PNP GENE MODIFICATION FOR IMPROVED XYLOSE UTILIZATION IN

ZYMOMONAS This application claims the benefit of United States Provisional

Application 61/577871 , filed December 20, 201 1 , and is incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States government support under Contract No. DE-FC36-07GO17056 awarded by the Department of Energy. The government has certain rights in this invention. FIELD OF THE INVENTION

The invention relates to the fields of microbiology and genetic engineering. More specifically, the pnp gene in the Zymomonas genome was identified as a target for modification to provide improved xylose utilization and ethanol production.

BACKGROUND OF THE INVENTION

Production of ethanol by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research. It is desirable that microorganisms producing ethanol, as well as other useful products, be capable of using xylose as a carbon source since xylose is the major pentose in hydrolyzed lignocellulosic biomass. Biomass can provide an abundantly available, low cost carbon substrate. Zymomonas mobilis and other bacterial ethanologens which do not naturally utilize xylose have been genetically engineered for xylose utilization by introduction of genes encoding 1 ) xylose isomerase, which catalyses the conversion of xylose to xylulose; 2) xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate; 3) transketolase; and 4) transaldolase (US 5514583, US 5712133, US 6566107, WO 95/28476, Feldmann et al. (1992) AppI. Microbiol. Biotechnol. 38: 354- 361 , Zhang et al. (1995) Science 267:240-243; Yanase et al. (2007) AppI. Environ. Mirobiol. 73:2592-2599). Typically the coding regions used were from E. coli genes.

Even with expression of this xylose utilization pathway, typically the engineered strains do not grow and produce ethanol as well on xylose as on glucose. Strains engineered for xylose utilization have been adapted by serial passage on xylose medium, resulting in strains with improved xylose utilization as described in U. S. Pat. 7,223,575 and U. S. Pat.

7,741 ,1 19. The latter also discloses inactivation of the GFOR locus encoding glucose-fructose oxidoreductase to improve xylose utilization. Disclosed in U.S. Pat. 7,998,722 is engineering for improved xylose utilization by expression of E. coli xylose isomerase from a mutated, highly active Zymomonas mobilis. glyceraldehyde-3-phosphate

dehydrogenase gene promoter (Pgap).

There remains a need for engineered strains of Zymomonas and other bacterial ethanolagens with improved xylose utilization and ethanol production in xylose-containing medium, and processes for using these strains to produce ethanol.

SUMMARY OF THE INVENTION

The invention provides recombinant xylose-utilizing Zymomonas or Zymobacter cells in which an endogenous pnp gene encoding

polynucleotide phosphorylase is modified. In addition the cells have increased ribose-5-phosphate isomerase (RPI) activity and non-limiting xylose isomerase activity. Xylose utilization and ethanol production in xylose-containing media are improved in these cells.

Accordingly, the invention provides a recombinant bacterial host cell comprising:

a) a xylose metabolic pathway comprising at least one polypeptide having xylose isomerase activity;

b) at least one genetic modification which increases ribose-5- phosphate isomerase activity in the host cell as compared with ribose-5-phosphate isomerase activity in the host cell lacking said genetic modification; and

c) at least one genetic modification in the sequence of an endogenous gene encoding polynucleotide phosphorylase that shortens the coding region resulting in expression of a C-terminal truncated protein;

wherein the bacterial host cell utilizes xylose to produce ethanol, xylose isomerase activity is not limiting in the bacterial host cell. In another embodiment the invention provides a process for producing ethanol comprising:

a) providing the recombinant host cell described above; and b) culturing the host cell of (a) in a medium comprising xylose whereby xylose is converted to ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure:

INFORMATION ON DEPOSITED STRAINS

Figure 1 shows a diagram of pathways for xylose metabolism and ethanol production.

Figure 2 shows a plasmid map of pMODIinker-Spec-GapRpi.

Figure 3 shows for strains B9, B1 1 , I, each with RPI

overexpression, and parent strain ZW801 -4: growth in mRM3-X10 (A); xylose consumption and ribulose production at 54 hours (B), and growth in mRM3-G10 (C). Figure 4 shows for seven I strain transformants with additional xylose isomerase (I(cm1 -5,8,9)) as well as I strain (1-1 ,2) and ZW801 (801 - 1 ,2) controls: growth in mRM3-X10 (A), and growth in mRM3-G10 (B).

Figure 5 shows growth in mRM3-X10 under pH-controlled conditions of two I strain transformants with additional xylose isomerase (I(cm1 ,9)), as well as I strain and ZW801 -4 controls:

Figure 6 shows growth in mRM3-X10 for ten B1 1 strain

transformants with additional xylose isomerase (B1 1 (cm1 -10)), along with B1 1 controls (B1 1 -1 ,2) (A); or ten B9 strain transformants with additional xylose isomerase (B9(cm1 -10)), as along with B9 controls (B9-1 ,2) (B) .

Figure 7 shows an alignment of amino acid sequences of the wild type Z. mobilis strain ZM4 pnp encoded polynucleotide phosphorylase (SEQ ID NO:2) and the fusion protein encoded by the modified pnp gene of the I strain (SEQ ID NO:9).

Figure 8 shows plasmid maps of pZX21 (A), pZX52 (B), and pZX6

(C).

Figure 9 shows graphs of growth, glucose used, and ethanol produced for cultures grown in mRM3-G10 of ZW1 -X109 (A), ZW1 -X210

(B), and control ZW1 (C).

Figure 10 shows graphs of growth, glucose used, and ethanol produced for cultures grown in mRM3-X10 of ZW1 -109 (A), ZW1 -210 (B), and control ZW1 (C).

Figure 1 1 shows plasmid maps of pPNP-l (A), pPNP-IN (B), pPNP-

C (C), and pPNP-M (D).

Figure 12 shows graphs of growth, glucose used, and ethanol produced for cultures grown in mRM3-G10 of ZW1 -X109-PNPi (A), ZW1 -

X109-PNPC (B), ZW1 -X109-PNPm (C), ZW1 -X109-PNPin (D), and control

ZW1 -X109 (E).

Figure 13 shows graphs of growth, glucose used, and ethanol produced for cultures grown in mRM3-X10 of ZW1 -X109-PNPi (A), ZW1 - X109-PNPC (B), ZW1 -X109-PNPm (C), ZW1 -X109-PNPin (D), and control ZW1 -X109 (E). Figure 14 shows graphs of growth, xylose used, and ethanol produced for cultures grown in mRM3-X10 of ZW801 -PNPi (A), ZW801 - PNPc (B), and control ZW801 (C). The following sequences conform with 37 C.F.R. 1 .821 -1 .825

("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures - the Sequence Rules") and are consistent with World Intellectual Property Organization (WlPO) Standard ST.25 (2009) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the

Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in

37 C.F.R. §1 .822.

SEQ ID NO:1 is the nucleotide sequence of the pnp coding region from Zymomonas mobilis strain ZM4.

SEQ ID NO:2 is the amino acid sequence of the pnp encoded polynucleotide phosphorylase from Zymomonas mobilis strain ZM4.

SEQ ID NO:3 is the nucleotide sequence of the pnp coding region from Zymomonas mobilis strain NCIMB 1 1 163.

SEQ ID NO:4 is the amino acid sequence of the pnp encoded polynucleotide phosphorylase from Zymomonas mobilis strain NCIMB 1 1 163.

SEQ ID NO:5 is the nucleotide sequence of the pnp coding region from Zymomonas mobilis strain ATCC 10988.

SEQ ID NO:6 is the amino acid sequence of the pnp encoded polynucleotide phosphorylase from Zymomonas mobilis strain ATCC 10988.

SEQ ID NO:7 is the nucleotide sequence of the pnp coding region from Zymomonas mobilis pomaceae ATCC 29192.

SEQ ID NO:8 is the amino acid sequence of the pnp encoded polynucleotide phosphorylase from Zymomonas mobilis strain ATCC 29192. SEQ ID NO:9 is the amino acid sequence of the modified pnp encoded fusion protein of the I strain, having 709 native N-terminal amino acids and 14 additional C-terminal amino acids.

SEQ ID NO:10 is the amino acid sequence of a modified pnp encoded fusion protein having 695 native N-terminal amino acids and 2 additional C-terminal amino acids.

SEQ ID NO:1 1 is the amino acid sequence of a modified pnp encoded fusion protein having 368 native N-terminal amino acids and 10 additional C-terminal amino acids.

SEQ ID NO:12 is the amino acid sequence of a modified pnp encoded fusion protein having 32 native N-terminal amino acids and 17 additional C-terminal amino acids.

SEQ ID NO:13 is the nucleotide sequence of the coding region for the Z. mobilis RPI protein.

SEQ ID NO:14 is the amino acid sequence of the Z. mobilis RPI protein.

SEQ ID NO:15 is the nucleotide sequence of the coding region for the E. coli RPI protein

SEQ ID NO:16 is the amino acid sequence of the E. coli RPI protein.

SEQ ID NO:17 is nucleotide sequence of the coding region for Actinoplanes missouriensis xylose isomerase that is codon-optimized for expression in Zymomonas mobilis.

SEQ ID NO:18 is the amino acid sequence of the Actinoplanes missouriensis xylose isomerase.

SEQ ID NO:19 is nucleotide sequence of the coding region for Zymomonas mobilis ribulose-phosphate 3-epimerase.

SEQ ID NO:20 is the amino acid sequence of the Zymomonas mobilis ribulose-phosphate 3-epimerase.

SEQ ID NO:21 is the nucleotide sequence of the P _gap s (also called

801 gap) mutant promoter. SEQ ID NO:22 is the nucleotide sequence of the RPI expression cassette that is in plasmid pZB188aadA Gap/Zymo RPI/EcoliSL, that is located between the unique Ncol and Notl sites.

SEQ ID NOs:23 and 24 are primers PPI-F and PPI-R-Sbfl.

SEQ ID NO:25 is the nucleotide sequence of plasmid pZX21 .

SEQ ID NO:26 is the nucleotide sequence of the GFO-L fragment.

SEQ ID NO:27 is the nucleotide sequence of the gfor coding sequence.

SEQ ID NO:28 is the nucleotide sequence of the GFO-R fragment. SEQ ID NO:29 is the nucleotide sequence of a 1 ,661 -bp chimeric xylA gene containing the 304-bp Z. mobilis Super GAP promoter, a 1 ,185- bp A. missouriensis xylA coding sequence, and a 166-bp E. coli araD 3'UTR with a 5' Xbal site.

SEQ ID NO:30 is the nucleotide sequence of a 1 ,960-bp chimeric xylB gene containing a 191 bp P _en o, a 1 ,455-bp E. coli xylB coding sequence and a 314-bp E.coli xylB 3'UTR.

SEQ ID NO:31 is the nucleotide sequence of a 1 ,014 bp aadA marker (for spectinomycin resistance; Spec-R) bounded by lox sites.

SEQ ID NO:32 is the nucleotide sequence of shuttle vector pZX52. SEQ ID NO:33 is the nucleotide sequence of the LDH-L fragment.

SEQ ID NO:34 is the nucleotide sequence of the LDH-R fragment.

SEQ ID NO:35 is the nucleotide sequence of the IdhA coding sequence.

SEQ ID NO:36 is the nucleotide sequence of a 3,339 bp P _gaP T-Tal- Tkt operon containing a 304-bp T-mutant of the Z. mobilis GAP promoter (Pgap-r), a 954-bp E. coli Tal coding region, a 1 ,992-bp E. coli Tkt coding region, and a 68-bp E. coli Tkt 3'UTR.

SEQ ID NO:37 is the nucleotide sequence of the P _gap T promoter. SEQ ID NO:38 is the nucleotide sequence of a 1 ,443 bp P _eno -Rpi- Rpe operon containing a 191 bp P _en o, a 471 bp Z. mobilis Rpi coding sequence, a 663 bp Z. mobilis Rpe coding sequence, and a 35 bp E.coli xylA 3'UTR. SEQ ID NO:39 is the nucleotide sequence of the DCO shuttle vector pZX6.

SEQ ID NO:40 is the nucleotide sequence of the PNP-L fragment. SEQ ID NO:41 is the nucleotide sequence of the PNP-R fragment. SEQ ID NOs:42 to 54 are PCR primers.

SEQ ID NO:55 is the nucleotide sequence of the DCO suicide vector pPNP-l.

SEQ ID NO:56 is the nucleotide sequence of the upstream flanking sequence for integration into the pnp gene.

SEQ ID NO:57 is the nucleotide sequence of the downstream flanking sequence for integration into the pnp gene.

SEQ ID NO:58 is the nucleotide sequence of the DCO suicide vector pPNP-IN.

SEQ ID NO:59 is the nucleotide sequence of the PNP-U fragment.

SEQ ID NO:60 is the nucleotide sequence of the PNP-D fragment.

SEQ ID NO:61 is the nucleotide sequence of the DCO suicide vector pPNP-C.

SEQ ID NO:62 is the nucleotide sequence of the DCO suicide vector pPNP-M.

SEQ ID NO:63 is the nucleotide sequence of the PNPm-L fragment. SEQ ID NO:64 is the nucleotide sequence of the PNPm-R

fragment.

SEQ ID NO:65 is the nucleotide sequence of the coding region for the Z. mobilis RPI protein with the start codon mutated to ATG.

