FORM 2
THE PATENT ACT 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION A XYLOSE FERMENTING YEAST
2. APPLICANT
(a) NAME: PRAJ INDUSTRIES LIMITED
(b) NATIONALITY: Indian Company
(c) ADDRESS: PRAJ Tower, 274-275, Bhumkar Chowk -
Hinjewadi Road, Hinjewadi, Pune-411057, INDIA
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

4. DESCRIPTION
FIELD OF THE INVENTION
The invention relates to a strain of yeast able to use xylose as a sote source of carbon and energy useful for utilization of a pentose sugar like xylose in preparation of ethanol and other bio-chemicals. More particularly, it relates to recombinant yeast comprising a bacterial gene having xylose isomerase activity effectively expressed in the yeast.
BACKGROUND
Ethanol has a number of industrial and fuel applications. Of particular interest is the use of ethanol as an additive to gasoline to boost octane value, reduce pollution and partially replace gasoline in the mixture. This composition of gasoline and ethanol is well known commercially as "gasohol". Beside pure ethanol is also used as a motor fuel without any additives. Ethanol added fuels produce considerably less air pollution due to reduced emissions of carbon monoxide and hydrocarbons.. Furthermore, ethanol is renewable chemical and can be prepared from a variety of natural vegetative materials like lignocellulosic biomass.
Presently a large portion of total global production of ethanol is achieved by using sugars obtained from food crops like sugarcane, sugar beet, maize,

etc. The use of food crops for the production of ethanol has been controversial for some time now due to challenges associated with food requirements. Lignocellulosic materials [LCM] iike wood, grasses, agricultural waste obtained from crops like corn, sugarcane, etc contains substantial amount of sugars in polymers of cellulose and hemicelluiose. However, LCMs are difficult to process compared with starchy materials due to hardy and complex nature. These LCMs besides having glucose as a major sugar also have pentose sugars like xylose as second a major sugar. To achieve an economic production of ethanol from LCM, it is necessary that all pentoses and hexoses present in LCM are being used effectively for the production of ethanol by the fermenting yeast. Towards this goal in the art, several recombinant yeasts have been developed that use xylose along with glucose for the production of ethanol from LCM. However, there is still need of novel and more efficient xylose utilizing yeasts that are able to ferment ethanol from pentose and hexose sugars present in LCM.
DESCRIPTION OF THE FIGURES
FIGURE1 depict the quantitative PCR determination of expression of the xylose isomerase of D. geothermalis in the recombinant yeast S. cerevisiae,
FIGURE 1A depicts the xylose isomerase activities of different types of xylose isomerases isolated and expressed in the yeast.

FIGURE 1B depicts the growth of the recombinant yeast of the invention on xylose as the sole source of carbon and energy.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to recombinant yeast strains that have xylose isomerase enzyme activity. A challenge for engineering yeast to utilize xylose, which is the second most predominant sugar obtained from cellulosic biomass, is to produce sufficient xylose isomerase activity in the yeast cell. The xylose isomerase catalyzes the conversion of xylose to xylulose, which is the first step in a xylose utilization pathway. Herein the expression of a bacterial xylose isomerase [from D. geothermalis] in a yeast cell results in enzymaticaily active xylose isomerase. This yeast cell expressing xylose isomerase activity provides a host cell for expression of a complete xylose utilization pathway, thereby enabling the engineering of a yeast cell that can produce bio-chemicals like ethanol using xylose derived from lignocellulosic biomass as a carbon source.
In one embodiment of the present invention, any yeast cells that either produce a bio-chemicals may be used as host cells. Examples of such yeasts include, but are not limited to, yeasts of the genera Kiuyveromyces, Candida, Pichia, Hansenula, Schizosaccharomyces, Kioeckera, Schwammiomyces, Yarrowia, and Saccharomyces. Engineering of a yeast cell for expression of xylose isomerase activity as disclosed herein and for

