MICROORGANISMS RESISTANT TO NONVOLATILE SIDE PRODUCTS FROM ACID HYDROLYSATE OF LIGNOCELLULOSIC BIOMASS

CROSS-REFERENCE TO A RELATED APPLICATION This application claims the benefit of U.S. Provisional Application Serial No. 62/278,078, filed January 13, 2016, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

The Sequence Listing for this application is labeled "Seq-List.txt" which was created on December 29, 2016 and is 948 KB. The entire content of the sequence listing is incorporated herein by reference in its entirety.

This invention was made with government support under Grant No. 2011-10006- 30358 awarded by USD A/National Institutes of Food and Agriculture; Grant No. DE- PI0000031 awarded by U.S. Department of Energy; Grant No. 020650 awarded by Florida Department of Agriculture & Consumer Services; and Grant No. AGR DTD 01-28-2016 awarded by BASF. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

Sugars derived from lignocellulosic residues have the potential to serve as carbohydrate substrates for microbial fermentation into bio-based metabolites of interest with minimal impact on food and feed. However, conversion of lignocellulose and hydrolysis to sugar monomers require harsh treatments such as dilute mineral acids at elevated temperatures. Inhibitory side products that retard growth and fermentation are formed during a dilute acid pretreatment, such as furfural, soluble fragments from lignin, acetic acid, and other products formed during a dilute acid pretreatment. Furfural, a dehydration product of pentose sugars (primarily xylose), is the dominant inhibitor in dilute acid hydrolysates of lignocellulose. Removal of inhibitors after dilute acid pretreatment typically involves additional process steps such as fiber separation, counter-current washing and over-liming, all of which increase the production cost. Despite the progress with furfural resistance, little progress has been reported with other inhibitors in acid hydrolysate of lignocellulosic biomass. BRIEF SUMMARY OF THE INVENTION

Genetic engineering to produce microorganisms resistant to inhibitors in acid hydrolysate of lignocellulosic biomass provides a cost-effective approach for reducing production cost of biologically produced metabolites. Accordingly, the invention provides genetic modifications in a microorganism that increase the microorganism's resistance to the nonvolatile compounds in an acid hydrolysate of lignocellulosic biomass, for example, sugarcane bagasse or an artificial hydrolysate. Such microorganisms are hereinafter referred to as the "acid hydrolysate resistant microorganism".

The genes identified to be associated with the increased resistance of a microorganism to the nonvolatile compounds in the hydrolyzed lignocellulosic biomass are LysR family putative transcriptional regulator (yafC) and N-ethylmaleimide reductase (nemA) genes. Accordingly, the invention provides microorganisms that contain one or more genetic modifications to these genes and that become resistant to vacuum-treated lignocellulosic hydrolysate (for example, vacuum-treated sugarcane bagasse hemicellulose hydrolysate) and artificial hydrolysate.

In certain embodiments, the invention provides an acid hydrolysate resistant microorganism, wherein the microorganism is genetically modified to increase the expression of NemA or to express a mutant YafC (hereinafter, YafC*). The genetic modifications that increase the expression of NemA or cause the expression of YafC* include the expression of a protein of interest via a plasmid, a mutation in the genomic DNA of a microorganism which results in the expression of YafC*, a mutation in the regulatory region of nemA that causes the overexpression of NemA, a mutation in the regulatory region of YafC which causes reduction or elimination of the expression of yafC or inactivation of YafC expression by way mutations introduced into the coding region of the yafC gene. In certain embodiments, the > <; /( ^' gene or its native/endogenous promoter is mutated, deleted, interrupted, or down regulated in such a way as to decrease the activity of the gene. In certain embodiments, the activity of YafC can be eliminated by knockout or removal of the entire genomic DNA sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, deletions or insertions into the YafC coding region, and the like, can be used to inactivate or eliminate YafC activity by completely preventing transcription and/or translation of active YafC. YafC* refers to a mutant of YafC which is incapable of effectively regulating, particularly, inhibiting, the expression of proteins regulated by the wild-type YafC. For example, a single base mutation in the C-terminus of yafC coding region that produces D275G substitution in the wild-type YafC to produce YafC*, a mutated protein that is incapable of regulating its target genes or has reduced ability to regulate its target genes. This results in altered expression of the genes regulated by YafC.

As such, the subject invention provides microorganisms, for example, Escherichia coli, that are useful in the production of metabolites in a medium containing an acid hydrolysate of lignocellulosic biomass, such as, sugarcane bagasse, or an artificial hydrolysate. Accordingly, the materials and methods of the subject invention can be used to produce metabolites for use in a variety of applications.

In certain embodiments, derivatives of E. coli can be used for the construction of an acid hydrolysate resistant microorganism. In various embodiments, E. coli KOl l (ATCC ^® 55124™) can be used as can any other strain of E. coli that can be obtained from various depositories or commercial sources. The engineered microorganisms of the invention, in some embodiments, also contain only native genes {i.e., contain no genetic material from other organisms). Additional advantages of this invention will become readily apparent from the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C. Effect of vacuum evaporation on the toxicity of artificial hydrolysate (PX) containing 50 g liter ^"1 xylose and 10 g liter ^"1 phosphoric acid (2 h at 140°C). A. Toxicity to LY180 (parent) and SL100 (mutant selected for resistance to sugarcane bagasse hydrolysate) without vacuum treatment to remove volatiles; B. Toxicity after vacuum treatment. C. Example of screening for LY180 clones harboring pUC19 derivatives that express SL100 genes and confer resistance to 80% vol/vol vacuum -treated artificial hydrolysate (PXV) in AMI medium. Clones with highest ethanol production contained SL100 nemA genes. Clones with medium ethanol production contained SL100 yafC* genes.

Figures 2A-2B. Two chromosomal regions of SL100 with genes that increased resistance to vacuum-treated artificial hydrolysate (PXV). Cloned fragments are shown between double forward slashes, nemR ' to Ihr ' and dkgB ' to yaflJ '. Nucleotide mutations are shown as boxed capital letters. A. Larger fragment with nemA. Although the cloned fragment did not contain any mutations, the adjacent repressor gene {nemR) contained a mutation in the upstream regulatory region. This mutation was present in strain SL100 (SEQ ID NO: 22 with 372nd nucleotide as adenine) and absent in LY180 (SEQ ID NO: 22). The nemR mutation could affect expression of nemA and other downstream genes. B. Smaller fragment with yafC*. The smaller fragments contained a single nucleotide mutation (boxed capital letters) in the carboxyterminal region of this predicted LYSR-type regulator, denoted yafC*. This mutation was present in SL100 (SEQ ID NO: 26) but absent in LY180 (SEQ ID NO: 25). This mutation could affect the expression and function of many genes. Deletion of this gene has been shown to decrease survival after ionizing radiation (33).

Figures 3A-3D. Plasmid expression of cloned SL100 genes increased resistance of LY180 to hydrolysates. Expression vector pLOI5883 is included as a control (empty vector). Inducer was added as indicated. A. Ethanol production by LY180 constructs with 80% vol/vol PXV in AMI medium; B. Ethanol production by LY180 constructs with 20% vol/vol SCBHzV in AMI medium. C. Ethanol production by LY180 constructs with 10 mM furfural in AMI medium; D. Ethanol production by SL100 constructs with 35% vol/vol SCBHzV in AMI medium.

Figures 4A-4C. Effects of vacuum -treated hydrolysates on NemA activity. A and B. Vacuum-treated artificial hydrolysate (PXV) (Fig. 4A, open symbols with broken line, right axis) and vacuum-treated sugarcane bagasse hydrolysate (SCBHzV) (Fig. 4B, broken line with open symbols, right axis) can serve as substrates for NADPH-dependent reduction by NemA. Vacuum-treated hydrolysates (Fig. 4A and 4B; solid line with filled symbols, left axes) also inhibit N-ethylmaleimide reduction by NemA. C. Induction of NemA activity (NEM as the electron acceptor). Activity induced in LY180 and SL100 during growth in AMI (control), by vacuum-treated PX and by vacuum-treated SCBHz (filled bars, left axis). IPTG-induced NemA activity in LY180 harboring pLOI5908 (open bar, right axis) is also included for comparison.

Figures 5A-5D. Plasmid expression of nemA and yafC* increase resistance to 20% vol/vol SCBHzV in AMI medium during pH-controlled fermentation. Glucose and xylose were added to adjust sugar concentration to 100 g liter ^"1 . An empty vector control (pLOI5883) in AMI with hydrolysate and without hydrolysate (labeled AMI) are included as controls. Fermentations were sampled for ethanol at 24-h intervals. A. Ethanol; B. Total sugars; C. Xylose; and D. Glucose.

Figure 6. Genes with ribosomal binding sites were inserted into the polylinker sites (EcoRI->HindIII) for expression from the Ptrc promoter. Figure 7. Isolation of the acid hydrolysate resistant clones.

Figure 8. Cells were washed to remove soluble color before measuring optical density. Some particulate color remains. Note that shapes of curves are similar for optical density and for ethanol production.

Figure 9. Description of various plasmid containing portions of the E. coli genome as compared to the genome of the reference E. coli W, GenBank: CP002967.1, ATCC 9637.

Figure 10. No mutation was found in the coding region of nemR in SL100 as compared to parent LY180. However, a mutation in upstream regulatory region of nemR was found which causes an increase in expression of nemA. Guanine present at 19 bp upstream from the translational start codon of nemR in LY180 is replaced with adenine in SL100, as indicated by red box around the mutated adenine (SEQ ID NO: 27).

Figure 11. Nucleotide and amino acid sequences (SEQ ID NOs: 26 and 24) of YafC*. Character in the square is the mutation. The mutation is at the position 824 of the sequence identified in the Figure. The nucleotide is adenine in LY180 (parental microorganism) and is guanine in SL100 (an example of the modified microorganism of the invention). The amino acid corresponding to the mutated codon at position 275 is D (aspartic acid) in LY180 and is G (glycine) in SL100.

