DESC:TECHNICAL FIELD
The present disclosure relates to the fields of chemistry, biotechnology and genetic engineering. In particular, the present disclosure relates to methods for production of ethylene glycol. The present disclosure also relates to non-naturally occurring microorganisms to produce tartaric acid and thereby ethylene glycol, and methods of producing the microorganism.

BACKGROUND
Mono ethylene glycol (MEG) is a colourless, odourless liquid with a sweetish taste which has several industrial applications. MEG has higher density than water and is soluble in water in all proportions. MEG is used as an anti-freeze reagent. It is also used as industrial coolants for gas compressors, heating, ventilating, and air-conditioning systems, etc. Further, MEG is employed as raw material for production of polymers such as polyesters and polyethylene terephthalate. Additional application of MEG includes its usage as hydraulic brake fluid, industrial solvent, component of printer inks and batteries, etc.

While prior art provides for production of ethylene glycol, there is a need for providing efficient means and methods for producing ethylene glycol.

OBJECTS OF THE DISCLOSURE
The present disclosure describes processes for producing ethylene glycol, particularly mono ethylene glycol.

The present disclosure describes a process for preparing ethylene glycol, said process comprising steps of:
a. alkylating tartaric acid to obtain dialkyl tartrate;
b. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
c. reducing the alkyl glycolate to obtain ethylene glycol.

In an embodiment, the process for preparing mono ethylene glycol comprises steps of:
a. methylating tartaric acid to obtain dimethyl tartrate;
b. oxidatively cleaving the dimethyl tartrate followed by reduction to obtain methyl glycolate; and
c. reducing the methyl glycolate to obtain mono ethylene glycol.

The present disclosure also describes a process for preparing alkyl glycolate optionally followed by preparing ethylene glycol, said process comprising steps of oxidatively cleaving dialkyl tartrate followed by reduction to obtain alkyl glycolate; and optionally reducing the alkyl glycolate to obtain ethylene glycol.

The present disclosure also describes a process for preparing ethylene glycol, said process comprising steps of:
a. hydrolyzing oxaloacetate to obtain tartaric acid;
b. alkylating the tartaric acid to obtain dialkyl tartrate;
c. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
d. reducing the alkyl glycolate to obtain ethylene glycol.

The present disclosure also describes a process for preparing ethylene glycol, said process comprising steps of:
a. acetylating syngas to obtain acetyl-CoA;
b. converting the acetyl-CoA to obtain pyruvate;
c. phosphorylating the pyruvate to obtain phosphoenol pyruvate;
d. dephosphorylating the phosphoenol pyruvate to obtain oxaloacetate;
e. hydrolyzing the oxaloacetate to obtain tartaric acid;
f. alkylating the tartaric acid to obtain dialkyl tartrate;
g. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
h. reducing the alkyl glycolate to obtain ethylene glycol.

In an embodiment, the conversion of syngas to tartaric acid in the aforesaid process is carried out in a non-naturally occurring microorganism.

In an embodiment, the acetylation of the syngas to obtain the acetyl-CoA is carried out by Wood-Ljungdahl pathway, conversion of the acetyl-CoA to obtain the pyruvate is carried out in presence of pyruvate synthetase, phosphorylation of the pyruvate to obtain the phosphoenol pyruvate is carried out in presence of phosphoenol pyruvate synthetase, dephosphorylation of the phosphoenol pyruvate to the obtain oxaloacetate is carried out in presence of phosphoenol pyruvate carboxylase, and hydrolysis of the oxaloacetate to obtain the tartaric acid is carried out in presence of fumarase B or tartrate dehydrogenase.

The present disclosure also describes non-naturally occurring microorganism comprising one or more ethylene glycol pathway gene selected from a group comprising pyruvate synthetase, phosphoenol pyruvate synthetase, phosphoenol pyruvate carboxylase, fumarase B and tartrate dehydrogenase or any combination thereof.

In an embodiment, the non-naturally occurring microorganisms of the present disclosure are capable of production of tartrate from a carbon source/starting material/substrate selected from group comprising syngas, acetyl-CoA, pyruvate, phosphoenol pyruvate and oxaloacetate. In embodiments of the present disclosure, the tartrate produced by the microorganism is further converted to ethylene glycol chemically.

The present disclosure further describes a process for preparing the said microorganisms.

In an embodiment, the microorganism of the present disclosure is a recombinant microorganism comprising one or more genes selected from a group comprising pyruvate synthetase, phosphoenol pyruvate synthetase, phosphoenol pyruvate carboxylase, fumarase B and tartrate dehydrogenase.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

Figure 1 depicts the pathway for production of tartrate as per the present disclosure.
Figure 2 depicts the gluconeogenic pathway for the conversion of acetyl-CoA to phosphoenol pyruvate.
Figure 3 depicts the pathway for production of ethylene glycol from tartrate by chemical synthesis.
Figure 4 depicts a. diagnostic PCR of pps in pET28a cloned in E. coli TOP10 F’; b. diagnostic PCR of ppc and ttdAB in pET28a cloned in E. coli TOP10 F’; c. diagnostic PCR of fumB in pET28a cloned in E. coli TOP10 F’; d. restriction enzyme digestion of pET28a_pps and pET28a_fumB; and e. restriction enzyme digestion of pET28a_ppc and pET28a_ttdAB.
Figure 5 depicts a. diagnostic PCR of pET28a_pps_ppc cloned in E. coli TOP10 F’; and b. restriction enzyme digestion of pET28a_pps_ppc.
Figure 6 depicts a. diagnostic PCR of pMTL0_T7-pps_ppc cloned in E. coli TOP10 F’; and b. restriction enzyme digestion of pMTL0_T7-pps_ppc.
Figure 7 depicts a. diagnostic PCR to confirm transformation of pET28a_fumB and pMTL0_T7-pps_ppc, E. coli BL21 (DE3); and b. diagnostic PCR to confirm transformation of pET28a_ttdAB and pMTL0_T7-pps_ppc, E. coli BL21 (DE3).
Figure 8 depicts a. SDS-PAGE gel to check expression of Ppc in E. coli BL21 DE3; b. SDS-PAGE gel to check expression of Pps and FumB in E. coli BL21 DE3; c. SDS-PAGE gel to check expression of TtdAB in E. coli BL21 DE3; d. SDS-PAGE gel to check expression of Pps, Ppc, TtdA, TtdB and FumB in E. coli BL21 DE3. Lane 1: ladder, Lanes 2-4: pET28a_fumB, pMTL0_T7-pps_ppc–col 3, 4, and 5. Lanes 5-7: pET28a_ttdAB, pMTL0_T7-pps_ppc –col 1, 2, and 3 in strain E. coli MCC 0129, Lane 8: pET28a_pps_ppc, Lane 9: pET28a_pps_ppc, pMTL0_ fumB in strain E. coli MCC 0128.
Figure 9 depicts the chromatogram of HPLC analysis of tartrate standard compared to samples from strain E. coli MMC 0128 or 0129 containing tartrate producing genes.
Figure 10 depicts mass spectrum analysed in negative mode ionization for tartrate in cell supernatant of E. coli MMC 0128 or 0129.
Figure 11 depicts mass spectrum analysed in negative mode ionization for tartrate in crude lysate reaction of E. coli MMC 0128 or 0129.
Figure 12 depicts GC-MS chromatogram depicting the formation of dimethyl tartrate at 26.3 min.
Figure 13 depicts GC-MS spectra depicting the fragmentation pattern of dimethyl tartrate.
Figure 14 depicts LC-MS chromatogram depicting the formation of DNPH derivative of methyl glycolate (molecular weight = 268) with m/z value t negative mode (H-1) at 267.03.
Figure 15 depicts GC-MS chromatogram depicting the formation of MEG at 12.65 min.
Figure 16 depicts GC-MS spectra depicting the fragmentation pattern of MEG.

DETAILED DESCRIPTION
The present disclosure overcomes the drawbacks of the prior art and provides for efficient and cost-effective methods for production of ethylene glycol. The present disclosure also provides for microorganism(s) for efficient production of tartrate which is processed to obtain ethylene glycol, methods of producing said microorganism(s) and applications thereof.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.

Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Throughout this specification, the term "about" means to be nearly the same as a referenced number or value. As used herein, the term "about" should be generally understood to encompass ± 10% of a specified amount or value.

As used herein, the terms ‘method’ and ‘process’ have the same scope and meaning and are used interchangeably.

As used herein, the term ‘ethylene glycol pathway’ refers to the pathway followed for preparation of ethylene glycol. The ethylene glycol pathway may be carried out chemically, biologically or combination thereof. The ethylene glycol pathway of the present disclosure may be carried out by employing a starting material selected from group comprising syngas, acetyl-CoA, pyruvate, phosphoenol pyruvate, oxaloacetate, tartrate, dimethyl tartrate, methyl 2-oxo acetate and methyl glycolate or any combination thereof.

As used herein, the term ‘non-naturally occurring’ is used in reference to the microorganism of the present disclosure and is intended to mean that the microorganism has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. The genetic alteration may be due to gene cloning, transformation, recombination, CRISPR-Cas9 etc. or a combination thereof. The non-naturally occurring microorganism of the present disclosure comprises at least one exogenous nucleic acid encoding enzymes required for the ethylene glycol pathway of the present disclosure.

As used herein, the term ‘ethylene glycol pathway gene(s)’ refers to the genes selected from group comprising pps (phosphoenol pyruvate synthetase), ppc (phosphoenol pyruvate carboxylase), ttdAB (tartrate dehydrogenase), fumB (fumarase B), or any combinations thereof. Within the purview of the present disclosure, the term ‘ethylene glycol pathway gene(s)’ may also include genes natively involved or engineered to take part in the Wood-Ljungdahl pathway for conversion of syngas to acetyl-CoA, and/or genes natively involved or engineered to take part in the conversion of acetyl-CoA to pyruvate. The ethylene glycol pathway gene(s) aid in converting a carbon source/ substrate selected from group comprising syngas, acetyl-CoA, pyruvate, phosphoenol pyruvate and oxaloacetate or any combination thereof to tartrate which is further processed by the methods of the present disclosure to obtain ethylene glycol.