production of a bio-chemical may occur simultaneously or in any order. In one embodiment, yeast cells that produce ethanol may be used as host cells in engineering to produce the present cells. In one embodiment, the yeast cells are capable of anaerobic alcoholic fermentation. The yeast cells may naturally produce ethanol, or may be engineered to produce ethanol, or to produce increased yields of ethanol.
In yet another embodiment of the present invention, the xylose isomerase of D. geothermalis is identified by bioinformatic methods and chemically synthesised. This synthetic gene, which is coding optimised for effective expression in yeast, is cloned in a cloning plasmid and retrieved when required to create yeast expression plasmids for expression of the gene in yeast cells. The yeast cells so engineered are then used for detection of xylose isomerase activity in vivo and grown on xylose as carbon source. So developed recombinant yeasts are then suitable for further developing strains that efficiently use xylose and product bio-chemicals like ethanol.
The coding region sequence for the xylose isomerase polypeptide is readily obtained from the genome of the bacterial strain in which it is natively expressed, as is well known to one skilled in the art. Native nucleotide sequences encoding each of these proteins may be codon optimized for expression in the yeast host cell to be engineered, as is well known to one skilled in the art. Example of DNA sequence that is codon optimized for expression in S. cerevisiae of the xylose isomerase from D. geothermalis is

identified as SEQ ID NO: 01 and the corresponding amino acid sequence is identified as SEQ ID NO: 02 and described in the examples below.
EXAMPLES
Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various experimental results is given in the examples and tables, which demonstrate the advantageous and novel aspects of the yeast expressing xylose isomerase of D. geothermalis and in its used for utilization of xylose as a carbon source by said yeast to produce bio-chemicals like ethanol.
EXAMPLE 1: SYNTHESIS OF XYLOSE ISOMERASE GENE OF D. GEOTHERMALIS
Dienococcus geothermalis is an extremophile bacteria recently described , having very high resistance to gamma radiation. It is also able to use xylose as sole source of carbon for growth and have classical bacterial xylose isomerase driven pathway for metabolism of xylose for energy and carbon requirements. A comparative homology analysis identified a xylose isomerase of D. geothermalis and is reported in GENBANK as such under ID NO: WP_011525878.1 [SEQ. ID NO: 01]. As the entire genome

sequence of this bacterium has been sequenced recently and reported. This gene was chemically synthesized by standard chemical method of DNA synthesis and cloned in a cloning plasmid for further applications. The synthetic gene had a size about 1212 base pairs and it was codon optimized for expression in Sacchromyces cerevisiae [SEQ. ID NO: 02]; using a standard method. This synthetic gene was moved from the cloning plasmid to expression plasmid when required using standard molecular biology tools.
EXAMPLE 2: CLONING XYLOSE ISOMERASE IN YEAST EXPRESSION PLASMID
The entire xylose isomerase (DgXI) gene was PCR amplified using the a set of two flanking primers from the cloning plasmid hosting the synthetic gene. The synthetic gene contained a restriction site for BamHI proximal to the ATG - start codon and a restriction site for XhoI distal to the stop codon flanking the DgXI gene. A template DgXI DNA was used in a concentration of about 1 mcg for the PCR reaction. The PCR was performed at 35 cycles of 30 seconds at 95 °C, 5 seconds at 50 °C and 90 seconds at 72 °C, followed by final incubation of 10 minutes at 72 °C using a heat stable DNA polymerase, [CLONTECH -Prime STAR HS DNA polymerase]. The PCR product was electrophoretically separated on 1.0 % agarose gel and a 1212-bp fragment was isolated. This fragment was ligated into a plasmid

p426TEF using T4-DNA iigase resulting in the plasmid p426TEF-DgXI. This plasmid was maintained in an E. coli strain and retrieved when required.The sequence of DgXI gene in the new plasmid was confirmed by PCR and DNA sequencing. Next, about 1 mcg of the p426TEF-DgXI plasmid and p426TEF control plasmid used for the transformation of Saccharomyces cerevisiae strain BY4741 using a standard method. This strain of the yeast contained the plasmid p426TEF-leu2-ScXKS1, which over-expressed the yeast xylulose kinase [Sc-XKS1] protein for effective utilization of xylose as a sole carbon source. The parent stain used for preparing the strain BY4741 was auxotropic for uracil and leucine and the plasmid p426TEF-DgXI and p426TEF-leu2-ScXKS1, respectively, contained URA3 and LEU2 genes as selection markers. This recombinant strain was designated was PMOD06. The PMOD06 strain was maintained on a standard yeast dropout medium lacking uracil and leucine for effective retention of the two plasmids in the strain. The standard medium [SCM] contained about 2 % glucose, 2 % xylose. 0.67 % yeast nitrogen base (without amino acids), 0.01 % of adenine, arginine, cysteine, lysine, threonine and tryptophan each, 0.005 % of aspartic acid, histidine, isoleucine, methionine, phenylalanine, proline, serine, tyrosine and valine each and without uracil and leucine.
EXAMPLE 3: EXPRESSION ANALYSIS OF DgXI IN YEAST BY Q-PCR

