PROCESS FOR THE PREPARATION OF R-HYDROXYCARBOXYLIC ACIDS FROM CELLULAR POLYHYDROXYALKANOATES BY INTRACELLULAR HYDROLYSIS EMPLOYING THE CELLULAR POLYHYDROXYALKANOATE DEPOLYMERASE ACTIVITY INVOLVING A STEP OF SHIFITNG THE PH OF THE CULTURE MEDIUM IN ORDER TO FAVOUR THE HYDROLYSIS REACTION

Technical Field

5 The present invention relates to a method for the production of enantiomerically pure .R-hydroxycarboxylic acids (RHAs) by in vivo degradation of poly- (R) -hydroxyalkanoates (PHAs) biopolymers , as well as to a plant for the production of RHAs. 10

Background Art

In the past decades enantiomerically pure compounds have become key synthons in industry. (R) -hydroxycarboxylic acids (RHAs) are such synthons which are widely used as chiral 15 precursors due to several reasons .

Presently, RHAs are produced mainly by the following methods: oxidation of aliphatic glycol by a biotechnological process (Ohashi and Hasegawa, 1992a and 1992b) ^' ; [R) -β-hydroxylation of carboxylic acids using 20 microorganisms (Utaka and Watabu, 1990) ; and hydrogenation ^' of β-diketone using a chiral catalyst (Noyori and Kitamura, 2004) .

An alternative approach to the above mentioned methods is the hydrolysis of biotechnologically synthesized

25 poly (R) -hydroxyalkanoates (PHAs). PHAs are microbial polyesters, which are accumulated in the form of intracellular granules as a carbon and energy reserve under unfavorable environmental conditions (Witholt and Kessler, 1999) . More than 140 different monomers with different functional groups

30 have been found to be incorporated into PHAs (Steinbύchel and

Valentin, 1995; Sudesh and Abe, 2000) . These functional groups, which are always located in the polyester side chains, encompass olefinic, ester-, cyano-, amino-, nitroso-, phenyl-, nitrophenoxy- , chloro- and carboxylic functions, among others

35 (Steinbύchel and Valentin, 1995) .

WO 99/29889 discloses a method- for producing hydroxycarboxylic acids by autodegradation of polyhydroxyalkanoates . The method includes the steps of biosynthesis and accumulation of PHAs in cells by cultivating a

microorganism, subsequent separation of the cells from the medium, contacting the cells with a degradation solution being free of carbon source, where the depolymerization of accumulated PHAs takes place inside cells, and finally separating the RHAs from the solution. RHAs produced by this in vivo depolymerization method can be excreted into the medium when the further intracellular metabolism of RHAs is inhibited. This method avoided purification of PHAs and simplified the RHAs production process. However, this method has the major drawback that it can only be applied to obtain (J?) -3- hydroxyburyric acid (R3HB) and [R) -3-hydroxyvaleric acid

(R3HV) , and is not suitable for production of other RHAs due to extremely low production efficiencies, e. g. (.R) -3- hydroxyoctanoic acid (R3HO) could only ^• be obtained with an efficiency of maximal 10% in 4 days (WO 99/29889) . The low yield and the suitability for a limited number of RHAs render the method less interesting for its industrial application.

Ren, et. al. (2005) reported a similar approach to produce RHAs other ^' than R3HB or R3HV. A set of RHAs was produced with high yields, by making use of the observation that the studied PHAs could be depolymerized most efficiently when bacterial cells were exposed to a pH of 10-11. However, the scale-up of this process is difficult. It is believed that the released RHAs are acidic and thus neutralize the extracellular pH, which leads to unfavorable conditions for PHA depolymerization. Again, in the disclosed in vivo depolymerization method, cells containing PHAs were collected after cultivation by a centrifugation or filtration step, and then resuspended in a suitable solution with an adequate pH for the depolymerization of PHAs into RHAs . The released RHAs needed to be further separated from the cells by a second centrifugation or filtration step. Centrifugation and the required resuspension are cumbersome, due to the time needed for the cell collection and the resuspension, especially for large scale cultivations or high cell density cultures .

