Title of Invention

A METHOD FOR CULTURING A MICROORGANISM HAVING A METHANOL METABOLIC PATHWAY

Abstract A method for culturing a microorganism having methanol metabolic pathway and into said microorganism an expression unit has been introduced wherein said expression unit comprises a target gene which is controlled by a promoter that can be derepressed by starvation on glycerol and induced with methanol or mixed feed of methanol and glycerol and said method comprising of; culturing the microorganism in a nutrient medium characterized in that maintaining the dissolved oxygen level between 50 % and 95% of saturation level in the presence of glycerol in growth phase; feeding a limiting amount of glycerol for derepression of the target gene wherein the dissolved oxygen fluctuates between 0% to 80%; inducing the target gene by feeding methanol or methanol/glycerol feed at a rate greater than the maximum methanol consumption rate of the microorganism wherein the dissolved oxygen level fluctuates synchronously between 0% and 100% with methanol addition.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETE SPECIFICATION
[See section 10]
A METHOD FOR CULTURING A MICROORGANISM HAVING A METHANOL METABOLIC PATHWAY;
SERUM INSTITUTE OF INDIA LIMITED, A COMPANY INCORPORATED UNDER THE COMPANIES ACT, 1956, WHOSE ADDRESS IS "SAROSH BHAVAN" 16-B/1, DR. AMBEDKAR ROAD, PUNE - 411 001, MAHARASHTRA, INDIA.
THE FOLLOWING SPECIFICATION
PARTICULARLY DESCRIBES THE
NATURE OF THIS INVENTION AND THE
1 APR 2004 MANNER IN WHICH IT IS TO BE
PERFOMED. 1


BACKGROUND OF INVENTION
1. Field of Invention:
Present invention, in general, utilizes refinements in the fermentation technology particularly directed to improvements in biomass and gene expression in a controlled high production manner so as to maximize the recovery of target gene product.
The preferred embodiments of the invention relates to a method for culturing yeast having a methanol metabolic pathway in a bioreactor and having an expression unit of a target gene linked downstream to a promoter that is able to be derepressed or induced by limiting supply of carbon source for efficient expression of the target gene. The oxygen transfer rate in the said bioreactor is increased by supplementation with gaseous oxygen in proportion to the biomass to enable higher metabolic rate of the culture for rapid utilization of carbon source enabling faster growth to high cell densities, in spite of carbon limitation. Methods of derepression and induction influence the target gene expression significantly.
2. Related (Prior) art:
Genetically engineered yeast expressing heterologous proteins is gaining increasing use for large-scale production of pharmaceutically useful proteins.
The most favorable and advantageous characteristic of Hansenula polymorpha is its ability to grow on simple chemical medium and have methanol utilization pathway. Upon induction with methanol, key enzymes of this pathway, including genes for methanol oxidase (MOX), dihydoxyacetone synthase (DHAS) and a formate dehydrogenase (FMDH) are transcribed at high levels, making them attractive promoter elements for heterologous gene expression. Detailed information on the culturing of methylotrophic yeasts and the induction conditions for the above three promoters is already described previously {Gellissen, G. in Murooka/ Imanaka (eds.) Recombinant microbes for industrial and agricultural applications, Marcel Dekker, NY 1993, 787-796 and Weydemann, U. et. al, Appl. Microbiol. Biotechnol. 1995., 44, 377-385). Furthermore, these control elements can also promote high-level expression of the inserted target gene in fermentations under glycerol limitation due to the presence of a derepression mechanism. Besides these, H. polymorpha displays other favorable characteristics such as its ability to grow on simple synthetic medium to high-cell densities at excellent growth rates giving high yields of expressed proteins thus making them the most attractive host system for number of biotech products (Hollenberg C.P. and G. Gellissen. 1997 Production of recombinant proteins by methylotrophic yeasts. Current Opinion in Biotechnol. 8: -554-560).
2