Figure 12. nemA and NemA sequences (SEQ ID NOs: 20 and 21) in LY180 and SL100. No mutations in coding region or regulatory region were observed in SL100 compared to LY180.

BRIEF DESCRIPTION OF THE SEQUENCES

Description Sequence SEQ ID

NO:

nemA AGTGAATTCAAGGAGATATACCATGTCATCTGAAAA 1 forward ACTGTATTCCCC

primer

nemA AGT A AGC TTTT AC A AC GTC GGGT A ATC GGT AT 2 reverse

primer

gloA AGTGAATTCAAGGAGATATACCATGCGTCTTCTTCA 3 forward TACCATGC

primer

gloA reverse AGTAAGCTTTTAGTTGCCCAGACCGCG 4 primer

rnt forward AGTGAATTCAAGGAGATATACCATGTCCGATAACGC 5 primer TCAACTTAC rnt reverse AGTAAGCTTATTACACCTCTTCGGCGGC 6 primer

nemA-gloA- AGTGAATTCAAGGAGATATACCATGTCATCTGAAAA 7 rnt forward ACTGTATTCCCC

primer

nemA-gloA- AGTAAGCTTATTACACCTCTTCGGCGGC 8 rnt reverse

primer

nemRA CATTAACGGGTCTGGTCGGT 9 forward

primer

Upstream GCAGAAATTTGCGTGGCTT 10 reverse

primer

M13 GT A A A AC GACGGC C AG 11 forward

primer

Ml 3 reverse CAGGAAACAGCTATGAC 12 primer

nemA AATGTGGTGTCCGGCATCA 13 sequencing

forward

primer

nemA CGCACGTCTGGTACTGGAA 14 sequencing

reverse

primer

yaj & AGTGAATTCAAGGAGATATACCATGAAAGCCACGTC 15 yafC* GGAAG

forward

primer

yaj & AGTAAGCTTTTAAGCCTCTCTGACAGCTCC 16 yafC*

reverse

primer

99 A GCAGGTCGTAAATCACTGC 17 forward

sequencing

primer

99 A CTTCTCTCATCCGCCAAAAC 18 reverse

sequencing

primer

nemA-yafC* GGATGGAAGCTTGGCTGTTTTGGCGG 19

SEQ ID NO: 20: The nucleotide sequence of the coding region of nemA gene from ATGTCATCTGAAAAACTGTATTCCCCACTGAAAGTGGGCGCGATCACGGCGGCA AACCGTATTTTTATGGCACCGCTGACGCGTCTGCGCAGTATTGAACCGGGTGACA TTCCTACCCCGTTGATGGCGGAATACTATCGCCAACGTGCCAGTGCCGGTTTGAT TATTAGTGAAGCCACGCAAATTTCTGCCCAGGCAAAAGGATATGCAGGTGCGCC TGGCATCCATAGTCCGGAGCAAATTGCCGCATGGAAAAAAATCACCGCTGGCGT TCATGCTGAAAATGGTCATATGGCCGTGCAGCTGTGGCACACCGGACGCATTTCT CACGCCAGCCTGCAACCTGGCGGTCAGGCACCGGTAGCGCCTTCAGCACTTAGC GCGGGAACACGTACTTCTCTGCGCGATGAAAATGGTCAGGCGATCCGTGTTGAA ACATCCATGCCGCGTGCGCTTGAACTGGAAGAGATTCCAGGTATCGTCAATGATT TCCGTC AGGCC ATTGCT AACGCGCGTGAAGCCGGTTTTGATCTGGTAGAGCTCC A CTCTGCTCACGGTTATTTGCTGCATCAGTTCCTTTCTCCTTCTTCAAACCATCGTA CCGATCAGTACGGCGGCAGCGTGGAAAATCGCGCACGTCTGGTACTGGAAGTGG TCGATGCCGGGATTGAAGAATGGGGTGCCGATCGCATTGGCATTCGCATTTCGCC AATCGGTACTTTCCAGAACACGGATAACGGCCCGAATGAAGAAGCCGATGCACT GTATCTGATTGAAC AACTGGGTAAACGCGGC ATTGCTTATCTGCATATGTCAGAA CCAGATTGGGCGGGGGGTGAACCGTATACTGATGCGTTCCGCGAAAAAGTACGC GCCCGTTTCCACGGTCCGATTATCGGCGCAGGTGCATACACAGTAGAAAAAGCT GAAACGCTGATCGGCAAAGGGTTAATTGATGCGGTGGCATTTGGTCGTGACTGG ATTGCGAACCCGGATCTGGTCGCCCGCTTGCAGCGCAAAGCTGAGCTTAACCCAC AGCGTGCCGAAAGTTTCTACGGTGGCGGCGCGGAAGGCTATACCGATTACCCGA CGTTGTAA

SEQ ID NO: 21: The amino acid sequence of NemA protein from E. coli.

MSSEKLYSPLKVGAITAA RIFMAPLTRLRSIEPGDIPTPLMAEYYRQRASAGLIISEA TQISAQAKGYAGAPGIHSPEQIAAWKKITAGVHAENGHMAVQLWHTGRISHASLQP GGQ AP VAP S ALS AGTRT SLRDENGQ AIRVET SMPRALELEEIPGI V DFRQ AI ANARE AGFDL VELHS AHGYLLHQFL SP S S TRTDQ YGGS VE RARL VLEVVD AGIEEWGAD RIGIRISPIGTFQNTDNGP EEADALYLIEQLGKRGIAYLHMSEPDWAGGEPYTDAFR EKVRARFHGPIIGAGAYTVEKAETLIGKGLIDAVAFGRDWIA PDLVARLQRKAELN PQRAESFYGGGAEGYTDYPTL

SEQ ID NO: 22: The nucleotide sequence of the regulatory region of nemR operon.

The 372 ^nd nucleotide (guanine) is underlined and bolded. The mutation of the guanine in the

372 ^nd position to adenine results in an increased expression of NemA. The underlined portion is the protein coding portion of nemR gene.

AATCGCTTCCTCTTATCAGATATGAGAGGAGTATACGCAAGATTAGGTTCAAAAG AGTGATGGTTGCTCCGGTTCGTCTGATGACGCTGGCTTATTTGCGCGTAATTTGCG CATTAATCGCTGCCGACAAAGGCGCAGCACCTCTTGTTTTTCGCCATCGCTCATTT TATTCCAGTTAAAACGCTCATCCCGACTACGAAAACAGCCGCGACAAAACCCGC GTTCGTCAGACTGGCAAATTCCCCGGCACGGGCTCTGGACGGGAAAGAACTCTA ATTGCTCCGCCACTTCGCCCTCCTCAGATAAGATTATTACCATTATTGAAGCTGTT AATGTCCAAAGTAGCAACTTTGCTTGCACTAGACCGACTGGTCTACTACACTCCA ACGCATGAACAAACACACCGAACATGATACTCGCGAACATCTCCTGGCGACGGG CGAGCAACTTTGCCTGCAACGTGGATTCACCGGGATGGGGCTAAGCGAATTACT

AAAAACCGCTGAAGTGCCGAAAGGGTCCTTCTATCACTACTTTCGCTCTAAAGAA GCGTTTGGCGTTGCCATGCTTGAGCGTCATTACGCCGCATATCACCAGCGACTGA

CTGAGTTGCTGCAATCCGGCGAAGGTAACTACCGCGACCGCATACTGGCTTATTA

CCAGCAAACACTGAACCAGTTTTGCCAACATGGAACCATCAGTGGTTGCCTGACA

GTAAAACTCTCTGCCGAAGTGTGCGATCTGTCAGAAGATATGCGCAGCGCGATG

GATAAAGGTGCTCGCGGCGTGATCGCCCTGCTCTCTCAGGCGCTGGAAAATGGCC

GTGAGAACCATTGTTTAACCTTTTGTGGCGAACCGCTACAACAGGCACAAGTGCT

TTACGCACTATGGCTGGGTGCGAATCTGCAGGCCAAAATTTCGCGCAGTTTCGAG

CCACTGGAAAACGCGCTGGCCCATGTAAAAAACATTATTGCGACGCCTGCCGTTT

AG

SEQ ID NO: 23: The amino acid sequence of a wild-type YafC protein.

MKATSEELAIFVSVVESGSFSRAAEQLGQANSAVSRAVKKLEMKLGVSLLNRTTRQL SLTEEGERYFRRVQSILQEMAAAESEIMETRNTPRGLLRIDAATPVVLHFLMPLIKPFR ERYPEVTLSLVSSETIINLIERKVDVAIRAGTLTDSSLRARPLFNSYRKIIASPDYISRY G KPETIDDLKQHVCLGFTEP ASLNTWPIAC SDGQLHEVK YGL S SNS GETLKQLCL SGN GI ACL SD YMIDKEIARGEL VELMADK VLP VAMPF S AVYYSDRAVSTRIRAFIDFL SEH VKTAPGGAVREA

SEQ ID NO: 24: The amino acid sequence of a YafC* protein.

MKATSEELAIFVSVVESGSFSRAAEQLGQANSAVSRAVKKLEMKLGVSLLNRTTRQL SLTEEGERYFRRVQSILQEMAAAESEIMETRNTPRGLLRIDAATPVVLHFLMPLIKPFR ERYPEVTLSLVSSETIINLIERKVDVAIRAGTLTDSSLRARPLFNSYRKIIASPDYISRY G KPETIDDLKQHVCLGFTEP ASLNTWPIAC SDGQLHEVK YGL S SNS GETLKQLCL SGN GI ACL SD YMIDKEIARGEL VELMADK VLP VAMPF S A VYY S GRA VS TRIR AFIDFL SEH VKTAPGGAVREA

SEQ ID NO: 25: The nucleotide sequence of a wild-type yafC gene.