Saccharomyces cerevisiae strains PMOD06 and BY4741 [the control strain without any XI] were both grown in minimal media with all amino acids omitting uracil and leucin and supplemented with 5 % dextrose as sugar source. Sampling was done at 8 h. Total RNA was purified and cDNA was synthesized through reverse transcription. EvaGreen dye based assay was carried out using the cDNA as template with both, biological and technical replicates. An average Cq [quantification cycle] value of 19 for appearance of fluorescence was seen in the case of PMOD06 using DgXI specific primers. No expression was detected in the control of BY4741 and "No template" and "No reverse transcriptase" controls and shown in FIGURE 1. The gene TAF10 was used a positive control in the experiment.
EXAMPLE 4: THE DgXI ACTIVITY IN RECOMBINANT YEAST
The four different strains PMOD06 (Dienococcus XI), PMOP01 (containing Piromyces XI instead of Dienococcus XI), PMOA06 (containing Agrobacterium XI instead of Dienococcus XI) AND PMOP01 (containing Lactobacillus XI instead of Dienococcus XI) along with control strain (BY4741) were grown on SCM media as above, for about 48 h at 200 RPM and 30 °C. The culture were harvested by centrifugation, washed by resuspension in water and centrifuged again. The yeast cell extracts were prepared by a standard method and xylose isomerase activity for each type of cell extract was assayed by measuring the decreasing of NADH in a 1

mL of reaction mixture at 340 nm by spectrophotometry using a standard sorbitol dehydrogenase based assay. The protein content was determined by the Lowery's method for protein estimation and XI activity was normalizedto per milligram of protein in the extracts. Results are shown in FIGURE 2A. The highest xylose isorrierase activity per mg of total yeast protein was obtained for PMOD06 expressing the xylose isomerase of D. geothermalis.
EXAMPLE 5: GROWTH OF YEAST ON XYLOSE AS SOLE CARBON SOURCE
Saccharomyces cerevisiae strain PMOD06 as described in EXAMPLE 2B, comprises an expression cassette containing D. geothermalis xylose isomerase with an expression cassette containing the nucleotide sequence encoding native xylulokinase. This strain was gradually adapted to growth on xylose by serial dilution of the glucose content in the SCM medium containing both the sugars initially at about 2 % by weight, followed by growth on xyiose as the only carbon source. FIGURE 2B shown the growth curve of PMOD06 growing on xylose only. The maximum growth rate achieved in this situation is about 0.03/h without any modifications in the strain.