Methylotropic yeast Hansenula polymorpha, has been finding increasing use as a host for the expression of a wide variety of recombinant proteins. Because of its favorable fermentation characteristics, it finds industrial applications in large-scale production of heterologous proteins. The expression of heterologous genes is very strong and tightly regulated by formate dehydrogenase (FMDH) promoter. This organism enables a faster high-yield fermentation process with its ability to grow to very high cell densities (>150 g/1 dry weight) on a simple synthetic medium.
Production of recombinant products using fermentation requires oxygen to convert the substrates (carbon source) into the desired product. Dissolved oxygen is therefore the most important control variables in an aerobic batch reactor.
Oxygen having poor solubility in aqueous condition couple with high oxygen demand exerted by certain aerobic microorganisms results in oxygen transfer the rate-limiting step during cultivation. Therefore it becomes essential to design bioreactors, which could provide high oxygen transfer to a highly demanding aerobic cultivation process. The oxygen usually comes from air-bubbles that is passed through the fermentation medium using a sparger and simultaneously broken up by impellers that are used to stir the medium. The oxygen transfer rate (OTR) in a reactor varies with air/oxygen-flow rate, the degree of agitation and mass transfer coefficient (k^a), the later being dependent on the design of the bioreactor.
Air is commonly used as a source of oxygen in liquid phase systems to increase the dissolved oxygen concentration. In some circumstances, it is desired to feed oxygen to the liquid phase independently from the air-feed port to improve the efficiency of oxygen utilization. (Kiyonaga, K, Litz, L., and T.J. Bergman. 1994. Oxygen enrichment method and system. U.S. Patent No. 5356600).
The object of the present invention is to gradually increase the volumetric ratio of oxygen to air proportional to the biomass in order to maintain a set value of DO at any given biomass thereby reducing the loss of added oxygen as occurs in applications in which oxygen-enriched air is added at constant proportions.
For high cell density fermentation, demand for dissolved oxygen increases necessitating oxygen-enriched air or substituting air with pure oxygen. However previous published reports have indicated that good cell biomass levels do not necessarily lead to good heterologous protein production. (Carty, C.E., F.X.Kovach, WJ.McAleer and R.Z.Maigetter. 1987. Fermentation of recombinant yeast producing Hepatitis B surface antigen. J. Industr. Microbiol. 2:117-121; Ichikawa, K., K. Komiya, K. Suzuki, T. Nakahara and H. Jigami 1989. The effects of culture conditions on the secretion of human lysozyme by Saccharomyces cerevisiae A2-1-1A. Agric.
3

Biol. Chem. 10:2687-2694: Sudbury P.U., M.A. Gleeson, R.A. Veale, A.M. Ledeboer and M. CM. Zoetmulder. 1988. Hansenula polymorpha as a novel yeast system for the expression of heterologous genes. Biochem. Soc. Trans. 16:1081-1083; Tikhomirova, LP., R.N. lkonomova, E.N. Kuznetsova, J.I.Fodor, L.V.Bystrykh, L.R.Aminova and Y.A.Tortsenko. 1988. Transformation of methylotrophic yeast Hansemda polymorpha; Cloning and expression of genes. ,/. Basic Microbiol. 28: 343-351). One must therefore differentiate between biomass production and antigen production and each parameter must be optimized separately as well as in terms of productivity per biomass yield.
The production of an intracellular heterogeneous protein in H. polymorpha can be correlated to the biomass if carbon source limitation is maintained during the derepression phase of fermentation {Hollenberg C.P. and G. Gellissen. 1997 Production of recombinant proteins by methylotrophic yeasts. Current Opinion in Biotechnol. 8: 554-560).
Another objective of the invention is to increase space-time-biomass yield and product titer for Hepatitis-B surface antigen production by a fed-batch fermentation process using the recombinant yeast Hansemda polymorpha. To achieve higher yield of biomass and bio-product, the key is to overcome limitation in the feed-rate of carbon source, imposed for the purpose of derepression of genes under the control of FMDH promoter or imposed for the prevention of toxicity on account of methanol accumulation. This limitation is challenged by compensating with higher carbon-utilization rate by increasing the metabolic activity as a consequence of improving the oxygen transfer rate in the fermentor using oxygen supplementation.
A limited study employing a mixture of air and oxygen in fermentation of recombinant Hansenula polymorpha expressing pre-S2 antigen (middle surface antigen) has shown volumetric increase in productivity to that of a fermentor aerated with air alone and DO-controlled feeding of methanol. However the substrate-feeding strategy and aeration conditions did not boost biomass productivity and fermentation yields beyond 35-40 gram dry cell weight per litre and 1.6 mg HBsAg per litre of culture, respectively {M.R.de Roubin, L. Bastein, S. -H. Shen and D. Groleau. 1991. Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeast Hansenula polymorpha. J. oflndustr. Microbiol. 8: 147-156).
In a more elaborate study of Pichia pastoris based fermentation of Thrombomodulin under the control of methanol-induced alcohol oxidase promoter was reported in which air, air-oxygen mixture and oxygen alone was sequentially fed. Both glycerol and methanol were used as substrates. During the initial growth phase, glycerol added at the start of fermentation gradually got depleted, and was supplemented by
4