ATGAAAGCCACGTCGGAAGAACTCGCCATTTTTGTTTCGGTCGTCGAAAGCGGCA GCTTTAGCCGGGCAGCGGAACAATTAGGGCAAGCAAACTCAGCGGTAAGCCGGG C AGTGAAAAAGCTGGAGATGAAACTTGGCGTT AGCCTGCTTAATCGGACC ACGC GACAACTTAGCCTGACGGAAGAAGGCGAGCGTTATTTTCGTCGCGTACAGTCAAT TTTGCAGGAGATGGCAGCGGCAGAATCAGAAATTATGGAGACGCGTAATACACC GCGTGGACTGTTGCGCATCGATGCCGCAACTCCAGTGGTGCTGCACTTTCTGATG CCGTTGATTAAGCCTTTCCGTGAACGCTATCCGGAAGTCACTTTGTCGCTAGTCTC CTCCGAAACGATTATT AATTTGATCGAAAGAAAAGTTGATGTCGCGAT ACGCGCC GGTACGTTAACGGATTCCAGCTTACGTGCCAGGCCGTTATTTAATAGTTATCGAA AAATTATCGCCTCCCCCGATTATATTTCCCGCTACGGGAAGCCAGAAACGATCGA CGATTTAAAGCAACATGTTTGCCTGGGATTCACTGAACCCGCTTCCCTCAATACC TGGCCGATAGCCTGTAGTGATGGACAATTACATGAGGTGAAGTACGGTTTGTCAT CCAATAGTGGGGAAACACTGAAACAGCTTTGCCTGAGTGGCAACGGGATTGCGT GTTTGTCCGACTACATGATCGACAAAGAAATCGCTCGCGGAGAATTGGTGGAGTT AATGGCAGATAAAGTGTTGCCAGTGGCAATGCCATTCAGTGCAGTCTATTACAGC GACCGTGCGGTAAGTACGCGCATCCGGGCTTTTATCGATTTCCTTAGCGAGCATG TAAAAACAGCTCCCGGAGGAGCTGTCAGAGAGGCTTAA SEQ ID NO: 26: The nucleotide sequence of a mutant yafC gene encoding for a YafC* protein.

ATGAAAGCCACGTCGGAAGAACTCGCCATTTTTGTTTCGGTCGTCGAAAGCGGCA GCTTTAGCCGGGCAGCGGAACAATTAGGGCAAGCAAACTCAGCGGTAAGCCGGG CAGTGAAAAAGCTGGAGATGAAACTTGGCGTTAGCCTGCTTAATCGGACCACGC GACAACTTAGCCTGACGGAAGAAGGCGAGCGTTATTTTCGTCGCGTACAGTCAAT TTTGCAGGAGATGGCAGCGGCAGAATCAGAAATTATGGAGACGCGTAATACACC GCGTGGACTGTTGCGCATCGATGCCGCAACTCCAGTGGTGCTGCACTTTCTGATG CCGTTGATTAAGCCTTTCCGTGAACGCTATCCGGAAGTCACTTTGTCGCTAGTCTC CTCCGAAACGATTATT AATTTGATCGAAAGAAAAGTTGATGTCGCGAT ACGCGCC GGTACGTTAACGGATTCCAGCTTACGTGCCAGGCCGTTATTTAATAGTTATCGAA AAATTATCGCCTCCCCCGATTATATTTCCCGCTACGGGAAGCCAGAAACGATCGA CGATTTAAAGCAACATGTTTGCCTGGGATTCACTGAACCCGCTTCCCTCAATACC TGGCCGATAGCCTGTAGTGATGGACAATTACATGAGGTGAAGTACGGTTTGTCAT CC AATAGTGGGGAAACACTGAAAC AGCTTTGCCTGAGTGGC AACGGGATTGCGT GTTTGTCCGACTACATGATCGACAAAGAAATCGCTCGCGGAGAATTGGTGGAGTT AATGGCAGATAAAGTGTTGCCAGTGGCAATGCCATTCAGTGCAGTCTATTACAGC GGCCGTGCGGTAAGTACGCGCATCCGGGCTTTTATCGATTTCCTTAGCGAGCATG TAAAAACAGCTCCCGGAGGAGCTGTCAGAGAGGCTTAA

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising". The transitional terms/phrases (and any grammatical variations thereof) "comprising", "comprises", "comprise", "consisting essentially of, "consists essentially of, "consisting" and "consists" can be used interchangeably.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system and on the parameter being measured. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 0-20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed. In the context of compositions containing amounts of ingredients where the terms "about" or "approximately" are used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0-10% around the value (X±10%).

In the present disclosure, ranges are stated in shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. For example, a range of 0.1 -1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1- 1 .0, such as 0.2-0.5, 0.2-0.8, 0.7-1 .0, etc. Values having at least two significant digits within a range are envisioned, for example, a range of 5-10 indicates all the values between 5.0 and 10.0 as well as between 5.00 and 10.00 including the terminal values. Also, when ranges are used herein, combinations and subcombinations of ranges (e.g., subranges within the disclosed range), and the specific embodiments therein, are included.

For the purpose of this invention the term "artificial hydrolysate" refers to the hydrolysates produced by heating xylose and mineral acids to provide a simpler mixture of inhibitors of microbial, particularly, bacterial growth. Furfural is the most abundant product and inhibitor in an artificial hydrolysate. Furfural also increased the toxicity of other inhibitors in binary mixtures. Additional products in the artificial hydrolysate include glycolaldehyde, formate, lactate, acetate, lactaldehyde, phenolics and pseudo-lignin. Vacuum evaporation has been shown to remove furfural from hemicellulose hydrolysates and to reduce but not eliminate toxicity. Toxic nonvolatile compounds remained after furfural evaporation. Full toxicity was regained by restoration of furfural.

For the purposes of the invention, an "acid hydrolysate resistant microorganism" refers to a microorganism that is resistant to the nonvolatile compounds in an acid hydrolysate of lignocellulosic biomass (for example, sugarcane bagasse) or an artificial hydrolysate.

Accordingly, the term "acid hydrolysate" as used herein refers to an acid hydrolysate of lignocellulosic biomass or an artificial hydrolysate as described above.

For the purposes of the invention, the term "metabolite produced by a microorganism" refers to a metabolite of commercial interest. Metabolites that can be produced from the microorganisms of the invention and commercially used depend on the parent microorganism genetically modified to produce the microorganism of the invention. For example, a microorganism producing ethanol can be genetically modified according to the invention to produce an acid hydrolysate resistant microorganism capable producing ethanol. The metabolites envisioned to be produced by the microorganism of interest include ethanol, succinate, malate, lactate, acetate and formate. Additional examples of metabolites that can be produced by a microorganism are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

For the purposes of this invention, the phrase "parental microorganism" or a synonymous phrase refers to the microorganism to which the genetic modifications according to the invention are performed to produce the acid hydrolysate resistant microorganism of the invention. Accordingly, the characteristics of an acid hydrolysate resistant microorganism of the invention are represented in relation to the parental microorganism. For example, a parental microorganism may exhibit a certain level of resistance to the acid hydrolysate; however, when genetically modified according to the invention, the resultant acid hydrolysate resistant microorganism exhibits higher resistance to the acid hydrolysate compared to the parental microorganism.

Accordingly, if the wild-type E. coli is genetically modified according to the invention to produce an acid hydrolysate resistant E. coli, the wild-type E. coli is the parental microorganism of the resultant acid hydrolysate resistant microorganism. Similarly, if an E. coli strain containing an initial genetic modification is further genetically modified according to the invention to produce an acid hydrolysate resistant strain of E. coli, the E. coli strain containing the initial genetic modification is the parental strain of the resultant acid hydrolysate resistant E. coli.

The term "microorganism" used herein refers to organisms recognized in the art as "microorganisms". Microorganisms contemplated in the invention include bacteria, filamentous fungi and yeast. Additional examples of microorganism that can be used according to the invention are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

A "native gene" or "an endogenous gene" is a gene that is naturally found in a host microorganism; whereas, an "exogenous gene" is a gene introduced into a host microorganism and which was obtained from a microorganism other the host microorganism. Likewise, a "native promoter" or "endogenous promoter" is a promoter that is naturally found in a host microorganism. In contrast, "exogenous promoter" or "heterologous promoter" is a promoter introduced into a host microorganism via a genetic construct and which was obtained from a microorganism different from host microorganism.

The non-italicized abbreviations as used herein refer to the corresponding protein; whereas italicized abbreviations used herein refer to the corresponding gene. For example, the term "NemA" refers to NemA protein and the term "nemA" refers to the gene encoding the NemA protein.

The subject invention provides materials and methods wherein unique and advantageous genetic modifications are used produce an acid hydrolysate resistant microorganism. The microorganisms and techniques of the subject invention can be used to obtain metabolites from native pathways as well as from recombinant pathways. Advantageously, the subject invention provides a versatile platform for the production of the metabolites by from a medium containing an acid hydrolysate.

Acid hydrolysate-resistant Escherichia coli SL100 was isolated from ethanologenic LY180 (a derivative of KOI 1 that ferments well on AMI mineral salts medium containing sugar) after sequential transfers in AMI containing dilute acid hydrolysate of sugarcane bagasse. A chromosomal library of this strain was used as a source of resistance genes. Many genes have been previously described that affect tolerance to furfural, the most abundant inhibitor of bacterial growth in lignocellulosic biomass. To identify genes associated with the resistance to inhibitors other than furfural, plasmid clones were selected in artificial hydrolysate that had been treated with vacuum to remove furfural. Two new resistance genes were discovered from Sau3A-libraries of SL100 genomic DNA: nemA (the gene encoding N-ethylmaleimide reductase) and a yafC* (the gene encoding an LysR family putative transcriptional regulator containing a mutation in the coding region).

