FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10, rule 13)
1. Title of the invention
A PROCESS FOR MICROBIAL PRODUCTION OF 1,3-PROPANEDIOL
2. Applicant(s)
Name Nationality Address
TATA CHEMICALS LTD. INDIA BOMBAY HOUSE, 24 HOMI MODI STREET,
MUMBAI-400001
3. Preamble to the description
PROVISIONAL SPECIFICATION
The following specification particularly describes the invention.

The present disclosure generally relates to a process for microbial production of 1,3-propanediol and more particularly the disclosure relates to a process for production of 1,3-propanediol using a genetically modified microorganism. The present disclosure also relates to a genetically modified microorganism for the production of 1,3-propanediol.
BACKGROUND
1,3-Propanediol is a monomer used in the production of polyester fibers of superior quality and in the manufacture of polyurethanes and cyclic compounds. It is also a solvent and used as an antifreeze and wood paint.
A variety of chemical routes for the manufacture of 1, 3- propanediol are known. 1,3-propanediol may be chemically synthesized by the hydration of acrolein, or by the hydroformylation of ethylene oxide to afford 3-hydroxypropionaldehyde. The 3-hydroxypropionaldehyde is hydrogenated to give 1,3-propanediol. However, these chemical methods are expensive and cause pollution.
Microbial production of 1,3-propanediol from glycerol is a viable alternative to the chemical methods. Several bacteria from the genus Clostridium, Klebsiella, Enterobacter and Citrobacter are able to convert glycerol to 1,3-propanediol.
However, the yield of 1, 3-propanediol by microbial production is low as a significant amount of glycerol is converted into by-products such as acetate, lactate, butyrate, ethanol, butanol and 2,3-butanediol. In the bacterial cell, glycerol can enter into two different pathways. Figure 1 illustrates the metabolism of glycerol in bacteria such as Citrobacter and, Klebsiella.
Through the first pathway a glycerol dehydratase (dhaBCE) removes a water molecule from glycerol to form 3-hydroxypropionadehyde. The 3-hydroxypropionadehyde is then reduced to 1,3- propanediol by a nicotinamide adenine dinucleotide (NAD+J linked 1,3


propanediol dehydrogenase (dhaT). The 1, 3- propanediol formed is not metabolised further and accumulates in the media.
Through the second pathway glycerol is dehydrogenated to dihydroxacetone and then to dihydroxyacetonephosphate that is further metabolised to produce various by-products such as acetate, lactate, butyrate, ethanol, butanol and 2,3-butanediol. The dehydrogenation of glycerol is carried out by a NAD+ linked glycerol dehydrogenase (dhaD) and the phosphorylation of dihydroxyacetone is carried out by dihydroxyacetone kinase (dhaK). Thus the yield of 1, 3 propanediol depends upon the amount of glycerol that is converted to by¬products.
In Citrobacter lactic acid is one of the by-products formed by the metabolism of glycerol. It has been observed that at an initial concentration of 250mM, glycerol is converted to 1,3-Propanediol by cells in log phase with no accompanying by-product lactic acid as illustrated in Fig. 2. However, at a higher concentration of 500mM, significant amount of the added substrate glycerol is converted into the by-product lactic acid (Fig. 3) by cells in the log phase. In stationary phase as well, metabolism of glycerol by cells results in the formation of lactic acid (Fig.4).
Therefore, there is a need for a process that would allow for a microbial production of 1, 3- propanediol in an efficient manner and at high yield.
BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
The accompanying drawing illustrates the preferred embodiments of the invention and together with the following detailed description serves to explain the principles of the invention.
Figure 1 illustrates the metabolic pathway for production of 1, 3- propanediol.


Figure 2 is a HPLC chromatogram of the supernatant obtained by culturing a sample of Citrobacter at initial glycerol concentration of 250mM in log phase.
Figure 3 is a HPLC chromatogram of the supernatant obtained by culturing a sample of Citrobacter at initial glycerol concentration of 500mM in log phase.
Figure 4 is a HPLC chromatogram of the supernatant obtained by culturing a sample of Citrobacter in stationary phase.
DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the process, and such further applications of the principles of the invention therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
A process for production of 1,3-propanediol using a genetically modified microorganism is disclosed. More particularly a process for production of 1,3-propanediol using a genetically modified microorganism engineered to maximise the metabolic efficiency of the pathway by blocking alternate pathways for glycerol metabolism is described.
The process as described involves genetically blocking the oxidative pathway for glycerol fermentation in bacteria such that the genetically modified bacteria is unable to produce by-products such as acetate, lactate, butyrate, ethanol, butanol and 2,3-butanediol.


The genes targeted for inactivation by genetic modification include the genes that encode for the enzymes glycerol dehydrogenase (dhaD), dihydroxyacetone kinase (dhaK) and lactate dehydrogenase (ldh).
Glycerol dehydrogenase (dhaD): The term glycerol dehydrogenase refers to a protein that catalysis the conversion of glycerol to dihydroxyacetone.
Dihydroxyacetone kinase (dhaK): The term dihydroxyacetone kinase refers to a protein that catalysis the conversion of dihydroxyacetone into dihydroxyacetone phosphate.
Lactate dehydrogenase (ldh): The term lactate dehydrogenase refers to a protein that catalysis the conversion of pyruvate into lactate.
Each of these genes may be inactivated individually. Alternatively any two or all three genes may be inactivated.
In accordance with an aspect, any one of the gene encoding for Glycerol dehydrogenase (dhaD) or Dihydroxyacetone kinase (dhaK) is targeted, as inactivation of any one of these genes will prevent the formation of by-products such as acetate, lactate, butyrate, ethanol, butanol and 2,3-butanediol.
Preferably the gene encoding for dihydroxyacetone kinase that catalyzes the conversion of dihydroxyacetone into dihydroxyacetone phosphate is targeted for inactivation. The inactivation of this gene is preferred as the cell is still able to carry out the conversion of glycerol to dihydroxyacetone catalysed by glycerol dehydrogenase that results in the formation of NADH+. The NADH+ produced in this step can be utilised in the 1, 3 -propanediol production by the bacterial cell.
The microorganism selected for genetic modification is said to be "wild type" that is it is not a laboratory mutant, the wild type microorganism may be any bacteria that has the ability to metabolise glycerol and produce 1,3-propanediol.


The bacteria selected for genetic modification include any bacteria from the genus Citrobacter, Klebsiella, Enterobacter, and Clostridium that has the ability to convert glycerol into 1, 3-propanediol. In accordance with an aspect, the wild type microorganism may be any bacteria of the genus Citrobacter having the ability to convert glycerol to 1, 3-propanediol.
The wild type microorganism may be isolated from the environment by any known microbial method. The wild type microorganism is selected for their ability to convert glycerol into 1, 3-propanediol. Alternatively commercially available strains of bacteria may also be used.
The targeted genes may be inactivated by any known mechanism including by insertion of transposon or by deletion of the gene sequence or a portion of the gene sequence.
By way of a specific example the inactivation of the gene is carried out by homologous recombination. The method comprises generating a linear deoxyribonucleic acid (DNA) fragment by polymerase chin reaction (PCR), the DNA fragment comprises of an antibiotic resistant marker flanked by regions having homology to the target gene. This fragment is then transformed into microbial cells harbouring plasmid encoded genes that promote homologous recombination. The transformed cells are then selected for resistance to the antibiotic marker.
To construct mutants with two inactivated genes, a mutant having a single inactivated gene is used and the second gene to be inactivated is then targeted by any known method. Similarly to construct mutant with all three inactivated genes, a mutant having two inactivated genes is used and the third gene to be inactivated is then targeted by any known method.
A method for production of 1, 3- propanediol from glycerol is described. The process comprises of culturing a genetically modified microbial stain, the genetically modified bacterial strain contains at least one inactivated gene encoding for one of glycerol


dehydrogenase (dhaD), dihydroxyacetone kinase (dhaK) or lactate dehydrogenase (ldh) in a suitable culture medium containing glycerol under predefined conditions for a predefined period of time till glycerol is converted into 1, 3-propanediol,
In accordance with an aspect the bacteria used for the production of 1,3- propanediol is a modified bacterial strain of Citrobacter containing at least one inactivated gene encoding for one of glycerol dehydrogenase (dhaD), dihydroxyacetone kinase (dhaK) or lactate dehydrogenase (ldh).
In accordance with an aspect, the culture medium in addition to glycerol may contain
an appropriate carbon source, suitable minerals, salts, cofactors, buffers and other
components suitable for the growth of the culture and promotion of the enzymatic pathway
neccssary for 1, 3- proporation production. The culture may be carried out for a predefined
period of time in either batch, fed-batch or continuous culture conditions.
The 1, 3- propanediol produced may be isolated from the culture medium by any known method.
INDUSTRIAL APPLICABILITY
The method described allows for higher yield of 1, 3. propanediol from glycerol and prevents the formation of by-product such as acetate, lactate butyrate, ethanol, butanol and 2,3-butanediol. The disruption of genes in the alternate Pathway will result in the entire glycerol available for conversion to 1, 3- propanediol. This will result in an increase in yield for the genetically modified bacteria over the wild type, for glycerol to 1, 3- propanediol conversion. For example, in wild type Citrobacter having a conversion yield of 40%, the yield increase expected for the modified Citrobacter is around 20% over the wild type. The process thus provides a simple method for decreasing the cost for microbial production of 1,3-propanediol from glycerol.


The method as described preferably uses modified Citrobacter. As Citrobacter is a facultative anaerobe, it does not require a completely anaerobic environmental for 1,3-propanediol production. This therefore helps in further reducing the cost for production of 1,3-propanediol.


Dated this 8th day of April 2009

Essenese Obhan Of Obhan & Associates Agent for the Applicant