continuous external feeding of glycerol. The amount of glycerol fed was gradually ramped up during the derepression phase. The pure oxygen supply to the fermentor was gradually increased parallel to the increase in biomass up to 100 % level. Glycerol was completely depleted from the fermentor prior to the start of induction phase with slow and continuous feeding of methanol. The feeding of methanol continued over a 10 days period in a recycle culture system leading to high expression levels of Thrombomodulin in the supernatant. {Chen, K, J. Krol, J. Cino and D. Freedman. 1996. Continuous production of Thrombomodulin from a Pichia pastoris fermentation. J. Chem. Tech. Biotechnol. 67: 143-148).
In a model and simulation study of expression of Hepatitis B surface antigen (HBsAg) using Saccharomyces cerevisiae, effect of number of state variables and process parameters under various experimental conditions have been defined. The study showed that by increasing the oxygen tension the expression of HBsAg and the specific growth rate (JJ.) was enhanced (Y. Shi, D.D.Y. Ryu and W-K. Yuan. 1992 Ejfects of oxygen and ethanol on recombinant yeast fermentation for Hepatitis B virus surface antigen production: Modeling and Simulation studies. Biotechnol. Bioeng. 41: 55-66.).
Similar observation of increase in the yield of heterologous protein has been reported by Porro et. al. with increase in the level of dissolved oxygen. (Porro, D., E. Marlegani, Rami, B. M, Alberghina, L. 1991. Heterologous gene expression in continuous cultures of budding yeast. Appl. Microbiol. Biotechnol. 34: 632 - 636).
Production of various recombinant pharmaceuticals using H. polymorpha has been successfully employed using a complex fermentation scheme based on glycerol and methanol as carbon sources {Gillissen G., K. Melber. 1996. Methylotrophich yeast Hansenula polymorpha as production organism for recombinant pharmaceuticals. Drug Research 46(11), 9, 943-948).
Invitrogen BV has in its Pichia fennentation process guidelines reported the use of oxygen-enriched air to increase Oxygen Transfer Rate during a three phases fermentation of Pichia: (1) Growth phase in batch mode with glycerol as carbon source (2) A brief (4 hours) glycerol fed-batch phase by continuous feeding of glycerol at 18.15 ml per hour per liter to maintain a limiting carbon source and (3) Induction phase with continuous methanol-feeding at 3.6 ml per hour per liter with DO level >20%, to induce the expression of recombinant protein under the methanol oxidase promoter (AOX1). In these guidelines, the use of alternating pulses of feeding and fasting during the derepression phase has not been practiced in presence of oxygen-enriched air and the amount of oxygen used is 0.1 to 0.3 wm only.
5

SUMMARY OF THE INVENTION:
The present invention is more fully to be understood as a description of the operation wherein higher productivity of recombinant protein is achieved under the control of promoters that require carbon source limitation and methanol induction. Both these conditions are not conducive for higher specific growth rate or cell densities. These compulsions are overcome by increasing the carbon utilization rate, by propelling higher metabolic rate of these carbon sources, by increasing the amount of dissolved oxygen in the culture vessel.
In fermentation processes such as those utilizing methylotropic yeast, it is desirable to have high oxygen transfer rates so as to effect high growth rates and consequently high cell densities in a short time resulting in higher volumetric yields of the product. Higher oxygen transfer rates have been achieved by conducting the fermentation process with mixing of oxygen in the air stream or sparging of oxygen without air. Fermentor designs vary widely in their ability to transfer oxygen to the culture. The overall aeration rates for a fermentor can vary over a considerable range. For example, fermentors that are efficient in oxygen transfer - the aeration with oxygen or oxygen-enriched air can be conducted at a rate of about 0.5 to 6 v/v-broth/minute, preferably about 0.5 to 1.5 at suitable temperature and pressure.
The required, detailed embodiments of the present invention are disclosed herein, however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, process details disclosed herein are not to be interpreted as limiting but merely as a basis for the claims and as a representative basis for one skilled in the art of employing the present invention in virtually any appropriate fermentation process employing methylotrophic yeast. Exemplary non-limiting possibilities are given below.
H. polymorpha is detailed herein as a model system for the use of methylotrophic yeast as hosts. Other useful methylotrophic yeasts can be taken from the four genera, namely Pichia, Hamenula, Candida and Torulopsis (Gleeson et. al., 1988. Yeast 4: 1). Primarily based upon their demonstrated characteristics of being able to grow on a single source of carbon and inducible by methanol. Species from these genera may also be used as hosts herein.
The present invention provides for a method for, but not limited to, fermenting recombinant methylotrophic yeast strains using a synthetic medium containing simple nutrient sources of carbon, nitrogen, minerals and vitamins such as: Ammonium dihydrogen phosphate, ferrous ammonium sulphate, magnesium sulphate, potassium chloride, sodium chloride, copper sulphate, zinc sulphate., manganese sulphate, EDTA,
6