A single mutation in the upstream regulatory region of nemR operon (nemR-nemA- gloA operon), was also found in SL100. This mutation increased nemA activity 20-fold over that of the parent organism (LY180) in AMI medium without hydrolysate and increased nemA mRNA by over 200-fold. Addition of hydrolysates induced nemA expression (mRNA and activity), consistent with transcriptional control. NemA activity was stable in cell free extracts (9 h, 37°C), eliminating a role for proteinase in regulation.

Further, LY180 with a plasmid expressing nemA or yafC* was more resistant to vacuum-treated sugarcane bagasse hydrolysate and to vacuum-treated artificial hydrolysate than LY180 (empty vector control). Neither gene affected furfural tolerance. Addition of vacuum -treated hydrolysate inhibited the reduction of N-ethylmaleimide by NemA while also serving as substrate. The expression of either the nemA plasmid or the yafC* plasmid in LY180 doubled the rate of ethanol production and sugar utilization from vacuum-treated sugarcane bagasse hydrolysate medium.

Accordingly, the invention provides a microorganism comprising one or more genetic modifications selected from:

i) a genetic modification resulting in an increased expression of NemA or an increased catalytic activity of NemA, and

ii) a genetic modification resulting in the expression of a nemR.

The one or more genetic modifications according to the invention produce a microorganism that exhibits, compared to the parental microorganism modified to produce the microorganism, an increased resistance to nonvolatile products present in an acid hydrolysate of lignocellulosic biomass or an artificial hydrolysate.

Non-limiting examples of NemA that can be used in the invention are provided by proteins identified by the UniProt entries A0A011NRS1 (SEQ ID NO: 28), A0A023XJZ2 (SEQ ID NO: 29), A0A024H6B9 (SEQ ID NO: 30), A0A024HB15 (SEQ ID NO: 31), A0A059KHF8 (SEQ ID NO: 32), A0A060Q203 (SEQ ID NO: 33), A0A061CU01 (SEQ ID NO: 34), A0A061MPM4 (SEQ ID NO: 35), A0A066PLS0 (SEQ ID NO: 36), A0A068QVH3 (SEQ ID NO: 37), A0A077SB63 (SEQ ID NO: 38), A0A078BPM3 (SEQ ID NO: 39), A0A078KXX5 (SEQ ID NO: 40), A0A078MSX1 (SEQ ID NO: 41), A0A083ZY79 (SEQ ID NO: 42), A0A085AL03 (SEQ ID NO: 43), A0A085F478 (SEQ ID NO: 44), A0A085FYS5 (SEQ ID NO: 45), A0A085GCC0 (SEQ ID NO: 46), A0A085HS73 (SEQ ID NO: 47), A0A085JE30 (SEQ ID NO: 48), A0A090K266 (SEQ ID NO: 49), A0A090K3U6 (SEQ ID NO: 50), A0A090TVL7 (SEQ ID NO: 51), A0A090U8C0 (SEQ ID NO: 52), A0A098BJ69 (SEQ ID NO: 53), A0A098FY07 (SEQ ID NO: 54), A0A098G2L4 (SEQ ID NO: 55), A0A098GBU0 (SEQ ID NO: 56), A0A099IMP8 (SEQ ID NO: 57), A0A0A0XHF2 (SEQ ID NO: 58), A0A0A1ME46 (SEQ ID NO: 59), A0A0A1PL56 (SEQ ID NO: 60), A0A0A1 S5C5 (SEQ ID NO: 61), A0A0A7PHP2 (SEQ ID NO: 62), A0A0A8UZ37 (SEQ ID NO: 63), A0A0B5BVW9 (SEQ ID NO: 64), A0A0B5F341 (SEQ ID NO: 65), A0A0B6F1V2 (SEQ ID NO: 66), A0A0B6FIJ7 (SEQ ID NO: 67), A0A0B6S2X0 (SEQ ID NO: 68), A0A0B7D4B5 (SEQ ID NO: 69), A0A0B7GCC5 (SEQ ID NO: 70), A0A0B7IV26 (SEQ ID NO: 71), A0A0B7JIX1 (SEQ ID NO: 72), A0A0B8YL12 (SEQ ID NO: 73), A0A0C6FGQ1 (SEQ ID NO: 74), A0A0C6P697 (SEQ ID NO: 75), A0A0D1CZ50 (SEQ ID NO: 76), A0A0D6HEU5 (SEQ ID NO: 77), A0A0D6J9U8 (SEQ ID NO: 78), A0A0E3KKB2 (SEQ ID NO: 79), A0A0E8TFR8 (SEQ ID NO: 80), A0A0E9FZM2 (SEQ ID NO: 81), A0A0F0KJP6 (SEQ ID NO: 82), A0A0F0L2T2 (SEQ ID NO: 83), A0A0F0L8S9 (SEQ ID NO: 84), A0A0F0LVR7 (SEQ ID NO: 85), A0A0F7DIH6 (SEQ ID NO: 86), A0A0F7KSF0 (SEQ ID NO: 87), A0A0F7RFL8 (SEQ ID NO: 88), A0A0F8WEK9 (SEQ ID NO: 89), A0A0G3BWF1 (SEQ ID NO: 90), A0A0G4Q4E4 (SEQ ID NO: 91), A0A0H2M751 (SEQ ID NO: 92), A0A0H3CXL1 (SEQ ID NO: 93), A0A0H3NGC2 (SEQ ID NO: 94), A0A0H4RCW4 (SEQ ID NO: 95), A0A0H4W8Z5 (SEQ ID NO: 96), A0A0I1BVT1 (SEQ ID NO: 97), A0A0J6D3M4 (SEQ ID NO: 98), A0A0J6VE38 (SEQ ID NO: 99), A0A0J6VRV4 (SEQ ID NO: 100), A0A0J6VZI4 (SEQ ID NO: 101), A0A0K0HAF4 (SEQ ID NO: 102), A0A0K2AT60 (SEQ ID NO: 103), A0A0K2G676 (SEQ ID NO: 104), A0A0K2ZEW2 (SEQ ID NO: 105), A0A0K3A6N9 (SEQ ID NO: 106), A0A0K6KZF9 (SEQ ID NO: 107), A0A0K6MJW9 (SEQ ID NO: 108), A0A0M0EDF9 (SEQ ID NO: 109), A0A0M2WF54 (SEQ ID NO: 110), A0A0M3SX02 (SEQ ID NO: 111), A0A0M6XPZ7 (SEQ ID NO: 112), A0A0M6XY07 (SEQ ID NO: 113), A0A0M6YXS4 (SEQ ID NO: 114), A0A0M7A4Y0 (SEQ ID NO: 115), A0A0N0V3R9 (SEQ ID NO: 116), A0A0N0XN16 (SEQ ID NO: 117), A0A0N1QSX7 (SEQ ID NO: 118), A0A0N1RWJ6 (SEQ ID NO: 119), A0A0N1 SJL9 (SEQ ID NO: 120), A0LXD5 (SEQ ID NO: 121), A2RXL6 (SEQ ID NO: 122), A4G514 (SEQ ID NO: 123), A4SNR4 (SEQ ID NO: 124), A9HQC9 (SEQ ID NO: 125), A9HXG7 (SEQ ID NO: 126), B0V401 (SEQ ID NO: 127), B1H5L9 (SEQ ID NO: 128), B2VEP7 (SEQ ID NO: 129), B3DZE9 (SEQ ID NO: 130), B3X5N2 (SEQ ID NO: 131), B5RAM3 (SEQ ID NO: 132), B5Y655 (SEQ ID NO: 133), B6ESH8 (SEQ ID NO: 134), B7LQM1 (SEQ ID NO: 135), C0Q5U6 (SEQ ID NO: 136), C0QL35 (SEQ ID NO: 137), C3LWQ6 (SEQ ID NO: 138), C5S1W7 (SEQ ID NO: 139), C8TAR3 (SEQ ID NO: 140), C9Y3L1 (SEQ ID NO: 141), C9Y514 (SEQ ID NO: 142), D0WZT4 (SEQ ID NO: 143), D0XC00 (SEQ ID NO: 144), D2TBI7 (SEQ ID NO: 145), D2THI8 (SEQ ID NO: 146), D2U9N4 (SEQ ID NO: 147), D2YAI4 (SEQ ID NO: 148), D3HJE2 (SEQ ID NO: 149), D3NSB8 (SEQ ID NO: 150), D3VFQ0 (SEQ ID NO: 151), D4E6W9 (SEQ ID NO: 152), D4QMG4 (SEQ ID NO: 153), D4XE22 (SEQ ID NO: 154), D5ATQ6(SEQ ID NO: 155), D6CSN5 (SEQ ID NO: 156), D6YQE2 (SEQ ID NO: 157), D8MRJ5 (SEQ ID NO: 158), E0MZ62 (SEQ ID NO: 159), E3DF79 (SEQ ID NO: 160), E5B513 (SEQ ID NO: 161), E5UH84 (SEQ ID NO: 162), E6PPT9 (SEQ ID NO: 163), F5RSA1 (SEQ ID NO: 164), F7U7W5 (SEQ ID NO: 165), F8GVS1 (SEQ ID NO: 166), F8JEY0 (SEQ ID NO: 167), G2ZJI2 (SEQ ID NO: 168), G2ZZL3 (SEQ ID NO: 169), G4CL50 (SEQ ID NO: 170), G4SY97 (SEQ ID NO: 171), G7Z8Z1 (SEQ ID NO: 172), G8AIZ0 (SEQ ID NO: 173), H1 SER3 (SEQ ID NO: 174), H3RCD2 (SEQ ID NO: 175), H5TCE5 (SEQ ID NO: 176), H5V6A9 (SEQ ID NO: 177), H5W9B2 (SEQ ID NO: 178), H6R9L7 (SEQ ID NO: 179), H6RKA6 (SEQ ID NO: 180), H8MYB1 (SEQ ID NO: 181), I1DTT2 (SEQ ID NO: 182), I2BBY3 (SEQ ID NO: 183), I3TI57 (SEQ ID NO: 184), I3X3Z1 (SEQ ID NO: 185), I4EQE5 (SEQ ID NO: 186), I6DED1 (SEQ ID NO: 187), J1Q8G0 (SEQ ID NO: 188), J2MYD9 (SEQ ID NO: 189), J7JHI8 (SEQ ID NO: 190), K0K5S2 (SEQ ID NO: 191), K0M8N7 (SEQ ID NO: 192), K0PS70 (SEQ ID NO: 193), K1LNA6 (SEQ ID NO: 194), K6X1I3 (SEQ ID NO: 195), K6Y496 (SEQ ID NO: 196), K6ZLV5 (SEQ ID NO: 197), K7A2B3 (SEQ ID NO: 198), K7ACK5 (SEQ ID NO: 199), L7E7T9 (SEQ ID NO: 200), M0Q5D8 (SEQ ID NO: 201), M1F938 (SEQ ID NO: 202), M4RGJ6 (SEQ ID NO: 203), M7NRK1 (SEQ ID NO: 204), M9YC33 (SEQ ID NO: 205), Q1I8Y3 (SEQ ID NO: 206), Q1LCA4 (SEQ ID NO: 207), Q3II98 (SEQ ID NO: 208), Q3J2H5 (SEQ ID NO: 209), Q484K6 (SEQ ID NO: 210), Q4FVA3 (SEQ ID NO: 211), Q57PK0 (SEQ ID NO: 212), Q5DYM7 (SEQ ID NO: 213), Q5P3H8 (SEQ ID NO: 214), Q5QZN5 (SEQ ID NO: 215), Q65QZ3 (SEQ ID NO: 216), Q6KFM2 (SEQ ID NO: 217), Q6LGS0 (SEQ ID NO: 218), Q7NVU7 (SEQ ID NO: 219), Q83RB3 (SEQ ID NO: 220), Q8D0L8 (SEQ ID NO: 221), Q8R5V6 (SEQ ID NO: 222), Q8Z6P3 (SEQ ID NO: 223), R4LKA2 (SEQ ID NO: 224), R4SZ37(SEQ ID NO: 225), R7XMB0 (SEQ ID NO: 226), SOAECl (SEQ ID NO: 227), S6BC14 (SEQ ID NO: 228), S6CW44 (SEQ ID NO: 229), T2L8S2 (SEQ ID NO: 230), U2ZIL0 (SEQ ID NO: 231), U4M0N4 (SEQ ID NO: 232), U5QMJ3 (SEQ ID NO: 233), V4YGE9 (SEQ ID NO: 234), V5AT08 (SEQ ID NO: 235), V5CB48 (SEQ ID NO: 236), V5ZCP1 (SEQ ID NO: 237), V6F716 (SEQ ID NO: 238), V7ZCA3 (SEQ ID NO: 239), W0B1D8 (SEQ ID NO: 240), W0V834 (SEQ ID NO: 241), W1IPZ5 (SEQ ID NO: 242), W1J7S6 (SEQ ID NO: 243), W5RUC0 (SEQ ID NO: 244), W6Q9S9 (SEQ ID NO: 245), W7WGC5 (SEQ ID NO: 246) and X5KPQ2 (SEQ ID NO: 247). These UniProt protein entries identify homologs of NemA. Additional homologs of NemA are well known to a person of ordinary skill in the art and use of such homologs is within the purview of the invention. Also, a person of ordinary skill in the art can identify homologs of NemA in additional microorganisms and use of such homologs is also within the purview of the invention.