Thus, the present invention aims at providing an improved method for the production of enantiomerically pure RHAs and a corresponding plant adapted to such method.

Disclosure of the Invention-

Hence, it is a general object of the present invention to provide a method for the production of one or more i?-hydroxycarboxylic acids. Now, in order to implement these and still further objects of the invention, which will become more readily apparent as the description proceeds, this method comprises the steps of a) cultivating in a culture at least one microorganism which is capable of synthesizing polyhydroxyalkanoates and having an intracellular PHA depolymerase activity, b) adjusting the pH of said culture to a level allowing the -enzymatic depolymerization of said PHAs into one or more corresponding monomeric RHAs and the secretion of said RHAs into the culture medium, c) incubating said culture for effecting enzymatic depolymerization of PHAs, d) adjusting the pH of said culture to a pH ≤ 1.0 and e) optionally purifying said RHA monomers.

Brief Description of the Drawings

The present invention will be described with reference to the figures, wherein: Figure 1 shows the in vivo degradation of intracellular PHAs produced in Pseudomonas putida GPoI in batch culture over time. Fig. 1 A shows the decrease of intracellular PHAs, as well as the pH change of the culture broth when the pH of the culture broth was not controlled. Fig. 1 B shows the decrease of intracellular PHAs when the pH of the culture broth was controlled at 10 with 2 N NaOH by a pH stat . The data were derived from the average of at least two independent measurements. Cdw: cell dry weight.

Figure 2 shows a schematic representation of a continuous process for production of RHAs out of bacterial PHAs. Cultivation unit 1, base tank reactor 2a, overflow tubing 2b, base tank reactor 2c, harvest tank 3, pH stat 4, medium 5, carbon source 6, NaOH inlet 7, air inlet 8.

Figure 3 shows the continuous degradation of PHAs accumulated in P. putida GPoI. Fig. 3 A shows the influence of incubation time on PHAs degradation. Fig. 3 B shows the residence time, adjusted by tube length, on further release of monomers. The data were derived from the average of at least two independent measurements.

Figure 4 shows continuous preparation of RHAs by readjusting pH. The data were derived from the average of at least 2 independent measurements.

The present invention will be described in more detail below. It is understood that the various embodiments, preferences and ranges may be combined at will. Further, depending on the specific embodiment, selected definitions, embodiments or ranges may not apply.

It ^• has surprisingly been found that the centrifugation step of the described state-of-the-art methods, where the cells are first separated from the culture supernatant and resuspended in a solution of defined chemical composition in order to allow the enzymatic in vivo depolymerization, can be omitted. Especially for the large scale production of RHAs, this is clearly an advantage, since an entire process step can be skipped. The present invention refers to a method wherein the culture is directly adjusted to the adequate pH, so that the cells in the culture medium are directly exposed to the optimal pH conditions for PHA degradation. Hence, compared to the state-of-the-art methods, no additional solution for . the resuspension needs to be prepared. Further .advantages include reduced cell collection times, no resuspension of cells is needed, and the method can be applied to high cell densities. The incubation step c) may be directly carried out in the culture vessel, so that the microbial culture does not need to be transferred.

The present, invention typically yields at least 50% (w/w) , 'often at least 80% (w/w) , of RHA monomer.. Moreover, a wide range of enantiomerically pure (R) -hydroxycarboxylic acid monomers .are prepared with high yields by in vivo degradation of PHAs by directly adjusting the culture broth to the proper pH. The term "proper pH" refers to the pH at which PHA

degradation and RHA secretion is optimal in a given system. Therefore, the present invention discloses a method for the production of RHAs from microbiologically synthesized PHAs, which overcomes the above described limitations. Even though the efficiency may sometimes be lower for ^' selected PHAs/microorganisms than of reported particular cases, the overall improvement of the method outweighs this potential drawback.

In one advantageous embodiment, the cultivation of one or more microorganisms occurs in liquid culture, so that the microorganism accumulates intracellular PHAs. The culture conditions, such as the composition of the culture medium, temperature, pH are known to the person skilled in the art.

In a ^' more advantageous embodiment of the present invention, step a) is performed by batch fermentation, preferably by fed-batch fermentation. Batch culture is defined here as a "closed" system, where no other nutrients are supplied than those added with the culture medium from the

^■ beginning. The fed-batch culture ^' is ^" basically a ^' batch culture ^{^} that is continuously supplemented with selected nutrients after it enters the late exponential phase.

In another advantageous embodiment, the herein disclosed method is adapted to a continuous process wherein step a) is performed by continuous fermentation, preferably in a chemostat . In this case it is necessary to transfer ^■ cells that are exposed to alkaline pH (step b) into a separate base tank reactor 2a. Preferably, the pH is kept constant in the base tank reactor 2a for incubation by repeated re-adjustment as described above. The culture is optionally transferred, e.g. via an overflow tubing 2b, to a second tank reactor 2c. Finally, the pH shift to acidic conditions (step d) is performed in an acid tank reactor.

The one or more microorganisms to be used in the present invention is able to synthesize PHAs. Either a pure culture using only one strain or a mixed culture of several microorganisms can be used. In an advantageous embodiment of the present invention, the microorganism used in the inventive method is a wild-type, mutant or recombinant strain, capable of biosynthesizing PHAs, most advantageously a wild-type

strain is used. A variety of wild-type strains are known to synthesize PHAs. Also a mutant strain that shows enhanced PHA. synthesis may be used to increase the yield of RHAs . Said mutant can be a naturally isolated strain, a strain that has undergone chemical treatment with mutagenic compounds and has been selected subsequently for enhanced PHA production, or a genetically engineered strain, in which genes for PHA synthesis and/or depolymerization have been regulated with respect to expression. The skilled person knows how to obtain such mutant derivatives using classical protocols in the field of molecular microbiology (c.f. Sambrook, et . al . (1989), which is incorporated by reference) .

All wild-type strains that synthesize PHAs contain an intracellular PHA depolymerase (Jendrossek and Handrick, 2002) . The intracellular PHA depolymerase is only active towards native (amorphous) PHAs (Jendrossek and Handrick,

2002) . Most depolymerases have an activity range of pH 8-10.5

(Jendrossek and Handrick, 2002) . The depolymerase activity may be enhanced in ^" mutant strains, or be artificially inserted into cells by genetic engineering.

In another advantageous embodiment, the microorganism for the method of the present invention is preferably selected from the group of microorganisms of the. genus Achromobacter, Acidovorax, Acinetobacter, Actinobacillus, Actinomyces, Aeromonas/ Alcaligenes, Alteromonas, Amoebobacter, Aphanocapsa, Apha.nothece, Aquaspirillum, Azorhizobium, Azospirillυm, Azotobacter, Bacillus, Beggiatoa, Beijerinckia, Beneckea, Bordetella, Bradyrhizobium, Caryophanon, Caulobacter, Chloroflexus, ' Chlorogloea, Chromatium, Chromobacterium, Clostridium, Comamonas, Corynebacterium, Cupriavidus, Derxia, Desulfococcus, Desulfonema, Desulfosarcina, Desulfovibrio, Ectothiorhodospira, Escherichia, Ferrobacillus, Flavobacterium, Haemophilus, Halobacterium, Haloferax, Hydroclathratus, Hydrogenomonas, Hydrogenophaga, Hyphomicrobium, Ilyobacter, Lactobacillus, Lactococcus, Lamprocystis, Lampropedia, Legionella, Leptothrix, Methylobacterium, Methylococcus, Methylocystis, Methylomonaε, Methylosinus, Methylovibrio, Micrococcus, Microcoleus, Microcystis, Moraxella, Myco-bacterium,

