. 7C-7E). The Lsm overexpression strains grew similarly to wild-type BY4741 without the addition of acetate or with 305 mM NaCl (FIGS. 7I, 7J), but each of the Lsm protein overexpression strains showed enhanced acetate tolerance compared to the wild-type strain with sodium, ammonium or potassium counter-ions (FIGS. 7K-7M).
Lsm Proteins and Yeast Tolerance to Vanillin, Furfural and HMF
The effect of Lsm proteins on S. cerevisiae tolerance to pretreatment inhibitors vanillin, furfural, and HMF was also investigated using the seven Lsm deletion mutants and six Lsm overexpression strains described above. Each yeast deletion mutant and each overexpression strain showed similar growth profiles compared to wild-type strain BY4741 in the absence of inhibitors (FIGS. 7A; 7I). Deletion mutants for Lsm1, 6 and 7 proteins were unable to grow or showed extended lag phases before recovery from the inhibitory effects of pretreatment inhibitors (FIGS. 7F-7H). Overexpression of Lsm proteins provided a slight growth advantage in the presence of 1.5 g/L HMF and furfural (FIGS. 7O-7P). However, a detrimental effect on growth was observed for overexpression strains when cultured in the presence of 0.75 g/L vanillin (FIG. 7N). The data indicated that Lsm proteins Lsm1, 6, and 7 especially Lsm6, which are the components of yeast RNA processing ring complex, play a role in tolerance to the model inhibitors used in this study.
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## Claims

1. A method of producing alcohol from a cellulosic biomass material, comprising:
a. adding a genetically modified fungus to a fermentation mixture comprising a cellulosic biomass material or fermentation substrates derived from said cellulosic biomass material, wherein said genetic modification to said fungus comprises introduction of an expression vector comprising the coding sequence of a yeast protein of the Sm-like superfamily, and wherein said genetic modification results in elevated tolerance to at least one inhibitory chemical compound formed during biomass pretreatment as compared to without the genetic modification;
b. allowing said microorganism to ferment and produce alcohol; and
c. recovering alcohol produced by fermentation with said fungus.

2. The method of claim 1, wherein said fungus is a fungal species selected from Saccharomyces sp., Kluyveromyces sp., Pichia sp., Candida sp., and Schizosaccharomycetes sp.

3. The method of claim 2, wherein said fungal species is yeast selected from S. cerevisiae or P. pastoris.

4. The method of claim 3, wherein said yeast protein is a yeast Sm or Lsm protein.

5. The method of claim 4, wherein said protein comprises an amino acid sequence selected from any one of SEQ ID NOS: 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50, or a protein sequence that shares at least 95% sequence identity therewith.

6. The method of claim 1, wherein said expression vector is a replicative vector or an integrative vector.

7. The method of claim 1, wherein said at least one inhibitory chemical compound formed during biomass pretreatment is selected from the group consisting of an acetate salt, vanillin, furfural, hydroxymethylfurfural (HMF) and H2O2.

8. The method of claim 7, wherein said acetate salt is selected from the group consisting of sodium acetate, ammonium acetate and potassium acetate.

9. The method of claim 1, wherein said enhanced tolerance is characterized by ability to grow in a media containing sodium acetate at a concentration of 195 mM.