Non-limiting examples of YafC that can be used in the invention are provided by proteins identified by the UniProt entries F1YR16 (SEQ ID NO: 248), F7SWM0 (SEQ ID NO: 249), E3HRW8 (SEQ ID NO: 250), V5V8M1 (SEQ ID NO: 251), F0KPE4 (SEQ ID NO: 252), N9TWR4 (SEQ ID NO: 253), A0KIT2 (SEQ ID NO: 254), A0A023RLF9 (SEQ ID NO: 255), A0A061QAE0 (SEQ ID NO: 256), A0XY93 (SEQ ID NO: 257), A0A0D6QPL4 (SEQ ID NO: 258), A0A0A2W477 (SEQ ID NO: 259), A0A099IRG2 (SEQ ID NO: 260), A0A023XH98 (SEQ ID NO: 261), A0A061M2V3 (SEQ ID NO: 262), A0A085GIV1 (SEQ ID NO: 263), A0A090UFR2 (SEQ ID NO: 264), A0A0J1MBK6 (SEQ ID NO: 265), A0A0A1RWL8 (SEQ ID NO: 266), A0A090TYY4 (SEQ ID NO: 267), G6F2K2 (SEQ ID NO: 268), H8MKP7 (SEQ ID NO: 269), K7ZY69 (SEQ ID NO: 270), K8B473 (SEQ ID NO: 271), K8C8M9 (SEQ ID NO: 272), K8AJK9 (SEQ ID NO: 273), A0A0F6VPG6 (SEQ ID NO: 274), K8DBL5 (SEQ ID NO: 275), E0SML9 (SEQ ID NO: 276), U6ZGR8 (SEQ ID NO: 277), G8LDH5 (SEQ ID NO: 278), F5RT70 (SEQ ID NO: 279), W7PAI4 (SEQ ID NO: 280), B7LW83 (SEQ ID NO: 281), H5V7G6 (SEQ ID NO: 282), A0A090UZK2 (SEQ ID NO: 283), A0A067Z800 (SEQ ID NO: 284), T2L206 (SEQ ID NO: 285), A0A060VEY1 (SEQ ID NO: 286), A0A0I9Q5V5 (SEQ ID NO: 287), A0A085I4U7 (SEQ ID NO: 288), C6SE56 (SEQ ID NO: 289), D4GCU4 (SEQ ID NO: 290), U2MY31 (SEQ ID NO: 291), H3R858 (SEQ ID NO: 292), D6YQH4 (SEQ ID NO: 293), A0A0H3I3R7 (SEQ ID NO: 294), J8PSY2 (SEQ ID NO: 295), V6AD39 (SEQ ID NO: 296), E2XY11 (SEQ ID NO: 297), A0A024HG34 (SEQ ID NO: 298), A0A061CVH0 (SEQ ID NO: 299), I7BRE6 (SEQ ID NO: 300), A0A0F7XTK0 (SEQ ID NO: 301), A0A098FK47 (SEQ ID NO: 302), K0Q2Y1 (SEQ ID NO: 303), A0A083ZI61 (SEQ ID NO: 304), W6RQA2 (SEQ ID NO: 305), A0A0K0H726 (SEQ ID NO: 306), Q57R49 (SEQ ID NO: 307), A0A0F7J7Q5 (SEQ ID NO: 308), B5R5K6 (SEQ ID NO: 309), A0A0H2WRE3 (SEQ ID NO: 310), C0Q6M5 (SEQ ID NO: 311), Q8Z987 (SEQ ID NO: 312), A0A0H3N7V5 (SEQ ID NO: 313), U2NQ98 (SEQ ID NO: 314), SOADFO (SEQ ID NO: 315), Q8EEC7 (SEQ ID NO: 316), E7SZS0 (SEQ ID NO: 317), E2XCH1 (SEQ ID NO: 318), A0A0F6MAJ9 (SEQ ID NO: 319), Q3Z5F5 (SEQ ID NO: 320), I2BC92 (SEQ ID NO: 321), C9XWD7 (SEQ ID NO: 322), 13X913 (SEQ ID NO: 323), M5D0B7(SEQ ID NO: 324), A0A031HFS3 (SEQ ID NO: 325), I3TTA6 (SEQ ID NO: 326), A0A085AD91 (SEQ ID NO: 327), Q5E752 (SEQ ID NO: 328), M7QUS0 (SEQ ID NO: 329), L8XZ72 (SEQ ID NO: 330), H1XKE3 (SEQ ID NO: 331), A0A077SFG9 (SEQ ID NO: 332), H8FJR7 (SEQ ID NO: 333), A0A068QSI4 (SEQ ID NO: 334), W8TWM8 (SEQ ID NO: 335), W8VA84 (SEQ ID NO: 336), A0A0H3NV03 (SEQ ID NO: 337), F4MZR3 (SEQ ID NO: 338), F4N2X2 (SEQ ID NO: 339), C4TVK1 (SEQ ID NO: 340) and C4SHW8 (SEQ ID NO: 341). These UniProt protein entries identify homologs of YafC. Additional homologs of YafC are well known to a person of ordinary skill in the art and use of such homologs is within the purview of the invention. Also, a person of ordinary skill in the art can identify homologs YafC in additional microorganisms and use of such homologs is also within the purview of the invention.

In one embodiment of the invention, the genetic modification resulting in the increased expression of NemA comprises introducing into the microorganism a nucleotide molecule, for example, DNA or RNA, comprising a nemA gene. In certain embodiments, a nemA gene present in a DNA molecule introduced into a microorganism is identical to the nemA gene present in the genome of the microorganism, i.e., the DNA molecule provides an extra copy of the endogenous nemA gene. In certain other embodiments, a nemA gene present in a DNA molecule is different from the nemA gene present in the genome of the microorganism, i.e., the DNA molecule provides a homolog of the nemA gene present in the genome of the microorganism.

Examples of DNA molecule comprising the nemA gene include a plasmid, cosmid, yeast artificial chromosome (YAC), 2-micron DNA. Additional examples of DNA molecule suitable for the expression of a gene of interest in a host microorganism are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

A typical DNA molecule suitable for the expression of a gene of interest into a host microorganism contains an origin of replication, a promoter which drives the expression of the nemA gene, one or more selectable markers and one or more restriction enzyme cleavage sites for cloning a gene of interest into the DNA molecule. The promoter can be an inducible promoter or a constitutive promoter. The selectable markers can be an antibiotic resistant gene or a gene providing for a missing biochemical function in the microorganism. Additional examples of promoters as well as selectable markers are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

In one embodiment, the DNA molecule comprising the nemA gene is incorporated into the genome of the microorganism. In another embodiment, the DNA molecule comprising the nemA gene is present as an extra-genomic genetic material.