DETAILED DESCRIPTION

Disclosed herein are xylose-utilizing bacterial cells, and particularly, Zymomonas or Zymobacter cells, that have genetic modifications of an endogenous pnp gene (pnp modification). The cells are in addition genetically modified to have increased expression of ribose-5-phosphate isomerase (RPI) activity, as compared to cells without this genetic modification. In addition the cells are not limited in xylose isomerase activity in the absence of the pnp modification. Cells with these properties have improved xylose utilization, which is desired for growth in media containing xylose including saccharified biomass, leading to increased ethanol production. Ethanol is an important compound for use in replacing fossil fuels, and saccharified biomass provides an abundantly available renewable carbon source for ethanol production by fermentation.

The following definitions may be used for the interpretation of the claims and specification:

As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term "invention" or "present invention" as used herein is a non- limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.

As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities. In one embodiment, the term "about" means within 10% of the reported numerical value, preferably within 5% of the reported numerical value,

The term "carbon substrate" or "fermentable carbon substrate" refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, and

polysaccharides.

"Gene" refers to a nucleic acid fragment that expresses a specific protein or functional RNA molecule, which may optionally include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" or "wild type gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.

"Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A "locus" is a region of a genome that contains a gene.

The term "genetic construct" refers to a nucleic acid fragment that encodes for expression of one or more specific proteins or functional RNA molecules. In a gene construct the gene may be native, chimeric, or foreign in nature. Typically a genetic construct will comprise a "coding sequence". A "coding sequence" refers to a DNA sequence that encodes a specific amino acid sequence.

"Promoter" or "Initiation control regions" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.

Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters".

The term "genetic modification" refers, non-inclusively, to any modification, mutation, base deletion, base addition, codon modification, gene over-expression, gene suppression, promoter modification or substitution, gene addition (either single or multicopy), antisense expression or suppression, or any other change to the genetic elements of a host cell or bacterial strain, whether they produce a change in phenotype or not.

The term "recombinant bacterial host cell" refers to a bacterial cell that comprises at least one heterologus gene or genetic construct or nucleic acid fragment.

The term "expression", as used herein, refers to the transcription and stable accumulation of coding (mRNA) or functional RNA derived from a gene. Expression may also refer to translation of mRNA into a protein. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. "Over- expression" refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. "Co-suppression" refers to the production of sense RNA transcripts or fragments capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. 5,231 ,020).The term "transformation" as used herein, refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. The transferred nucleic acid may be in the form of a plasmid maintained in the host cell, or some transferred nucleic acid may be integrated into the genome of the host cell. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

The terms "plasmid" and "vector" as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double- stranded DNA molecules. Such elements may be autonomously

replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the

transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term "selectable marker" means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest. As used herein the term "codon degeneracy" refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded protein. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term "codon-optimized" as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the protein encoded by the DNA.

The term "lignocellulosic" refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose.

The term "cellulosic" refers to a composition comprising cellulose and additional components, including hemicellulose.

The term "saccharification" refers to the production of fermentable sugars from polysaccharides.

The term "pretreated biomass" means biomass that has been subjected to physical, chemical and/or thermal pretreatment to increase accessibility of polysaccharides in the biomass prior to saccharification.

"Biomass" refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat straw, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.

"Biomass hydrolysate" refers to the product resulting from

saccharification of biomass. The biomass may also be pretreated or pre- processed prior to saccharification.

The term "xylose metabolic pathway" or "xylose utilization

pathway" refers to a series of enzymes (encoded by genes) that

metabolize xylose through to fructose-6-phosphate and/or

glyceraldehyde- 6- phosphate and include 1 ) xylose isomerase, which catalyses the conversion of xylose to xylulose; 2) xylulokinase, which phosphorylates xylulose to form xylulose 5-phosphate; 3) transketolase; and 4) transaldolase.

The term "xylose isomerase" refers to an enzyme that catalyzes the interconversion of D-xylose and D-xylulose. Xylose isomerases (XI) belong to the group of enzymes classified as EC 5.3.1 .5.

The term "E-value", as known in the art of bioinformatics, is

"Expect-value" which provides the probability that a match will occur by chance. It provides the statistical significance of the match to a sequence. The lower the E-value, the more significant the hit.

The term "ribose-5-phosphate isomerase" or "RPI" refers to an enzyme that catalyzes the interconversion of ribulose-5-phosphate and ribose-5-phosphate. Ribose-5-phosphate isomerases belong to the group of enzymes classified as EC 5.3.1 .6.

The term "ribulose-phosphate 3-epimerase" or "RPE" refers to an enzyme catalyzes the interconversion of D-ribulose 5-phosphate and D- xylulose 5-phosphate and is classified as EC 5.1 .3.1 .

The term "Z. mobilis RPI-A" refers to the Z. mobilis RPI which has been labeled in the art as RPI-A. However, the Z. mobilis RPI protein has closer sequence identity to the E. coli RPI-B protein (36%) than to the E. coli RPI-A protein (20%) and further analysis of RPIs described in commonly owned and co-pending US Patent Application 13/161734, published as WO2012/006061 , which is incorporated herein by reference, places the Z mobilis RPI in the RPI-B group. However, herein the Z.

mobilis RPI is called RPI-A to be consistent with its publicly known name.

The term "pnp gene" refers to a gene encoding polynucleotide phosphorylase, also called polyribonucleotide nucleotidyltransferase. This enzyme is bifunctional with a phosphorylitic 3' to 5' exoribonuclease activity and a 3' terminal oligonucleotide polymerase activity. It is involved with mRNA processing and degradation and is classified as EC 2.7.7.8.

The term "native amino acids" refers to amino acids as they occur in positions of the peptide sequence that is encoded by an endogenous gene.

The term "non-native amino acids" refers to amino acids in positions that are not encoded by an endogenous gene.

The term "N-terminal amino acid sequence" refers to amino acid sequence starting at the N-terminus of a polypeptide. The first N-terminal amino acid is counted as "1 ".

The term "heterologous" means not naturally found in the location of interest. For example, a heterologous gene refers to a gene that is not naturally found in the host organism, but that is introduced into the host organism by gene transfer. For example, a heterologous nucleic acid molecule that is present in a chimeric gene is a nucleic acid molecule that is not naturally found associated with the other segments of the chimeric gene, such as the nucleic acid molecules having the coding region and promoter segments not naturally being associated with each other.

As used herein, an "isolated nucleic acid molecule" is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is "hybridizable" to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989), particularly Chapter 1 1 and Table 1 1 .1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms).

Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45 °C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50 °C for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60 °C. Another preferred set of highly stringent conditions uses two final washes in 0.1 X SSC, 0.1 % SDS at 65 °C. An additional set of stringent conditions include hybridization at 0.1 X SSC, 0.1 % SDS, 65 °C and washes with 2X SSC, 0.1 % SDS followed by 0.1X SSC, 0.1 % SDS, for example.

Hybridization requires that the two nucleic acids contain

complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability

(corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51 ). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of

mismatches becomes more important, and the length of the

oligonucleotide determines its specificity (see Sambrook et al., supra, 1 1 .7-1 1 .8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more

polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: 1 .) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputinq:

Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in

Molecular Biology (von Heinje, G., Ed.) Academic (1987); and

5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991 ). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.

Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl). Multiple alignment of the sequences is performed using the "Clustal method of alignment" which encompasses several varieties of the algorithm including the "Clustal V method of alignment" corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151 -153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. Additionally the "Clustal W method of alignment" is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151 -153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8:189-191 (1992)) and found in the MegAlign™ v6.1 program of the

LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB ). After alignment of the sequences using the Clustal W program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities may include, but are not limited to: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 25% to 100% may be useful in describing the present invention, such as 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, and more preferably at least 150 amino acids.

The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1 .) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wl); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc.

Madison, Wl); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Ml); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 1 1 1 -20. Editor(s): Suhai, Sandor. Plenum: New York, NY). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J. and Russell, D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001 ); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et. al., Short Protocols in Molecular Biology, 5 ^th Ed. Current Protocols, John Wiley and Sons, Inc., N.Y., 2002.

The present invention relates to engineered cells of xylose-utilizing bacteria, and particularly Zymomonas or Zymobacter that have improved xylose utilization when fermented in xylose containing media. A challenge for improving ethanol production by fermentation of a biocatalyst in media that includes biomass hydrolysate, produced typically by pretreatment and saccharification of biomass, is obtaining optimal utilization of xylose.

Xylose is one of the predominant pentose sugars in hydrolyzed

lignocellulosic biomass, the other being arabinose. Applicants have discovered that modification of the endogenous pnp gene, in combination with increased expression of ribose-5-phosphate isomerase and in the presence of non-limiting xylose isomerase activity, in xylose-utilizing cells leads to increased efficiency in xylose utilization, and thus to higher ethanol yields when fermentation is in xylose containing media.

Endogenous pnp Gene Modification

The present invention is directed to cells of engineered xylose utilizing Zymomonas or Zymobacter that have a modified endogenous pnp gene. The coding region of the endogenous pnp gene encodes a protein with polynucleotide phosphorylase activity. The encoded protein is also called polyribonucleotide nucleotidyltransferase. Modifications in the coding region of the endogenous pnp gene were found herein to improve xylose utilization in cells of engineered Zymomonas that are additionally engineered as described below.

Any gene of Zymomonas or Zymobacter that is identified as encoding a protein with polynucleotide phosphorylase or polyribonucleotide nucleotidyltransferase activity may provide the target endogenous pnp gene for modification as described herein. The pnp coding region of Zymomonas mobilis strain ZM4 has the sequence of SEQ ID NO:1 . Known endogenous pnp coding regions from other strains of Zymomonas have sequences with identities to SEQ ID NO:1 of 99% (Z. mobilis NCIMB 1 1 163; SEQ ID NO:3), 98% (Z. mobilis ATCC 10988; SEQ ID NO:5), and 83% (Z. mobilis pomaceae ATCC 29192; SEQ ID NO:7). Any of these sequences, or any sequence with at least about 95%, 96%, 97%, 98%, or 99% identity to any one of these sequences and identified as encoding a polynucleotide phosphorylase or polyribonucleotide nucleotidyltransferase may be used as the target for modification as described below. Additional target endogenous pnp gene sequences may be identified using BLAST analysis or other sequence comparison analyses that are well known to one skilled in the art.

In the present cells the pnp coding region is modified to shorten the coding region at the 3' end resulting in expression of a C-terminal truncated protein, as compared to the naturally encoded protein. The native encoded polynucleotide phosphorylase of Zymomonas mobilis is a protein of about 748 amino acids, which is any of SEQ ID NOs:2, 4, 6, 8 or any sequence with at least about 95%, 96%, 97%, 98%, or 99% identity to any one of these sequences and identified as a polynucleotide

phosphorylase or polyribonucleotide nucleotidyltransferase. In one embodiment the truncated protein expressed from the modified pnp coding region retains at least about 350 amino acids of the N-terminal amino acid sequence encoded by the endogenous gene encoding polynucleotide phosphorylase, which are native N-terminal amino acids. The truncated protein retains at least about 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550. 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, or 710 of the native N-terminal amino acids.

In one embodiment, the genetic modification to the endogenous pnp coding region adds coding sequence for non-native amino acids adjacent to and in frame with the truncated native coding region so that a fusion protein is produced. For example, coding region for additionally between about 1 and about 20 amino acids, which are not encoded by the endogenous gene, may be added adjacent to and in frame with the truncated native coding region producing a fusion protein having up to about 20 non-native amino acids at the C-terminus. The C-terminal truncated protein is then part of the fusion protein. In various

embodiments there may be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more non-native amino acids at the C-terminus. Some non-limiting examples of fusion proteins that may result from genetic modification of an endogenous gene encoding polynucleotide phosphorylase include proteins that: 1 ) retain 709 native N-terminal amino acids and include 14 additional C-terminal amino acids (SEQ ID NO:9); 2) retain 695 native N-terminal amino acids and include 2 additional C- terminal amino acids (SEQ ID NO:10); 3) retain 368 native N-terminal amino acids and include 10 additional C-terminal amino acids (SEQ ID NO:1 1 ); and 4) retain 32 native N-terminal amino acids and include 17 additional C-terminal amino acids (SEQ ID NO:12).

The endogenous pnp coding region may be modified as described above by any method known to one skilled in the art. Typically the coding region is targeted by recombination targeting DNA sequences that are portions of the pnp coding region and may include surrounding adjacent genomic DNA. The recombination targeting DNA sequences direct insertion of DNA sequences bounded by them into the endogenous pnp gene by homologous recombination. In one embodiment the bounded DNA sequence includes a coding sequence for up to about 20 amino acids that is designed to be in reading frame with a position in the native pnp coding region, as described above, following integration by

homologous recombination. Alternatively, the entire native pnp coding region may be replaced, using homologous recombination, with a coding region designed to produce a protein with a C-terminal truncation as described above. In addition, the replacement coding region may encode additional non-native amino acids at the C-terminus of the protein as described above, resulting in production of a fusion protein. Engineered xylose utilizing Zymomonas or Zymobacter

The endogenous pnp gene is modified in a Zymomonas or

Zymobacter cell that contains a xylose metabolic pathway conferring the ability to utilize xylose for production of ethanol. Zymobacter palmae is an ethanol-producing bacterium that has been engineered for xylose utilization by expressing genes for xylose utilization as described below for Zymomonas, using Z. mobilis glyceraldehyde-3-phosphate

dehydrogenase and enolase promoters (Yanase et al. Applied and

Environmental Microbiology (2007) 73:2592-2599).