Mycoplana, Nitrobacter, Nitrococcus, Nocardia, Oscillator±a, Paracoccus, ^' Pediococcus, Penicillium, Photobacterium, Physarum, Protomonas, Pseudomonas, Ralstonia, Rhizobium, Rhodobacillus, Rhodobacter, Rhodococcus, Rhodo-cyclus, Rhodomicrobium, Rhodopseudomonas, Rhodospirillum, Sphaerotilus, Sphingomonas, Spirillum, Spirulina, Staphylococcus, Stella, Streptococcus, Streptomyces, Synechococcus, Syntrophomonas, Thiobacillus, Thiocapsa, Thiocystis, Thio- dictyon, Thiosphaera, Trichodesmimum, Vibrio, Xanthobacter, Xanthomonas and Zooglea, most preferably a Pseudomonas strain.

The PHAs to be used in the present invention are any biosynthesized PHAs known in the field, including hetero- PHAs and homo-PHAs. Suitable PHAs can generally be classified into three groups based on the number of carbon atoms in the monomer units: short-chain-length PHAs (scl, 3-5 carbon atoms), medium-chain-length PHAs (me1, 6-14 carbon atoms) and long- chain-length PHAs (IcI, more than 14 carbon atoms) . Due to the asymmetric carbon atom, e.g. in the beta position, PHAs are optically active ^■ polymers containing only the J?-enantiomer. Especially good yields are achieved by the method of the present invention for scl- or mcl-PHAs. Furthermore, such PHAs may contain additional functional groups. These functional groups, which are always located in the polyester side chains, encompass olefinic, ester-, cyano-, amino-, nitroso-, phenyl-, nitrophenoxy- , halogenic- and carboxylic functions.

In another advantageous embodiment, the pH of the culture is adjusted after the cultivation of the microorganism to be above 7.0, preferably between 9.5 and 10.5. Typically, the pH will be adjusted by adding a solution of sodium hydroxide to the culture. In a more advantageous embodiment, the pH adjustment of step b) is continued during step c) . It was found that with the release of monomers the pH of the culture continuously dropped and that the degradation rate of PHAs was faster at the beginning than later. Without being bound to theory, it is ^' believed that the released monomer acids constantly lower -the environmental pH of the culture broth, resulting in unfavorable conditions for PHAs depolymerase, whose activity is highest in the alkaline range. In order to prevent the acidification of the medium during

incubation due to secretion of the RHAs, which is more critical at longer times of incubation, in an advantageous embodiment of the invention the method includes the readjustment of the alkaline pH during step c) . This re- adjustment of the pH is then followed by a further incubation. This cycle can be repeated multiple times, i.e. the pH adjustment can be stepwise. Ideally and most advantageously, the pH is kept constant during step c) by a pH- stat . Since the pH regulation allows for optimal in vivo depolymerization conditions and a maximal release of RHAs, in a most advantageous embodiment , the pH is kept constant until PHA degradation has reached a maximum.

In another advantageous embodiment, the incubation time of step c) is preferably at least 5 minutes, and more preferably 10-24 hours. Longer incubation times usually lead to higher product yields.

In another advantageous embodiment of the present invention, the temperature during step c) is kept at 10 ⁰ C to

65°C. Preferably, the temperature ^' is between 15°C and 40 ⁰ C, most preferably between 22 ⁰ C and 37°C. If step a) was performed in continuous culture, it may be preferable to run step b) to d) at lower temperatures, e.g. at room temperature, due to process design restrictions, whereas for processes where step a) was performed by batch cultivation, higher temperatures, e.g. the temperature at which the microorganism (s) grew, might be more suitable.

The second pH shift introduced in step d) leads to precipitation of various compounds from the medium and from lysed cells. The second pH to be reached in the method of the present invention is 1 or preferably below 1.

The generated RHAs are optionally purified. The precipitates generated due to the second pH shift may be removed from the supernatant by centrifugation or filtration, and the RHAs are further purified from the supernatant. Further purification of RHAs can be done by classical biochemical and chemical methods known to the skilled person. In one advantageous embodiment, purification involves cristallation, salt formation, chromatographic and/or distillation steps, preferably at least one chromatographic

step. Purification can also involve the extraction with organic solvents such as with tertbutyl methyl ether.