nickel sulphate, cobalt chloride, boric acid, potassium iodide, calcium chloride, d-biotin, thiamine hydrochloride. The composition of nutrients could vary with one or more of the nutrients excluded from the list or in addition to the list.
The fermentation is in three phases: (i) an initial growth phase in which less than 10.0 % v/v of glycerol is present from the start of fermentation, (ii) a derepression phase with intermittent feeding of glycerol (0.5 to 2.0% v/v-broth/h) as a carbon source and (iii) an induction phase with methanol feeding to give a final concentration of methanol ranging from 1.0 to 10.0% v/v either alone or mixed with glycerol (50-80% methanol v/v in induction solution). Modification of the above fermentation principle, for one skilled in the art of fermentation, such as by way of glucose starvation instead of glycerol during the derepression phase or entire fermentation based only on methanol (without the derepression phase), or continuous feeding instead of intermittent feeding of carbon source(s) or varying the degree of starvation of culture by controlling glycerol feed during the derepression phase, are some non-limiting examples.
Product-specific complex fermentation schemes that use glucose/glycerol and methanol have already been developed. The optimal product yield depends on the interplay of number of factors such as gene product itself, gene copy number, cell metabolic rate influencing the energy status, chaperon and protease levels and their influence on the product, and others. The objective of the present invention is to improve fermentation conditions by increasing dissolved oxygen so as to increase the efficiency of substrate utilization thereby challenging the metabolic rate restriction, imposed by the feeding restrictions during derepression and induction.
It is to be understood that the increase in the metabolic rate of the culture by increasing dissolved oxygen, leads to higher growth rate and efficient biomass formation while maintaining the derepression and induction conditions for optimum productivity. The invention, as so described, provides advancement in the prior art of fermentation, enabling desirably higher dissolved oxygen utilization, leading to high substrate consumption and corresponding biomass yield than has ever been obtainable in the conventionally practiced art of fermentation using methylotrophic yeast. Hence the adjustment of rate of methanol addition equal to or less than the maximum methanol consumption rate of the microorganism is not necessary because of higher metabolic activity contrary to any prior art. The present invention reveals the significance of degree of starvation achieved by employing different mode of DO dependant substrate feeding to adjust the number of derepression cycles per unit time to influence the product yield of HBsAg polypeptide and target gene expression.
7