Strains of Zymomonas, such as Z. mobilis have been engineered for xylose fermentation to ethanol. Typically four genes have been introduced into Z mobilis for expression of four enzymes involved in xylose metabolism forming a xylose metabolic pathway in the cell (Figure 1 in bold) as described in U.S. Pat. No. 5514583, U.S. Pat. No. 5712133, U.S. Pat. No. 6566107, WO 95/28476, Feldmann et al. ((1992) Appl Microbiol Biotechnol 38: 354-361 ), and Zhang et al. ((1995) Science 267:240-243). These include genes encoding xylose isomerase which catalyzes the conversion of xylose to xylulose, and xylulokinase which phosphorylates xylulose to form xylulose 5-phosphate. Additionally expressed are transketolase and transaldolase, two enzymes of the pentose phosphate pathway that convert xylulose 5-phosphate to intermediates that couple pentose metabolism to the glycolytic Entner-Douderoff pathway permitting the metabolism of xylose to ethanol (see Figure 1 ). DNA sequences encoding these enzymes may be obtained from any of numerous microorganisms that are able to metabolize xylose, such as enteric bacteria, and some yeasts and fungi. Sources for the coding regions may include Xanthomonas, Klebsiella, Escherichia, Rhodobacter,

Flavobacterium, Acetobacter, Gluconobacter, Rhizobium, Agrobacterium, Salmonella, Pseudomonads, and Zymomonas. The coding regions of E. coli are typically used.

Endogenous genes may provide part of a xylose fermentation pathway, or may be altered by any known genetic manipulation technique to provide a protein with enzyme activity useful for xylose metabolism. For example, the endogenous transketolase may complement other introduced enzyme activities in creating a xylose utilization pathway.

Zymomonas or Zymobacter strains that are additionally engineered to utilize other sugars that, like xylose, are not natural substrates, may also be used in the present process. An example is a strain of Z. mobilis engineered for arabinose utilization is described in U.S. Pat. No. 5843760, which is incorporated herein by reference. Strains may be modified in other additional ways to improve xylose utilization and ethanol production. Increased Expression of RPI

The present cells are additionally engineered for increased expression of ribose-5-phosphate isomerase (RPI) activity. RPI catalyzes the interconversion of ribulose-5-phosphate and ribose-5-phosphate (see Figure 1 ). Increased expression of RPI is disclosed in commonly owned and co-pending US Patent Application 13/161734, published as

WO2012/006061 , which is incorporated herein by reference, which discloses increased RPI expression conferring increased efficiency of xylose utilization associated with reduced production of ribulose.

Increased RPI expression may be accomplished using any protein or polypeptide with ribose-5-phosphate isomerase activity in Zymomonas as disclosed in US Patent Application USSN 13/161734, published as WO2012006061 . Polypeptides having ribose-5-phosphate isomerase activity have the EC classification EC 5.3.1 .6. There are two groups of ribose-5-phosphate isomerase enzymes that are called RPI-A and RPI-B. The RPI-B enzymes belong to the RpiB/LacA/LacB family of sugar- phosphate isomerases. E. coli has both types of RPI proteins. Z. mobilis has a single RPI protein that is annotated as RPI-A. However, the Z.

mobilis RPI protein has closer sequence identity to the E. coli RPI-B protein (36%) than to the E. coli RPI-A protein (20%). Further analysis of RPIs disclosed in US Patent Application 13/161734, published as

WO2012/006061 , placed the Z mobilis RPI in the RPI-B group.

The sequences of RPI proteins that may be used in the present cells are very diverse as exemplified by the Z. mobilis and E. coli RPI proteins (SEQ ID NOs:14 and 16, respectively; coding regions SEQ ID NOs:13 and 15, respectively). RPI proteins that may be used in the present microorganisms may be identified using bioinformatics analysis. Identification using a structure/function bioinformatics analysis based on Profile Hidden Markov Modeling (using the hmmsearch algorithm of the HMMER software package; Janelia Farm Research Campus, Ashburn, VA), active site residue identification, and additional identifying amino acid screening is described in Example 8 of US Patent Application USSN 13/161734, published as WO2012/006061 .

Examples of RPI-A and RPI-B proteins that fit these criteria and that may be used in the present microorganisms are described in US Patent

Application USSN 13/161734, published as US20120156746A1 , which is incorporated herein by reference. Additional RPIs may be readily identified in the literature and in bioinformatics databases as is well known to the skilled person and as described above. Identification of protein and/or coding sequences using bioinformatics is typically through BLAST

(described above) searching of publicly available databases with RPI amino acid sequences or encoding sequences, such as those provided herein. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH

PENALTY=0.1 , and Gonnet 250 series of protein weight matrix.

In the present cell, a genetic modification is made which increases ribose-5-phosphate isomerase activity as compared to ribose-5-phosphate isomerase activity in the cell lacking the genetic modification. Increased expression of RPI activity may be obtained by expressing an isolated DNA molecule encoding a protein having ribose-5-phosphate isomerase activity that is active in the host cell. Useful proteins with ribose-5-phosphate isomerase activity belong to the EC classification EC 5.3.1 .6 and include ribose-5-phosphate isomerase A and ribose-5-phosphate isomerase B proteins that are described above.

Any method for increasing activity of an enzyme in a cell may be used to increase RPI activity. Such methods are well known to one skilled in the art and include increasing the encoding gene copy number and/or expression by a gene containing a high expression promoter. The present strains may be engineered for increased expression of an endogenous RPI coding region, and/or expression of an introduced heterologous RPI coding region to give increased enzyme activity. In addition, RPI activity may be increased by mutation and screening of expressed mutated genes to identify cells having increased activity.

Typically, increased expression of RPI is achieved by transforming with a DNA molecule encoding RPI that is operably linked to a promoter in a chimeric gene or operon. Coding sequences for RPIs that may be used include any sequences encoding the RPI-A and RPI-B proteins described above.

When using a heterologous coding region, the sequence may be codon-optimized for maximal expression in the target host cell, as well known to one skilled in the art. If the native start codon is GTG, it may be changed to ATG for increased protein expression. Methods for gene expression in bacteria are well known in the art. Expression of genes in bacteria typically requires a promoter, operably linked to a coding region of interest, and a transcriptional terminator. Promoters that may be used are promoters that are expressed in Zymomonas or Zymobacter cells such as the promoters of Z. mobilis glyceraldehyde-3-phosphate dehydrogenase (GAP promoter; P _gap ) gene, of Z. mobilis enolase (ENO promoter; Peno) gene, and of the Actinoplanes missouriensis xylose isomerase encoding gene (Gl promoter, Pgi). Particularly high expression promoters that may be used are the P _gap promoters with mutations causing high expression as disclosed in US Pat. 7,998,722, which is incorporated herein by reference.

A chimeric gene or operon for RPI expression is typically

constructed in or transferred to a vector for further manipulations. Vectors are well known in the art. Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors: pRK437, pRK442, and pRK442(H) are available. These derivatives have proven to be valuable tools for genetic manipulation in gram-negative bacteria (Scott et al., Plasmid 50(1 ):74-79 (2003)). Particularly useful for expression in Zymomonas are vectors that can replicate in both E. col Ί and Zymomonas, such as pZB188 which is described in U.S. Pat. 5,514,583, which is incorporated herein by reference. Vectors may include plasmids for autonomous replication in a cell, and plasmids for carrying constructs to be integrated into bacterial genomes. Plasmids for DNA integration may include transposons, regions of nucleic acid sequence homologous to the target bacterial genome, or other sequences supporting integration. An additional type of vector may be a transposome produced using, for example, a system that is commercially available from EPICENTRE ^® . It is well known how to choose an appropriate vector for the desired target host and the desired function.

Bacterial cells may be engineered by introducing a vector having a chimeric gene comprising an RPI coding region by well known methods, such as using freeze-thaw transformation, calcium-mediated

transformation, electroporation, or conjugation. Any bacterial cell to be engineered for improved xylose utilization by increasing expression of an RPI enzyme is a target host cell for transformation to engineer a strain as described herein. Particularly suitable host cells are Zymomonas and Zymobacter. The introduced chimeric gene may be maintained in the cell on a stably replicating plasmid, or integrated into the genome following introduction.

For engineering a strain with an integrated RPI chimeric gene or operon in the bacterial cell genome, methods may be used that are well known in the art such as homologous recombination, transposon insertion, or transposome insertion. In homologous recombination, DNA sequences flanking a target integration site are placed bounding a spectinomycin- resistance gene, or other selectable marker, and RPI chimeric gene leading to insertion of the selectable marker and the RPI chimeric gene into the target genomic site. In addition, the selectable marker may be bounded by site-specific recombination sites, so that after expression of the corresponding site-specific recombinase, the resistance gene is excised from the genome. In addition, the promoter of the endogenous RPI expressing gene may be replaced with a more highly expressed promoter to increase RPI activity in the cell. This may be accomplished by homologous

recombination using vectors and methods as described above.

Xylose isomerase activity

The present cells have a level of xylose isomerase activity that is not limiting. Xylose isomerase (XI) activity is not limiting when it is not the rate-limiting step for function of the xylose utilization and ethanol production pathways as shown in Figure 1 . Xylose isomerase activity is not limiting when an increase in xylose isomerase activity does not improve xylose utilization and ethanol production. This situation indicates that one or more other pathway steps are limiting. As found herein the xylose isomerase activity in ZW658 cells, measured using an assay including xylose, NADH, MgSO ₄ , triethanolamine, and sorbitol dehydrogenase that is described in Example 1 1 herein, is about 0.25 μπηοΐβε product/mg protein/minute. This level of xylose isomerase activity is not limiting in the ZW658 strain as described in example 3 herein. The ZW658 strain is a precursor to the ZW801 -4 strain used herein as described in the Zymomonas mobilis strain construction section, herein. In one embodiment, non-limiting XI activity is greater than about 0.25 μπηοΐβε product/mg protein/minute as measured in a cell free extract using the assay described in Example 1 1 herein. Non-limiting XI activity may be greater than about 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5 μηηοΐβε product/mg protein/minute.

When XI activity is limiting in a cell, the cell may be engineered to have non-limiting XI activity by increasing expression level of an XI enzyme or introducing expression of a more highly active XI enzyme.

Increasing expression level may be by any method known to one skilled in the art such as increasing copy number of a gene encoding XI or using a more highly active promoter to express an XI enzyme.

For example, an XI coding region may be expressed using a mutated promoter that has increased expression level as compared to the non-mutated promoter. An example of a mutated high expression promoter is the mutated promoter of the Z. mobilis glyceraldehyde-3- phosphate dehydrogenase gene that is disclosed in US 7,989,206 called herein the Super GAP promoter (P _gap s)-

Xylose isomerase enzymes that may provide high activity by expression in multiple copy or from a mutated high expression promoter are any of those described in US 7,998,722, which is incorporated herein by reference. As disclosed therein, xylose isomerases enzymes belong to EC 5.3.1 .5 and may be identified using a Profile HMM (described above for RPI) and in addition four catalytic site amino acids found to be characteristic of xylose isomerases.

Alternatively or in addition, high XI activity may be obtained by expressing an XI enzyme having higher activity in Zymomonas cells than the commonly used E. co// XI. Commonly owned and co-pending US Patent Application Publication US201 10318801 , which is incorporated herein by reference, discloses that xylose isomerase enzymes identified by EC 5.3.1 .5 and belonging to Group I have higher activity than the E. coli XI, which belongs to Group II. In addition, a Group I XI may be expressed from a mutated high expression promoter, as described above, to obtain high XI activity in a host cell.

Group I xylose isomerases as disclosed in US Patent Application

Publication US201 10318801 refers to a xylose isomerase protein that belongs to Group I as defined by at least one of the following criteria: a) it falls within a 50% threshold sequence identity grouping that includes the A. missouriensis XI that is prepared using molecular phylogenetic bioinformatics analysis as in Example 4 of US Patent Application

Publication US201 10318801 ; b) it substantially fits the amino acids for Group I in the specificity determining positions (SDP) identified using GroupSim analysis of the Group I and Group II XI sets determined from molecular phylogenetic analysis in Example 4 of US Patent Application Publication US201 10318801 ; and/or c) it has an E-value of 1 E-15 or less when queried using a Profile Hidden Markov Model prepared as described in Example 4 of US Patent Application Publication US201 10318801 . For example XI from Actinoplanes missouriensis (for example, SEQ ID NO:18) was identified as belonging to Group I and when expressed in

Zymomonas mobilis cells it provides higher activity than similarly expressed E. co// ^' XI.

Chimeric genes, vectors, transformation, integration, codon- optimization, and expression of XI is as described for RPI above and is well-known to one skilled in the art.

Increased Expression of RPE

In one embodiment the present cells are additionally engineered for increased expression of ribulose-phosphate 3-epimerase (RPE) activity. RPE catalyzes the interconversion of D-ribulose 5-phosphate and D- xylulose 5-phosphate (see Figure 1 ) and is classified as EC 5.1 .3.1 . At least one genetic modification is made that increases RPE activity in the cell as compared to RPE activity in the cell lacking the genetic

modification. Modifications for increased expression are as described above for RPI, and may use any enzyme belonging to EC 5.1 .3.1 with RPE activity. For example, additional copies of the Z. mobilis RPE (SEQ ID NO:20; coding sequence SEQ ID NO:19) may be expressed in the cell, or an RPE coding region may be expressed from a high expression promoter.

Fermentation of improved xylose-utilizing strain

The present engineered xylose-utilizing Zymomonas or Zymobacter cells may be used in fermentation to produce ethanol. As an example, production of ethanol by a Z. mobilis strain of the invention is described.

For production of ethanol, recombinant xylose-utilizing Z. mobilis having increased RPI activity, non-limiting XI activity, and endogenous pnp gene modification is brought in contact with medium that contains xylose. Xylose may be the sole sugar, but typically the medium contains a mixture of sugars including xylose and glucose. The medium may contain biomass hydrolysate that includes these sugars that are derived from treated cellulosic or lignocellulosic biomass.

When the mixed sugars concentration is high such that growth is inhibited, the medium includes sorbitol, mannitol, or a mixture thereof as disclosed in commonly owned US 7,629,156. Galactitol or ribitol may replace or be combined with sorbitol or mannitol. The Z. mobilis cells grow in the medium where fermentation occurs and ethanol is produced. The fermentation is run without supplemented air, oxygen, or other gases (which may include conditions such as anaerobic, microaerobic, or microaerophilic fermentation), for at least about 24 hours, and may be run for 30 or more hours. The timing to reach maximal ethanol production is variable, depending on the fermentation conditions. Typically, if inhibitors are present in the medium, a longer fermentation period is required. The fermentations may be run at temperatures that are between about 30 ° C and about 37 ⁰ C, at a pH of about 4.5 to about 7.5.

The present Z. mobilis cells may be grown in medium containing mixed sugars including xylose in laboratory scale fermenters, and in scaled up fermentation where commercial quantities of ethanol are produced. Where commercial production of ethanol is desired, a variety of culture methodologies may be applied. For example, large-scale production from the present Z. mobilis cells may be produced by both batch and continuous culture methodologies. A classical batch culturing method is a closed system where the composition of the medium is set at the beginning of the culture and not subjected to artificial alterations during the culturing process. Thus, at the beginning of the culturing process the medium is inoculated with the desired organism and growth or metabolic activity is permitted to occur adding nothing to the system. Typically, however, a "batch" culture is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the culture is terminated. Within batch cultures cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase are often responsible for the bulk of production of end product or intermediate in some systems.

Stationary or post-exponential phase production can be obtained in other systems. A variation on the standard batch system is the Fed-Batch system. Fed-Batch culture processes are also suitable for growth of the present Z. mobilis cells and comprise a typical batch system with the exception that the substrate is added in increments as the culture progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH and the partial pressure of waste gases such as CO2. Batch and Fed-Batch culturing methods are common and well known in the art and examples may be found in Biotechnology: A Textbook of Industrial Microbiology, Crueger, Crueger, and Brock, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA, or Deshpande, Mukund V., Appl. Biochem.

Biotechnol., 36, 227, (1992), herein incorporated by reference.

Commercial production of ethanol may also be accomplished with a continuous culture. Continuous cultures are open systems where a defined culture medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous cultures generally maintain the cells at a constant high liquid phase density where cells are primarily in log phase growth. Alternatively, continuous culture may be practiced with immobilized cells where carbon and nutrients are continuously added, and valuable products, by-products or waste products are continuously removed from the cell mass. Cell immobilization may be performed using a wide range of solid supports composed of natural and/or synthetic materials as is known to one skilled in the art.

Continuous or semi-continuous culture allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by medium turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to medium being drawn off must be balanced against the cell growth rate in the culture. Methods of modulating nutrients and growth factors for continuous culture processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial

microbiology and a variety of methods are detailed by Brock, supra.

Particularly suitable for ethanol production is a fermentation regime as follows. The desired Z. mobilis cells of the present invention are grown in shake flasks in semi-complex medium at about 30 °C to about 37 °C with shaking at about 150 rpm in orbital shakers and then transferred to a 10 L seed fermentor containing similar medium. The seed culture is grown in the seed fermentor anaerobically until OD ₆ oo is between 3 and 6, when it is transferred to the production fermentor where the fermentation parameters are optimized for ethanol production. Typical inoculum volumes transferred from the seed tank to the production tank range from about 2% to about 20% v/v. Typical fermentation medium contains minimal medium components such as potassium phosphate (1 .0 - 10.0 g/L), ammonium sulfate (0- 2.0 g/L), magnesium sulfate (0 - 5.0 g/L), a complex nitrogen source such as yeast extract or soy based products (0 - 10 g/L). A final concentration of about 5 mM sorbitol or mannitol is present in the medium. Mixed sugars including xylose and at least one additional sugar such as glucose (or sucrose), providing a carbon source, are continually added to the fermentation vessel on depletion of the initial batched carbon source (50-200 g/L) to maximize ethanol rate and titer. Carbon source feed rates are adjusted dynamically to ensure that the culture is not accumulating glucose in excess, which could lead to build up of toxic byproducts such as acetic acid. In order to maximize yield of ethanol produced from substrate utilized, biomass growth is restricted by the amount of phosphate that is either batched initially or that is fed during the course of the fermentation. The fermentation is controlled at pH 5.0 - 6.0 using caustic solution (such as ammonium hydroxide, potassium hydroxide, or sodium hydroxide) and either sulfuric or phosphoric acid. The temperature of the fermentor is controlled at 30 °C - 35 °C. In order to minimize foaming, antifoam agents (any class- silicone based, organic based etc) are added to the vessel as needed. An antibiotic, for which there is an antibiotic resistant marker in the strain, such as kanamycin, may be used optionally to minimize contamination.

Any set of conditions described above, and additionally variations in these conditions that are well known in the art, are suitable conditions for production of ethanol by xylose-utilizing recombinant Zymomonas cells. EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

GENERAL METHODS

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989)

(hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience, Hoboken, NJ (1987).

The meaning of abbreviations is as follows: "kb" means

kilobase(s), "bp" means base pairs, "nt" means nucleotide(s), "hr" means hour(s), "min" means minute(s), "sec" means second(s), "d" means day(s), "L" means liter(s), "ml" means milliliter(s), "μί" means microliter(s), "μ9" means microgram(s), "ng" means nanogram(s), "mM" means millimolar, "μΜ" means micromolar, "nm" means nanometer(s), "μηηοΙ" means micromole(s), "pmol" means picomole(s), "Cm" means chloramphenicol, "Cm ^r or "Cm-R" means chloramphenicol resistant, "Cm ^s " means chloramphenicol sensitive, "Spec ^r " or "Spec-R" means spectinomycin resistance, "Sp ^s " means spectinomycin sensitive, "XI" is xylose isomerase, "XK" is xylulokinase, "TAL" is transaldolase, "TKT" is transketolase, "OD600" means optical density measured at a wavelength of 600 nm, "PCR" means polymerase chain reaction, "kDa" means kilodaltons, "g" means the gravitation constant, "bp" means base pair(s), "kbp" means kilobase pair(s), "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography, "RM" means rich medium containing 10 g/L yeast extract plus 2 g/L KH ₂ PO ₄ , "MM" means mating medium containing 10 g/L yeast extract, 5 g/L tryptone, 2.5 g/L (NH ₄ ) ₂ SO and 0.2 g/L KH ₂ PO ₄ .

Transformation of Z. mobilis

Replicating and non-replicating plasmid DNA was introduced into Z. mobilis using electroporation, essentially as described in US 5,514,583, which is incorporated herein by reference. Briefly, the 50-μΙ transformation reactions contained ~10 ¹⁰ cells/ml in 10% (v/v) glycerol and -0.5-2.0 μg of non-methylated plasmid DNA that was isolated from E. coli SCS1 10.

Control reactions were treated identically, but did not receive any plasmid DNA. The settings for the electroporator were 16 kv/cm, 200 Ω, and 25 μΡ, and the gap width of the cuvette was 0.1 cm. Following electroporation, the transformation reactions were diluted with MMG medium (50 g/L glucose, 10 g/L yeast extract, 5 g/L of tryptone, 2.5 g/L of (NH ₄ ) ₂ SO , 0.2 g/L K ₂ HPO , and 1 mM MgS0 ) and the cells were allowed to recover at 30 °C before they were plated on MMG medium that contained 1 .5% agar (MMG agar plate) with or without antibiotics as indicated. Plates were incubated in an anaerobic chamber at 30 °C, until colonies appeared. Additional details are described in the Examples below.

Shake flask experiments

Unless otherwise noted, all experiments described below were conducted at 30 °C in shake flasks (15-ml loosely-capped, conical shaped test tubes) using synthetic media that contained glucose or xylose as the sole carbon source. mRM3-G10 medium contains 10 g/L yeast extract, 2 g/L KH ₂ PO ₄ , 1 g/L MgSO ₄ (7H ₂ 0) and 100 g/L glucose. mRM3-X10 medium is identical but it contains 100 g/L xylose instead of glucose. Cell growth was monitored spectrophometrically by following changes in optical density at 600 nm as a function of time. In the text and figure legends "OD" or "OD600" means optical density at 600 nm. At indicated times during the shake flask growth studies, 1 .0-ml aliquots of the cultures were removed for HPLC analysis using an Agilent 1 100 equipped with a refractive index detector (Hewlett-Packard, Palo Alto, CA) to determine the concentrations of glucose, xylose, ribulose, and ethanol that were present in the fermentation broth. Prior to HPLC analysis, cells were removed by centrifugation and the supernatant was filtered through a 0.22 μηη

cellulose acetate Spin-X centrifuge tube filter (Costar, catalog number 8160) to remove small particles. Compounds were separated on an

Aminex HPX-87H column (Bio-Rad) that was run at 55 °C under isocratic conditions using a flow rate of 0.6 ml/min and 0.01 N H ₂ S0 as the mobile phase. Authentic standards of known concentration were used to quantify the peaks of interest and all results are expressed in g/L.

Zymomonas mobilis strain construction

A detailed description of the construction of the xylose-utilizing recombinant strain, ZW801 -4, starting from the wild type parent strain, ZW1 , is provided in US 7,741 ,1 19, which is herein incorporated by reference. Strain ZW801 -4 was derived from strain ZW800, which was derived from strain ZW658, all as described in US 7,741 ,084, which is herein incorporated by reference. ZW658 was constructed by integrating two operons, P _gap xylAB and P _gap taltkt, containing four xylose-utilizing genes encoding xylose isomerase (xylA), xylulokinase (xylB),

transaldolase (tal), and transketolase (tkt), with coding regions from E. coli genes, into the genome of ZW1 (rename of strain ZM4; ATCC 31821 ) via sequential transposition events followed by adaptation on selective medium containing xylose to produce strain X13L3, which was renamed ZW641 . Further adaptation of ZW641 on xylose-containing growth media gave rise to ZW658, which grows much better in xylose and was deposited under the Budapest Treaty as ATCC PTA-7858. As disclosed in commonly owned US 7,989,206, which is herein incorporated by reference, ZW658 has much more xylose isomerase activity due to a point mutation in the promoter (P _gap ) expressing the xylA coding region. This promoter (SEQ ID NO:21 ), herein called either the 801 GAP promoter or the Super GAP promoter or P _gap s, has a "T" instead of "G" in position 1 16 in SEQ ID NO:21 , when compared to the native P _gap in ZW641 (the 641 GAP promoter). The P _gap s has expression strength 3 to 4 times higher than the P _gap in Z. mobilis.

In ZW658, the gene encoding glucose-fructose oxidoreductase was insertionally-inactivated using host-mediated, double-crossover, homologous recombination and spectinomycin resistance as a selectable marker to create strain ZW800. The spectinomycin resistance marker, which was bounded by loxP sites, was removed by site specific

recombination using Cre recombinase to create strain ZW801 -4.

Preparation of Cell-Free Extracts of Zymomonas for Enzymatic Assay

Cells were grown in 50 ml of RM + 2% glucose at 30°C overnight to an OD ₆ oo of 1 .0-1 .2. Cells were harvested by centrifugation at 4500 rpm for 10 min at 4°C. The supernatant was discarded and the cell pellet washed with 25 ml ice-cold sonication buffer (10 mM Tris, pH 7.6, 10 mM MgC ), followed by centrifugation at 4500 rpm for 10 min. The pellet was resuspended in 2.0-2.5 ml sonication buffer plus 1 mM dithiothreitol. A 500 μΙ_ aliquot was centrifuged for 1 min in an eppendorf centrifuge at 4°C. Most of supernatant was discarded, leaving about 10-20 μΙ_ behind to keep the pellet from drying out. The cells were frozen and stored at about 80 °C until assayed. Prior to assay, the cells were thawed and

resuspended with 500 μΙ_ of sonication buffer plus 1 mM dithiothreitol. The mix was sonicated 2x for 45 seconds at 62% duty cycle and an output control of 2 using a Branson sonifier 450, letting samples cool about 3-5 min between sonications. Samples were centrifuged at14,000 rpm for 60 min in a Beckman microfuge at 4°C. The supernatant was transferred to a new tube and kept at 4°C. The Pierce BCA assay was used for determining protein concentrations.

Example 1

Construction of pMODIinker-Spec-GapRPi pMODLinker-Spec-GapRpi (Figure 2) is a plasmid that can be used to generate a transposon that contains a Z. mobilis RPI expression cassette and a lox-flanked Spec-resistance cassette. The Z. mobilis RPI expression cassette was generated by PCR using plasmid

pZB188aadA/Gap/Zymo RPI/EcoliSL as a template. The latter plasmid is described in US 7,989,206, which is incorporated herein by reference. It contains an expression cassette for the Z. mobilis ribose 5-phosphate isomerase (RPI), that consists of (from 5' to 3') a full-length 641 GAP promoter sequence, the entire open reading frame of the Z. mobilis RPI gene (SEQ ID NO:13), and the XylA stem-loop region that is present in the intergenic region of the E. coli XylA B operon. The entire RPI expression construct described above (SEQ ID NO:22) is located between the unique Ncol and Notl sites of plasmid pZB188aadA/Gap/Zymo RPI/EcoliSL.

A PCR-generated DNA fragment containing this expression cassette was inserted into pMOD-Linker-Spec, which is also described in US 7,989,206. pMOD-Linker-Spec was derived from the commercially available vector pMOD™-2<MCS> Transposon Construction Vector (Cat. No. MOD0602; EPICENTRE ^® Biotechnologies, Madison, Wl). The original multi-cloning site was replaced with unique restriction sites for AsiSi, Fsel and Sbfl. A DNA fragment that confers resistance to spectinomycin (Spec ¹ ) and has a wild type loxP site at both ends was inserted between the AsiSI and Fsel sites creating pMOD-Linker-Spec.

pMOD-Linker-Spec was sequentially digested with Sbfl and Fsel, and the 3.6 kb large vector fragment was purified from a 1 % agarose gel. Next, the Z. molilis RPI chimeric gene with its associated 641 GAP promoter and E. coli Xyl A terminator was PCR amplified from plasmid pZB188aadA/Gap/Zymo RPI/EcoliSL using primer PPI-F (SEQ ID NO:23) and primer PPI-R-Sbfl (SEQ ID NO:24). The resulting 0.96 kbp PCR product was also cut with Fsel and Sbfl, and the purified DNA fragment was then ligated to the Fsel/Sbfl-digested, pMOD-Linker-Spec vector fragment to yield pMODIinker-Spec-GapRpi, shown in Figure 2.

Example 2

Overexpression of RPI in ZW801 -4: Generation and

Characterization of the I strain

A transposon generated from pMODIinker-Spec-GapRpi (Example 1 ) was introduced into strain ZW801 -4 (see General Methods) to increase RPI expression. The transposable element in this plasmid that randomly integrates into DNA after conversion to a transposome is the entire DNA fragment that is situated between two mosaic ends (ME) in the vector, which includes both the Z. mobilis RPI expression cassette and the Spec ^r - cassette. The transposome was generated in vitro essentially as

described in US 7, 989,206 using the general protocol that is outlined in the EPICENTRE ^® instruction manual for the EZ::TN™pMOD™-2<MCS> Transposon Construction Vector (Cat. No. MOD0602). The resulting transposome was electroporated into ZW801 -4 cells and transformants were recovered on MMG and MMX (same medium with 50 g/L xylose instead of 50 g/L glucose) agar plates that contained spectinomycin (200 Mg/ml). Since the transposable element randomly inserts into DNA, which can result in detrimental gene disruption events and/or position effects that alter the expression level of the integrated RPI gene, a preliminary experiment was conducted to determine which transformants were best to pursue. Thus, 30 colonies from the mRM3-G10/Spec plates and 6 from the mRM3-X10/Spec plates were screened for growth in shake flask experiments (see General Methods) using mRM3-X10 as the test medium, and the three strains that grew to the highest final OD values (strains B9, B1 1 and I) were selected for further characterization. Strains B9 and B1 1 were recovered from the MMG/Spec plates and the I strain was recovered from a MMX Spec plate.

In commonly owned and co-pending US Patent Application USSN 13/161734, published as US20120156746A1 , which is incorporated herein by reference, RPI was disclosed as being the rate-limiting enzyme for xylose metabolism in ZW801 -4, and expressing RPI from a multicopy plasmid in this strain resulted in better growth and faster xylose utilization in shake flask experiments with mRM3-X10. Although cells grew to a higher final OD value with additional RPI there was little or no change in the exponential growth rate. Plasmid overexpression of RPI in ZW801 -4 also reduced the amount of ribulose that was produced, and this resulted in a higher metabolic yield for ethanol production from xylose.

As shown in Figure 3, similar results were obtained for the tested strains having a single copy of the RPI expression transposon randomly integrated into the ZW801 -4 genome. Seed cultures of the B9, B1 1 , and I strains, along with the ZW801 -4 control, were grown in mRM3-X10 and used to inoculate mRM3-X10 in shake flasks to an initial OD600 value of -0.1 for each culture. The cultures were grown at 30 °C and assayed over time for growth by OD600, and for xylose, ribulose, and ethanol as described in General Methods. As shown in Figure 3A, the three strains that contain the RPI expression transposon grew to a nearly 50% higher OD than the parent strain, ZW801 -4. As shown in Figure 3B, they also consumed xylose at a much faster rate and produced less ribulose than the control strain: at the 54-hr time point, nearly all of the xylose was gone for B9, B1 1 and the I strain, while the parent strain ZW801 -4 had only used -75% of the xylose. At a 71 -hr time point there was still -8 g/L residual xylose for ZW801 -4. The three strains with the RPI expression transposon also produced less ribulose than ZW801 -4 despite the fact that they consumed more xylose; the final concentration of ribulose in the growth media at the 72-hr time point for ZW801 -4 was 5.3 g/L versus 3.2- 3.6 g/L for the other three strains.

The same 4 strains were grown in mRM3-G10 medium at 30 °C and assayed over time for growth by OD600 (Figure 3C). In contrast to the results obtained with xylose, all four strains showed similar growth rates when grown on glucose, reaching an OD600 of about 1 at 8 hr and 5.2 - 5.5 at 20 hr. This result suggests that the stimulatory effect of RP1 overexpression on growth in the xylose shake flask experiments with B9, B1 1 and the I strain is carbon source dependent. Example 3

Overexpression of E. coli xylose isomerase in B9, B1 1 and I strains Although B9, B1 1 and I strains performed very similarly in the shake flask experiments with glucose and xylose as described above, subsequent experiments revealed an important difference between the I strain and the other two strains that also have an integrated copy of the RPI expression transposon. This difference was found by assessing whether higher expression of xylose isomerase in the 3 strains would result in a further increase in the rate of carbon flux through the

engineered xylose pathway.

Xylose isomerase was the rate-limiting enzyme for xylose metabolism in strain ZW641 , which was overcome in strain ZW658 by a point mutation in the P _gap promoter that drives the E. coli XylA/B operon, which resulted in increased expression of xylose isomerase (see strain construction in General Methods). As disclosed in commonly owned and co-pending US Patent Application USSN 13/161734, published

US20120156746A1 subsequent experiments established that the new rate-limiting step for xylose metabolism in both ZW658 and ZW801 -4 (a ZW658 derivative having glucose-fructose oxidoreductase gene

inactivation) was RPI.

Based on these observations it was of interest to determine whether elimination of the RPI bottleneck, as provided by increased RPI expression in the B9, B1 1 and I strains, would allow a higher level of expression of xylose isomerase to result in a further increase in the rate of carbon flux through the xylose pathway. To test this hypothesis we used a transposon that was generated from the plasmid pMODIinker-Cm- 801 GapXylA. This plasmid is identical to pMOD-Linker-Spec-801 GapXylA that is described in US 7,989,206 except that it has a Cm-resistance cassette between the two lox sites instead of a Spec-resistance cassette. pMOD-Linker-Spec-801 GapXylA is pMOD-Linker-Spec with an added DNA fragment that was obtained from ZW801 -4 that contains the Pgap, the XylA coding region, and the stem-loop region that is between the XylA and XylB open reading frames. Thus the promoter that drives XylA is the 801gap promoter (SEQ ID NO:21 ), which has the G to T point mutation at position 1 16 that makes it more active, and is the same mutant promoter that drives the E. coli XylA/B operon in ZW658.

In addition to the Cm-resistance cassette, the transposon that was generated from pMODIinker-Cm-801 GapXylA (referred to below as the "801 GapXylA-Cm transposon") contains the above xylose isomerase expression cassette that consists of the 801 GAP promoter, the E. coli XylA open reading frame and the stabilizing XylA stem-loop region that immediately follows the stop codon as described in detail in US 7,989,206. The 801 GapXylA-Cm transposon was converted to a transposome as described in Example 2 and the latter was electroporated into the I strain. Transformants were selected on MMG agar plates that contained chloramphenicol (120 μg ml).

In a preliminary study, ten randomly selected Cm ^r colonies that were recovered from the transformation reaction with the 801 GapXylA-Cm transposon were tested for growth in mRM3-X10 at 30 °C in a shake flask experiment where OD600 was measured periodically. Although all ten strains grew better in xylose than the parent strain (based on initial rates of growth and final OD values), only seven of them were selected for further characterization: I(cm1 ), I(cm2), I(cm3), I(cm4), I(cm5), I(cm8) and I(cm9). To examine these strains in greater detail, the shake flask experiment was repeated using seed cultures that were grown in mRM3- X10, and ZW801 -4 and the I strain were included as controls. The initial OD was 0.075 in all cases and the two control strains were run in duplicate.

Consistent with previous experiments the I strain, which has a higher expression level of RPI, grew much better in xylose than ZW801 -4, as shown in Figure 4A. Note that growth curves for the I strain duplicates were superimposable and cannot be distinguished. The final

concentration of ribulose in the growth medium, at the 56-hr time point, was lower for the I strain than for ZW801 -4: 1 .8 andl .9 g/L versus 3.4 and 4.2 g/L. Over-expression of xylose isomerase in the I strain greatly improved growth on xylose. All seven strains that contained the

801 GapXylA-Cm transposon grew much faster than ZW801 -4 and the I strain, and their growth curves were virtually identical despite the fact that they have different transposon insertion sites. This was actually confirmed by DNA sequencing of the insertion sites for I(cm1 )-l(cm6). Although the transposon insert sites were not determined for the other four strains they are probably also in different genomic locations due to the extremely low transformation frequency of Z. mobilis and the EPICENTRE ^® procedure that was used.

Increased expression of xylose isomerase enzyme activity in the I strain also increased the exponential growth rate, and this resulted in a more rapid build up of biomass and faster xylose utilization. End point values for ribulose for the seven strains that contained the 801 GapXylA- Cm transposon ranged from 2.9-3.7 g/L. Although these values are similar to the amount of ribulose that ZW801 -4 produced, they are actually smaller on a per sugar used basis since at the 56-hr time point the strains that over-expressed xylose isomerase had consumed all of the xylose in the medium while ZW801 -4 had used less than 80%.

These results clearly demonstrate that xylose isomerase is the rate- limiting enzyme for xylose metabolism in the I strain. They also reveal that the I strain has sufficient RPI enzyme activity to support a much higher expression level of xylose isomerase. The fact that all seven I strain derivatives that contained the 801 GapXylA-Cm transposon in different locations in the chromosome grew with the same kinetics in xylose containing medium strongly suggests that gene disruption events and/or position effects caused by the transposon did not significantly contribute to the observed phenotype (i.e. better growth in xylose). This conclusion is also supported by the experiment that is shown Figure 4B where the seven strains that contained the transposon all grew just as well as ZW801 -4 and the I strain in shake flask experiments with mRM3-G10 at 30 °C.

The above observations were confirmed using pH-controlled conditions as follows. During shake flask experiments the pH of the growth medium can drop by greater than 1 pH unit, and this has an inhibitory effect on Z. mobilis growth especially when xylose is the sole carbon source. To circumvent this problem the pH-controlled bioreactor experiment that is shown in Figure 5 was performed. Since all of the I strain derivatives that over-express xylose isomerase behaved the same in shake flasks, only two of them were used for this experiment, I(cm1 ) and l(Cm9). Seed cultures were grown in mRM3-X10 at 30 °C in shake flasks to an OD of -0.5, and the starting OD in the bioreactors was 0.035. The bioreactors also contained mRM3-X10, and temperature and pH were held constant at 30 °C and 5.8, respectively, using 2N KOH for pH control.

The growth curves for the I strain and ZW801 -4 were very similar under pH-controlled conditions (Figure 5), although it was clear that the I strain did grow slightly better toward the end of the experiment.

Interestingly, however, neither strain produced much ribulose under these conditions; endpoint values for ZW801 -4 and the I strain were 1 .92 and 1 .16 g/L, respectively. These observations suggest that RPI is not as big a bottleneck for xylose metabolism in ZW801 -4 when the pH of the growth is maintained at 5.8. They also indicate that the native RPI gene in ZW801 -4 is almost able to keep pace with the existing level of xylose isomerase activity under such conditions. Nevertheless, it was clear from the growth curves that are shown in Figure 5 that over-expressing xylose isomerase in the I strain greatly improved xylose metabolism even when the pH of the growth medium was held constant at pH 5.8.

The 801 GapXylA-Cm transposon was also electroporated into the B9 and B1 1 strains using the same procedure as used for the I strain, and ten primary transformants for each of these strains were tested for growth in xylose in shake flask experiments with mRM3-X10 at 30 °C (Figure 6). Although some of the transformants did grow better in xylose than the parent strain, in both cases the results were not nearly as dramatic as those that were obtained with the I strain. There was also a high degree of variation between the different transformants, suggesting that gene disruption events and/or insertional position effects were contributing to the observed phenotypes. These observations suggested that there might be something significant about the chromosomal location of the RPI expression transposon in the I strain that is beneficial to xylose growth and metabolism in addition to an elevated expression level of RPI.

The site of insertion for the RPI expression transposon in the I strain was determined by DNA sequencing to be between nts 543506 and 543507 of the Z. mobilis genome (GenBank accession number

AE008692). Sequencing of the insertion region showed that integration of the transposon caused a frame shift at the 3' end of the open reading frame of the pnp gene that codes for polyribonucleotide nucleotidyl transferase. An alignment of the wild type pnp gene product and the predicted pnp gene product for the I stain is shown in Figure 7. Note that the mutant protein is missing the last 39 amino acid residues of the native protein (retains 709 amino acids starting from the N-terminus) and has 14 new amino acids at its C-terminus (SEQ ID NO:9). It is not known if this hypothetical protein is functional, and if it is, to what extent it might contribute to the I strain phenotype in addition to RPI overexpression. Regardless of the answer, the above results indicate that overexpressing RPI at the same location as in the I strain, in the pnp coding region, resulted in better growth in xylose and allowed a higher expression level of xylose isomerase to be effective.

Example 4

Vector constructs for building xylose utilizing Z. mobilis strains using targeted integration

A new xylose utilizing Z. mobilis strain was constructed by introducing chimeric xylA, xylB, tal, and tkt genes into the ZW1 strain. The xylB, tal, and tkt coding regions were from E. coli genes as in the ZW658 strain described in General Methods. The xylA coding region was from Actinoplanes missouriensis (AMxylA) which is disclosed in commonly owned and co-pending US Patent Application Publication

US201 10318801 , which is incorporated herein by reference, as encoding an enzyme having higher activity than the E. coli xylose isomerase in Z. mobilis. The coding region for the AMxylA was codon optimized for expression in Z. mobilis (SEQ ID NO:17). Additional copies of Z. mobilis rpi and rpe genes were also introduced in order to increase ribose-5- phosphate-isomerase (RPI) and ribulose-phosphate 3-epimerase (RPE) activities. Double crossover (DCO) transformation vectors were designed to specifically integrate the chimeric genes into target regions in Z. mobilis genome.

Standard molecular recombination methods were used to construct DCO (double cross over) suicide integration vectors. To express xylose isomerase and xylulose kinase in Z. mobilis, a 10,250-bp DCO suicide vector pZX21 (SEQ ID NO:25; Figure 8A) was constructed. This vector has a pBluescript backbone which contains a replication origin for E. coli but no Z. mobilis replication origin, thus it cannot be propagated in Z.

mobilis making it a suicide vector. It contains DNA sequences from the Z mobilis gene encoding glucose-fructose oxidoreductase, GFO-L and GFO- R, flanking the sequences to be integrated. Both fragments were synthesized by PCR, using Z. mobilis genomic DNA as template. The 1 ,186-bp GFO-L fragment (SEQ ID NO:26) includes the first 654 bp (from nt-1 to nt-653) of the gfor coding sequence (SEQ ID NO:27) and 533 bp of upstream genomic sequence. The 1 ,446-bp GFO-R fragment (SEQ ID NO:28) includes the last 480 bp (from nt-823 to nt-1302) of the GFOR coding sequence and 966 bp of downstream genomic sequence. The GFO-L and GFO-R sequences direct integration into the gfor locus, replacing a segment of the gfor coding sequence (from nt-655 to nt-822) in the Z. mobilis genome. This disrupts expression of glucose-fructose oxidoreductase, which reduces xylitol production and increases ethanol production as disclosed in US 7,741 ,1 19, which is incorporated herein by reference.

The region in pZX21 between GFO-L and GFO-R includes three chimeric genes. One is a 1 ,661 -bp chimeric xylA gene (SEQ ID NO:29) containing the 304-bp Z. mobilis Super GAP promoter (P _gap s; described in US 7,989,206), a 1 ,185-bp A. missouriensis xylA coding sequence

(AMxylA) and a 166-bp E. coli araD 3'UTR with a 5' Xbal site (ECaraD 3'UTR). The AMxylA coding region was optimized for expression in Z. mobilis according to codon bias of Z. mobilis ZM4 (SEQ ID NO:17). The ECaraD 3'UTR was from the E. coli araBAD operon. The second gene is a 1 ,960-bp chimeric xylB gene (SEQ ID NO:30) containing a 191 bp P _en o, a 1 ,455-bp E. coli xylB coding sequence (ECxylB) and a 314-bp E.coli xylB 3'UTR (ECxylB 3'UTR). P _eno is a strong constitutive promoter from the Z. mobilis genomic DNA having approximately 28% activity of P _gap . The third gene is a 1 ,014 bp aadA marker (for spectinomycin resistance; Spec-R) bounded by lox sites (SEQ ID NO:31 ). The marker can be removed after integration by expressing Cre recombinase.

To express transaldolase, transketolase, ribose-5-P-isomerase, and D-ribulose-P-3-epimerase in Z. mobilis, a 12,198-bp DCO shuttle vector pZX52 (SEQ ID NO:32; Figure 8B) was constructed. This vector is a Zymomonas-E. coli shuttle vector which is based on the vector pZB188 (Zhang et al. (1995) Science 267:240-243; US 5,514,583), which includes a 2,582 bp Z. mobilis genomic DNA fragment containing a replication origin allowing the vector to replicate in Zymomonas cells, and a 909-bp E. coli replication origin (Ori). It has a 91 1 bp chloramphenicol resistance marker (Cm-R) for selection of either E. coli or Z. mobilis transformants. pZX52 contains DNA sequences from the Z mobilis IdhA gene encoding lactate dehydrogenase, LDH-L (875 bp; SEQ ID NO:33) and LDH-R (1 ,149 bp; SEQ ID NO:34), flanking the sequences to be integrated.

These sequences direct integration into the IdhA coding sequence (SEQ ID NO:35) in the Z. mobilis genome between nucleotides 493 and 494, thereby disrupting expression of lactate dehydrogenase.

The region in pZX52 between LDH-L and LDH-R includes two chimeric operons. The first one is a 3,339 bp P _gap T-Tal-Tkt operon (SEQ ID NO:36) containing a 304-bp T-mutant of the Z. mobilis GAP promoter (Pgap-r), a 954-bp E. coli Tal coding region (ECTal), a 1 ,992-bp E. coli Tkt coding region, and a 68-bp E. coli Tkt 3'UTR (ECTkt 3'UTR). This operon is identical to the naturally existing E. coli Tal-Tkt operon except for the PgapT promoter (SEQ ID NO:37), which is a P _gap with a "G" to an "A" change at position 83 in SEQ ID NO:21 and a "T" missing at position 285. The other chimeric operon is a 1 ,443 bp P _eno -Rpi-Rpe operon (SEQ ID NO:38), containing a 191 bp P _en o, a 471 bp Z. mobilis Rpi coding sequence with first codon changed to ATG (SEQ ID NO:65) (ZMRpi), a 663 bp Z. mobilis Rpe coding sequence (ZMRpe), and a 35 bp E.coli xylA 3'UTR (ECxylA 3'UTR).

Another DCO shuttle vector named pZX6 (SEQ ID NO:39; Figure

8C) was constructed. This 12,704 bp vector is a modification of pZX52 having LDH-L and LDH-R sequences replaced with sequences from the Z mobilis pnp gene encoding polynucleotide phosphorylase. The 1 ,318 bp PNP-L fragment (SEQ ID NO:40) is a segment of the pnp coding sequence (SEQ ID NO:1 ) from nt-767 to nt-2,084, while the 1 ,225 bp

PNP-R fragment (SEQ ID NO:41 ) includes the last 59 bp (from nt-2189 to nt-2247) of the pnp coding sequence and 1 ,166 bp of downstream genomic sequence. Therefore, pZX6 is able to direct integration of the PgapT-Tal-Tkt operon and the P _eno -Rpi-Rpe operon into the endogenous pnp gene near the end of the pnp coding sequence and replace a segment of the pnp coding sequence (from nt-2,084 to nt-2,188) in the Z. mobilis genome.

Example 5

Development of xylose utilizing Z. mobilis strains

The ZW1 strain was transformed with two plasmids in two steps. Competent cells of ZW1 were prepared by growing seed cells overnight in mRM3-G5 (1 % yeast extract, 15 mM KH ₂ PO ₄ , 4 mM MgSO ₄ , and 50 g/L glucose) at 30 °C with 150 rpm shaking, to an OD ₆ oo value near 5. Cells were harvested and resuspended in fresh medium to an OD ₆ oo value of 0.05. The cells were grown under the same conditions to early to middle log phase (OD ₆ oo near 0.5). Cells were harvested and washed twice with ice-cold water and then once with ice-cold 10% glycerol. The resulting competent cells were collected and resuspended in ice-cold 10% glycerol to an OD ₆ oo value near 100. Since transformation of Z. mobilis requires non-methylated DNA, DCO plasmids pZX21 , pZX52, and pZX6 were each transformed into E. coli SCS1 10 competent cells (Stratagene, La Jolla, CA). For each transformation, one colony of transformed cells was grown in 10 mL LB-Amp100 (LB broth containing 100 mg/L ampicillin) overnight at 37 °C. DNA was prepared from the 10 mL culture, using QIAprep Spin DNA Miniprep Kit (Qiagen).

Approximately 1 μg non-methylated pZX21 DNA was mixed with 50 μΐ ZW1 competent cells in a 1 MM Electroporation Cuvette (VWR, West Chester, PA). The plasmid DNA was electroporated into the cells at 2.0 KV using a BT720 Transporater Plus (BTX-Genetronics, San Diego, CA). Transformed cells were recovered in 1 mL MMG5 medium (10 g/L glucose, 10 g/L yeast extract, 5 g/L tryptone, 2.5 g/L (NH ₄ ) ₂ SO ₄ , 2 g/L K ₂ HPO ₄ , and 1 mM MgSO ₄ ) for 4 hours at 30 °C and grown on MMG5- Spec250 plates (MMG5 with 250 mg/L spectinomycin and 15 g/L agar) for 3 days at 30 °C, inside an anaerobic jar with an AnaeroPack (Mitsubishi Gas Chemical, New York, NY).

Since pZX21 is a DCO suicide vector, surviving Spec ^R colonies had the Pga _P s-AMxylA::Peno-ECxylB::Spec-R segment integrated into the gfor locus. The colonies were streaked and grown on a fresh MMG5-Spec250 plate, and then subjected to PCR to inspect chimeric gene integration. The first PCR used forward primer ara285 (SEQ ID NO:42) and reverse primer ara120 (SEQ ID NO:43) to inspect double crossover recombination mediated by the GFO-L fragment in pZX21 . The ara285 primer matches a segment of Z. mobilis genomic sequence that is 494 bp upstream of the GFO-L fragment in the genome, while ara120 complements the last 18 bp of Pgaps and the first 17 bp of AMxylA in pZX21 . If integration had occurred as designed, PCR would amplify a 1 ,903 bp fragment from the transformants. The 2nd PCR used forward primer ara46 (SEQ ID NO:44) and reverse primer ara274 (SEQ ID NO:45) to inspect double crossover recombination mediated by the GFO-R fragment in pZX21 . The ara46 primer is a sequence near the end of the Spec ^R gene in pZX21 , while ara274 complements a segment of Z. mobilis genomic DNA that is 83 bp downstream of the GFO-R fragment. This PCR would amplify a 1 ,864-bp fragment from the colonies having successful integration. Both

inspections produced the expected PCR products and thus confirmed accurate transgene integration. The resulting strain was named ZW1 - pZX21 .

In the second step, ZW1 -pZX21 was transformed with pZX52 and selected on a MMG5-Spec250-CM120 (MMG5-Spec250 with 120 mg/L of chloramphenicol) plate. Because pZX52 is a DCO shuttle vector having the Cm ^R marker for plasmid selection and a markerless integration segment (PgapT-ECTal-ECTkt::P _e no-ZMRpi-ZMRpe), the recovered colonies should contain not only the previously integrated construct P _gap s- AMxylA: : Peno-ECxylB::Spec-R in the Z. mobilis genome, but also the non- integrated construct PgapT-ECTal-ECTkt::P _e no-ZMRpi-ZMRpe in the propagated pZX52 plasmid. These transformants should have all required genes for the xylose utilization pathway. To demonstrate that all transgenes were functional in Z. mobilis, ten selected colonies were subjected to a 48-hour growth assay in xylose. In the assay, 2 ml_ of mRM3-G5-Spec200-CM120 (mRM3-G5 with 200 mg/L spectinomycin and 120 mg/L chloramphenicol) in a 14 mL Falcon polypropylene round- bottom tube was inoculated with a selected colony and cultured overnight at 30 °C with 150 rpm shaking. Tubes were tightly capped, but a hole was punched in the top of the cap using a 23G1 needle for pressure release during cell growth and fermentation. Cells were harvested, washed with MRM3X10 (MRM3 with 100 g/L xylose), and resuspended in mRM3-X10- Spec200-CM120 (mRM3-X10 containing 200 mg/L spectinomycin and 120 mg/L chloramphenicol) to have a starting OD ₆ oo of 0.1 . Five mL of the suspension was placed in a new 14 mL Falcon polypropylene round- bottom tube. Tubes were capped with a hole on the top. Cells were grown for 48 hrs at 30 °C with 150 rpm shaking and OD ₆ oo was measured on a Shimadzu UV-1201 Spectrophotometer. Then, 1 mL of culture was centrifuged at 10,000x g to remove cells. The supernatant was filtered through a 0.22 μηη Costar Spin-X Centrifuge Tube Filter (Corning Inc, Corning, NY) and analyzed for xylose and ethanol by running through a BioRad Aminex HPX-A7H ion exclusion column (BioRad, Hercules, CA) with 0.01 N H ₂ SO ₄ at a speed of 0.6 mL/min at 55 °C on an Agilent 1 100 HPLC system (Agilent Technologies, Santa Clara, CA). Results indicated that all 10 of the transformants had acquired the xylose utilization pathway for ethanol production. The new strain was named ZW1 -pZX21 -pZX52 and one of the cultures was used in further experiments.

ZW1 -pZX21 -pZX52 then went through three post-transformation procedures sequentially for integration of the P _ga pT-ECTal-ECTkt: : Peno- ZMRpi-ZMRpe construct.

(1 ) The strain was adapted on xylose. In this procedure, ZW1 -pZX21 - pZX52 was suspended in a 5-mL mRM3-G1 X9-Spec200-CM120 medium (MRM3 with 10 g/L glucose, 90 g/L xylose, 200 mg/L spectinomycin and 120 mg/L chloramphenicol) with a starting OD ₆ oo value of 0.2 and grown for 3 to 4 doublings at 30°C (OD ₆ oo value from 0.2 to 2; one passage). The culture was then diluted to the starting OD ₆ oo value and grown for another passage. Totally, 4 passages (approximately 15 doublings) were completed.

(2) Plasmid curing and integration of the P _ga pT-ECTal-ECTkt: : Peno-ZMRpi- ZMRpe construct were carried out by growing 10 μΙ_ of the adaptation cell pool in 2 ml_ mRM3-G5-Spec200 medium at higher temperature (37 °C) for overnight. The 10 μΙ_ culture was then diluted in 2 ml_ mRM3-G5- Spec200 medium and grown for another passage. Totally, 5 passages were performed at 37 °C in glucose medium. As a result of the high temperature growth, the majority of the population should not host the pZP52 plasmid any more, but the PgapT-ECTal-ECTkt::P _e no-ZMRpi-ZMRpe construct (lacking a selective marker) should have been integrated into the IdhA gene of the Z. mobilis genome. A minority of the population may maintain pZX52, without integration.

(3) The population was enriched by growing 50 μΙ_ of the cell pool in 2 ml_ mRM3-X10-Spec200 at 30 °C for overnight. The enriched population was grown on a MMG5-Spec250 plate at 30 °C for overnight. Individual colonies were selected and streaked on MMG5 plates and MMG5-CM120 plates in replica. After incubating at 30 °C for overnight, those colonies that grew on MMG5 but not on MMG5-CM120 were selected for further PCR inspection. The first PCR used forward primer ara45 (SEQ ID NO:46) and reverse primer ara356 (SEQ ID NO:47) to inspect double crossover recombination mediated by the LDH-L fragment in pZX52. The ara45 primer matches a segment of Z. mobilis genomic DNA that is 86 bp upstream of the LDH-L fragment in the genome, and ara356 complements a fragment (from nt-91 to nt-1 12) of the ECTal coding region in pZX52. The PCR would amplify a 1 ,383-bp fragment from the colonies if integration had occurred as designed. The 2nd PCR used forward primer ara354 (SEQ ID NO:48) and reverse primer ara43 (SEQ ID NO:49) to inspect double crossover recombination mediated by the LDH-R fragment in pZX52. The ara354 primer is a sequence near the 3' end of ZMRpe in pZX52. The ara43 primer complements a segment of Z. mobilis genomic DNA that is 122 bp downstream of the LDH-R fragment. This PCR would amplify a 1 ,468 bp fragment from the colonies when recombination was as expected. Both PCRs produced DNA fragments with the expected sizes, which confirmed that the PgapT-ECTal-ECTkt::P _e no-ZMRpi-ZMRpe construct had been accurately integrated as designed in all inspected colonies. The resulting colonies were named ZW1 -X109.

In a second approach, the ZW1 -pZX21 strain was transformed with the pZX6 DCO shuttle vector and the three post-transformation

procedures were performed as described above for ZW1 -X109, except that adaptation was for 10 passages rather than 4 passages. Therefore, the PgapT-ECTal-ECTkt: : Peno-ZMRpi-ZMRpe construct was targeted to the endogenous pnp gene. As described for construction of ZW1 -X109, the 48-hour growth assay was preformed prior to the three post- transformation procedures to make sure that all transgenes were functioning as expected. After the three post-transformation procedures, the integration was also inspected by PCR. The first PCR used forward primer ara340 (SEQ ID NO:50) and reverse primer ara356 (SEQ ID NO:47) to inspect double crossover recombination mediated by the PNP-L fragment in pZX6. The ara340 primer matches Z. mobilis genomic DNA that is 75 bp upstream of the PNP-L fragment. The ara356 primer used here complements a fragment (from nt-91 to nt-1 12) of ECTal in pZX6. The PCR produced a 1 ,815-bp fragment from the transformants, as expected for an accurate integration event. The 2nd PCR used forward primer ara354 (SEQ ID NO:48) and reverse primer ara339 (SEQ ID NO:51 ) to inspect double crossover recombination mediated by PNP-R fragment in pZX6. In this case, the ara354 primer matches a sequence near the 3' end of ZMRpe in pZX6, and the ara339 primer complements a segment of Z. mobilis genomic DNA that is 59 bp downstream of the PNP- R fragment sequence. This PCR amplified a 1 ,549 bp fragment from the transformants, a size that was expected for successful integration.

Therefore, PCR inspection confirmed that the P _ga pT-ECTal-ECTkt: : Peno- ZMRpi-ZMRpe construct had been accurately integrated in all inspected colonies. This new strain was named ZW1 -X210.

In summary, two xylose utilizing Z. mobilis strains were rebuilt de novo from wild type ZW1 . They both had a P _ga ps-AMxylA: : Peno- ECxylB::Spec-R construct integrated into the gfor locus. The ZW1 -X109 strain had a PgapT-ECTal-ECTkt::P _e no-ZMRpi-ZMRpe construct integrated into the IdhA locus, while the ZW1 -X210 strain had the same construct integrated in the endogenous pnp gene. Both strains had one marker gene in the integrated Pgaps-AMxylA::P _e no-ECxylB::Spec-R construct, which could be removed by introduction of Cre recombinase.

Example 6

Characterization of new xylose utilizing Z. mobilis strains

The ability of the ZW1 -X109 and ZW1 -X210 strains to ferment xylose was demonstrated by a standard growth assay as described in Example 5. To quantitatively determine the growth and metabolic profiles of these new strains and compare them to ZW1 , these strains were characterized in shake flask fermentation assays. First they were subjected to the shake flask fermentation using MRM3G10 in order to determine their basal glucose metabolism. The strains were grown overnight in 2 ml_ mRM3-G5-Spec250 at 30 °C, with 150 rpm shaking. Cells were harvested, washed with mRM3-G10, and resuspended in mRM3G10 to have a starting OD ₆ oo of 0.1 . Twenty ml_ of the suspension were placed in a 45 ml_ screw capped VWR centrifuge tube and grown at 30 °C with 150 rpm shaking. To prevent pressure buildup due to ethanol evaporation during fermentation, the caps were closed tightly and then loosened for one turn. During the time course, OD ₆ oo was measured on a Shimadzu UV-1201 Spectrophotometer at 0, 10, and 24 hour time points. At each time point, 1 mL of culture was centrifuged at 10,000x g to remove cells. The supernatant was filtered through a 0.22 μηη Costar Spin-X Centrifuge Tube Filter and analyzed by running through a BioRad Aminex HPX-A7H ion exclusion column with 0.01 N H ₂ SO ₄ in a speed of 0.6 mL/min at 55 °C on an Agilent 1 100 HPLC system. Wild type ZW1 was grown without antibiotics and analyzed as a control. Results given in Figure 9 show that each strain quickly exhausted available glucose in 24 hours; both ZW1 -X109 and ZW1 -X210 strains (Figure 8A, 8B,

respectively) utilized glucose similarly to ZW1 (Figure 8C). For example, after 10 hours of fermentation, ZW1 -X109 had utilized approximately 34.8% of glucose (a reduction from 102.7 g/L to 66.9 g/L) to support an ethanol titer of 16.4 g/L and a biomass growth to OD ₆ oo value of 4.88; ZW1 -X210 had utilized approximately 32.1 % of glucose (a reduction from 102.7 g/L to 69.7 g/L) to support an ethanol titer of 15.2 g/L and a biomass growth to OD ₆ oo value of 4.68; ZW1 had utilized approximately 33.9% glucose (a reduction from 103.1 g/L to 68.2 g/L) to support an ethanol titer of 16.4 g/L and a biomass growth to OD ₆ oo value of 4.6. Therefore, both new strains have robust basal glucose metabolism.

Shake flask fermentation was carried out in 20 mL mRM3-X10 in order to determine each strain's ability to use xylose. OD ₆ oo value and both xylose and ethanol concentrations were measured at 0, 24, 48, and 72 hours. Figure 10 is a summary of the results for ZW1 -X109 (A), ZW1 - 210 (B) and ZW1 (C). The results also confirm that both new strains were able to ferment xylose. After 72 hours of fermentation, ZW1 -X109 had utilized approximately 64.2% of xylose (a reduction from 105.6 g/L to 37.8 g/L) to support an ethanol titer of 31 .5 g/L and biomass growth to OD ₆ oo value of 3.51 ; ZW1 -X210 had utilized almost all available xylose (a reduction from 105.6 g/L to 1 .6 g/L) to support an ethanol titer of 48.5 g/L and a biomass growth to OD ₆ oo value of 5.22. However, ZW1 could not grow in mRM3-X10 due to lacking the xylose metabolic pathway.

Therefore, among new strains, ZW1 -X210 could ferment xylose faster than ZW1 -X109, in the xylose-containing single sugar medium. The major difference between ZW1 -X109 and ZW1 -X210 is that the P _gap T-ECTal- ECTkt: : Peno-ZMRpi-ZMRpe construct was inserted into the IdhA locus in ZW1 -X109, and into the endogenous pnp gene in ZW1 -X210. This result iindicates that interruption of the pnp gene may benefit xylose metabolism in Z. mobilis.

Example 7

Vector constructs for interruption of the endogenous pnp gene in Z.

mobilis strains

To directly test whether interruption of the endogenous pnp gene benefits xylose metabolism, four DCO suicide vectors shown in Figure 4 were constructed by standard molecular recombination methods.

pPNP-l (SEQ ID NO:55; Figure 1 1A) is a 5,548-bp pUC18 based DCO vector. Its backbone contains a 653 bp replication origin (pUC Ori) and a 1 ,144 bp ampicillin resistance marker (Amp-R) which allow the vector to propagate and be selected for in E. coli. pPNP-l contains a 91 1 bp chloramphenicol-resistance gene (Cm-R) and flanking sequences that target integration of this gene into the endogenous pnp gene. The 891 bp upstream flanking sequence (SEQ ID NO:56) consists of a 775 bp PNPi-L sequence (pnp coding region (SEQ ID NO:1 ) from nt-1 ,345 to nt-2,1 19), a 9 bp directly reversed segment (DR), a 19-bp ME (mosaic end) element, and a 34 bp Lox element. The 1 ,030 bp downstream flanking sequence (SEQ ID NO:57) consists of a 19 bp ME element, a 9 bp directly reversed segment (DR), and a 916 bp PNPi-R sequence. The PNPi-R sequence includes the last 1 16 bp of the pnp coding region (from nt-2,129 to nt- 2,244) and an 800 bp DNA sequence downstream of the pnp coding sequence. Both PNPi-L and PNPi-R were amplified from genomic DNA of Z. mobilis ZW1 . In pPNP-l, the PNPi-L and PNPi-R sequences direct integration of the DNA sequence between them into the Z. mobilis genome between nt-2,1 19 and nt-2,129 of the pnp coding region through double cross over homologous recombination. The integration interrupts the endogenous pnp gene and is similar to the genotype of the I strain described in Example 3, with the integrated transgene as Cm-R rather than Rpi::Spec-R. It results in a truncated 723-aa pnp fusion protein product as described in Example 3, which is 25 amino acid shorter than the wild-type pnp protein product (a 748 amino acid polynucleotide phosphorylase; SEQ ID NO:2). The truncated protein shares the first 709 amino acid residues with the wild-type, but has a new 14-aa sequence attached at the C-terminus (SEQ ID NO:9).

pPNP-IN (Figure 1 1 B; SEQ ID NO:58) is also a pUC18 based DCO vector with a size of 6,471 bp. It was directly derived from pPNP-l by replacing PNPi-L with PNP-U and PNPi-R with PNP-D. PNP-U (SEQ ID NO:59) is a 1 ,369-bp genomic DNA fragment of Z. mobilis which consists of the first 96 bp of the pnp coding region and a 1 ,273-bp sequence upstream of the pnp coding region. PNP-D (SEQ ID NO:60) is a 1 ,251 -bp genomic DNA fragment of Z. mobilis which includes a part of the pnp coding sequence, from nt-97 to nt-1 , 347. In vector pPNP-IN, PNP-U and PNP-D are homologous recombination fragments that direct integration of the sequence between two 9 bp DR elements into the Z. mobilis genome between nt-96 and nt-97 of the pnp coding region. The integration interrupts the endogenous pnp gene and results in a truncated pnp protein product with only 49 aa residues (SEQ ID NO:12), which is 699 aa shorter than the wild-type pnp protein product. This short protein shares the first 32 aa residues with the wild-type, then has 17 new amino acid residues attached at the C-terminus.

pPNP-C (SEQ ID NO:61 , Figure 1 1 C) is a 6,342-bp pBluescript based vector. Its backbone consists of an f1 (+) replication site (f1 (+) Ori), an ampicillin resistance marker (Amp-R), and a pUC replication site (pUC Ori) for propagation and selection in E. coli. In the vector, a 91 1 -bp chloramphenicol resistance gene (Cm-R) is flanked by a 1 ,318-bp PNP-L fragment and a 1 ,225-bp PNP-R fragment. The PNP-L and PNP-R fragments were amplified from Z. mobilis ZW1 genomic DNA and are identical to those in pZX6 described in Example 4. The PNP-L fragment is a segment of the pnp coding sequence, from nt-767 to nt-2,084, while the PNP-R fragment includes the last 56 bp (from nt-2189 to nt-2244) of the pnp coding sequence, its stop codon, and the 1 ,166-bp downstream sequence. Therefore, pPNP-C directs integration of the Cm-R marker into the pnp coding region between nt-2,085 and nt-2,188. This integration site is near the 3' end of the pnp coding sequence and 34 bp upstream of the target integration site of pPNP-l. The truncated pnp coding region produces a 697-aa protein (SEQ ID NO:10), which is 51 amino acid residues shorter than the wild-type and shares 695 amino acid residues with the wild-type, with 2 new amino acids at the C-terminus.

Similar to pPNP-C, pPNP-M (SEQ ID NO:62; Figure 1 1 D) is a 6,322-bp pBluescript based vector, which also has a backbone sequence consisting of a f1 (+) Ori, an Amp-R, and a pUC Ori. However, in pPNP-M, the 91 1 bp chloramphenicol-resistance transgene (Cm-R) is flanked by a 1 ,200 bp PNPm-L fragment and a 1 ,324 bp PNPm-R fragment. Both flanking fragments were amplified from Z. mobilis ZW1 genomic DNA. PNPm-L (SEQ ID NO:63) includes a 96 bp genomic sequence upstream of the pnp coding sequence and the first 1 ,104 bp of the pnp coding sequence (from nt-1 to nt-1 104), while PNPm-R (SEQ ID NO:64) includes the last 1 140 bp (from nt-1 ,105 to nt-2244) of the pnp coding sequence, its stop codon, and the 181 bp downstream sequence. Therefore, pPNP-M is able to direct integration of the Cm-R marker into the endogenous pnp gene between nt-1 ,104 to nt-1 ,105 in Z. mobilis genome. The integration site is near the middle of the pnp coding sequence and 1 ,015 bp upstream of the integration site for pPNP-l. It results in a truncated 378-aa pnp protein product (SEQ ID NO:1 1 ), 370 aa shorter than the wild-type pnp protein product. The truncated protein shares the first 368 aa residues with the wild-type, but has a new 10 aa sequence attached at the C- terminus.

Example 8

Interruption of the endogenous pnp gene in strain ZW1 -X109

To determine whether interruption of the endogenous pnp gene benefits xylose utilization in Z. mobilis, ZW1 -X109 was transformed separately with pPNP-l, pPNP-C, pPNP-M, and pPNP-IN as described in Example 5. Since all four vectors are suicide vectors, transformants were directly selected on a MMG5-CM120 plate (MMG5 with 120 mg/L chloramphenicol and 15 g/L agar). The resulting strains were named ZW1 -X109-PNPi, ZW1 -X109-PNPc, ZW1 -X109-PNPm, and ZW1 -X109- PNPin. A few colonies of each strain were streaked and grown on fresh MMG5-CM120 plates. Transgene integration was confirmed by PCR inspection.

Five primers were employed in the PCR inspection. Forward primer ara448 (SEQ ID NO:52) is a sequence located at the beginning of the Cm-R marker in all pPNP plasmids. Reverse primer ara339 (SEQ ID NO:51 ) complements a Z. mobilis genomic sequence segment

downstream of the pnp gene. They were used to inspect double crossover recombination mediated by the following recombination fragments: PNPi-R in pPNP-l, PNP-R in pPNP-C, PNPm-R in pPNP-M, and PNP-D in pPNP-IN. If the integration was successful, these two primers would amplify a PCR product of 2,393 bp from ZW1 -X109-PNPi strain, 2,256 bp from ZW1 -X109-PNPc strain, 3,361 from ZW1 -X109- PNPm strain, and 4,565 bp from ZW1 -X109-PNPin strain. Forward primer ara340 or 4R0 (SEQ ID NOs:50 and 53, respectively) and reverse primer ara449 (SEQ ID NO:54) were used to inspect double crossover

recombination mediated by the following recombination fragments: PNPi-L in pPNP-l, PNP-L in pPNP-C, PNPm-L in pPNP-M, and PNP-U in pPNP- IN. The ara340 primer matches a segment (from nt-702 to nt-724) of the pnp coding sequence located upstream of the PNPi-L and PNP-L sequences in Z. mobilis genomic DNA. The 4R0 primer is a segment of Z. mobilis genomic sequence outside of the pnp gene and upstream of PNPm-L and PNP-U sequences. The ara449 primer complements a sequence at the end of the Cm-R marker in pPNP plasmids. Therefore, when integration was successful, ara340 and ara449 could amplify a 2,551 -bp PCR product from ZW1 -X109-PNPi strain and a 2,306-bp PCR product from ZW1 -X109-PNPc strain, while 4R0 and ara449 could amplify a 3,424-bp PCR product from ZW1 -X109-PNPm strain, and a 2,430-bp PCR product from ZW1 -X109-PNPin strain. Standard PCR reactions using Invitrogene's PCR Supermix were performed directly on freshly grown strains. Results demonstrated accurate integration.

Example 9

Characterization of the pnp-integration strains

ZW1 -X109-PNPi, ZW1 -X109-PNPc, ZW1 -X109-PNPm, and ZW1 - X109-PNPin strains were further characterized in shake flask

fermentations to determine their growth and metabolic profiles. The parental ZW1 -X109 strain, which contains an uninterrupted endogenous pnp gene, was used as a control. The fermentation followed a standard protocol as described in Example 6, except that the volume of cell culture was reduced from 20 mL to 10 ml_, therefore capped 14 ml_ Falcon round- bottom tubes with a punched hole in the top was used instead of the 45 mL VWR centrifuge tubes. The first shake flask fermentation was carried out in mRM3-G10. At 0, 10, and 24 hour of fermentation OD ₆ oo was measured on a Shimadzu UV-1201 Spectrophotometer, while glucose and ethanol concentrations were determined by an Agilent 1 100 HPLC system using a BioRad Aminex HPX-A7H ion exclusion column. Results are graphed in Figure 12, showing that all four pnp-interrupted strains have a basal glucose metabolism similar to the parental ZW1 -X109 strain. For example, after 10 hours of fermentation, ZW1 -X109-PNPi (A) utilized approximately 37.7 % of glucose (a reduction from 120.8 g/L to 75.3 g/L) to support an ethanol titer of 22.9 g/L and a biomass growth to OD ₆ oo value of 5.36; ZW1 -X109-PNPC (B) utilized approximately 37.7 % of glucose (a reduction from 120.8 g/L to 75.3 g/L) to support an ethanol titer of 23.0 g/L and a biomass growth to OD ₆₀ o value of 4.98; ZW1 -X109-PNPm (C) utilized approximately 39.1 % of glucose (a reduction from 120.8 g/L to 73.6 g/L) to support an ethanol titer of 23.5 g/L and a biomass growth to OD ₆ oo value of 5.14; ZW1 -X109-PNPin (D) utilized approximately 36.8 % of glucose (a reduction from 120.8 g/L to 76.4 g/L) to support an ethanol titer of 22.6 g/L and a biomass growth to OD ₆ oo value of 5.32. The parental ZW1 -X109 strain (E) utilized approximately 39.5 % of glucose (a reduction from 120.8 g/L to 73.4 g/L) to support an ethanol titer of 24.0 g/L and a biomass growth to OD ₆ oo value of 5.62. After 24 hours of

fermentation, all of the pnp-interrupted strains exhaust glucose to support an ethanol titer near 58.1 g/L and biomass growth to OD ₆ oo value of around 7.90, while ZW1 -X109 also exhausted glucose to support an ethanol titer of 57.3 g/L and a biomass growth to OD ₆ oo value of 7.64.

The shake flask fermentation was then carried out in 10 mL mRM3- X10 as described above. OD ₆ oo value and both xylose and ethanol concentrations were measured at 0, 24, 48, and 72 hours. Figure 13 shows graphs of the results. It shows that all four pnp-interrupted strains utilized xylose better in fermentation than the parental ZW1 -X109 strain. After 48 hours of fermentation, ZW1 -X109-PNPi (A) had already utilized approximately 95.5 % of xylose (a reduction from 1 17.9 g/L to 5.3 g/L) to support an ethanol titer of 54.7 g/L and a biomass growth to OD ₆ oo value of 5.34. After another 24 hours of fermentation, it had almost used up the xylose with only 1 .4 g/L left to support an ethanol titer of 56.0 g/L and a biomass growth to OD ₆ oo value of 5.62. In the same 72 hours period, ZW1 -X109-PNPC (B) utilized approximately 94.2 % of xylose (a reduction from 1 17.9 g/L to 6.8 g/L) to support an ethanol titer of 53.6 g/L and a biomass growth to OD ₆₀ o value of 4.64; ZW1 -X109-PNPm (C) utilized approximately 83.3 % of xylose (a reduction from 1 17.9 g/L to 19.7 g/L) to support an ethanol titer of 47.5 g/L and a biomass growth to OD ₆ oo value of 4.24; ZW1 -X109-PNPin (D) utilized approximately 79.8 % glucose (a reduction from 1 17.9 g/L to 23.8 g/L) to support an ethanol titer of 45.5 g/L and a biomass growth to OD ₆ oo value of 4.04. The parental ZW1 -X109 strain (E) utilized approximately 61 .7 % of xylose (a reduction from 1 17.9 g/L to 45.1 g/L) to support an ethanol titer of 34.8 g/L and a biomass growth to OD ₆ oo value of 3.26. The results also indicate that the strains with less truncation of the pnp encoded protein, such as ZW1 -X109-PNPi and ZW1 -X109-PNPc, use xylose more efficiently than the strains with larger truncations, such as ZW1 -X109-PNPm and ZW1 -X109-PNPin.

Ranking of these strains in order of their ability to utilize xylose, with best first, was: ZW1 -X109-PNPi, ZW1 -X109-PNPc, ZW1 -X109-PNPm, ZW1 - X109-PNPin, and finally ZW1 -X109. In summary, this example demonstrates that integration in the endogenous pnp gene of Z. mobilis does not impact glucose metabolism but improves xylose utilization in fermentation. Example 10 (comparative)

Interruption of endogenous pnp gene in ZW801 -4 in the absence of RPI overexpression

The effects of endogenous pnp gene modifications were assayed in ZW801 -4 (see General Methods) using the two suicide constructs that had the greatest impact on xylose growth and metabolism in ZW1 -X109 (e.g. pPNP-l and pPNP-C, described in Example 7). ZW801 -4 was transformed with each of these suicide vectors separately, as described in Example 8. Two transformants for each construct were evaluated in shake flask experiments with xylose, and ZW801 -4 was included as a control (also done in duplicate). The growth medium was mRM3-X10 and the temperature was 33 °C. Samples of each culture were assayed for OD600, xylose, and ethanol as described previously. The results of this experiment are shown in Figure 14. Only the transformant prepared with the pPNP-l construct showed slight improvement as compared to the control. Note that double-crossover homologous recombination of the pPNP-l suicide construct with the ZW801 -4 chromosome results in the exact same pnp gene modification that is present in the I strain.

Example 1 1

Assay of xylose isomerase activity

The activity of xylose isomerase in ZW658 (see General Methods) was measured in a reaction containing 20 μΙ_ of cell free extract (see General Methods), 0.256 mM NADH, 50 mM xylose, 10 mM MgSO ₄ , 10 mM triethanolamine, and 1 U/ml SDH (sorbitol dehydrogenase) at 30°C. The A _{3 0} was read on a plate reader for 3-5 min. XI activity was calculated as follows:

1 unit of XI corresponds to the formation of 1 μηηοΐβ of D-xylulose per minute at 30°C U (μηηοΐθ/ηηίη) = slope (dA ₃₄ o/min) ^* volume of reaction (μΙ_) / 6220 / 0.55 cm (moles of NADHP^NAD is 6220 A _{3 0} per mole per L in a 1 cm cuvette) (pathlength of 200 μΙ_ per well in microplate=0.55 cm)

Specific Activity ^mole/min-nng) = μηηοΐβ/ηηίη / protein concentration (mg) The activity measured for ZW658 was 0.25 +/-0.033 μηηοΐβε product/mg protein/minute.