The RHA monomers obtainable by the method of the present invention depend on the composition of the biosynthesized PHA. The composition of the PHAs, in turn, depends - inter alia - on the microorganism and the culture conditions used. The selection of an appropriate microorganism and culture conditions is known in the art. In an advantageous embodiment of the present invention, the RHA monomers generated by the method of the present invention are selected from the group consisting of 3-hydroxypropionic acid, R-3- hydroxybutyric acid, (R) -3-hydroxyvaleric acid, (R) -3- hydroxyhexanoic acid, (R) -3-hydroxyheptanoic acid, (R) -3- hydroxyoctanoic acid, (R) -3-hydroxynonanoic acid, (R) -3- hydroxydecanoic acid, (R) -3-hydroxyundecanoic acid, (R) -3- hydroxydodecanoic acid, (R) -3-hydroxytetradecanoic acid, (R) -3- hydroxyhexadecanoic acid, 4-hydroxybutyric acid, (R) -4- hydroxyvaleric acid, (R) -4-hydroxyhexanoic acid, (R) -4- hydroxyheptanoic acid, (R) -4 -hydroxyoctanoic acid', (R) -4- hydroxydecanoic acid, 5-hydroxyvaleric acid, (R) -5- hydroxyhexanoic acid, (R) -6-hydroxydodecanoic acid, (R) -3- hydroxy-4-pentenoic acid, (R) ~3-hydroxy-4-trans-hexenoic acid, (R) -3-hydroxy-4-cis-hexenoic acid, (R) -3-hydroxy-5-hexenoic acid, (R) -3-hydroxy-5-hexynoic acid, (R) -3-hydroxy-6-heptynoic acid, (R) -S-hydroxy-G-trans-octenoic acid, (R) -3hydroxy-6-cis- octenoic acid, (R) -3-hydroxy-7-octenoic acid, (R) -3-hydroxy-7- octynoic acid, (R) -3-hydroxy-8-nonenoic acid, (R) -3 -hydroxy-8- nonynoic acid, (R) -3-hydroxy-9-decenoic acid, (R) -3 -hydroxy-9- decynoic acid, (R) -3 -hydroxy-10-undecynoic acid, (R) -3- hydroxy-5-cis-dodecenoic acid, (R) -3-hydroxy-6-cis-dodecenoic acid, (R) -3-hydroxy-5-cis-tetradecenoic acid, (R) -3 -hydroxy-7- cis-tetradecenoic acid, (R) -3-hydroxy-5, 8-cis-cis- tetradecenoic acid, (R) -3 -hydroxy-4-methylvaleric acid, (R) -3- hydroxy-4-methylhexanoic acid, (R) -3-hydroxy-5-methylhexanoic acid, (R) -(-) 3 -hydroxy- 6-methylheptanoic acid, (R) -3 -hydroxy-

4-methyloctanoic acid, (R) -3-hydroxy-5-methyloctanoic acid,

(R) -3-hydroxy-6-methyloctanoic acid, (R) -3 -hydroxy- 7- methyloctanoic acid, (R) -3-hydroxy-6-methylnonanoic acid, (R)-

3-hydroxy-7-methylnonanoic acid, (R) -3-hydroxy-8-methylnonanoic

acid, (R) -3 -hydroxy-7- tnethyldecanoic acid, (R) -3 -hydroxy- 9- methyldecanoic acid, (R) -3-hydroxy-7-methyl-6-octenoic acid, malic acid, (R) -3-hydroxysuccinic acid methyl ester, (R) -3- hydroxyadipinic acid methyl ester, (R) -3-hydroxysuberic acid methyl ester, (R) -3-hydroxyazelaic acid methyl ester, (R) -3- hydroxysebacic acid methyl ester, (R) -3-hydroxysuberic acid ethyl ester, (R) -3-hydroxysebacic acid ethyl ester, (R) -3- hydroxypimelic acid propyl ester, (R) -3-hydroxysebacic acid benzyl ester, (R) -3-hydroxy-8-acetoxyoctanoic acid, (R) -3- hydroxy-9-acetoxynonanoic acid, phenoxy- (R) -3-hydroxybutyric acid, phenoxy- (R) -3 -hydroxyvaleric acid, phenoxy- (R) -3- hydroxyheptanoic acid, phenoxy- (R) -3 -hydroxyoctanoic acid, para-cyanophenoxy- (R) -3-hydroxybutyric acid, para- cyanophenoxy- (R) -3 -hydroxyvaleric acid, para-cyanophenoxy- (R) - 3-hydroxyhexanoic acid, para-nitrophenoxy- (R) -3- hydroxyhexanoic acid, (R) -3-hydroxy-5-phenylvaleric acid, (R)- 3 -hydroxy-5-cyclohexylbutyric acid, (R) -3,12- dihydroxydodecanoic acid, (R) -3 , 8-dihydroxy-5-cis- tetradecenoic acid, (R) -3-hydroxy-4, 5-epoxydecanoic acid, (R)- 3-hydroxy-6, 7-epoxydodecanoic acid, (R) -3-hydroxy-8, 9-epoxy-5 , 6-cis-tetradecanoic acid, 7-cyano- (R) -3 -hydroxyheptanoic acid, 9-cyano- (R) -3-hydroxynonanoic acid, (R) -3 -hydroxy-7 fluoroheptanoic acid, (R) -3-hydroxy- 9-fluorononanoic acid, (R) -3-hydroxy-6-chlorohexanoic acid, (R) -3 -hydroxy- 8- chlorooctanoic acid, (R) -3 -hydroxy-6-bromohexanoic acid, (R) -3- hydroxy-8-bromooctanoic acid, (R) -3 -hydroxy-1, 1-bromoundecanoic acid, 3-hydroxy-2-butenoic acid, (R) -6-hydroxy-3-dodecenoic acid, (R) -3-hydroxy-2-methylbutyric acid, (R) -3-hydroxy-2- methylvaleric acid, and (R) -3-hydroxy-2 , 6-dimethyl-5-heptenoic acid.

More preferably, the monomers are selected from the group consisting of 3-hydroxypropionic acid, R-3- hydroxybutyric acid, (R) -3 -hydroxyvaleric acid, (R) -3- hydroxyhexanoic acid, (R) -3 -hydroxyheptanoic acid, (R) -3- hydroxy-6-heptynoic acid, (R) -3 -hydroxyoctanoic acid, (R) -3- hydroxynonanoic acid, (R) -3-hydroxy-8-nonynoic acid, (R) -3- hydroxydecanoic acid, (R) -3-hydroxyundecanoic acid, (R) -3- hydroxy-10-undecynoic acid, (R) -3-hydroxydodecanoic acid, (R)- 3-hydroxy-5-hexenoic acid, (R) -3-hydroxy-7-octenoic acid, (R)-

3-hydroxy-8-nonenoic acid, (R) -s-hydroxy-δ-decenoic acid, and (R) -3 -hydroxy-5-phenylvaleric acid.

According to the present invention, the prepared RHAs can exist as a mixture of two or more RHAs, which can be further separated and purified using known methods, such as column chromatography and, if required, solvent extraction.

The entire method in the present invention requires only one step of either centrifugation or filtration instead of 2 or 3 steps of centrifugation, as reported previously. More than 90% PHAs can be degraded into RHAs. The prepared RHAs reached a purity of more than 97%. The yield of RHA monomers by this method is usually above 50% and at least 10% weight of PHA polymer .

A second aspect of the present invention concerns a plant for the production of R-hydroxycarboxylic acids from a continuous culture, preferably a chemostat culture by in vivo depolymerization of PHAs, comprising a cultivation unit 1, at least one depolymerization unit 2 and optionally a harvest unit 3. Thereby, steps a) to e) of the above method can be performed in separate units. This allows adapting the process parameters for each unit individually.

In an advantageous embodiment, the depolymerization unit comprises a first tank reactor 2a, which is connected via an overflow tubing 2b with a second tank reactor 2c. Preferably, the length of said overflow tubing 2b is adjusted to allow for a sufficient residence time for depolymerization and can thus be optimized according to the set process parameters and the available equipment. Typically, a residence time of 0,5 hours to 24 hours is considered to be sufficient. It is believed that the residence time depends on the given system, and can be determined by routine experiments.

The microorganism capable of synthesizing PHAs and having an intracellular PHA polymerase activity is grown in continuous culture in said cultivation unit 1. The outflow of the continuous culture is transferred into the depolymerization unit 2. Preferably, the outflow is pooled in a first tank reactor 2a, where the pH of the culture broth is adjusted to the desired value. Said first tank reactor 2a is connected via an overflow tubing 2b with a second tank reactor

2c, wherein the pH of the culture broth is again adjusted to a desired value. One or more repeats of the depolymerization unit 2 may be used. Finally, the cell broth may be pumped into a harvest unit 3, preferably a tank, where the cell broth is acidified to a pH ≤ 1. The released RHAs monomers can optionally be purified by the above mentioned methods.

Modes for Carrying Out the Invention The present invention is further illustrated by the following ^" examples that are not intended to be in any way limiting the scope of the invention as claimed.

Example 1 Batch preparation of RHAs by adjusting the initial pH of the culture broth

Pseudomonas putida GPoI was cultivated at 30 ⁰ C under double-nutrient (carbon and nitrogen) limited conditions in continuous culture in a chemically defined medium supplemented with octanoic acid at a carbon to nitrogen ratio (C/N) of 15 g-g ^"1 (Hartmann, 2004). PHAs containing (J?) -3- hydroxyoctanoic acid (R3H0) and (R) ~3-hydroxyhexanoic acid (R3HH) were produced to 40-45% (weight/weight (w/w) ) of the cell dry weight (CDW) at the steady state. The culture broth was collected and then exposed to PHAs degradation conditions. Degradation of PHAs and the monomers released into the culture medium were measurement by GC and HPLC (Ren, et. al. 2005) .

With the initial pH of 10 at room temperature (22 ⁰ C) and no further controlling of pH, it was detected that PHAs degraded from 43% (w/w) to 26% (w/w) in 9 hours (Fig. IA) . The monomer release reached almost maximum after 12 hours , even though there was still half of the original accumulated PHAs left in the cell (Fig. IA) . Further incubation up to 24 hours did not lead to much more PHAs degradation (Fig. IA) . These results are better than those reported previously by Lee and co-workers (WO 99/29889) who showed that the maximal yield of only 9.7% (w/w) was obtained after 4 days.

Example 2

Batch preparation of RHAs by controlling pH at a constant value

In this experiment the collected culture broth was adjusted to pH 10 and maintained at pH 10 with 2 N NaOH by a pH stat. The PHAs degradation and monomer release were followed for 24 hours at the room temperature (22 ⁰ C) (Fig.

IB) . It was found that PHAs immediately started to degrade exponentially from 44% (w/w CDW) to 13% (w/w CDW) in 12 hours.

After 1-6 hours 80% (w/w) of total PHAs were found to be degraded. Further incubation to 24 hours led to only 5% (w/w

CDW) PHAs left in the cells.

Example 3

Continuous preparation of RHAs by in vivo degradation of PHAs

In this experiment the culture broth at steady state was continuously channeled out to a stirred tank 2a where the pH value was controlled to be 10 with 2 W NaOH by a pH stat 4 (Fig. 2) . The cells remained in the tank 2a at room temperature (22 ⁰ C) for different time periods (5, 7.5, 10 and 15 hours) and were then pumped out through an overflow tube 2b (Fig. 3A) . It was found that only about 50% (w/w) PHAs were degraded into RHAs after 10 hours incubation in the tank 2a (Fig. 3A) . Prolonged incubation to 15 hours did not lead to much better yield of PHAs degradation. To maximize the RHAs production, the length of the overflow tube 2b was designed in such a manner that the cells had more time (1, 3, 6, 9, or 12 hours) to further degrade the remaining PHAs. A yield of about 90% (w/w) was obtained after the pH of the tank 2a was controlled at 10, cells remained in the .tank for 10 hours and went through the overflow tube 2b in 9 hours (Fig. 3B) . The pH of the cell suspension changed from 10 to 8.4 after 9 hours' going through the overflow tube 2b (Fig. 3B) .