EXAMPLE 6: DNA AND AMINO ACID SEQUENCES OF D. GEOTHERMALIS XYLOSE ISOMERASE
The DNA sequence of the xylose isomerase from D. geothermalis is [herein
is called SEQ ID NO: 01]:
ATGCCAGATTATACTCCAACTCCAGCTGATAAGTTTACTTTCGGTTTGT
GGACTGTTGGTCAAACTGGTAGAGATCCATTTGGTGAAGCTACTAGAC
CAGGTTTTTCTGCTCCATATATCGTTCAAAAGTTGGCTGAATTGGGTG
CTTATGGTGTTAACTTGCATGATAACGATTTGGTTCCAATTGATGCTAC
CCCACAACAAAGAGATAGAATCGTTGCTGAATTCAAGCAAGCCTTGTC
TGATCATGGTTTGGTTGTTCCAATGGCTACTACTAATTTGTTCACCGAT
CCAGI I I I IAAGGATGGTGCTTTTACTTCTGCTGATGCTAGAGTTAGA
GCTTACGCATTGCAAAAGACTATGCAATCTATGGATTTGGGTCATGAA
TTAGGTGCTCAAACTTACGTTTTTTGGGGTGGTAGAGAAGGTACTGAA
GTTGATGCTTCTTCTAAGTTGTTGGATGCTTTGGCTTGGTTTAGAGACT .
CTTTGAATTTCTTGGCCGAATACTCTCAATCTCAAGGTTACGGTTATAG
ATTCGCTTTGGAACCTAAACCTAATGAACCTAGAGGTGATATTTTCTTC
CCAACTGCTGGTTCTATGTTGGGTTTTATTGCTACTTTGGATCAACCAG
ATTTGTTCGGTGTTAATCCAGAATTTGCTCATGATACAATGGCCGGTTT
GAATTTCACTCATGCTGTTGCTCAAGTTATTGATGCTGGTAAGTTGTTC
CACATCGATTTGAACGATCAAAAGATGGGTAGATTCGACCAAGATTTG
AGATTTGGTGCCGAAAATTTGAAAACCGCTTTCTTCTTGGTCAAATTAT
TGGAAGATTCCGGTTACGATGGTCCAAGACATTTTGATGCTCATGCTT
TGAGAACCGAAGATGAAGAAGGTGTTTGGGCTTTTGCTAGAGGTTGTA

TGAGAACCTACTTGATCTTGAAAGAAAAGGCCCAACAATTCGACGAAG
ATCCAGAAATTCAAGCTGCTTTGGCTGCTTATAGAGTTGATGATGAAG
AATTGTCTAGATTGACCGCTAAGTTCTCTCCAGAAAATGCTAAAGCTTT
GAAGGCTAGAACCTTCGATAGAGAATTATTGGGTAGAAGAGGTCCAG
GTTTGGAACAATTGGATCAATTGACTGTTGAATTGCTATTGGGTTTGAG
AGGTCATACTTCTACTACTACCCATCCAGAAATTGAAGTCACCAGATA.
This is reported in the GENBANK with NCBI reference sequence as
WP_011525878.1.
The amino acid sequence of the xylose isomerase from D. geothermalis is
[herein is called SEQ ID NO: 02]:
MPDYTPTPADKFTFGLWTVGQTGRDPFGEATRPGFSAPYIVQKLAELGA
YGVNLHDNDLVPIDATPQQDRIVAEFKQALSDHGLVVPMATTNLFTDPVF
KDGAFTSADARVRAYALQKMQSMDLGHELGAQTYVFWGGREGTEVDA
SSKLLDALAWFRDSLNFLAESQSQGYGYRFALEPKPNEPRGFFPTAGSL
GFIATLDQPDLFGVNPEFAHDTMAGLNFTHAVAQVIDAGKLFHIDLNDQK
MGRFDQDLRFGAENLKTAFFLVKLLEDSGYDGPRHFDAHLRTEDEEGV
WAFARGCMRTYLILKEKAQQFDEDPEIQAALAAYRVDDEELSRLTAKFSP
ENAKALKARTFDRELLGRRGPGLEQLDQLTVELLLGLRGHTSTTTHPEIE
VTR.

5. CLAIMS
WE CLAIM:
1. A recombinant yeast with xylose isomerase activity comprising:
(a) a gene having DNA sequence of SEQ ID NO: 1;
(b) said gene expressing an amino acid of SEQ ID NO: 2;
(c) said gene codon-optimised for effective expression in said yeast; and
(d) said yeast utilizing a pentose sugar for growth.

2. The yeast according to claim 1 is a Saccharomyces sp.
3. The gene of claim 1, wherein said DNA sequence of SEQ ID NO: 1 is at least 50 % identical to xylose isomerase gene of Dienococcus geothermalis.
4. The gene of claim 1, wherein said amino acid sequence of SEQ ID NO: 2 is at least 60 % identical to xylose isomerase protein of Dienococcus geothermalis.
5. The gene of claim 1, which upon translation in said yeast gives xylose isomerase activity.
6. The sugar according to claim 1 is xylose.

7. The yeast according to claim 1 is able to convert xylose to xylulose.
8. The yeast according to claim 1 is able to use xylose as the sole source of carbon.