DETAILED DESCRIPTION
The present invention provides a method for adjusting the feeding rate of carbon source in a culture medium consisting of methylotropic yeast grown under conditions wherein dissolved oxygen concentration is not the limiting factor. The following provides a detailed description of the present invention using Hamenula polymorpha as and example of a yeast having a methanol metabolic pathway and the target gene refers to a gene coding for Hepatitis B surface antigen under the control of formate dehydrogenase promoter.
A study of culturing conditions was conducted using glycerol and methanol as the carbon sources during fermentation. Fermentation process was divided into growth, derepression and induction phase characterized by 40-50 h of derepression and 20-30 h of induction.
During the derepression phase, the rate of oxygen transfer is controlled by varying the level of gaseous oxygen introduced into the fermentor from 0 to 100%, to control the DO between 0 to 80%, largely dictated by the feeding rate of the carbon source and consequently biomass, which is optimized on a case by case basis depending on factors such as the product itself, the type of promoter, gene copy number, energy levels, etc. For any feed rate of carbon source, the oxygen transfer rate is adjusted by varying the gaseous oxygen supplementation to the fermentor such that the dissolved oxygen (DO) concentration will be above 0%. The feeding rate of the carbon source can be adjusted by the following strategies: (1) by controlled continuous feeding at a set flow rate of 5-10 ml/L medium/hour which could be kept constant or increased in periodic steps or as a gradient proportional to the increase in cell density with time. (2) By intermittent feeding based on time-based starting and stopping of the feed-pump wherein the length of feeding-period (time during which the feed-pump remains ON) and the time-interval between feeds (time interval during which the feed-pump remains OFF) can be programmed. (3) By DO-controlled feeding wherein the level of DO in the medium controls the start and stop of the feed-pump, measured using a dissolved oxygen electrode. A set threshold values of DO signals the start and stop of the pump. In the 1st feeding strategy equilibrium is reached in which the amount of glycerol fed is equal to the amount consumed by the yeast. In the 2nd and 3rd feeding strategy the DO levels synchronously fluctuates with intermittent feeding. The intermittent feeding could vary from 3 to 8 pulses per hour. The difference between the 2nd and 3rd strategy is that in the case of 2nd strategy the number of feeding pulses is time based (that is externally programmed) while in the 3rd strategy it is culture dependent (that is metabolic rate and cell density dependant). Adjusting the flow-rate of the feed-pump could further vary the level of carbon source feeding from 3 to 10 ml / L medium / hour leading to more DO demand, thus requiring higher supplementation
8

of gaseous oxygen (from 0 to 1.5 volumes of air per volume of liquid per minute (vvm) to the fermentor. The rate of carbon source feeding is also dependent on biomass content and increases from 3 to 10 ml / L medium / hour with increase in biomass from 1700 to 12250 gram dry cell weight (g.d.c.w.), which is calculated by multiplying the OD 60o value of the culture medium with a factor determined in advance based on the relationship between the two.
During the induction phase, methanol is fed for induction of the promoter either alone or mixed with glycerol (50% to 80% v/v - induction solution). The feeding of induction solution is based on time, with methanol addition rate per cycle = 0.01 to 0.03 v/v-medium, irrespective of biomass. The difference in this strategy from any of the prior art (US Patent No. 6171828) is that the methanol addition is time based instead of DO-controlled and exceeded the maximum consumption rate (Fig.1 shows a build-up of methanol with every feed). Notwithstanding, the accumulation of methanol in the culture medium, the DO fluctuated synchronously, with the similar drop in DO with each feeding cycle, indicating that the toxic effect of methanol is not manifested even though the concentration of methanol exceeded 7.0% v/v. the rate of methanol addition during induction is greater than the maximum methanol consumption rate of the. microorganism in contrast to patents EP Patent No. 0794256A2 and US Patent No. 6171828 wherein the methanol addition is equal to or less than the maximum consumption rate. Fluctuations in the level of dissolved oxygen concentration synchronous with methanol or methanol mixed feed addition cycle continue over a prolonged time till the end of induction phase. Even after accumulation of methanol up to 10%, the culture is responsive and metabolically active, hence the adjustment of the rate of methanol addition equal to or less than the maximum methanol consumption rate of the microorganism is not necessary.
The present invention provides a culturing method that is efficient both in terms of growth of the microorganism and production of the target product while also offering a high degree of reproducibility of maintaining metabolic-rate-dependent alternating feeding and starvation, which enables strong derepression and subsequent induction of the promoter. It is found that the mode of controlled substrate feed affects the level of gene expression critically. In present invention, method of derepression of target gene promoter is different than that cited in patent WO 90/03431. such method of dissolved oxygen dependant substrate feed in tandem with different modes of controlled substrate feed employed for starvation of a culture during derepression is n ot disclosed in any of the prior art. This method discloses significance of degree of starvation achieved by setting constant or varying number of derepression cycles per unit time throughout derepression phase influencing the biomass and expression of target gene.
It was determined that much better dissolution rates of oxygen in the medium could be achieved by increasing the partial pressure of oxygen in the bubbles.
9