As used herein, the term ‘ethylene glycol pathway enzyme(s)’ refers to the protein/enzymes selected from group comprising Pps (phosphoenol pyruvate synthetase, encoded by the gene pps), Ppc (phosphoenol pyruvate carboxylase, encoded by the gene ppc), TtdAB (tartrate dehydrogenase, encoded by the gene ttdAB), FumB (fumarase B, encoded by the gene fumB), or any combinations thereof. Within the purview of the present disclosure, the term ‘ethylene glycol pathway enzyme(s)’ may also include enzymes natively involved or engineered to take part in the Wood-Ljungdahl pathway for conversion of syngas to acetyl-CoA, and/or enzymes natively involved or engineered to take part in the conversion of acetyl-CoA to pyruvate. The ethylene glycol pathway enzyme(s) aid in converting a carbon source/ substrate selected from group comprising syngas, acetyl-CoA, pyruvate, phosphoenol pyruvate and oxaloacetate or any combination thereof to tartrate which is further processed by the methods of the present disclosure to obtain ethylene glycol.

As used herein, the term ‘ttdAB’ refers to the genes encoding the enzyme tartrate dehydrogenase and the term ‘TtdAB’ refers to the enzyme tartrate dehydrogenase. ttdAB consists of two genes ttdA and ttdB coding for 2 polypeptides that combine together to form the functional enzyme tartrate dehydrogenase.

As used herein, ‘syngas’ is a fuel gas mixture comprising predominantly carbon monoxide and hydrogen, and also comprising carbon dioxide. Besides its use as a fuel, it can be valorised to make chemicals either through chemical catalysis or biological fermentation route. Syngas is obtained through gasification of feedstocks such as biomass, municipal solid waste or any other trash. It is also a component of the waste gas from steel mills, gasification plant etc.; and on-purpose production is possible from gasification of refinery residue such as coke. In embodiments of the present disclosure, syngas is metabolized biologically using anaerobic bacteria of the present disclosure to produce tartrate. The tartrate thus produced is further converted to ethylene glycol as per the process of the present disclosure.

As used herein, the terms ‘phosphoenol pyruvate’ and ‘PEP’ are used interchangeably.

As used herein, the terms ‘oxaloacetate’ and ‘OAA’ are used interchangeably.

As used herein, the terms ‘tartrate’ and ‘tartaric acid’ are used interchangeably.

As used herein, the term ‘ethylene glycol’ also includes ‘mono ethylene glycol’.

As used herein, the terms ‘mono ethylene glycol’ and ‘MEG’ are used interchangeably.

In embodiments of the present disclosure, the tartaric acid employed for production of ethylene glycol may be produced chemically or through biological sources. Tartaric acid produced through biological sources (also referred to as biologically produced tartaric acid) refers to tartaric acid produced by microorganism(s) by capturing syngas or by employing starting material / carbon source / substrate selected from group comprising acetyl-CoA, pyruvate, phosphoenol pyruvate, oxaloacetate and tartrate.

The ethylene glycol pathway of the present disclosure comprises conversion of tartrate to ethylene glycol.

The present disclosure also relates to a process for preparing ethylene glycol comprising steps of:
a. converting tartaric acid to dialkyl tartrate;
b. converting the dialkyl tartrate to alkyl glycolate; and
c. converting the alkyl glycolate to ethylene glycol.

In an embodiment, the conversion of tartaric acid to dialkyl tartrate is carried out by alkylating the tartaric acid in presence of alkylating agent.

In another embodiment, the conversion of dialkyl tartrate to alkyl glycolate is carried out by oxidatively cleaving dialkyl tartrate in presence of oxidizing agent followed by reduction in presence of reducing agent.

In an embodiment, the conversion of alkyl glycolate to ethylene glycol is carried out by reducing the alkyl glycolate in presence of reducing agent.

In an embodiment, the process for preparing ethylene glycol comprises:
a. alkylating tartaric acid to obtain dialkyl tartrate;
b. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
c. reducing the alkyl glycolate to obtain ethylene glycol.

The present disclosure also relates to a process for preparing alkyl glycolate optionally followed by preparing ethylene glycol. In an embodiment, the process for preparing alkyl glycolate optionally followed by preparing ethylene glycol comprises steps of oxidatively cleaving dialkyl tartrate followed by reduction to obtain alkyl glycolate; and optionally reducing the alkyl glycolate to obtain ethylene glycol.

In an embodiment, the process for preparing ethylene glycol comprises steps of oxidatively cleaving dialkyl tartrate followed by reduction to obtain alkyl glycolate; and reducing the alkyl glycolate to obtain ethylene glycol.

In an embodiment, converting/alkylating the tartaric acid to dialkyl tartrate in the aforesaid processes is carried out by contacting the tartaric acid with alkylating agent and acid, and heating at a temperature ranging from about 0oC to about 90°C, preferably about 90°C for a period of about 30 minutes to about 9 h preferably about 4 h.

In an embodiment of the present disclosure, the alkylating agent is selected from a group comprising methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol and pentanol or any combination thereof; and the acid is selected from a group comprising sulphuric acid, nitric acid, tosic acid, orthophosphoric acid, methane sulphonic acid and glacial acetic acid or any combination thereof.

In an embodiment, the converting/oxidative cleavage of dialkyl tartrate in the aforesaid processes is carried out in presence of oxidizing agent and base at temperature ranging from about 0°C to about 90°C, preferably about 0°C for a period of about 30 minutes to about 4 hours preferably about 1 h.

In an embodiment of the present disclosure, the oxidizing agent is selected from a group comprising sodium periodate (NaIO4), potassium nitrate, potassium permanganate, nitric acid, hydrogen peroxide and halogens or any combination thereof; and the base is selected from a group comprising sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, ammonia and bromine or any combination thereof.

In an embodiment, the conversion/reduction to obtain alkyl glycolate in the aforesaid processes is carried out in presence of reducing agent at temperature ranging from about 0°C to about 90°C, preferably about 0°C for a period of about 30 minutes to about 4 hours preferably about 1 h.

In an embodiment of the present disclosure, the reduction to obtain alkyl glycolate is carried out in presence of reducing agent selected from a group comprising sodium borohydride (NaBH4), sodium hydride, calcium hydride, lithium hydride, lithium aluminium hydride, sodium sulphite and formic acid or any combination thereof.

In an embodiment, conversion/reduction to obtain ethylene glycol in the aforesaid processes is carried out in presence of a reducing agent at temperature ranging from about 0°C to about 90°C, preferably about 0°C for a period of about 30 minutes to about 4 hours preferably about 1 h.

In an embodiment of the present disclosure, the reduction to obtain ethylene glycol is carried out in presence of a reducing agent selected from a group comprising lithium aluminium hydride, sodium hydride, calcium hydride, lithium hydride, sodium borohydride, sodium sulphite and formic acid or any combination thereof.

In a preferred embodiment, the dialkyl tartrate is dimethyl tartrate, the alkyl glycolate is methyl glycolate and the ethylene glycol is mono ethylene glycol.

In a preferred embodiment, the process for preparing ethylene glycol comprises steps of:
a. methylating tartaric acid to obtain dimethyl tartrate;
b. oxidatively cleaving the dimethyl tartrate followed by reduction to obtain methyl glycolate; and
c. reducing the methyl glycolate to obtain MEG.

In an embodiment, the methylation of tartaric acid to dimethyl tartrate is carried out by contacting the tartaric acid with methylating agent and acid. In an embodiment of the present disclosure, the methylating agent is methanol; and the acid is selected from a group comprising sulphuric acid, nitric acid, tosic acid, orthophosphoric acid, methane sulphonic acid and glacial acetic acid or any combination thereof.

In an embodiment, the oxidative cleavage of dimethyl tartrate is carried out in presence of oxidizing agent selected from a group comprising sodium periodate (NaIO4), potassium nitrate, potassium permanganate, nitric acid, hydrogen peroxide and halogens or any combination thereof; and base selected from a group comprising sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, ammonia and bromine or any combination thereof.

In an embodiment, the reduction to obtain methyl glycolate is carried out in presence of reducing agent selected from a group comprising sodium borohydride (NaBH4), sodium hydride, calcium hydride, lithium hydride, lithium aluminium hydride, sodium sulphite and formic acid or any combination thereof.

In an embodiment, the reduction to obtain mono ethylene glycol in the aforesaid processes is carried out in presence of a reducing agent selected from a group comprising lithium aluminium hydride, sodium hydride, calcium hydride, lithium hydride, sodium borohydride, sodium sulphite and formic acid or any combination thereof.

In an exemplary embodiment, converting tartaric acid to dialkyl tartrate comprises contacting tartaric acid with alkylating agent and acid and refluxing the mixture at a temperature ranging from about 70oC to about 90oC. The mixture obtained is cooled at a temperature ranging from about 10oC to about 35oC additional reactants or solvents may be removed from the reaction mixture by conventional techniques including but not limiting to evaporation, filtration, sedimentation or distillation. The additional acid is neutralized by neutralizing agent such as sodium bicarbonate, sodium hydroxide, potassium hydroxide, or ammonia, till pH is about 7.0. The dialkyl tartrate thus obtained is extracted using solvents such as but not limiting to ethyl acetate, methyl acetate, diethyl ether or acetone or combinations thereof. The dialkyl tartrate in the organic layer is isolated by techniques such as evaporation, filtration, sedimentation or distillation. In an embodiment, the evaporation is done over sodium sulfate as per standard protocols.

In an exemplary embodiment, converting dimethyl tartrate to methyl glycolate comprises dissolving dimethyl tartrate in solvents such as but not limited to dichloromethane, methyl acetate, diethyl ether, or acetone. The obtained mixture is reacted with about 1 to about 10 equivalents of oxidizing agent such as NaIO4. The reaction was carried out at 0°C for about 30 minutes to about 4 h, preferably about 1 h. The organic layer is evaporated over sodium sulfate to obtain methyl glycolate. In an embodiment, the product is confirmed by creating a 2,4-Dinitrophenylhydrazine (DNPH) derivative and using LC-MS.

In an exemplary embodiment, converting methyl glycolate to MEG comprises mixing methyl glycolate, with solvent such as tetrahydrofuran (THF), dichloromethane, methyl acetate, diethyl ether, or acetone and about t 1 to about 10 equivalents of reducing agent such as Lithium aluminium hydride at about t 1 to about 10 equivalents, preferably 0°C for about 30 minutes to about 4 h, preferably 1 h. The reaction is quenched carefully using quenching agents such as but not limiting to NaOH, potassium hydroxide, calcium hydroxide or ammonia. The reaction mixture is analysed for the presence of MEG in GC-MS.
In an exemplary embodiment of the present disclosure, the conversion of tartrate to ethylene glycol occurs as per the scheme illustrated in Figure 3(a).

In an exemplary embodiment of the present disclosure, the conversion of tartrate to ethylene glycol occurs as per the scheme illustrated in Figure 3(b) which shows formation of an intermediate ethyl 2-oxo acetate which further forms methyl glycolate.

The present disclosure also relates to a process for obtaining ethylene glycol from oxaloacetate, said process comprises steps of:
a. hydrolyzing oxaloacetate to obtain tartaric acid;
b. alkylating the tartaric acid to obtain dialkyl tartrate;
c. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
d. reducing the alkyl glycolate to obtain ethylene glycol.

In an embodiment, the hydrolysis of the oxaloacetate to obtain the tartaric acid in the aforesaid process is carried out in presence of fumarase B or tartrate dehydrogenase.

In an embodiment, the steps of alkylation, oxidative cleavage and reduction in the aforesaid process for obtaining ethylene glycol from oxaloacetate is as detailed above.

The present disclosure also relates to a process for preparing ethylene glycol from syngas, said process comprising steps of:
a) converting syngas to acetyl-CoA;
b) converting the acetyl-CoA to pyruvate;
c) converting the pyruvate to phosphoenol pyruvate;
d) converting the phosphoenol pyruvate to oxaloacetate;
e) converting the oxaloacetate to tartaric acid;
f) converting the tartaric acid to dialkyl tartrate;
g) converting the dialkyl tartrate to alkyl glycolate; and
h) converting the alkyl glycolate to ethylene glycol.

In an embodiment, the conversion of syngas to acetyl-CoA is carried out by Wood-Ljungdahl pathway, the conversion of acetyl-CoA to pyruvate is carried out in presence of pyruvate synthetase, the conversion of pyruvate to phosphoenol pyruvate is carried out in presence of phosphoenol pyruvate synthetase, the conversion of phosphoenol pyruvate to oxaloacetate is carried out in presence of phosphoenol pyruvate carboxylase, and/or the conversion of oxaloacetate to tartaric acid is carried out in presence of fumarase B or tartrate dehydrogenase. In an embodiment, the conversion of tartaric acid to ethylene glycol via the intermediates dialkyl tartrate and alkyl glycolate in the aforesaid process for obtaining ethylene glycol from syngas is as detailed above.

In an embodiment, the process for preparing ethylene glycol comprises:
a) acetylating syngas to obtain acetyl-CoA;
b) converting the acetyl-CoA to obtain pyruvate;
c) phosphorylating the pyruvate to obtain phosphoenol pyruvate;
d) dephosphorylating the phosphoenol pyruvate to obtain oxaloacetate;
e) hydrolyzing the oxaloacetate to obtain tartaric acid;
f) alkylating the tartaric acid to obtain dialkyl tartrate;
g) oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
h) reducing the alkyl glycolate to obtain ethylene glycol.

In an embodiment, the acetylation of the syngas to obtain the acetyl-CoA is carried out by Wood-Ljungdahl pathway, conversion of the acetyl-CoA to obtain the pyruvate is carried out in presence of pyruvate synthetase, phosphorylation of the pyruvate to obtain the phosphoenol pyruvate is carried out in presence of phosphoenol pyruvate synthetase, dephosphorylation of the phosphoenol pyruvate to the obtain oxaloacetate is carried out in presence of phosphoenol pyruvate carboxylase, and/or hydrolysis of the oxaloacetate to obtain the tartaric acid is carried out in presence of fumarase B or tartrate dehydrogenase. In an embodiment, the steps of alkylation, oxidative cleavage and reduction in the aforesaid process for obtaining ethylene glycol from syngas is as detailed above.

In an embodiment, the conversion of syngas to tartaric acid in the aforesaid process for preparing ethylene glycol is carried out in a microorganism.

In an embodiment, the conversion of syngas to tartaric acid in the aforesaid process is carried out in microorganism selected from a group comprising Clostridium sp., Moorella sp., Acetitomaculum sp., Acetobacterium sp., Blautia sp., Eubacterium sp., Sporomusa sp., Syntrophococcus sp., Butyribacterium sp., Escherichia sp., Desulfobacter sp., Methanothermobacter sp., Methylobacterium sp., Pseudomonas sp., Rhodobacter sp., Euglena sp., Helicobacter sp., Methanococcus sp., Mycobacterium sp., Panteoa sp., Bacillus sp., Yarrowia sp., and Trichoderma sp.

In an exemplary embodiment, the aforesaid microorganism is selected from a group comprising Acetitomaculum ruminis, Acetobacterium carbinolicum, Acetobacterium woodii, Blautia producta, Clostridium formicaceticum, Clostridium ljungdahlii, Eubacterium limosum, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa malonica, Sporomusa termitida, Syntrophococcus sucromutans, Butyribacterium methylotrophicum, Desulfobacter hydrogenophilus, Escherichia coli, Desulfobacter hydrogenophilus, Megathyrsus maximus, Methanothermobacter thermoautotrophicus, Methylobacterium extorquens, Pseudomonas putida, Rhodobacter sphaeroides, Euglena gracilis, Helicobacter pylori, Methanococcus maripaludis, Mycobacterium tuberculosis, Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica and Trichoderma reesei or any combination thereof.

In an embodiment, the microorganism is non-naturally occurring microorganism comprising one or more genes selected from a group comprising pyruvate synthetase, phosphoenol pyruvate synthetase, phosphoenol pyruvate carboxylase, fumarase B and tartrate dehydrogenase.

In an embodiment, the conversion of syngas to tartaric acid in the aforesaid process for preparing ethylene glycol is carried out when the microorganism comes in contact with the syngas.

In embodiments of the present disclosure, the aforesaid process(es) is/are carried out in presence of solvent selected from a group comprising methanol, ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, ethanol, glycerol, purified water and methyl acetate or any combination thereof.

In embodiments of the present disclosure, one or more step(s) of the aforesaid process(es) is/are carried out at temperature ranging from about 0°C to about 90°C, and/or for a time period ranging from about 30 minutes to about 4 hours.

In embodiments of the present disclosure, the aforesaid process(es) further comprise acts selected from a group comprising isolation and/or purification of the corresponding compound; wherein the said isolation is carried out by acts selected from a group comprising addition of solvent, quenching, cooling, heating, removal of solvent, drying, filtration, extraction and combination of acts thereof; and wherein the purification is carried out by water washing or solvent(s) washing or a combination thereof.

In an alternate embodiment, the conversion of tartrate to ethylene glycol in the process or microorganism of the present disclosure can occur in the microorganism by a) a single step double decarboxylation reaction of tartrate to directly yield ethylene glycol with the evolution of two mole of carbon dioxide, in presence of enzymes such as but not limiting to decarboxylases eg. tartrate decarboxylase, b) sequential decarboxylation of tartaric acid to obtain glycerate followed by conversion of glycerate to ethylene glycol, and c) oxidation of tartrate to form 2-hydroxy-3-oxosuccinate in presence of D-malate/3-isopropylmalate dehydrogenase (decarboxylating) and tartrate dehydrogenase, followed by decarboxylation in presence of oxaloglycolate reductase to give glycerate which is further converted to ethylene glycol. In an embodiment, decarboxylation of glycerate to ethylene glycol is carried out in presence of enzymes such as but not limiting to Pyruvate decarboxylase, Malic enzyme, or Acetoacetate decarboxylase.

In an embodiment, the ethylene glycol pathway comprises: capturing of syngas by the microorganism, conversion of syngas to acetyl-CoA, conversion of acetyl-CoA to pyruvate, conversion of pyruvate to phosphoenol pyruvate (PEP), conversion of PEP to oxaloacetate, conversion of oxaloacetate to tartrate and conversion of tartrate to ethylene glycol by the process of the present disclosure. In an alternate embodiment, the ethylene glycol pathway comprises: conversion of acetyl-CoA to pyruvate, conversion of pyruvate to PEP, conversion of PEP to OAA, conversion of oxaloacetate to tartrate and conversion of tartrate to ethylene glycol. In another embodiment, the ethylene glycol pathway comprises: conversion of pyruvate to PEP, conversion of PEP to OAA, conversion of oxaloacetate to tartrate and conversion of tartrate to ethylene glycol. In yet another embodiment, the ethylene glycol pathway comprises: conversion of phosphoenol pyruvate to oxaloacetate, conversion of oxaloacetate to tartrate and conversion of tartrate to ethylene glycol. In still another embodiment, the ethylene glycol pathway comprises: conversion of oxaloacetate to tartrate and conversion of tartrate to ethylene glycol. In still another embodiment, the ethylene glycol pathway consists of conversion of tartrate to ethylene glycol. In an embodiment, all the aforesaid conversions may be carried out chemically (i.e. by carrying out the chemical reactions sequentially in vitro) or biologically (i.e. in a biolological system such as the microorganism of the present disclosure) or a combination thereof. Thus, the present disclosure envisages conducting the ethylene glycol pathway partially in a biological system and partially chemically. In a preferred embodiment, the ethylene glycol pathway of the present disclosure involves conversion of syngas/acetyl-CoA/pyruvate/PEP/OAA to tartrate in a microorganism, obtaining tartrate from the microorganism and conversion of the tartrate to ethylene glycol chemically.

In a preferred embodiment, the ethylene glycol pathway comprises: capturing of syngas by the microorganism, conversion of syngas to acetyl-CoA, conversion of acetyl-CoA to pyruvate, conversion of pyruvate to phosphoenol pyruvate (PEP), conversion of PEP to oxaloacetate, conversion of oxaloacetate to tartrate; obtaining tartrate from the microorganism and chemical conversion of the tartrate to ethylene glycol by the process of the present disclosure.

In embodiments of the present disclosure, the ethylene glycol pathway comprises: capturing of syngas by the microorganism, conversion of syngas to acetyl-CoA by Wood-Ljungdahl pathway of the microorganism, conversion of acetyl-CoA to phosphoenol pyruvate by initial gluconeogenesis reactions in presence of the enzyme pyruvate synthase, conversion of phosphoenol pyruvate to oxaloacetate with the consumption of CO2 (which is optionally taken up from syngas) in presence of the enzyme phosphoenol pyruvate carboxylase, conversion of OAA to form tartrate by hydration reaction in presence of the enzyme fumB or ttdAB. The final summation of formation of tartrate from syngas (CO + H2) is shown below:
CO + H2 + CO2 + 2 H2O + 3 ATP + NADPH + Fdred + NADH --> NAD+ + 3 ADP + 3 PO43- + NADP+ + Fdox + 5 H+ + Tartrate

The tartrate thus produced by the microorganism is extracted from the microorganism and processed chemically as per the process of the present disclosure to obtain ethylene glycol. The ethylene glycol pathway also encompasses production of tartrate by the microorganism by replacing the carbon source from syngas to acetyl-CoA, pyruvate, PEP or OAA.

In embodiments of the present disclosure, tartrate produced by the microorganism is obtained by conventional techniques including but not limiting to sonication, solvent extraction or combination thereof.

The present disclosure also relates to non-naturally occurring microorganism(s) having an ethylene glycol pathway. The non-naturally occurring microorganisms of the present disclosure are capable of producing tartrate and comprise at least one exogenous nucleic acid encoding one or more ethylene glycol pathway enzyme.

In embodiments of the present disclosure, the said microorganism(s) comprises one or more ethylene glycol pathway gene(s).

In a preferred embodiment, the microorganism of the present disclosure comprises at least one exogenous ethylene glycol pathway gene selected from selected from a group comprising but not limiting to ydbK (pyruvate synthase), pps (phosphoenol pyruvate synthetase), ppc (phosphoenol pyruvate carboxylase), ttdAB (tartrate dehydrogenase), fumB (fumarase B), genes involved in Wood-Ljungdahl pathway, or any combinations thereof.

In an exemplary embodiment of the present disclosure, the microorganism of the present disclosure comprises exogenous ethylene glycol pathway genes: pps (phosphoenol pyruvate synthetase), ppc (phosphoenol pyruvate carboxylase) and ttdAB (tartrate dehydrogenase)/ fumB (fumarase B).

In a preferred embodiment, the microorganism of the present disclosure comprises exogenous ethylene glycol pathway gene selected from a group comprising pyruvate synthetase, phosphoenol pyruvate synthetase, phosphoenol pyruvate carboxylase and fumarase B or any combination thereof.

In another preferred embodiment, the microorganism of the present disclosure comprises exogenous ethylene glycol pathway gene selected from a group comprising pyruvate synthetase, phosphoenol pyruvate synthetase, phosphoenol pyruvate carboxylase and tartrate dehydrogenase or any combination thereof.

The microorganism of the present disclosure is genetically engineered to overexpress one or more of the ethylene glycol pathway enzymes. In an embodiment, the microorganism of the present disclosure express ethylene glycol pathway enzymes in a sufficient amount to produce tartrate.

The microorganism of the present disclosure is prokaryotic or eukaryotic microorganism.

In an embodiment, the microorganism of the present disclosure is selected from a group comprising but not limiting to Clostridium sp., Moorella sp., Acetitomaculum sp., Acetobacterium sp., Blautia sp., Eubacterium sp., Sporomusa sp., Syntrophococcus sp., Butyribacterium sp., Escherichia sp., Desulfobacter sp., Methanothermobacter sp., Methylobacterium sp., Pseudomonas sp., Rhodobacter sp., Euglena sp., Helicobacter sp., Methanococcus sp., Mycobacterium sp., Panteoa sp., Bacillus sp., Yarrowia sp., and Trichoderma sp.
In an exemplary embodiment, the microorganism of the present disclosure is selected from, but not limiting to, group comprising Acetitomaculum ruminis, Acetobacterium carbinolicum, Acetobacterium woodii, Blautia producta, Clostridium formicaceticum, Clostridium ljungdahlii, Eubacterium limosum, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa malonica, Sporomusa termitida, Syntrophococcus sucromutans, Butyribacterium methylotrophicum, Desulfobacter hydrogenophilus, Escherichia coli, Desulfobacter hydrogenophilus, Megathyrsus maximus, Methanothermobacter thermoautotrophicus, Methylobacterium extorquens, Pseudomonas putida, Rhodobacter sphaeroides, Euglena gracilis, Helicobacter pylori, Methanococcus maripaludis, Mycobacterium tuberculosis, Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica and Trichoderma reesei or any combination thereof.

In a preferred embodiment, the microorganism is from Clostridium sp.

In an exemplary embodiment, the microorganism is Clostridium ljungdahlii.

In an exemplary embodiment, the microorganism of the present disclosure is of Clostridium sp. such as Clostridium ljungdahlii which has the capability to use CO in syngas as carbon source through the Wood-Ljungdahl pathway, where acetyl-CoA is the central intermediate. The said microorganism is further modified to express the genes encoding the enzymes Pps and Ppc (optionally Ps); and at least one of FumB and TtdAB.

In another preferred embodiment, the microorganism is from Escherichia sp.

In an exemplary embodiment, the microorganism is Escherichia coli such as but not limiting to Escherichia coli K-12 substr. MG1655, E. coli BL21 DE3, E. coli ATCC 8739 etc.

The microorganisms of the present disclosure comprising pps-ppc-fumB or pps-ppc-ttdAB genes have been deposited with the International Depository National Centre for Microbial Resource (NCMR), Pune, India, and has been accorded the accession number MCC 0128 [E. coli (SIBD 1978); pps-ppc-fumB] and MCC 0129 [E. coli (SIBD 1735); pps-ppc-ttdAB].

In an exemplary embodiment, the present disclosure provides for microorganism and methods for formation of tartaric acid from syngas as per the pathway depicted in Figure 1.

In an embodiment, the microorganism of the present disclosure is naturally capable of capturing syngas and converting it to acetyl-CoA by Wood-Ljungdahl pathway and optionally converting the acetyl-CoA to pyruvate by initial gluconeogenesis reactions/pyruvate synthase. The said microorganism is further engineered to comprise genes selected from group comprising pps, ppc, fumB and ttdAB or combinations thereof, for production of tartrate. The tartrate produced by the microorganism may be chemically converted as described above.

In an embodiment, the conversion of syngas to tartaric acid is carried out when the microorganism comes in contact with the syngas. The microorganism may capture syngas which is naturally occurring in the environment or syngas which has been industrially/artificially produced. In another preferred embodiment, the microorganism is capable of producing ethylene glycol from syngas.

In an embodiment, the Wood-Ljungdahl Pathway can generate acetyl-CoA from syngas naturally using anaerobic cultivation of microorganisms such as but not limiting to microorganisms of Clostridium sp., Morella sp., Acetitomaculum sp., Acetobacterium sp., Blautia sp., Eubacterium sp., Sporomusa sp., Syntrophococcus sp., Butyribacterium sp. etc. In an exemplary non-limiting embodiment of the present disclosure, the microorganisms capable of capturing syngas are Acetitomaculum ruminis, Acetobacterium carbinolicum, Acetobacterium woodii, Blautia producta, Clostridium formicaceticum, Eubacterium limosum, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa malonica, Sporomusa termitida, Syntrophococcus sucromutans and Butyribacterium methylotrophicum. The acetyl-CoA is further converted to pyruvate, then to tartaric acid and then to ethylene glycol.
In another embodiment, pyruvate is converted to phosphophenol pyruvate in the gluconeogenetic pathway by the phosphophenol pyruvate synthetase enzyme. The gene which expresses this enzyme is pps found in microorganisms such as but not limiting to microorganisms of Desulfobacter sp. like Desulfobacter hydrogenophilus, Escherichia sp. like Escherichia coli, etc. The reaction converts pyruvate to PEP under the influence of ATP with the release of AMP and one mole of phosphate ion.

In another embodiment, conversion of PEP to OAA occurs naturally in multiple organisms and is catalysed by phosphoenol pyruvate carboxylase. This reaction replenishes the OAA pools in the cells which is required for the TCA cycle activity. The following strains contain probable genes for expression of the required enzyme: Amaranthus sp., Arabidopsis sp. (ppc3), Desulfobacter sp., Escherichia sp. (ppc), Megathyrsus sp., Methanothermobacter sp. (ppcA), Methylobacterium sp. (ppcA), Panicum sp., Zea sp. (pep1). In an exemplary embodiment, the strains containing the probable genes for expression of the required enzyme may be selected from group comprising but not limiting to Amaranthus hypochondriacus, Arabidopsis thaliana col (ppc3), Desulfobacter hydrogenophilus, Escherichia coli K-12 substr. MG1655 (ppc), Megathyrsus maximus, Methanothermobacter thermoautotrophicus Delta H (ppcA), Methylobacterium extorquens AM1 (ppcA), Panicum miliaceum, Zea mays (pep1), etc.

In another embodiment, conversion of OAA to tartrate is not directly seen in nature. Enzymes with promiscuous activity for the hydration and reduction reaction have been used for this conversion. The genes required for carrying out these reactions are fumB encoding for fumarase B. Alternately, these reactions are carried out by L-tartrate dehydrogenase encoded by the genes ttdA and ttdB. The gene fumB encoding for fumarase B is found in strains such as but not limiting to Escherichia sp., Arabidopsis sp., Ascaris sp., Desulfobacter sp., Euglena sp., Helicobacter sp., Methanococcus sp., Mycobacterium sp. For example, the genes fumB may be present in microorganisms such as Arabidopsis thaliana, Ascaris suum, Desulfobacter hydrogenophilus, E. coli like Escherichia coli K-12 substr. MG1655, Euglena gracilis, Helicobacter pylori like Helicobacter pylori 26695, Homo sapiens, Methanococcus maripaludis, Mycobacterium tuberculosis like Mycobacterium tuberculosis H37Rv, etc. The genes ttdA and ttdB encoding for L-tartrate dehydrogenase are found in strains including but not limiting to Escherichia sp., Pseudomonas sp. and Rhodobacter sp. For example, the genes ttdA and ttdB may be present in microorganisms such as E. coli, Pseudomonas putida, Rhodobacter sphaeroides, etc.

In an embodiment, tartrate can be intracellularly utilized by the microorganism to produce ethylene glycol by a recombinant microorganism expressing enzymes selected from a group comprising but not limiting to Tdc (tartaric acid decarboxylase, encoded by the gene tdc), DmlA (D-malate/3-isopropylmalate dehydrogenase, encoded by the gene dmlA), Tdh (tartrate dehydrogenase, encoded by the gene tdh), oxaloglycolate reductase, Pdc (pyruvate decarboxylase, encoded by the gene pdc), MaeB (malic enzyme, encoded by the gene maeB) and Adc (acetoacetate decarboxylase, encoded by the gene adc) or any combination thereof. Preferably, the tartrate produced by the microorganism is converted to ethylene glycol chemically by a) alkylating tartaric acid to obtain dialkyl tartrate; b) oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and c) reducing the alkyl glycolate to obtain ethylene glycol.

The microorganism of the present disclosure is capable of producing tartrate from a substrate/starting material selected from any of syngas, acetyl-CoA, pyruvate, PEP or OAA.

The present disclosure also relates to methods of production of microorganism(s) having ethylene glycol pathway.

The method of producing the microorganism of the present disclosure comprises incorporating one or more exogenous ethylene glycol pathway genes in the microorganism. In an embodiment, one or more ethylene glycol pathway genes are cloned and/or transformed into the microorganism.

In an embodiment, the microorganisms which can be produced as per the method of the present disclosure to have ethylene glycol pathway are selected from but not limiting to microorganisms of Clostridium sp., Moorella sp., Acetitomaculum sp., Acetobacterium sp., Blautia sp., Eubacterium sp., Sporomusa sp., Syntrophococcus sp., Butyribacterium sp., Escherichia sp., Desulfobacter sp., Methanothermobacter sp., Methylobacterium sp., Pseudomonas sp., Rhodobacter sp., Euglena sp., Helicobacter sp., Methanococcus sp., Mycobacterium sp., Panteoa sp., Bacillus sp., Yarrowia sp., and Trichoderma sp. In an exemplary embodiment, the microorganism is selected from, but not limiting to, group comprising Acetitomaculum ruminis, Acetobacterium carbinolicum, Acetobacterium woodii, Blautia producta, Clostridium formicaceticum, Clostridium ljungdahlii, Eubacterium limosum, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa malonica, Sporomusa termitida, Syntrophococcus sucromutans, Butyribacterium methylotrophicum, Desulfobacter hydrogenophilus, Escherichia coli, Desulfobacter hydrogenophilus, Megathyrsus maximus, Methanothermobacter thermoautotrophicus, Methylobacterium extorquens, Pseudomonas putida, Rhodobacter sphaeroides, Euglena gracilis, Helicobacter pylori, Methanococcus maripaludis, Mycobacterium tuberculosis, Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica and Trichoderma reesei or any combination thereof. In a preferred embodiment, the microorganism is from Clostridium sp. In an exemplary embodiment, the microorganism is Clostridium ljungdahlii. In another preferred embodiment, the microorganism is from Escherichia sp. In an exemplary embodiment, the microorganism is Escherichia coli such as but not limiting to Escherichia coli K-12 substr. MG1655, E. coli BL21 DE3, E. coli ATCC 8739 etc.

In an embodiment, the ethylene glycol pathway genes that are incorporated into the microorganism of the present disclosure, by means such as but not limiting to cloning and/or transformation, are selected from: at least one of ttdAB (tartrate dehydrogenase) or fumB (fumarase B), along with pps (phosphoenol pyruvate synthetase) and ppc (phosphoenol pyruvate carboxylase).

In an embodiment, the ethylene glycol pathway genes that are incorporated into the microorganism such as but not limiting to microorganism of the Clostridium sp., by means such as but not limiting to cloning and/or transformation, are selected from: at least one of ttdAB (tartrate dehydrogenase) or fumB (fumarase B), along with pps (phosphoenol pyruvate synthetase) and ppc (phosphoenol pyruvate carboxylase).
In an embodiment, the ethylene glycol pathway genes that are incorporated into the microorganism such as but not limiting to microorganism of the Escherichia sp., by means such as but not limiting to cloning and/or transformation, are selected from: at least one of ttdAB (tartrate dehydrogenase) or fumB (fumarase B), along with pps (phosphoenol pyruvate synthetase) and ppc (phosphoenol pyruvate carboxylase).

In a preferred embodiment, the ethylene glycol pathway genes pps (phosphoenol pyruvate synthetase), ppc (phosphoenol pyruvate carboxylase) along with either of ttdAB (tartrate dehydrogenase) and fumB (fumarase B) are all cloned and transformed into the microorganism such as but not limiting to E. coli, Clostridium formicaceticum, Clostridium ljungdahlii, etc.

In an embodiment, the cloning is confirmed by diagnostic PCR and/or restriction enzyme double digestion reactions.

In an embodiment, the method for producing the microorganism of the present disclosure comprises acts of:
- Individually cloning the ethylene glycol pathway genes ydbK (optionally cloned), pps and ppc, and genes selected from fumB or ttdAB into a suitable expression vector. Positive clones are inoculated, followed by isolation of the plasmids,
- The plasmids comprising the fumB or ttdAB genes, and vector(s) comprising ydbK (optionally cloned), pps and ppc or combination thereof are co-transformed using suitable molecular biology techniques into the microorganism,
- The transformants are selected on a suitable selection medium,
- Optionally the requisite genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol are individually clined into a suitable expression vector. Positive clones are inoculated, followed by isolation of the plasmids; the plasmids are transformed using suitable molecular biology techniques into the transformant, and
- The transformant thus produced is selected on a suitable selection medium.

In an embodiment, the method for producing the microorganism of the present disclosure comprises acts of:
- Individually cloning the ethylene glycol pathway genes ydbK, pps and ppc, and genes selected from fumB or ttdAB, and optionally genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol, into a suitable expression vector. Positive clones are inoculated, followed by isolation of the plasmids,
- The plasmids comprising the fumB or ttdAB genes, and vector comprising ydbK, pps and ppc, and optionally the vector(s) comprising the genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol are co-transformed using suitable molecular biology techniques into the microorganism, and
- The transformants are selected on a suitable selection medium.

In an embodiment, the method for producing the microorganism of the present disclosure comprises acts of:
- Individually cloning the ethylene glycol pathway genes pps and ppc, and genes selected from fumB or ttdAB, and optionally genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol, into a suitable expression vector. Positive clones are inoculated, followed by isolation of the plasmids,
- The plasmids comprising the fumB or ttdAB genes, and vector comprising pps and ppc or combination thereof, and optionally the vector(s) comprising the genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol are co-transformed using suitable molecular biology techniques into the microorganism, and
- The transformants are selected on a suitable selection medium.

In an embodiment, the method for producing the microorganism of the present disclosure comprises acts of:
- Individually cloning the ethylene glycol pathway gene ppc, and genes selected from fumB or ttdAB, and optionally genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol, into a suitable expression vector. Positive clones are inoculated, followed by isolation of the plasmids,
- The plasmids comprising the fumB or ttdAB genes, vector comprising ppc, and optionally the vector(s) comprising the genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol are co-transformed using suitable molecular biology techniques into the microorganism, and
- The transformants are selected on a suitable selection medium.

In an exemplary embodiment, the method for producing the microorganism of the present disclosure comprises acts of:
- Individually cloning the ethylene glycol pathway genes such as pps, ppc, fumB/ttdAB, and optionally genes for conversion of tartrate to ethylene glycol, into a suitable expression vector. Positive clones are inoculated, followed by isolation of the plasmids,
- The vector-ppc is used as a template plasmid to clone the pps gene through restriction sites to obtain the vector-pps-ppc.
- The expression cassettes of genes pps and ppc are amplified from the above mentioned vector and cloned into a shuttle through restriction sites,
- The plasmids comprising the fumB or ttdAB genes, vector-pps-ppc, and optionally the vector(s) comprising the genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol are co-transformed using techniques such as but not limited to electroporation into the microorganism, and
- The transformants are selected on a suitable selection medium.

In an exemplary embodiment, the method for producing the microorganism of the present disclosure comprises acts of:
- Cloning the ethylene glycol pathway genes such as pps, ppc, fumB/ttdAB, and optionally the genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol, individually, into a suitable expression vector (such as but not limiting to pET28a) through restriction endonucleases sites (such as but not limiting to NdeI and HindIII). The cloning is confirmed by diagnostic PCR and/or restriction enzyme double digestion reactions. Positive clones are inoculated, followed by isolation of the plasmids and the cloning is reconfirmed by restriction enzyme double digestion for the presence of the respective inserts.
- The vector-ppc is used as a template plasmid to clone the pps gene through restriction sites (such as but not limiting to XbaI and HindIII). The cloning for vector-pps-ppc is confirmed using the aforesaid methods.
- The expression cassettes of genes pps and ppc (T7 prom-RBS-pps-ppc-T7 term) are amplified from the above mentioned vector and cloned into a shuttle vector (such as the pMTL-0 vector, shuttle vector for C. ljungdahlii) through restriction sites (such as AscI and SbfI). The cloning is confirmed as described earlier.
- The plasmids comprising fumB or ttdAB, vector-pps-ppc and optionally the vector(s) comprising the genes encoding enzyme(s) required for conversion of tartrate to ethylene glycol are co-transformed using electroporation into the microorganism and the transformants are selected on a suitable selection medium (such as but not limiting to Luria-Bertani (LB) medium with kanamycin and chloramphenicol). The cloning is confirmed as described earlier.

In an embodiment, the protein expression profile for all the constructed strains with the ethylene glycol pathway genes is studied. All strains expressed Pps, Ppc, TtdA, TtdB/FumB and optionally genes encoding enzymes required for conversion of tartrate to ethylene glycol, when induced.

In an embodiment, the analytical method employed for tartrate and/or ethylene glycol is chromatographic techniques such as HPLC and spectroscopy techniques such as Mass Spectroscopy, GC-MS and LC-MS.

In an embodiment, the microorganism cell culture is lysed for determining production of tartrate/ethylene glycol. In an embodiment, the cell lysis is performed using the pulse sonication technique. The sample lysis is performed by incubating the sample in an ice bath. The parameters used in the sonication are: Amplitude of about 50; Pulse time of about 3 sec; Total pulse time of about 2 min; Rest time between pulses of about 1 min.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves overexpressing one or more ethylene glycol pathway genes in the microorganism produced by the present disclosure.

The microorganisms produced as per the method of the present disclosure express the ethylene glycol pathway genes and efficiently produce tartrate and/or ethylene glycol by employing starting material / carbon source / substrate selected from group comprising syngas, acetyl-CoA, pyruvate, phosphoenol pyruvate, oxaloacetate and methyl glycolate or any combinations thereof.

The present disclosure also relates to use of the microorganism of the present disclosure for producing tartare and thereby ethylene glycol.

Although the pathway of Figures 1 and 3 depict formation of ethylene glycol from syngas and tartrate, respectively, a person skilled in the art, in view of the teachings of the present disclosure, will be able to suitably modify the methods and microorganisms taught by the present disclosure to obtain ethylene glycol by employing any of syngas, acetyl-CoA, pyruvate, PEP, OAA, tartrate, dimethyl tartrate or methyl glycolate as the starting material / carbon source / substrate. Hence, the present disclosure also envisages methods and microorganisms for production of ethylene glycol from syngas, acetyl-CoA, pyruvate, PEP, OAA, tartrate, dimethyl tartrate or methyl glycolate or any combination thereof.

The present disclosure also relates to methods for producing ethylene glycol by culturing a microorganism described herein under conditions suitable to produce tartrate, optionally isolating tartrate and chemically converting tartrate to ethylene glycol.

The present disclosure relates to method for production of tartrate from syngas as the carbon source, using the microorganism described herein.
In an embodiment of the present disclosure, the method for producing tartrate comprises culturing strains containing overexpressed genes for tartrate production under ambient conditions comprising syngas.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the steps: capturing of syngas by the microorganism of the present disclosure; generation of acetyl-CoA from the syngas by the microorganism; conversion of the acetyl-CoA to pyruvate which is further converted to phosphoenol pyruvate by the microorganism; conversion of the PEP to OAA by the microorganism; conversion of the OAA to form tartrate; optionally obtaining tartrate from the microorganism and conversion of tartrate to ethylene glycol by any of the afore-described reactions.

In an embodiment of the present disclosure, the method for producing ethylene glycol comprises the steps of:
capturing syngas by the microorganism of the present disclosure;
generation of acetyl-CoA from syngas by anaerobic cultivation of the microorganism;
conversion of the acetyl-CoA to phosphoenol pyruvate by initial gluconeogenesis reactions (Figure 2);
conversion of the PEP to OAA by the phosphoenol pyruvate carboxylase enzyme;
conversion of the OAA to tartrate by the enzymes Fumarase B or TtdAB; and
conversion of tartrate obtained from the microorganism to ethylene glycol (preferably MEG) by any of the afore-described reactions.

The present disclosure relates to method for production of tartrate from acetyl-CoA as the carbon source, using the microorganism described herein.

In an embodiment of the present disclosure, the method for producing tartrate comprises culturing strains containing overexpressed genes for tartrate production on a media comprising acetyl-CoA at ambient conditions.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the steps: conversion of the acetyl-CoA to pyruvate by the microorganism of the present disclosure; conversion of the pyruvate to PEP; conversion of the PEP to OAA; conversion of the OAA to tartrate by the microorganism; optionally obtaining tartrate from the microorganism and conversion of tartrate to ethylene glycol by any of the afore-described reactions.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves conversion of acetyl-CoA by the microorganism of the present disclosure to pyruvate by the enzyme pyruvate synthase, conversion of pyruvate to phosphoenol pyruvate by the enzyme phosphoenol pyruvate synthase; conversion of the PEP to OAA by the enzyme phosphoenol pyruvate carboxylase; conversion of the OAA to form tartrate by the enzyme Fumarase B or TtdAB; and conversion of tartrate obtained from the microorganism to ethylene glycol (preferably MEG) by any of the afore-described reactions.

The present disclosure relates to method for production of tartrate from pyruvate as the carbon source, using the microorganism described herein.

In an exemplary embodiment of the present disclosure, the method for producing tartrate comprises culturing strains containing overexpressed genes for tartrate production in medium comprising pyruvate at ambient conditions.

In an exemplary embodiment of the present disclosure, the method for producing tartaric acid comprises culturing strains containing overexpressed genes for the tartaric acid production in LB medium with 50 mM pyruvate. When the OD600 reached 0.6, the samples were induced with 0.2 mM IPTG and were incubated overnight at 20°C. The cultures were centrifuged at 8000 rpm for 10 min and the supernatants were analysed for tartaric acid.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the steps: culturing microorganism on a substrate comprising pyruvate at ambient conditions, wherein the microorganism is capable of conversion of pyruvate to phosphoenol pyruvate; conversion of the PEP to OAA, conversion of the OAA to form tartrate, optionally obtaining tartrate from the microorganism and conversion of tartrate to ethylene glycol by any of the afore-described reactions.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves conversion of pyruvate by the microorganism of the present disclosure to phosphoenol pyruvate by the enzyme phosphoenol pyruvate synthase; conversion of the PEP to OAA by the enzyme phosphoenol pyruvate carboxylase; conversion of the OAA to form tartrate by the enzyme Fumarase B or TtdAB; and conversion of tartrate obtained from the microorganism to ethylene glycol (preferably MEG) by any of the afore-described reactions.

The present disclosure relates to method for production of tartrate from PEP as the carbon source, using the microorganism described herein.

In an embodiment of the present disclosure, the method for producing tartrate comprises culturing strains containing overexpressed genes for tartrate production on a media comprising PEP at ambient conditions.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the steps: conversion of the PEP to OAA; conversion of the OAA to tartrate; optionally obtaining tartrate from the microorganism and conversion of tartrate to ethylene glycol by any of the afore-described reactions.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves conversion of phosphoenol pyruvate to oxaloacetate by the enzyme phosphoenol pyruvate carboxylase by the microorganism; followed by conversion of the OAA to form tartrate by the enzyme FumaraseB or TtdAB; and conversion of tartrate obtained from the microorganism to ethylene glycol (preferably MEG) by any of the afore-described reactions.

The present disclosure relates to method for production of tartrate from OAA as the carbon source, using the microorganism described herein.

In an embodiment of the present disclosure, the method for producing tartrate comprises culturing strains containing overexpressed genes for tartrate production on a media comprising oxaloacetate at ambient conditions.

In an exemplary embodiment of the present disclosure, the method for producing tartaric acid comprises culturing strains containing overexpressed genes for the tartaric acid production in LB medium with 50 mM oxaloacetate. When the OD600 reached 0.6, the samples were induced with 0.2 mM IPTG and were incubated overnight at 20°C. The cultures were centrifuged at 8000 rpm for 10 min and the supernatants were analysed for tartaric acid.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the step of conversion of the OAA to tartrate, optionally obtaining tartrate from the microorganism and conversion of tartrate to ethylene glycol by any of the afore-described reactions.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the step of conversion of the OAA to tartrate by the microorganism, by enzymes selected from FumB or TtdAB, and conversion of tartrate obtained from the microorganism to ethylene glycol (preferably MEG) by any of the afore-described reactions.

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the step of conversion of tartrate to ethylene glycol, either biologically or chemically directly or via intermediates such as glycerate, dimethyl tartrate, methyl glycolate etc..

In an embodiment of the present disclosure, the method for producing ethylene glycol involves the step of conversion of tartrate to ethylene glycol (preferably MEG) by any of the afore-described reactions

The present disclosure provides unique means for efficient production of industrially important ethylene glycol using a combined strategy applying biological and chemical techniques.

Ethylene glycol such as MEG is conventionally produced from ethylene as substrate. Ethylene is a product of steam cracking of hydrocarbons. The present disclosure provides alternate means for production of ethylene glycol employing renewable sources. Further, naturally occurring microbes do not produce tartrate / ethylene glycol from syngas/oxaloacetate. The present disclosure employs extensive metabolic engineering to create strains capable of carrying out the reactions in the pathway and employ alternate starting materials such as syngas, acetyl CoA, pyruvate, PEP, OAA to produce tartrate. Tartrate is a versatile building block and has wide range of industrial applications. Synthetic tartrate is known to be cheaper than natural tartaric acid. The present invention provides for efficient means for producing tartrate biologically via microorganisms as an efficient alternate to synthetic tartaric acid. Tartrate is further converted to MEG using chemical techniques of the present disclosure.

Further, the present disclosure allows conversion of waste gases such as syngas to value added chemicals, reducing the carbon footprint which helps in controlling pollution and producing high value chemicals. The methods of the present disclosure thus provide an economic way to produce chemicals since the starting material syngas is a cheap feedstock.

The foregoing descriptive matter is illustrative of the disclosure and not a limitation. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

Any possible combination of two or more of the embodiments described herein is comprised within the scope of the present disclosure.

Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

Further, the disclosure herein provides for examples illustrating the above described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples provided herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES:
Example 1:
The requisite ethylene glycol pathway genes are individually cloned into suitable expression vectors. Positive clones are inoculated, followed by isolation of the vector. The vectors are co-transformed using suitable molecular biology techniques into the microorganism, such as E. coli or C. ljungdahlii. The transformants are selected on a suitable selection medium. The transformants are further cultured under ambient conditions for production of ethylene glycol. The parent strain E. coli ATCC 8739 employed in the examples was acquired from American Type Culture Collection (ATCC), USA. This strain was employed to generate two new strains (i) E. coli containing pps, ppc and ttdAB: Accession number MCC 0129; and (ii) E. coli containing pps, ppc and fumB: Accession number MCC 0128.

Example 1(A): Cloning of genes in E. coli
The ethylene glycol pathway genes pps (phosphoenol pyruvate synthetase), ppc (phosphoenol pyruvate carboxylase) and ttdAB (tartrate dehydrogenase) / fumB (fumarase B) are cloned and transformed into E. coli (including but not limiting to E. coli ATCC 8739 and its derivatives). The cloning is confirmed by diagnostic PCR and restriction enzyme double digestion reactions. The protocol followed for plasmid isolation was given by the manufacturer. The steps involved are summarised below:

Step 1 - The pps, ppc, fumB and ttdAB genes are individually cloned into the expression vector pET28a (Sigma-Aldrich, USA) through NdeI and HindIII restriction endonucleases sites. The cloning is confirmed by diagnostic PCR. Positive clones are inoculated in LB media with respective antibiotics, followed by isolation of the plasmids using Plasmid Isolation kit (Qiagen, USA). The protocol followed for plasmid isolation was given by the manufacturer. The cloning is reconfirmed by restriction enzyme double digestion for the presence of the respective inserts (Figure 4). Figure 4a depicts results of diagnostic PCR of pps in pET28a cloned in E. coli TOP10 F’. Two of the colonies screened for pps are positive (2.3 kb). Figure 4b depicts results of diagnostic PCR of ppc and ttdAB in pET28a cloned in E. coli TOP10 F’. Out of the 2 colonies screened for ppc, one is positive (2.6 kb) and out of the 3 colonies screened for ttdAB, 2 are positive (1.5 kb). Figure 4c depicts results of diagnostic PCR of fumB in pET28a cloned in E. coli TOP10 F’. All five colonies screened for fumB are positive (1.6 kb). Figure 4d depicts results of restriction enzyme digestion of pps-pET28a and fumB-pET28a. Insert release for pps and fumB is observed at 2.4 kb and 1.6 kb respectively. Figure 4e depicts results of restriction enzyme digestion of ppc-pET28a and ttdAB-pET28a. Insert release for ppc and ttdAB is observed at 2.7 kb and 1.6 kb respectively.

Step 2 - The vector pET28a-ppc is used as a template plasmid to clone the pps gene through XbaI and HindIII restriction sites. The cloning for pET28a-pps-ppc is confirmed using the same methods described earlier and the results are illustrated in Figure 5. Figure 5a depicts the results of diagnostic PCR of pps-ppc in pET28a cloned in E. coli TOP10 F’. 12 out of 14 colonies screened are positive (5.5 kb). Figure 5b depicts the results of restriction enzyme digestion of pps-ppc-pET28. Two cuts in the plasmid backbone show the expected band sizes at 1.2 kb and 9 kb.

Step 3 - The expression cassettes of genes pps and ppc (T7 prom-RBS-pps-ppc-T7 term) are amplified by PCR using T7 promoter forward and T7 terminator reverse primers from the above mentioned vector and cloned into the pMTL-0 vector (CHAIN Biotech, UK), a shuttle vector for C. ljungdahlii, through AscI and SbfI restriction sites. The cloning is confirmed as described earlier and the results are illustrated in Figure 6. Figure 6a depicts the results of diagnostic PCR of PT7-RBS-pps-ppc-T7-term in pMTL-0 cloned in E. coli TOP10 F’. Of the 14 colonies screened 4 are positive. Figure 6b depicts the results of restriction enzyme digestion of pps-ppc-pMTL-0. The inserts are observed at 5.5 kb along with the cut vector band at 1.5 kb.

Step 4 – The plasmids pET28a-fumB or pET28a-ttdAB and pMTL0-pps-ppc are co-transformed using electroporation into E. coli BL21 DE3 and the transformants are selected on LB medium with kanamycin and chloramphenicol. The cloning is confirmed as described earlier and the results are illustrated in Figure 7. Figure 7a depicts the results of diagnostic PCR to confirm transformation of pET28a_fumB and pMTL0_pps_ppc, E. coli BL21 (DE3). Of the eight colonies screened for fumB three are positive. Figure 7b depicts the results of diagnostic PCR to confirm transformation of pET28a_ttdAB and pMTL0_pps_ppc, E. coli BL21 (DE3). Of the 14 colonies screened for ttdAB, 13 are positive.

The strains thus obtained comprising the pps-ppc-fumB or pps-ppc-ttdAB genes have been deposited with the International Depository National Centre for Microbial Resource (NCMR), Pune, India, and have been accorded the accession number MCC 0128 [E. coli (SIBD 1978); pps-ppc-fumB] and MCC 0129 [E. coli (SIBD 1735); pps-ppc-ttdAB].

The primers used for diagnostic PCR reactions in this study are summarized in table 1 below:

Table 1:
Sequence ID No. Primer sequence 5’-3’ Description
1 ctacgtCATATGtccaacaatggctcgtc EC pps Forward with NdeI tail
2 acgactAAGCTTacttgcACTAGTttatttcttcagttcagccag EC pps Reverse with SpeI and HindIII tail
3 acgactCATATGaacgaacaatattccgc EC ppc Forward with NdeI tail
4 agtctgACTAGTattagccggtattacgcatac EC ppc Reverse with SpeI tail
5 ctactgCATATGatgagcgaaagtaataagc EC ttdA Forward with NdeI tail
6 gtcatcACTAGTtccgggagggttatttgatg EC ttdB Reverse with SpeI tail
7 agcatcCATATGtcaaacaaaccctttatc EC fumB Forward with NdeI tail
8 acgactAAGCTTacttgcACTAGTttacttagtgcagttcgcgc EC fumB Reverse with SpeI and HindIII tail
9 Attaatacgactcactataggg T7 promoter Forward primer from pET
10 ttgactCCTGCAGGattaatacgactcactataggg T7 promoter Forward with Sbf1 tail
11 ttgactGGCGCGCCatccggatatagttcctcctttcagc T7 terminator Reverse with AscI tail

List of the plasmids/vectors employed in the present disclosure are provided in Table 2 below.
Table 2:
Plasmid Name Description
pET28a_ppc Kanamycin resistance, ColE1 ori, T7 promoter, Expresses Ppc enzyme
pET28a_fumB Kanamycin resistance, ColE1 ori, T7 promoter, Expresses FumB enzyme
pET28a_ttdAB Kanamycin resistance, ColE1 ori, T7 promoter, Expresses enzymes TtdA and TtdB
pET28a_pps Kanamycin resistance, ColE1 ori, T7 promoter, Expresses Pps enzyme
pET28a_pps_ppc Kanamycin resistance, ColE1 ori, T7 promoter, Expresses enzymes Pps and Ppc
pMTL0_T7-pps_ppc Chloramphenicol resistance, p15A ori, T7 promoter, Expresses enzymes Pps and Ppc

Example 1(B): Protein expression studies
The protein expression profile for both the strains constructed with the genes as per Example 1(A) is studied. The cells grown till OD600 reached 0.6 units and are induced with about 0.2mM Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and grown overnight on a shaker at about 20°C. The cells are harvested at 8000g for 10 minutes at 4°C by centrifugation. The supernatant is discarded and the pelleted cells are resuspended in 5 ml of Tris buffer (pH 7.4). The cells are then lysed by sonication (50 amplitude; 2s pulse; 8s rest; 2-3 minutes) to result in whole cell lysate. 1 ml of the whole cell lysate is subjected to centrifugation at 18000 g for 10 min at 4°C to result in a clear supernatant. These samples are checked and analysed on 12% SDS-PAGE gels (Figure 8).

Figure 8a depicts results of the SDS-PAGE gel to check expression of Ppc in E. coli. Ppc is expressed when induced and the expected sized band is observed at 99.1 kDa. Figure 8b depicts results of the SDS-PAGE gel to check expression of Pps and FumB in E. coli. Pps and FumB are expressed when induced and the expected sized bands are observed at 87.4 kDa and 60 kDa respectively. Figure 8c depicts results of the SDS-PAGE gel to check expression of TtdA and TtdB in E. coli BL21 DE3. TtdA and TtdB are expressed when induced and the expected sized bands are observed at 32.7 kDa and 22.7 kDa. Figure 8d depicts results of the SDS-PAGE gel to check expression of Pps, Ppc, TtdA, TtdB and FumB in E. coli. In figure 8(d), lanes 5, 6 and 7 depict E. coli MCC 0129 containing Pps, Ppc and TtdAB. In figure 8(d), lane 9 depicts E. coli MCC 0128 containing Pps, Ppc and FumB. All strains expressed when induced and the expected sized bands are observed at 87.4, 99.1, 32.7, 22.7, 60.1 kDa for Pps, Ppc, TtdA, TtdB, and FumB, respectively.

Example 2: Production of tartaric acid
The E. coli strains of Example 1(A) expressing the ethylene glycol pathway genes, viz. (i) E. coli comprising Pps, Ppc, TtdA and TtdB; and (ii) E. coli comprising Pps, Ppc, and FumB, are analysed for production of tartrate. Strains containing the overexpressed genes for the tartrate production are grown in LB medium with about 50 mM pyruvate. When the OD600 reached about 0.6, the samples are induced with about 0.2 mM IPTG and are incubated overnight at about 20°C. The cultures are centrifuged at about 8000 rpm for about 10 min and the supernatants are analysed for tartrate.

The analytical method for tartrate was HPLC. The protocol for HPLC was as follows:
Column temperature: about 50oC
Column: Biorad 87-H anion exchange column
Mobile phase: about 5mM H2SO4
Flow rate: about 0.6 ml/min

The results obtained for both the strains are similar and as depicted in Figures 9-11.

The standard tartrate showed the retention time of 10.8 min in the resultant chromatogram. The supernatant from both the strains also showed a peak at the retention time of 10.8 min as shown in Figure 9.

The samples are also analysed for the production of tartaric acid using LC-MS. LC-MS analysis is carried out using Agilent 6530, Accurate Mass Q-TOF LCMS, direct mode of injection; and water: ACN (50:50) as solvent system. The supernatant containing the enzymes FumB as well as TtdAB showed the presence of mass corresponding to that of tartaric acid in negative mode of LC-MS analysis.

The molecular weight of tartaric acid is 150, and will be seen as 149 in negative mode of LC-MS. From the mass spectrum obtained (Figure 10), presence of tartrate in the supernatant of the cultivated strains is confirmed.

The culture was lysed by sonication and was analysed for enzyme activity of Pps, Ppc, and FumB. The reaction was carried out using pyruvate as the substrate and the expected product was tartrate from the culture lysate. The enzyme mixture (crude cell free extract) was incubated in about 37oC for about 2h and then was analysed for the formation of tartrate by LC-MS. The crude mass spectrum (Figure 11) indicated the presence of the peak (molecular weight 149) corresponding to tartrate in negative mode.

Similar experiment was conducted for assessing production of tartrate by the culture for enzyme activity of Pps, Ppc, and TtdAB. The reaction was carried out using pyruvate as the substrate and the expected product was tartrate from the culture lysate. The enzyme mixture (crude cell free extract) was incubated in about 37oC for about 2h and then was analysed for the formation of tartrate by LC-MS. The crude mass spectrum indicated the presence of the peak corresponding to tartrate.

Example 3: Production of Mono ethylene glycol
1g of tartrate produced by E. coli strain comprising Pps, Ppc, TtdA and TtdB as per Example 2 was subjected to chemical reaction to synthesize MEG as illustrated in Figure 3(a). Briefly, the method involves methylating tartaric acid to obtain dimethyl tartrate which is converted to methyl glycolate by oxidative cleavage and reduction reaction. The methyl glycolate is reduced using Lithium Aluminium Hydride (LAH) to yield MEG.

Example 3(A): Conversion of tartaric acid to dimethyl tartrate
1 g tartaric acid, 20 ml methanol and 2 ml of concentrated sulphuric acid was refluxed for 4 h at 90°C. The reaction mixture was cooled to room temperature by using ice bath and methanol was evaporated from the reaction mixture by vaporising the solvent using IKA RotoVap RV- 10 instrument at 40°C under vacuum. 50 ml water was added to extract the product. The acid was neutralized by the addition of sodium bicarbonate till pH reached around 7. The product was extracted using 50 ml ethylacetate. The organic layer was evaporated over sodium sulfate to obtain dimethyl tartrate. The formation of dimethyl tartrate was confirmed using GC-MS. The chromatogram and fragmentation pattern of the dimethyl tartrate thus obtained are shown in Figures 12 and 13 respectively.

Example 3(B): Conversion of dimethyl tartrate to methyl glycolate
1 g of the dimethyl tartrate was dissolved in 20 ml dichloromethane. 2 equivalents of Sodum bicarbonate with 5 equivalents of NaIO4 was added to the mixture along with 2 equivalents of NaBH4. NaOI4, DCM and NaBH4 were added simultaneously. The reaction was carried out at 0°C for 1 h. The organic layer was evaporated over sodium sulfate to obtain methyl glycolate. The sample obtained was treated with 1 mL of DNPH in concentrated sulphuric acid and incubated for 10 minutes to create a 2,4-Dinitrophenylhydrazine (DNPH) derivative for confirmation of the product methyl glycolate. The DNPH derivative of molecular weight 268 was extracted in methanol. The compound was confirmed using LC-MS. The spectra showed m/z compound of molecular weight 267 in negative mode (H-1) analysis. The LC-MS spectra is illustrated in Figure 14, depicting the formation of DNPH derivative of methyl glycolate (molecular weight = 268) with m/z value t negative mode (H-1) at 267.03. Carrying out the above reaction in the presence of 2 equivalents of NaBH4 allowed reduction of the product giving methyl glycolate.

Example 3(C): Conversion of methyl glycolate to MEG
100 mg of methyl glycolate, 2 ml THF and 25 equivalents of Lithium aluminium hydride are mixed at 0°C for 1 h. The reaction was quenched carefully using NaOH (2N solution in water, 10 ml). The reaction mixture was analysed for the presence of MEG in GC-MS. The chromatogram depicting the formation of MEG is provided in Figure 15 and the fragmentation pattern of MEG is shown in Figure 16.

SEQUENCE LISTING

<110> RELIANCE INDUSTRIES LIMITED

<120> PROCESS FOR PRODUCTION OF ETHYLENE GLYCOL, INTERMEDIATES AND
MICROORGANISM THEREOF

<130> IP42092

<140> Indian Patent Application No. 201821012033
<141> 2019-09-29

<160> 11

<170> PatentIn version 3.5

<210> 1
<211> 29
<212> DNA
<213> Artificial Sequence

<220>
<223> Primer sequence 5'-3'

<220>
<221> misc_feature
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<400> 1
ctacgtcata tgtccaacaa tggctcgtc 29

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<213> Artificial Sequence

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<223> Primer sequence 5'-3'

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<223> EC pps Reverse with SpeI and HindIII tail
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acgactaagc ttacttgcac tagtttattt cttcagttca gccag 45

<210> 3
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acgactcata tgaacgaaca atattccgc 29

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<213> Artificial Sequence

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<223> Primer sequence 5'-3'

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<223> EC ppc Reverse with SpeI tail

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agtctgacta gtattagccg gtattacgca tac 33

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<223> Primer sequence 5'-3'

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<223> EC ttdA Forward with NdeI tail

<400> 5
ctactgcata tgatgagcga aagtaataag c 31

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<223> Primer sequence 5'-3'

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gtcatcacta gttccgggag ggttatttga tg 32

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agcatccata tgtcaaacaa accctttatc 30

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acgactaagc ttacttgcac tagtttactt agtgcagttc gcgc 44

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<223> Primer sequence 5'-3'

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ttgactcctg caggattaat acgactcact ataggg 36

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ttgactggcg cgccatccgg atatagttcc tcctttcagc 40
,CLAIMS:1. A process for preparing ethylene glycol, said process comprising steps of:
a. alkylating tartaric acid to obtain dialkyl tartrate;
b. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
c. reducing the alkyl glycolate to obtain ethylene glycol.
2. A process for preparing alkyl glycolate optionally followed by preparing ethylene glycol, said process comprising steps of oxidatively cleaving dialkyl tartrate followed by reduction to obtain alkyl glycolate; and optionally reducing the alkyl glycolate to obtain ethylene glycol.
3. A process for preparing ethylene glycol, said process comprising steps of:
a. hydrolyzing oxaloacetate to obtain tartaric acid;
b. alkylating the tartaric acid to obtain dialkyl tartrate;
c. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
d. reducing the alkyl glycolate to obtain ethylene glycol.
4. A process for preparing ethylene glycol, said process comprising steps of:
a. acetylating syngas to obtain acetyl-CoA;
b. converting the acetyl-CoA to obtain pyruvate;
c. phosphorylating the pyruvate to obtain phosphoenol pyruvate;
d. dephosphorylating the phosphoenol pyruvate to obtain oxaloacetate;
e. hydrolyzing the oxaloacetate to obtain tartaric acid;
f. alkylating the tartaric acid to obtain dialkyl tartrate;
g. oxidatively cleaving the dialkyl tartrate followed by reduction to obtain alkyl glycolate; and
h. reducing the alkyl glycolate to obtain ethylene glycol.
5. The process as claimed in any of claims 1, 3 or 4, wherein alkylation of the tartaric acid is carried out in presence of alkylating agent and acid; wherein the alkylating agent is selected from a group comprising methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol and pentanol or any combination thereof; and wherein the acid is selected from a group comprising sulphuric acid, nitric acid, tosic acid, orthophosphoric acid, methane sulphonic acid and glacial acetic acid or any combination thereof.
6. The process as claimed in any of claims 1-4, wherein oxidative cleavage is carried out in presence of oxidizing agent and base; wherein the oxidizing agent is selected from a group comprising sodium periodate (NaIO4), potassium nitrate, potassium permanganate, nitric acid, hydrogen peroxide and halogens or any combination thereof; and wherein the base is selected from a group comprising sodium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium carbonate, ammonia and bromine or any combination thereof.
7. The process as claimed in any of claims 1-4, wherein the reduction to obtain alkyl glycolate or ethylene glycol is carried out in presence of reducing agent selected from a group comprising sodium borohydride (NaBH4), sodium hydride, calcium hydride, lithium hydride, lithium aluminium hydride, sodium sulphite and formic acid or any combination thereof.
8. The process as claimed in any of claims 1-4, wherein the dialkyl tartrate is dimethyl tartrate, the alkyl glycolate is methyl glycolate and the ethylene glycol is monoethylene glycol.
9. The process as claimed in claim 4, wherein acetylation of the syngas to obtain the acetyl-CoA is carried out by Wood-Ljungdahl pathway by anaerobic cultivation of the microorganism, conversion of the acetyl-CoA to obtain the pyruvate is carried out in presence of pyruvate synthetase, phosphorylation of the pyruvate to obtain the phosphoenol pyruvate is carried out in presence of phosphoenol pyruvate synthetase, and dephosphorylation of the phosphoenol pyruvate to the obtain oxaloacetate is carried out in presence of phosphoenol pyruvate carboxylase.
10. The process as claimed in claim 3 or claim 4, wherein hydrolysis of the oxaloacetate to obtain the tartaric acid is carried out in presence of fumarase B or tartrate dehydrogenase.
11. The process as claimed in any of the preceding claims, wherein said process is carried out in presence of solvent selected from a group comprising methanol, ethyl acetate, dichloromethane, tetrahydrofuran, acetonitrile, ethanol, glycerol, purified water and methyl acetate or any combination thereof.
12. The process as claimed in any of the preceding claims, wherein one or more step(s) of the process is carried out at temperature ranging from about 0°C to about 90°C, and/or for a time period ranging from about 30 minutes to about 4 hours.
13. The process as claimed in any of the preceding claims, wherein the process further comprises acts selected from a group comprising isolation and/or purification of the corresponding compound; wherein the said isolation is carried out by acts selected from a group comprising addition of solvent, quenching, sonication, cooling, heating, removal of solvent, drying, filtration, extraction and combination of acts thereof; and wherein the purification is carried out by water washing or solvent(s) washing or a combination thereof.
14. The process as claimed in claim 4 or claim 9, wherein the acetylation of syngas to tartaric acid is carried out in microorganism selected from a group comprising Clostridium sp., Moorella sp., Acetitomaculum sp., Acetobacterium sp., Blautia sp., Eubacterium sp., Sporomusa sp., Syntrophococcus sp., Butyribacterium sp., Escherichia sp., Desulfobacter sp., Methanothermobacter sp., Methylobacterium sp., Pseudomonas sp., Rhodobacter sp. , Euglena sp., Helicobacter sp., Methanococcus sp., Mycobacterium sp., Panteoa sp., Bacillus sp., Yarrowia sp., and Trichoderma sp.
15. The process as claimed in claim 14, wherein the microorganism is selected from a group comprising Acetitomaculum ruminis, Acetobacterium carbinolicum, Acetobacterium woodii, Blautia producta, Clostridium formicaceticum, Clostridium ljungdahlii, Eubacterium limosum, Moorella thermoacetica, Moorella thermoautotrophica, Sporomusa malonica, Sporomusa termitida, Syntrophococcus sucromutans, Butyribacterium methylotrophicum, Desulfobacter hydrogenophilus, Escherichia coli, Desulfobacter hydrogenophilus, Megathyrsus maximus, Methanothermobacter thermoautotrophicus, Methylobacterium extorquens, Pseudomonas putida, Rhodobacter sphaeroides, Euglena gracilis, Helicobacter pylori, Methanococcus maripaludis, Mycobacterium tuberculosis, Panteoa citrea, Bacillus subtilis, Yarrowia lipolytica and Trichoderma reesei or any combination thereof.
16. The process as claimed in claim 14 or claim 15, wherein the microorganism is non-naturally occurring microorganism comprising one or more genes selected from a group comprising pyruvate synthetase, phosphoenol pyruvate synthetase, phosphoenol pyruvate carboxylase, fumarase B and tartrate dehydrogenase.
17. The process as claimed in any one of claims 14-16, wherein the acetylation of syngas to tartaric acid is carried out when the microorganism comes in contact with the syngas.
18. A microorganism comprising one or more ethylene glycol pathway gene selected from a group comprising pyruvate synthetase, phosphoenol pyruvate synthetase, phosphoenol pyruvate carboxylase, fumarase B and tartrate dehydrogenase or any combination thereof.
19. The microorganism as claimed in claim 19, wherein the microorganism is selected from a group comprising Clostridium sp., Moorella sp., Acetitomaculum sp., Acetobacterium sp., Blautia sp., Eubacterium sp., Sporomusa sp., Syntrophococcus sp., Butyribacterium sp., Escherichia sp., Desulfobacter sp., Methanothermobacter sp., Methylobacterium sp., Pseudomonas sp., Rhodobacter sp., Euglena sp., Helicobacter sp., Methanococcus sp., Mycobacterium sp., Panteoa sp., Bacillus sp., Yarrowia sp., and Trichoderma sp.