Example 4

Continuous preparation of RHAs by in vivo degradation of PHAs by readjusting pH

The culture broth at the steady state was continuously channeled out to a stirred tank where the pH of

the culture broth was brought to 10. Cells were then channeled out through a tube. When the extracellular pH dropped to 9, the culture broth was once more brought to pH 10 (Fig. 4) .

This process was repeated several times until the intracellular PHAs were degraded to maximum (Pig. 4) . Here it was found that after about 15 hours PHAs degraded from 43%

(w/w CDW) to 9% (w/w CDW) , resulting an efficiency of about

80%.

Example 5

Separation of monomers using column chromatography

The cell suspension containing the mixture of R3H0 and R3HH was acidified to pH 1 or below with 37% HCl. Acidification facilitates the separation of RHAs from other cell components and further purification of RHAs (Ruth, et. al . 2007) . The monomers present in the supernatant were collected by centrifugation and subjected to the separation process (Ruth, et. al . 2007). In detail, separation of different monomers was accomplished with a hydrophobic interaction column packed with silica gel 100 C18-reversed phase (particle size 0.040-0.063 mm; Fluka) . During operation, the column was cooled to <10°C and set under pressure (-80 kPa) . After washing with 0.1 M HCl (> 5x bed volume), the mixture of monomers was fractionated using a stepwise elution with 0.1 M HCl/acetonitrile mixtures as mobile phase. The collected fractions were analyzed for the presence of monomers by HPLC and GC (Ren, et. al. 2005) . The yield of the separation step reached 80% (w/w) for both R3HO and R3HH.

Example 6

Purification of RHAs

The monomers, which were separated by the method shown in example 5, can be further purified by conventional chemical methods or by the method described below to make final products for appropriate use.

The fractions containing only one type of RHA were pooled and acetonitrile was removed by rotary evaporation. Subsequently, the solutions were further acidified by addition of 1 M HCl (- acid/sample = l/l (vol%) ) , saturated with KCl,

and extracted three times with the same volume of tert-butyl methyl ether (aqueous phase:organic phase = 1/1 (vol%) ) . The combined organic phases were dried over Na ₂ SO ₄ , filtered and the solvent evaporated. The yield of the purification step reached more than 95%.

Example 7

Identification of purified 3-hydroxyoctanoic acid and examination of its optical purity To determine purities and structures of RHA, the purified 3-hydroxyoctanoic acid was analyzed by ¹ H NMR spectroscopy (Ruth, et. al . 2007). The purity of the obtained 3-hydroxyoctanoic acid was found to be higher than 95% (w/w) .

The configuration of the purified 3-hydroxyoctanoic acid was examined by chiral GC (Ren, et. al . 2005). The racemic standard (R, S) -3-hydroxyoctanoafce methyl ester showed two peaks with the retention times (t _R ) of 27.3 and 27.7 minutes which refer to the two . enantiomers. As previously reported, the later- peak can be assigned to the (R) -enantiomer (Ren, et. al. 2005) . Only one peak was detected with the prepared 3 -hydroxyoctanoate methyl ester, which revealed the high enantiomerical purity of this substance. By mixing 3- hydroxyoctanoate methyl ester and the racemic standard the area of the later peak at 27.7 min increased, confirming the absolute (R) -configuration of purified 3-hydroxyoctanoic acid. Thus, in vivo depolymerization and subsequent isolation is a feasible approach to produce enantiomerically pure RHAs.

While there are shown and described presently preferred embodiments of the invention, it is to be distinctly understood that the invention is not limited thereto but may be otherwise variously embodied and practiced within the scope of the following claims.

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