In a typical fermentor system, air only was fed until the oxygen demand of growing yeast resulted in the dissolved oxygen level dropping from the initial saturating concentration. To make -up for the drop, pure oxygen was mixed with air such that the proportion of oxygen to air gradually increases from 0 to 100% and maintaining the dissolved oxygen level between 50% and 95% of saturation level. During the derepression phase, the dissolved oxygen level fluctuates synchronously between 0 to 80% during glycerol feeding and starvation, respectively. The DO level increased with the increase in oxygen to air ration and was highest when pure oxygen was used. Increasing supplemented oxygen to air ratio proportional to the increase in the biomass, resulted in nearly doubling of biomass in terms of OD 6oo (800 - 900) compared to fermentation with air alone that resulted in an increase in the maximum specific growth rate from 0.03 to 0.1 h"1 during fermentation.
The process described herein, can be effectively used to determine the optimum limitation of carbon-source during derepression phase that yields maximum product formation in terms of biomass. Glycerol concentration is a parameter that is normally monitored for culture control. The level of glycerol limitation during derepression phase was controlled by two different modes: (1) Manually modifying the rate of feeding or (2) culture-dependent auto regulation of feed rate, as described below:
Mode (1) : Manually modifying the rate of feeding: this was carried out by varying the flow rate (that is adjusting the speed of pump) of the glycerol feed so that a constant number of feeding pulses per hour is maintained throughout the derepression phase. The volume of glycerol fed/g.d.c.w. was kept constant throughout the derepression phase by varying the pump speed. A number of individual fermentation batches were run, each with a different number of feeding pulses per hour in order to determine the optimum glycerol feed rate during derepression phase that gives (a) maximum product yield (grams of HbsAg/g.d.c.w.) and (b) that gives maximum expression of HbsAg (grams/liter of culture) irrespective of biomass yield. In this manual control of feed-rate, the volume of glycerol feeding / hour varies with the age of the culture and increases from 500 ml/hour to 1000 ml/ hour with increase in the biomass. The relationship between the required culturing condition for optimal derepression.
From the results shown in Fig 2., the optimum glycerol-feeding rate during derepression that gave the highest yield in terms of grams of HbsAg/g.d.c.w. was determined to be 7 cycles/hour. The optimum feed rate that gave maximum product yield as well maximum expression of HbsAg was 0284ml/100 g.d.c.w./hour.
10

Mode (2): Culture dependent auto-regulation of feed rate: The rate of glycerol feeding can also be auto-regulated using a DO controller which senses the DO-levels. The level of dissolved oxygen in the medium, signals the starting and stopping of the glycerol feed-pump, which is set at a constant flow-rate (pump speed). As the culture ages, the numbers of feeding pulses per hour increases gradually with increase in cell density as shown in FIG, 3. A number of individual fermentations were run at different flow-rates of the feed-pump to determine the optimum glycerol-feeding rate that gives: (a) maximum yield in terms of grams of HBsAg/g.d.c.w. and (b) that which gives the maximum expression of HBsAg (gramsAiter of culture) irrespective of biomass. From the results shown in FIG.2, the optimum glycerol-feeding rate during derepression that gave the maximum expression in terms of grams of HBsAg / L culture was determined to be 0.305 ml/100 gd.c.w. /hour.
On the basis either of the above feeding modes, the optimum glycerol limiting conditions that gives maximum expression of a target protein (grams/liter of culture), irrespective of the biomass yield, and that which gives maximum yield of the target protein (grams/g.d.c.w.) can be optimized.
In another mode of induction strategy, methanol alone can be fed separately in a continuous or periodic manner, while continuing the glycerol feeding pattern of derepression phase (derepression cycles characterized by alternate glycerol feed and starvation) further during the induction phase. This helps in maintaining the culture active even in the presence of accumulated methanol with necessary gene expression.
The present invention provides a culturing method that is efficient in terms of biomass and target product at the methanol addition rate greater than the maximum methanol consumption rate of the microorganism. Use of programmed oxygen enriched air supply during derepression and induction and the methods employed for derepression result into highly active culture at the end of derepression resisting higher methanol feed rates. Method of d02 dependant substrate feed controlled by various modes to affect the number of cycles per unit time has a bearing on the degree of starvation during derepression phase influencing biomass, yield and expression of target gene. This culturing method offers a high degree of reproducibility, greater simplicity and improved productivity.