In a particular embodiment, the microorganism is a bacterium and the DNA molecule is a plasmid carrying the nemA gene.

In one embodiment, the genetic modification resulting in the increased expression of NemA comprises a mutation in the genome of the microorganism, particularly, in the regulatory region of the HTH-type transcriptional repressor (nemR) operon. The nemR operon comprises three genes, namely, nemR, nemA and lactoglutathione lyase (gloA).

In a particular embodiment, the microorganism is E. coli and the regulatory region of the nemR operon comprises the sequence of nucleotides 1 to 390 of SEQ ID NO: 22 and the mutation which results in the increased expression of NemA is the substitution of guanine (G) at the position 372 of SEQ ID NO: 22 with a nucleotide different from G, for example, adenine (A), cytosine (C) or thymine (T), preferably, A.

In a given microorganism having a specific sequence of the regulatory region of nemR operon, a person of ordinary skill in the art can determine G in the position of corresponding to G at the position 372 of SEQ ID NO: 22 and mutate the corresponding G with a nucleotide different from G, for example, A, C or T, preferably, A.

Techniques of mutating a single nucleotide of interest in the genome of an organism are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

In a further embodiment, the activity of NemA is increased by introducing a mutant form of NemA into the microorganism, wherein the mutant NemA has higher catalytic activity than wild-type NemA. The mutant NemA can be introduced in a plasmid (as discussed above) or the endogenous nemA gene can be mutated to introduce a mutant nemA gene that encodes a mutant NemA protein. A person of ordinary skill in the art can identify a mutant NemA that has a higher catalytic activity than wild-type NemA by mutagenesis techniques known in the art.

In another embodiment of the invention, the genetic modification resulting in the mutated gene expression of YafC* comprises introduction of a nucleotide molecule, for example, DNA or RNA, comprising the yafC* gene into the microorganism. In one embodiment, the wild-type YafC has the amino acid sequence of SEQ ID NO: 23 and YafC* contains the substitution of the aspartic acid at position 275 of SEQ ID NO: 23. In a certain embodiment, the wild-type YafC has the amino acid sequence of SEQ ID NO: 23 and YafC* contains the substitution of the aspartic acid at position 275 of SEQ ID NO: 23 with glycine. In one embodiment, the wild-type YafC has the amino acid sequence of SEQ ID NO: 23 and is encoded by a wild-type gene yafC gene having the nucleotide sequence of SEQ ID NO: 25 and YafC* contains the substitution of the aspartic acid at position 275 of SEQ ID NO: 23 with glycine and is encoded by yafC* gene having the nucleotide sequence of SEQ ID NO: 26. In a further embodiment, the wild-type yafC comprises SEQ ID NO: 25 and the mutation resulting in the expression of YafC* causes the substitution of adenine at position 824 of SEQ ID NO: 25 with a nucleotide different from adenine.

The claimed invention can be practiced with YafC corresponding to any of the YafC proteins identified in the UniProt entries provided above as well as any homolog in a given microorganism that can be identified by a person of ordinary skill in the art. In a YafC protein of a given amino acid sequence, a person of ordinary skill in the art can identify the aspartic acid corresponding to the aspartic acid at position 275 of SEQ ID NO: 23, for example, using the sequence alignment, and produce a mutation in the gene encoding the YafC protein to encode the amino acid at the aspartic acid corresponding to the aspartic acid at position 275 of SEQ ID NO: 23 to be different from aspartic acid. For example, in a YafC protein of a given amino acid sequence, a person of ordinary skill in the art can identify the aspartic acid corresponding to the aspartic acid at position 275 of SEQ ID NO: 23, for example, using the sequence alignment, and produce a mutation in the gene encoding the YafC protein to encode glycine at the position corresponding to the aspartic acid at position 275 of SEQ ID NO: 23.

The discussion above with respect to the materials and methods for introducing a DNA molecule comprising the nemA gene into a microorganism is also applicable to the introduction of a DNA molecule comprising the yafC* gene into the microorganism. Accordingly, the techniques discussed with respect to introducing a DNA molecule comprising the nemA gene can be applied to introduce a DNA molecule comprising the yafC* gene into the microorganism and such embodiments are within the purview of the invention.

In an embodiment, the yafC gene in the genomic DNA of a microorganism is mutated to encode a YafC* protein. In one embodiment, the endogenous yafC gene of a microorganism has the sequence of SEQ ID NO: 25, which encodes YafC having the amino acid sequence of SEQ ID NO: 23, where the yafC gene is mutated to encode YafC* having the aspartic acid at the position 275 of SEQ ID NO: 23 mutated with an amino acid different from aspartic acid, for example, glycine.

A person of ordinary skill in the art can design and create a mutation in the genomic DNA of a microorganism where a y /C gene encoding a YafC protein is mutated so that the aspartic acid of the YafC corresponding to the aspartic acid at position 275 of SEQ ID NO: 23 is replaced by an amino acid different from aspartic acid, for example, glycine. In one embodiment, the microorganism comprises a genetic modification resulting in the increased expression of NemA or the increased catalytic activity of NemA and a genetic modification resulting in the expression of YafC*.

Numerous embodiments of genetic modifications resulting in an increased expression of NemA or the increased catalytic activity of NemA are described herein. Also, numerous embodiments of genetic modifications resulting in the expression of YafC* are described herein. Accordingly, any combination of a genetic modifications resulting in an increased expression of NemA or the increased catalytic activity of NemA and a genetic modification resulting in the expression of YafC* can be introduced into a microorganism to produce the microorganism having both the increased expression of NemA or the increased catalytic activity of NemA and the expression of YafC*. In one embodiment, a DNA carrying genes for both NemA and YafC* is introduced into a microorganism. The DNA can be incorporated into the genome of the organism or can remain as an extra-genomic genetic material. In a further embodiment, a microorganism comprises a plasmid encoding NemA and contains a mutation in the genome that results in the expression of YafC*.

In some embodiments, the bacterium that is genetically modified may be Escherichia coli, or a particular strain thereof, such as E. coli B, E. coli C, E. coli W, or the like. In some other embodiments of the invention, bacteria that can be modified according to the present invention include, but are not limited to, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Coryne bacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, Xanthomonas citri and so forth.

In certain embodiments, the subject invention provides acid hydrolysate resistant bacterial strains (such as E. coli) lacking plasmids, antibiotic resistance genes and/or material from other organisms. Unlike other microbial systems, the microorganisms of the subject invention can be employed in a single step production of a metabolite of interest using a growth medium containing the acid hydrolysate. In certain embodiments, the acid hydrolysate resistant microorganism of the invention is metabolically evolved for a particular characteristic, for example, synthesis of a metabolite. In one embodiment, the acid hydrolysate resistant microorganism of the invention does not contain an exogenous gene or fragment thereof or only contains native genes. In a further embodiment, the acid hydrolysate resistant microorganism of the invention only contains mutations in the genomic DNA of the microorganism.

The microorganism of the invention, when cultured in a medium containing the nonvolatile products present in an acid hydrolysate of lignocellulosic biomass or an artificial hydrolysate, produces an increased amount of a metabolite compared to the amount of the metabolite produced by the parental microorganism cultured in the medium containing the nonvolatile products present in the acid hydrolysate of lignocellulosic biomass or the artificial hydrolysate. Accordingly, a further embodiment of the invention provides a method of culturing or growing a microorganism of the invention in a medium under conditions that allows the production of a metabolite of interest. The culturing or growing can be performed in a batch process, a fed batch process or a continuous process. In certain embodiments, the medium comprises in which the acid hydrolysate resistant microorganism of the invention comprises nonvolatile products present in an acid hydrolysate of lignocellulosic biomass or an artificial hydrolysate. Method of producing different culture/growth media and conditions that allow for the culturing/growing of the microorganism of interest are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

MATERIALS AND METHODS

Strains and media

Two strains of ethanologenic E. coli were used, LY180 and SL100. SL100 is a hydrolysate-resistant derivative of LY180, selected by serial transfers for more than a year in AMI mineral salts medium containing sugarcane bagasse hydrolysate. Except for the plasmid constructions using Luria broth, strains were grown and maintained on AMI medium, AMI mixed with artificial hydrolysate, or AMI mixed with sugarcane bagasse hydrolysate. Xylose was added as needed to provide 5% sugars. Media were adjusted to pH 6.5 prior to inoculation (incubated at 37°C).

SL100 chromosomal library

SL100 chromosomal DNA was partially digested with Sau3Al and ligated (2-8 kbp fragments) into the dephosphorylated BamHl site of pUC19. TOP10F'™ cells (ThermoFisher Scientific) was used as the host (100 mg ampicillin liter ^"1 ). Colonies were pooled and used to prepare a chromosomal library of plasmids.

Preparation of hydrolysates

Artificial hydrolysate (PX) was prepared by autoclaving a mixture of xylose (50 g liter ^"1 ) and phosphoric acid (10 g liter ^"1 ) for 2 h at 140°C (Hirayama model HA-305M; Amerex Instruments, Inc., Lafayette, CA). Sugarcane bagasse hydrolysate (SCBHz) was prepared using a Metso-Valmet continuous screw digester (185°C, 7.5 min, 8 kg phosphoric acid per dry ton sugarcane bagasse, 3 ton h ^"1 ) as previously described. Where indicated, volatiles such as furfural were removed from hydrolysates by evaporation (55°C) to half the original weight. Weight loss was replaced with distilled water, designated PXV and SCBHzV, respectively.

Construction of pLOI5883 derivatives for gene expression

Plasmids, strains, and primers are listed in Table 1. Coding regions (ATG to TAA) of individual genes (nemA, gloA, rnt) were amplified using SL100 chromosomal DNA as a template unless specified otherwise. An artificial ribosomal binding site was supplied on the primers. Amplified genes were ligated into the expression vector pLOI5883 (EcoKI to HindlH; Fig. 6) between the Ptrc promoter and rrnB terminator to construct pLOI5908 (nemA), pLOI5909 (gloA), and pLOI5910 (rnt). These three adjacent genes were also amplified together and ligated to construct pLOI5911 (nemA-gloA, rnt). Similar plasmids were constructed for yafC (pLOI5913), yafC* (pLOI5914), and a combination of nemA- yafC* (pLOI5926). Sequences were confirmed by Sanger sequencing. Inducer (10 μΜ IPTG) was added where indicated.

Table 1. Bacterial strains, plasmids and primers

Enrichment for clones conferring resistance to PXV

The SL100 chromosomal library was transformed into LY180 (parent), grown overnight in 100 ml AMI mineral salts medium (250 ml flask) with 5% xylose and 100 μg ml ^"1 ampicillin (37°C, 50 rpm), and inoculated (0.1 OD ₅₅₀ ) into 100 ml AMI broth with 80% vol/vol PXV. Although little growth was observed after 48 h, cells were harvested by centrifugation (10 min, 2000 g) and transferred to fresh AMI broth containing 80% vol/vol PXV. Growth was abundant after 24 h. Plasmids were extracted, transformed into LY180, and tested for resistance to AMI broth with 80% vol/vol PXV by measuring ethanol production (Fig. 7).

Measuring toxicity

Toxicity of hydrolysates was tested by measuring ethanol production in tube cultures (13 x 100 mm; 4 ml broth) containing mixtures of water, hydrolysate and the constituents of AMI medium. Cultures were inoculated to an initial density of 0.1 OD ₅₅ onm and incubated for 48 h (37°C). Due to color, ethanol production (determined by gas chromatography) and visual observation of cells were used as a measure of fermentation and confirm growth. In some experiments, cells were harvested by centrifugation, washed twice in AMI medium and resuspended in AMI medium. This removed soluble color and allowed an estimation of cell growth by turbidity. In all cases, the shapes of the curves for OD ₅₅ onm were very similar to measurements of ethanol indicating a close relationship (Fig. 8).

Assay of NemA reductase activity

NemA reductase activity was measured as the N-ethylmaleimide (NEM) dependent oxidation of NADPH unless stated otherwise. For enzyme assays, cells (50 ml) grown in AMI xylose broth containing 100 mM MOPS (pH 7) were harvested by centrifugation at approximately 1 OD ₅₅ onm, washed twice with 10 ml of cold potassium phosphate buffer (50 mM, pH 7.0), and resuspended in phosphate buffer (3 ml). After disruption with 0.1 mm glass spheres for 20 sec using a FastPrep-24 (MP Biomedical LLC, Santa Ana, US), cell debris was removed by centrifugation (20 min, 14,000 g). The soluble protein fraction was used for assays of NEM-dependent activity at 22°C in 50 mM potassium phosphate (pH 7.0) with 0.2 mM NADPH, 0.1 mM NEM, cell extract, and vacuum-treated hydrolysates (PXV or SCBHzV) as indicated. Protein was measured with the BCA reagent using bovine serum albumin as a standard. Vacuum-treated hydrolysates were tested as inhibitors of NemA activity using NEM as the electron acceptor. Vacuum-treated hydrolysates were also tested as inducers of NemA activity in LY180, and as a source of electron acceptors (without NEM) for NemA reductase using protein lysates of LY180 (pLOI5908). One unit of activity is defined as the amount of enzyme that converts 1 μΜ of NADPH to NADP ⁺ min ^"1 .

Additional experiments were conducted to explore potential proteolysis of NemA. Disrupted cell extracts were prepared from the LY180 grown with 5% SCBHzV (induced) and AMI alone (uninduced). Extracts were diluted 1 : 1 with phosphate buffer, and a mixture of equal amounts of induced and uninduced extracts was also made. All three samples were incubated for 9 h at 37°C. NemA activity was measured at TO, T3, T6, and T9 h. No loss of activity was observed with LY180 (uninduced) or LY180 (induced).

Expression of item A as measured by Real-Time PCR

Expression of nemA mRNA was determined using polA as reference gene. RNA was isolated as previously described. Message abundance was compared for the parent LY180 and the mutant SL100 after growth in the presence and in the absence of vacuum-treated 6% SCBHzV (inducer).

Bench scale fermentations

Acid hydrolysate resistance genes were expressed from pLOI5883 derivatives in LY180 during bench-scale fermentations (300 ml broth) in AMI medium containing 20% vol/vol SCBHzV. Results were compared to controls (empty vector) grown in the same medium and in AMI without hydrolysate. Sufficient xylose and glucose were added to provide 100 g total sugar liter ^"1 (75 g glucose liter ^"1 and 25 g xylose liter ^"1 ), similar to hydrolysis of cellulose and hemicellulose. Fermentations were maintained at 37°C and pH 6.5 (automatic addition of 2M potassium hydroxide). These were inoculated with a 24-h broth culture to provide an initial OD ₅₅ onm of 0.10 (approximately 0.05 g dew liter ^"1 ).

Analyses

Ethanol was measured as described previously using an Agilent 6890N gas chromatograph equipped with flame ionization detectors and a 15-m HP -Plot Q Megabore column. Furfural and xylose were measured using an Agilent 1200 FIPLC with an Aminex FIPX-87P column. Experiments were conducted at least twice with three replicates each. Results are reported as averages with standard deviations. Significance was inferred (p < 0.05) from a two-tailed Student's t-tests using GraphPad Prism software for computations.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 - VACUUM TREATMENT REMOVED FURFURAL AND DECREASED

THE TOXICITY OF ARTIFICIAL HYDROLYSATE

Heating xylose in dilute sulfuric acid can be used to produce an artificial hydrolysate. However, the process described in Nieves et al. (2011) (Bioresour Technol., 102:5145-5152) for lignocellulose fermentation uses phosphoric acid pretreatment instead of sulfuric to avoid the need for exotic metallurgy. A similar brown, toxic artificial hydrolysate (PX) can be made from xylose and phosphoric acid (2 h at 140°C) in the absence of lignin and cellulose. Resulting PX contained 13.6 mM furfural (Table 2). AMI broth containing 40% vol/vol PX was sufficiently toxic to completely inhibit the growth (visual) and fermentation of the parent, LY180 (Fig. 1A). SL100, a mutant of LY180 selected for resistance to sugarcane bagasse hydrolysate (SCBHz), was also more resistant to PX than LY180 and required 60% vol/vol PX for a similar degree of inhibition. Furfural removal by vacuum evaporation (PXV; Table 2) reduced the toxicity of hydrolysates to both LY180 and SL100 (Fig. IB). Ethanol production by LY180 was partially inhibited by 60% vol/vol PXV and fully inhibited by 80% vol/vol PXV. Ethanol production by SL100 was inhibited by less than 1/3 with 80% vol/vol PXV. Prior selection for resistance to SCBHz with SL100 also co-selected for resistance to volatile (primarily furfural) and nonvolatile inhibitors in artificial hydrolysate (heated xylose in phosphoric acid solution).

Table 2. Concentrations of xylose and furfural in artificial and sugarcane bagasse

EXAMPLE 2 - CLONING AND SEQUENCING GENES FROM SL 100 THAT INCREASE

TOLERANCE TO VACUUM-TREATED HYDROLYSATES Differences in the resistance to PXV between LY180 and SL100 provided a basis for selection of the genes conferring resistance (Fig. 1; Fig. 7). Strain LY180 was transformed with a plasmid library of SL100 chromosomal fragments and selected for growth in AMI broth (5% xylose) containing sufficient PXV (80% vol/vol) to inhibit ethanol production and growth (visual). The cells grew well in the second transfer and were harvested for plasmid purification. Resulting plasmids were back-transformed into LY180, diluted, and spread on AMI solid media (2% xylose) with 100 μg/ml ampicillin. A total of 475 clones were screened from three independent plasmid libraries of SL100. Each clone was tested for ethanol production in tube cultures containing 80% vol/vol PXV. Examples of colony screening are shown in Fig. 1C. Positive clones produced 6-fold to 10-fold higher levels of ethanol than empty pUC19 and negative clones.

DNA fragments in 15 clones (highest ethanol titers) were sequenced. Many were siblings with identical fragments of SL100 DNA confirming the rigor of the selection. All clones fell into two groups (Fig. 2; also Fig. 9). Twelve clones contained a large fragment with 'nemR-nemA-gloA rnt Ihr ' (6 unique fragments plus siblings) and three contained smaller fragments with 'dkgB yaf yaflJ ' (2 unique fragments plus one sibling). Sequencing revealed that the nemR was incomplete in the plasmids with the large fragments and unlikely to function. The nemA, gloA, and rnt genes were complete and did not contain mutations. The nemA and gloA genes are part of the cellular defense system for cytoplasmic detoxification. The rnt gene encodes RNAse T, an exonuclease involved in trimming stable RNAs and t-RNA maturation. Although no mutations were found in the cloned nemRA-gloA, rnt, or Ihr ' regions, a chromosomal mutation in SL100 was found in the upstream regulatory region of nemR. This mutation was absent in LY180 and could affect nemRA-gloA expression (Fig. 2A). The yafC gene encodes a putative transcriptional regulator of unknown function. A single base mutation was found in the C-terminus of yafC coding region (D275G), designated yafC* (Fig. 2B) (present in SL100).

EXAMLE 3 - TESTING SUBCLONES WITH SINGLE GENES RELATED TO

HYDROLYSATE TOLERANCE

Each of the genes in the large nemA fragment nemR-nemA- gloA, rnt), yafC(LYl&0), and ¾2/C*(SL100) were cloned into expression vector pLOI5883 (Fig. 6). An artificial ribosomal binding site was supplied by the primers used for amplification (nemA, gloA, rnt, and yafC*). SL100 served as a template for all except yafC (wild type from LY180). Each gene was ligated into pLOI5883 (RSFlOlO-based expression vector; See Fig. 6) to produce pLOI5908, pLOI5909, pLOI5910, pLOI5913 and pLOI5914, respectively (Table 1). Two combinations of genes were also constructed, nemA-gloA rnt with intergenic regions (pLOI5911) and nemA yafC* (pLOI5926).

Expression plasmids were transformed into LY180 and tested for ethanol production in AMI containing 80% v/v PXV (Fig. 3A) and in AMI containing 20% vol/vol SCBHzV (Fig. 3B). With AMI containing 80% vol/vol PXV (no IPTG), ethanol production was significantly increased (p < 0.05) by plasmids expressing nemA (pLOI5908), the 3-gene combination (pLOI5911), and the nemA yafC* combination (pLOI5926) in comparison to the empty vector (pLOI5883). Addition of IPTG caused a small but significant (p < 0.05) increase in ethanol production by LY180(pLOI5908) expressing nemA and the 3-gene combination (pLOI5911). When tested in AMI containing 20% vol/vol SCBHzV, ethanol production was significantly increased (p < 0.05) by plasmids expressing nemA (pLOI5908), yafC* (pLOI5914), the 3-gene combination (pLOI5911), and the nemA yafC* combination (pLOI5926) in comparison to the empty vector control (pLOI5883) and yafC (wild type), with or without IPTG. Adding inducer provided a small benefit for nemA constructs but was detrimental for yafC* constructs (Fig. 3 A and 3B). Plasmids expressing gloA, rnt, or yafC individually were of little benefit for 80% vol/vol PXV or 20% vol/vol SCBHzV. However, the combination of the three genes (pLOI5911) and the combination of two genes (pLOI5926; nemA yafC*) were significantly better (p < 0.05) than nemA alone when tested with 20% vol/vol SCBHzV (Fig. 3B). This plasmid-mediated increase in resistance was specific for nonvolatile components of hydrolysates and did not increase resistance to furfural (Fig. 3C). LY180 expressing nemA and all combinations with nemA exhibited an increase in resistance to vacuum treated hydrolysates (PXV and SCHzV). None of the expression vectors increased furfural tolerance in LY180 (Fig. 3C).

Plasmids with various genes were also transformed into SL100 and tested for resistance to 35% v/v SCBHzV (Fig. 3D). Although the effects were small, SL100 containing plasmids with nemA alone, gloA alone, and the combination produced more ethanol (p < 0.05) than the vector control, rnt alone, yafC* alone, or the nemA-yafC* combination. None of the constructs with yafC* increased the resistance of SL100 to SCBHzV since t eya/C* mutation is already present on the SL100 chromosome. In contrast, the nemA-yafC* combination was the most beneficial for ethanol production by LY180 in SCHzV (Fig 3 A and 3B).

Addition of IPTG was generally beneficial for ethanol production with plasmid constructs lacking yafC or yafC*, a putative transcriptional regulator. Addition of IPTG decreased ethanol production in all constructs containing yafC or yafC*. Increasing the expression of this putative regulator appears to hinder cellular functions.

EXAMPLE 4 - VACUUM-TREATED HYDROLYSATES CONTAIN SUBSTRATES FOR

NEMA

NemA is a versatile NADPH-dependent flavoprotein reductase (old yellow enzyme) capable of reducing a broad range of organic compounds including electrophiles (quinones, glyoxals, trinitrotoluene) and even inorganic substrates such as nitrates and chromates. Considering the diversity of compounds formed by acid treatment of xylose it is not surprising that some components in PXV (Fig. 4A) and SCBHzV (Fig. 4B) can serve as electron acceptors for NemA/NADPH (Table 3). Although activity with LY180 was low with hydrolysates as sole source for electron acceptors, the activity in LY180 harboring the nemA expression vector (pLOI5908) was twice that of the respective vector controls, consistent with measurements of a nemA -encoded activity. With vacuum treated hydrolysates as a potential substrate, activity plateaued or declined with increases in PXV and SCBHzV (Figures 4A and 4B). These kinetics can be attributed in part to the dual action of hydrolysate components as both substrates for NemA and inhibitors of NemA activity with NEM as substrate.

Table 3. Hydrolysate as substrate and inhibitor of NemA activity in protein extracts of LY180.

*NEM specific activity indicates that NEM (0.1 mM) served as the substrate EXAMPLE 5 - VACUUM-TREATED HYDROLYSATES CONTAIN INHIBITORS OF N-

ETHYLMALEIMIDE REDUCTION BY NEMA

NemA activity is typically measured with NEM as the electron acceptor, although the physiological substrate for this enzyme is unknown. The NEM-dependent activity of this enzyme was inhibited by the addition of vacuum-treated hydrolysates, PXV (Table 3 and Fig. 4A) or SCBHzV (Fig. 4B). SCBHzV was a more potent inhibitor. Addition of 1.25% vol/vol SCBHzV in the reaction mixture was sufficient to inhibit 99% of the NemA activity with NEM as substrate (1.1 U mg ^"1 protein without inhibitor). In contrast, addition of 25% vol/vol PXV to the reaction mixture inhibited only 90% of NemA activity. Both hydrolysates appear to contain a combination of substrates and inhibitors that affect NemA activity. The dose-dependent inhibition by hydrolysate may be responsible for the unusual kinetics observed when hydrolysates were tested as a source of electron acceptors for NemA activity (Fig. 4A and 4B).

EXAMPLE 6 - VACUUM-TREATED HYDROLYSATES AS INDUCERS OF NEMA

ACTIVITY

The nemR operon contains three genes (nemR-nemA-gloA) that are repressed by nemR in the absence of inducer. In AMI medium without inducer, NemA activity (NEM as electron acceptor) in SL100 was more than 20-fold higher (0.09 U/mg protein) than in lysates from LY180 (parent), consistent with a role for NemA in the acid hydrolysate resistance (Fig. 4C). NemA activity in LY180 harboring the nemA expression plasmid pLOI5908 (1.1 U mg ^{" l} , right axis) was 13-fold higher than in SL100 (left axis) and over 250-fold higher than LY180. NemA activity in LY180 was induced 8-fold and 2.5-fold (0.12 U) by growth in the presence of 50% vol/vol PXV and 5% vol/vol SCBHzV, respectively. In contrast, a high level of NemA activity was produced in SL100 without hydrolysate (0.09 U), increasing by only 41% when 5% vol/vol SCBHzV was included during growth (Fig. 4C). This partial derepression of nemA in SL100 is presumed to result from the base mutation in the upstream regulatory region of nemR in SL100, a transcriptional regulator.

Transcriptional regulation of nemA was confirmed by measuring message levels

(Real-Time PCR) with polA as a the reference gene. Induction with 5% SCBHzV increased mRNA in LY180 by 5-fold in comparison to uninduced LY180. In the resistant mutant SL100, nemA mRNA was over 200-fold more abundant than in the parent strain.

Potential regulation by proteinases was also investigated. Disrupted cell extracts were prepared from the LY180 grown with 5% SCBHzV (induced) and AMI alone (uninduced). Extracts were diluted 1 : 1 with phosphate buffer, and a mixture of equal amounts of induced and uninduced extracts was also made. All three samples were incubated for 9 h at 37°C. NemA activity was measured at TO, T3,T6, and T9 h. No loss ofNemA activity was observed with LY180 (uninduced), LY180 (induced) or the mixture.

SL100 was more resistant to PXV and SCBHzV than the parent, and had higher uninduced-levels of NemA activity. Expression of nemA from plasmids in LY180 (parent) increased NemA activity, increased nemA mRNA and increased tolerance to vacuum-treated hydrolysates. This effect was specific for vacuum treated hydrolysate and did not increase tolerance to furfural. SL100 exhibited a further increase in resistance to vacuum-treated hydrolysate when nemA and gloA genes were co-expressed from a single plasmid, pLOI5911 (Fig. 3D). EXAMPLE 7 - PLASMID EXPRESSION OF RESISTANCE GENES IMPROVED

FERMENTATION PERFORMANCE

Plasmids containing nemA or yafC* were expressed individually in LY180 during batch fermentations of 20% (vol/vol) SCBHzV in AMI (supplemented with glucose and xylose to make 100 g total sugar liter ^"1 ). Under these conditions, the maximum rate of ethanol production with LY180 (pLOI5883) empty vector control was approximately half that observed without added hydrolysate (Fig. 5A). Production of 30 g ethanol liter ^"1 required 8 days with hydrolysate and empty vector but required only 2 days in AMI medium without hydrolysate. Expression of yafC* and nemA (individually) substantially improved ethanol production, reducing the time required to produce 30 g ethanol liter ^"1 to three days.

These two genes may be useful for engineering the acid hydrolysate resistance biocatalysts for renewable products. Plasmid expression yafC has been shown to increase survival to ionizing radiation. Both genes improved the rates of growth (observations of turbidity) and sugar utilization (Fig. 5B, 5C, 5D). With either nemA or yafC*, fermentation of glucose was complete after 3 days but required more than 8 days with the empty vector control. The fermentation of xylose was partially inhibited by the addition of hydrolysate. In hydrolysate medium, xylose was used concurrently with glucose but at a much slower rate. After 2 days, the rate of xylose utilization was increased by plasmids expressing nemA and yafC*. However, fermentations failed to completely utilize xylose even after 8 days. Without hydrolysate, xylose fermentation was completed after 4 days with near theoretical yields from both sugars.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated within the scope of the invention without limitation thereto. REFERENCES

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