WE CLAIM
1. A method for culturing a microorganism having methanol metabolic pathway
and into said microorganism an expression unit has been introduced wherein
said expression unit comprises a target gene which is controlled by a promoter
that can be derepressed by starvation on glycerol and induced with methanol or
mixed feed of methanol and glycerol and said method comprising of;
culturing the microorganism in a nutrient medium characterized in that maintaining the dissolved oxygen level between 50 % and 95% of saturation level in the presence of glycerol in growth phase;
feeding a limiting amount of glycerol for derepression of the target gene wherein the dissolved oxygen fluctuates between 0% to 80%;
inducing the target gene by feeding methanol or methanol/glycerol feed at a rate greater than the maximum methanol consumption rate of the microorganism wherein the dissolved oxygen level fluctuates synchronously between 0% and 100% with methanol addition.
2. A method as claimed in claim 1 wherein the degree of starvation during
derepression phase is controlled by feeding a limiting amount of glycerol on
number of derepression cycles per unit time characterized in that alternating
periods of feeding and starvation that influences the target gene expression.
\12

3. A method as claimed in claim 1 or 2 wherein the glycerol feeding in derepression phase is controlled by manually modifying the rate of feeding.
4. A method as claimed in claim 1 or 2 wherein the glycerol feeding in derepression phase is controlled by culture dependent auto regulation of feed rate using a dissolved oxygen controller.
5. A method as claimed in any of the preceding claims wherein periods of feeding and starvation is achieved by dissolved oxygen-dependent start and stop feeding of glycerol.
6. A method as claimed in claim 1 wherein oxygen is mixed with air or separately introduced into the fermentor to increase the consumption of carbon source during fermentation under derepression phase by 50% than in the fermentation where oxygen is not supplemented with air.
v7. A method as claimed in any of the preceding claims wherein the
dissolved oxygen concentration is controlled by sparging air and oxygen in varying proportion through the medium.
8. A method as claimed in claim 6 wherein the proportion of oxygen mixed with the air is controlled from 0 % to 100% depending on the biomass 9. A method as claimed in claims 1 to 5 wherein the level of aeration is controlled in the range of 0.5 to 1.5 vvm.
10. A method according to any of the preceding claims wherein the said microorganism is Methylotrophic yeast.
13

11. A method according to any of the preceding claims wherein the said methylotrophic yeast includes Pichia, Hansenula, and Candida.
12. A method according to any of the preceding claims wherein the said methylotrophic yeast includes Pichia pastoris, Hansenula polymorpha.
13. A method according to any of the preceding claims wherein said target gene codes for a polypeptide.
14. A method as claimed in claim 13 wherein the said polypeptide is Hepatitis B surface antigen.
Dated this 29th day of August 2003
FOR SERUM INSTITUTE OF INDIA LIMITED By their Agent


(MANISH SAURASTRI) KRISHNA & SAURASTRI

Documents:

859-mum-2003-cancelled pages (02-12-2004).pdf

859-mum-2003-claims(granted)-(02-12-2004).doc

859-mum-2003-claims(granted)-(02-12-2004).pdf

859-mum-2003-correspondence (21-05-2007).pdf

859-mum-2003-correspondence(ipo)-(21-10-2004).pdf

859-mum-2003-drawing(02-12-2004).pdf

859-mum-2003-form 1(29-08-2003).pdf

859-mum-2003-form 19(29-08-2003).pdf

859-mum-2003-form 2(granted)-(02-12-2004).doc

859-mum-2003-form 2(granted)-(02-12-2004).pdf

859-mum-2003-form 3(29-08-2003).pdf

859-mum-2003-form 5(29-08-2003).pdf

859-mum-2003-power of attorney (01-12-2003).pdf

abstract1.jpg


Patent Number 207049
Indian Patent Application Number 859/MUM/2003
PG Journal Number 42/2008
Publication Date 17-Oct-2008
Grant Date 21-May-2007
Date of Filing 29-Aug-2003
Name of Patentee SERUM INSTITUTE OF INDIA LIMITED
Applicant Address "SAROSH BHAVAN" 16-B/1, DR. AMBEDKAR ROAD, PUNE - 411 001,
Inventors:
# Inventor's Name Inventor's Address
1 DR. RUSTOM MODY FLAT NO 902, BUILDING "W", SACRED HEART CO-OPERATIVE HOUSING SOCIETY NO 75/2/2B, 9TH FLOOR, WANAWADI, PUNE 411 040
PCT International Classification Number C12N 1/16
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA