AU707659B2 - A recombinant Escherichia coli producing poly-3-hydroxybutyrate (PHB) and a process for preparing PHB employing the same - Google Patents

Description

A RECOMBINANT Escherichia coli PRODUCING POLY-3- HYDROXYBUTYRATE(PHB) AND A PROCESS FOR PREPARING PHB EMPLOYING THE SAME Field of the Invention The present invention relates to a recombinant Escherichia coli producing poly-3-hydroxybutyrate(PHB) from lactose or whey and a process for preparing PHB employing the same, more specifically, to a recombinant Escherichia coli transformed with a recombinant plasmid containing a PHB synthesis gene of Alcallgenes eutrophus, which produces effectively PHB from lactose or whey and a process for preparing PHB which comprises a step of culturing the recombinant E. coli in a medium containing lactose or whey.

Background of the Invention Poly-3-hydroxybutyrate(PHB) is an energy storage material which is synthesized and accumulated intracellularly in numerous microorganisms. In recent years, PHB has drawn much attention as a candidate for biodegradable plastic, since its physical and chemical nature are similar to those of the conventional synthetic polymers and it can be completely degraded into water and carbon dioxide by microorganisms due to its excellent biocompatibility and biodegradability.

Especially, it has been applied in drug delivery system or suture, since 3-hydroxybutyrate, a degraded product of PHB, is also a metabolite present in human and animals. However, it has not been practically applied in packaging materials or containers due to its high cost.

In order to overcome the cost problem, much attention have been paid to Alcaligenes eutrophus and Alcaligenes latus, which are known to produce PHB(see: USP 4,433,053; Anderson and Daws, Micorbiol. Rev., 54:450-472(1990)). Furthermore, it has been reported that PHB can be produced by recombinant Escherichia coli which contains a PHB synthesis gene of Alcaligenes eutrophus(see: WO 89/00202; Slater et al., J. Bacteriol., 170:4431-4436(1988); Schubert et al., J. Bacteriol., 170:5837- 5847(1988); Peoples and Sinskey, J. Biol. Chem., 264:15298- 15303(1989)).

In line with these activities, the present inventor has also made an effort to develop high copy number plasmids, highly stabilized plasmids, host microorganisms for transformation and culture techniques for the transformed microorganism, and established culturing conditions to facilitate PHB synthesis, as a promising approach to produce effectively PHB employing the recombinant E. coli. In particular, the capability of producing PHB was compared with each other, employing a series of recombinant E. coli, transformed with plasmids pSYL101 and pSYL102 having different S 25 copy numbers, and plasmids pSYL103 and pSYL104 prepared by cloning parB region of plasmid Rl(see: Gerdes, Bio/Technol., 6:1402(1988)) into the plasmids for the purpose of improving stabilization, was compared each others, and it was found that the use of plasmid with a high copy number enables increased production of PHB(see: Lee et al., Ann. NY Acad. Sci., 721:43- 53(1994); Lee et al., J. Biotechnol., 32:203-211(1994)).

Moreover, when the production of PHB was investigated on a variety of E. coli strains, E. coli XL1-blue, JM109, B, W and K12 which contain a stable plasmid pSYL105 having a high copy number, it was determined that the production rate and yield are closely related to the characteristics of each microorganism(see: Lee et al., Biotechnol. Bioeng., 44:1337-1347(1994)).

In addition, since microorganisms grown in an expensive complex medium were found to produce more PHB than those grown in a minimal medium, a method for producing PHB by culturing microorganisms in a minimal medium supplemented with a small amount of complex nitrogen source has been proposed. In S: accordance with this method, a complex nitrogen source such as tryptone, casamino acid or casein hydrolysate was added in a small amount(0.5 to 5g/L) to a growth medium for microorganism, which resulted in the increase of PHB production(see: Lee and Chang, J. Environ. Polymer Degrad., 2:169-176(1994)). Also, it was found that the addition of amino acid or oleic acid, instead of the complex nitrogen source, may increase PHB production(see: Lee et al., J. Ferment. Bioeng., 79:177- 25 180(1995) Meanwhile, a fed-batch culture method was developed to increase PHB production, which resulted in 101g/L of dry cell weight and 81g/L of PHB productivity in 39 hours(see: Lee et 4 al., J. Biotechnol., 32:203-211(1994)). Also, even though a small amount of complex nitrogen source was added into the fedbatch culture of E. coli, a high productivity of PHB, e.g., can be realized(see: Lee and Chang, J. Environ. Polymer Degrad., 2:169-176(1994)).

On the other hand, filamentation during PHB production by a recombinant E. coli was found to decrease or stop the growth of cells which, in turn, lowers culture efficiency and decreases PHB production. Therefore, in order to inhibit the filamentation of recombinant E. coli, a recombinant plasmid pSYL107 containing a PHB synthesis gene and a cell division related protein gene(ftsZ) preventing filamentation, was proposed to transform E. coli, which resulted in high production of PHB while eliminating the filamentation phenomenon(see: S. Y. Lee, Biotechnol. Lett., 16:1247- 1252(1994); S. Y. Lee, Kor. J. Appl. Microbiol. Biotechnol., 22:614-620(1994); Lee and Lee, J. Environ, Polymer Degrad., so 4:131-134(1996)).

However, all of the methods mentioned above, has revealed a critical shortcoming that they essentially employ a carbon source of glucose whose price is very high. Therefore, there has been a continuous need to lower the cost for PHB production S" by developing an alternative carbon sources.

In this regard, whey, a liquid by-product arising from the 25 manufacturing process of cheese or casein, may be a promising candidate for a major carbon source for PHB production by recombinant microorganisms. Whey contains abundant lactose and whey protein, and it is produced in an amount of about 8kg 5 while manufacturing 1kg of cheese. Moreover, the whey, since it has a high BOD value, may be a major pollutant of rivers, which naturally accelerates its utilization in this area.

On the other hand, Alcaligenes eutrophus, Alcaligenes latus and methanol autotrophs have been widely used for PHB production. However, they cannot utilize lactose which is abundant in whey, as a carbon and energy source, while some E.coli strains are know to grow on a medium containing lactose.

SAccordingly, there are strong reasons for exploring and developing an improved method for producing PHB from lactose or whey, which enables universal use of 15 PHB in various fields.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", means 20 "including but not limited to" and is not intended to exclude other additives, components, integers or steps.

Summary of the Invention In this regard, the present inventor has screened a series of Escherichia coli strains which can utilize lactose or whey as a carbon source, and developed recombinant E.coli strains which are transformed with a recombinant plasmid pSYL 105 or pSYL 107 containing a PHB synthesis gene, and successfully produced PHB in a high yield by culturing the recombinant E.coli in a minimal medium containing lactose or whey.

Accordingly the present invention provides a recombinant Escherichia coli selected from the group consisting of E.coli/pSYL107, GCSC 4401 (KCTC 0350BP) and E.coli/pSYLl07 GCSC6576 (KCTC 0351BP), wherein said E.coli H:\Bkrot\Keep\speci\48351-97.doc 15/03/99 .k> 6 produces poly-3-hydroxybutyrate (PHB) from lactose or whey.

The present invention further provides a process for preparing PHB which comprises the steps of culturing a recombinant E.coli as described herein in a minimal medium containing lactose or whey, and recovering PHB from the culture.

Brief Description of Drawings The above and the other aspects and features of SS. the present invention will become apparent from the following description given in conjunction with the accompanying drawings, in which: 0@ Figure 1 is a gene map of a recombinant plasmid pSYL 105.

Figure 2 is a gene map or a recombinant plasmid S 20 pSYL 107.

Detailed Description of the Invention The present inventor first screened a series of 25 Escherichia coli strains which can utilize lactose or whey as a carbon source as followings: a variety of E.coli stains were grown in a lactose of whey supplemented minimal medium containing 13.5g/L of KH 2

PO

4 4g/L of (NH 4 2 HP0 4 1.4g/L of MgSO 4 .7H20, 1.7g/L of citric acid, 0.1g/L of FeSO 4 .7H 2 0, 0.02g/L of CaC1 2 .2H 2 0, 0.022g/L of ZnSO 4 .7H 2 0, 0.005g/L of MnSO 4 .4H 2 0, 0.01g/L of CuSO 4 .5H 2 0, 0.001g/L of

(NH

4 6M0 7 0 24 .4H 2 0, 0.002g/L of Na 2 B40 7 .10H 2 0 and 0.01g/L of thiamine to compare their growth rates, and E.coli B(DSM 499), E.coli TG1 (DSM 6056) and E.

H:\Bkrot\Keep\speci\48351-97.doc 15/03/99 V 1 coli W3110(KCTC 2223) which showed superior growth in a lactose supplemented minimal medium and E. coli K12(a wild-type strain, E. coli HMS174(X-, recAl, IN(rrnD-rrnE), rpoB331, hsdR19, E. coli MG1047(F-, X6(3X)::Tnl000, recA56), E. coli B(a wild-type strain, GCSC 6572), E. coli C-1(a wild-type strain, and E. coli B(a wild-type strain, GCSC 2507) which showed superior growth in a whey supplemented minimal medium, were finally selected as potential microorganisms for PHB production.

Each of the screened E. coli strains was transformed with a recombinant plasmid pSYL105 comprising -lactamase gene, PHB synthesis gene and parB region of plasmid Rl(see: Figure 1) or pSYL107 prepared by inserting a cell division related protein gene(ftsZ) into the plasmid pSYL105(see: Figure 2), respectively.

The transformed strains thus obtained were grown in a minimal medium containing 10 to 80g/L, preferably 30 to of lactose or whey or in a minimal medium containing 10 to of lactose or whey and 0.1 to 10g/L of complex nitrogen 20 source and then, dry cell weight, PHB concentration and PHB content(as a percentage of PHB concentration to dry cell .i weight) were determined, respectively. At this time, the .I transformed strains harboring pSYL105 were grown at 20 to 40 0

C,

preferably at 25 to 38°C, most preferably at 37°C, while the 25 transformed strains harboring pSYL107 were grown at 20 to 400C, preferably at 25 to 350C, most preferably at 300C to prevent from formation of minicell.

As a result, the transformed E. coli B(DSM 499), E. coli TG1(DSM6056) and E. coli W3110(KCTC 2223) were found to be able to produce PHB from lactose or whey. And, the transformed

E.

coli K12(a wild-type strain, F E. coli HMS174(-, recAl, IN(rrnD-rrnE), rpoB331, hsdR19, E. coli MG1047(F-, X6(3X)::Tnl000, recA56), E. coli B(a wild-type strain, F-, GCSC 6572), E. coli C-l(a wild-type strain, and E. coli B(a wild-type strain, GCSC 2507) were resulted to produce effectively PHB from whey, and all the said transformed strains showed more PHB production in a minimal medium supplemented with a complex nitrogen source than in a minimal medium. In this regard, the complex nitrogen source includes tryptone, yeast extract, peptone, casamino acid, cotton seed hydrolysate, beef extract, casein hydrolysate, corn steep liquor and soybean hydrolysate.

*S

The present invention is further illustrated in the following examples, which should not be taken to limit the *scope of the invention.

Example 1: Comparison of growth rates of a variety of Escherichia coli strains growing in a medium containing lactose as a carbon source

S.

25 To screen E. coli strains showing an excellent growth in a minimal medium containing lactose as a carbon source, 7 strains of E. coli which have been known to utilize lactose 9 were grown in a flask containing a minimal medium(see: Table 1 below) which was supplemented with 20g/L of lactose, and comparison of their growth rates followed(see: Table 2).

Table 1: Composition of minimal medium Component Amount(g/L) Component Amount(g/L)

KH

2

PO

4 13.5 (NH 4 2 HPO4 4 MgSO 4 -7H 2 0 1.4 citric acid 1.7 FeSO 4 '7H 2 O 0.1 CaC 2 ,2H20 0.02 ZnS04-7H 2 0 0.022 MnS04-4H20 0.005 2 0 0.01 (NH 4 6 Mo 24 4H20 0.001 Na 2

B

4 0 7 10H 2 0 0.002 thiamine 0.01 Table 2: Comparison of growth rates of E. coli strains 15 Microorganisms Growth rates Remark E. coli(ATCC 9492) Not good E. coli W(ATCC 9637) Mean E. coli K12(ATCC 23716) Good E. coli B(DSM 499) Excellent 20 E. coli TG1(DSM 6056) E. coli K12(KCTC 1116) E. coli W3110(KCTC 2223) As shown in Table 2, E. coli B(DSM 499), E. coli TG1(DSM 25 6056) and E. coli W3110(KCTC 2223) were found to be good or excellent at growth in a medium containing lactose as a carbon source. Accordingly, these three strains were finally screened as potential microorganisms for PHB production from lactose.

Example 2: Preparation of recombinant E. coli for PHB production from lactose Each of three E. coli strains selected in Example 1 was transformed with a recombinant plasmid pSYL105 containing Plactamase gene, PHB synthesis gene and parB region of plasmid Rl(see: Figure 1) and a recombinant plasmid pSYL107 prepared by inserting a cell division related protein gene(ftsZ) into the plasmid pSYL105(see: Figure respectively, by the aid of the conventional electroporation technique(see: Dower et al., Nucleic Acids Rev., 16:6127-6145(1988)).

Analysis of restriction pattern of plasmids isolated from the recombinant microorganisms revealed that the following six i strains contain pSYL105 or pSYL107: DSM 499(pSYL105), DSM 15 499(pSYL107), DSM 6056(pSYL105), DSM 6056(pSYL107),

KCTC

2223(pSYL105) and KCTC 2223(pSYL107).

Example 3: Production of PHB from lactose Example 3-1: Determination of optimal lactose concentration

S

oS Prior to investigating the PHB production from lactose, lactose was added in five concentrations to the minimal medium described in the Table 1 above, to prepare 5 types of media and each of six transformed strains obtained in Example 2 were inoculated into each medium. Each strain was grown at 37°C(in case of the strains transformed with pSYL105) and 30°C(in case 11 of the strains transformed with pSYL107) for 60 hours. Then, the growth of each strain was determined by measuring an absorbance at 600nm(see: Table 3).

Table 3: Comparison of growth of strains depending on concentrations of lactose(unit:

OD

600 *r *5 .5,

*S.

S

*5 Recombinant Lactose Concentration(g/L) E. coli strains 20 30 45 DSM 499(pSYL105) 4.5 4.7 4.7 4.7 5.1 DSM 499(pSYL107) 4.7 4.4 7.7 11.0 19.4 DSM 6056(pSYL105) 0.7 0.8 0.79 0.72 0.62 DSM 6056(pSYL107) 1.67 0.7 0.78 1.0 0.83 KCTC 2223(pSYL105) 14.3 19.8 26.3 17.8 18.3 KCTC 2223(pSYL107) 2.8 3.4 3.3 3.1 As shown in Table 3, optimal lactose concentration for the maximum growth of each strain was found to be quietly different from each other, depending on the kinds of strains as follows: 30g/L for DSM 499(pSYL105), 60g/L for DSM 499(pSYL107), for DSM 6056(pSYL105), 10g/L for DSM 6056(pSYL107), 30g/L for KCTC 2223(pSYL105), and 20g/L for KCTC 2223.

Example 3-2: Production of PHB by recombinant E. coli growing in a minimal medium containing lactose Each transformed strain was inoculated into a 250ml flask filled with 50ml of a minimal medium containing an optimal lactose concentration determined in Example 3-1 and cultured while shaking for 60 hours, to determine the PHB productivity.

At this time, the strains with pSYL105 were cultured at 37 0

C,

while the other strains with pSYL107 were cultured at 300C.

PHB productivity was determined by measuring dry cell weight, PHB concentration and PHB content(percentage), in accordance with the conventional methods in the art(see: Lee et al., Biotechnol. Bioeng., 44:1337-1347(1994)), and the results are summarized in Table 4.

Table 4: Microbial growth and production of PHB in a minimal medium containing lactose

C..

C.

Recombinant Dry cell PHB conc. PHB E. coli strains weight(g/L) content(%) DSM 499(pSYL105) 0.53 0.02 3.77 DSM 499(pSYL107) 3.30 1.58 47.9 DSM 6056(pSYL105) 0.52 0.05 9.6 DSM 6056(pSYL107) 0.86 0.10 11.6 KCTC 2223(pSYL105) 6.78 2.16 31.9 KCTC 2223(pSYL107) 1.15 0.05 4.35 As shown in Table 4, it was clearly determined that all six transformed strains were able to produce PHB from lactose, in particular, 2 strains of DSM 499(pSYL107) and KCTC 2223(pSYL105) produced PHB more efficiently than any other one.

Example 3-3: Production of PHB by recombinant E. coli growing in a minimal medium containing lactose supplemented with a complex nitrogen source Since DSM 499(pSYL107) and KCTC 2223(pSYL105) which 13 produced PHB more efficiently as shown in Table 4, they were cultured in minimal media containing lactose, in an amount of for DSM 499(pSYL107) and 30g/L for KCTC 2223(pSYL105), which were supplemented with a small amount of various complex nitrogen sources listed in the Table 5 below(see: Lee and Chang, J. Environ. Polymer Degrad., 2:169-176(1994)), in an analogous manner as in Example 3-2. PHB productivities of recombinant strains in minimal media containing lactose which were supplemented with various complex nitrogen sources(i.e., A, B, C, D, E, F, G, H and I) were determined by measuring dry cell weight, PHB concentration and PHB content(percentage), whose results are summarized in the Table 6 below.

Table 5: Complex nitrogen sources and their concentration 15 added to minimal medium 0 Ce 20 0 0 Complex Nitrbgen Sources Concentration(g/L) Tryptone 2 Yeast extract Peptone 1 Casamino acid 1 Cotton seed hydrolysate 2 Beef extract Casein hydrolysate 2 Corn steep liquor Soybean hydrolysate a.

a a..

a a a.

14 Table 6: Microbial growth and production of PHB in minimal media containing lactose supplemented with a variety of complex nitrogen sources A B C D E F G H I DSM 499(pSYL107) Dry cell weight(g/L) 3.98 3.4 4.08 4.73 5.24 3.59 3.44 8.81 4.09 PHB conc.(g/L) 1.45 2.06 2.48 2.93 1.75 2.12 2.03 4.82 2.19 PHB content(%) 36.4 60.6 60.8 61.9 33.4 59.1 59.0 54.7 53.5 KCTC 2223(pSYL105) Dry cell weight(g/L) 6.6 6.9 8.2 6.98 7.03 5.43 6.6 8.7 7.05 PHB conc.(g/L) 2.36 2.65 3.6 2.61 2.9 2.16 2.93 2.13 3.37 PHB content(%) 35.8 38.4 43.9 37.4 41.3 39.8 44.4 34.1 38.7 As shown in Table 6, the addition of complex nitrogen source in a small amount increased the PHB production by the said two transformed strains, especially, DSM 499(pSYL107) showed a much higher productivity. Furthermore, E. coli B DSM 499(pSYL107) showed the highest PHB productivity of 4.82g/L by the addition of corn steep liquor, while E. coli W3110 KCTC 2223(pSYL105) showed the highest productivity of 3.6g/L by the addition of peptone.

Example 4: Production of PHB from whey Example 4-1: Determination of optimal whey concentration Since the recombinant E. coli can produce PHB from lactose, the production of PHB from the strains was investigated, by employing a carbon source of whey which contains abundant lactose.

Prior to investigating the PHB production, whey was added in four concentrations to the minimal medium described in the Table 1 above, to prepare 4 types of media and each of six of the transformed strains obtained in Example 2 were inoculated into each medium. Each strain was grown at 37°C(in case of the strains transformed with pSYL105) and 30 0 C(in case of the strains transformed with pSYL107) for 60 hours. Then, the growth of each strain was determined by measuring an absorbance at 600nm(see: Table At this time, a whey in powder(Sigma, USA) was added to distilled water to make a final concentration of 200g/L and then, completely dissolved by stirring for minutes while boiling. The mixture was autoclaved at 121 0 C for minutes and centrifuged at 6,000rpm for 15 minutes, and the supernatant thus obtained was employed for the further 15 experiment.

Table 7: Comparison of growth of strains depending on concentrations of whey(unit: ODo 00 20 20* 9 25 r a• Recombinant Whey Concentration(g/L) E. coli Strains 10 20 40 DSM 499(pSYL105) 6.1 6.5 4.9 4.8 DSM 499(pSYL107) 6.1 5.9 7.0 12.4 DSM 6056(pSYL105) 1.12 1.36 1.64 DSM 6056(pSYL107) 1.3 1.36 1.71 KCTC 2223(pSYL105) 12.0 20.3 23.5 20.2 KCTC 2223(pSYL107) 5.5 4.6 5.3 2.9 As shown in Table 7, all six E. coli strains, especially DSM 499(pSYL105), DSM 499(pSYL107), KCTC(pSYL105), KCTC 2223(pSYL107), were found to be able to utilize whey as a 16 carbon source. The optimal whey concentration for the maximum growth of each strain was quietly different from each other, depending on the kinds of strains as follows: 20g/L for DSM 499(pSYL105), 40g/L for DSM 499(pSYL107), 80g/L for DSM 6056(pSYL105), 80g/L for DSM 6056(pSYL107), 40g/L for KCTC 2223(pSYL105), and 10g/L for KCTC 2223.

Example 4-2: Production of PHB by recombinant E. coli strain growing in a minimal medium containing whey Each transformed strain was inoculated into a minimal medium containing an optimal whey concentration determined in Example 4-1 and cultured to determine the PHB productivity in an analogous manner as in Example 3-2. The results are listed in Table 8.

9 9 9 .9 999 9*9 99 9 9 99999 *r 9 Table 8: Microbial growth and production of PHB medium containing whey in a minimal Recombinant Dry cell PHB conc. PHB E. coli Strains weight(g/L) content(%) DSM 499(pSYL105) 1.05 0.07 6.67 DSM 499(pSYL107) 6.33 2.64 41.7 DSM 6056(pSYL105) 0.83 0.02 2.41 DSM 6056(pSYL107) 2.65 0.20 7.55 KCTC 2223(pSYL105) 5.98 0.48 8.03 KCTC 2223(pSYL107) 2.31 0.06 2.60 As shown in Table 8, it was clearly determined that all 17 six transformed strains were able to produce PHB from whey and, in particular, 2 strains of DSM 499(pSYL107) and KCTC 2223(pSYL105), produced PHB more efficiently than any other one.

Example 4-3: Production of PHB by recombinant E. coli growing in a minimal medium containing whey supplemented with a complex nitrogen source Since DSM 499(pSYL107), DSM 6056(pSYL107) and KCTC 2223(pSYL105) produced PHB more efficiently as shown in Table 8, they were cultured for 60 hours in a minimal medium containing whey supplemented with a small amount of complex nitrogen source listed in the Table 5 above, in an analogous 15 manner as in Example 3-2. PHB productivity was determined by measuring dry cell weight, PHB concentration and PHB content (percentage), and the results are summarized in the Table 9 below. At this time, whey was added to a minimal medium in an optimal concentration as listed in the Table 7 above.

SS

S*.o

S*«

o 18 Table 9: Microbial growth and production of PHB in a minimal medium containing whey supplemented with a variety of complex nitrogen sources A B C D E F G H I DSM 499(pSYL107) Dry cell weight(g/L) 4.95 10.6 4.38 5.38 5.63 3.83 4.56 6.70 4.60 PHB conc.(g/L) 3.15 6.52 2.90 3.41 3.28 2.50 3.14 3.47 3.26 PHB content(%) 63.6 61.5 66.2 63.4 58.3 65.3 68.9 51.8 70.9 a.

a DSM 6056(pSYL107) Dry cell weight(g/L) 5.23 3.08 4.25 3.95 4.60 4.10 3.95 3.23 4.95 PHB conc.(g/L) 2.14 0.78 0.80 1.15 0.83 1.15 0.48 0.20 0.33 PHB content(%) 40.9 25.3 18.8 29.1 18.0 28.0 12.2 6.2 10.5 KCTC 2223(pSYL105) Dry cell weight(g/L) 7.9 6.85 6.98 7.23 7.65 5.85 6.7 6.32 7.78 PHB conc.(g/L) 2.22 0.90 1.10 1.62 1.48 0.34 0.67 0.75 0.69 PHB content(%) 28.1 13.1 15.8 22.4 19.3 5.81 10.0 11.9 8.87 As shown in Table 9, a small amount of complex nitrogen source was added in a medium, and the PHB production was 20 dramatically increased by the said transformed strains.

Especially, DSM 499(pSYL107) was found to produce more than of PHB content, which means that the complex nitrogen sources are very effective in increasing the PHB productivity.

25 Example 5: Preparation of recombinant E. coli synthesizing

PHB

from whey To select E. coli strains which show superior growth by employing whey as a carbon source, various strains of E. coli strains were cultured in a minimal medium(see: Table 1) containing 30g/L of whey, and six E. coli strains were finally selected for this invention as follows(see: Table 10 below): 19 E. coli K12(a wild-type strain, F) E. coli HMS174 recAl, IN(rrnD-rrnE), rpoB331, hsdR19, E. coli MG1047(F-, X6(3X)::Tn1000, recA56), E. coli B(a wild-type strain, F-, GCSC 6572), E. coli C-l(a wild-type strain, and E. coli B(a wild-type strain, GCSC 2507). The selected strains were transformed with a recombinant plasmid pSYL107 by the aid of conventional electroporation technique. Analysis of the restriction patterns of plasmid isolated from cells, revealed that the strains contain pSYL107, which are named as listed in Table Table 10: E. coli strains showing high growth on a carbon source of whey and transformed ones

S,

15 *5 E. coli strains transformed E. coli strains E. coli K12(GCSC* No. 4401) E. coli 4401(pSYL107) E. coli HMS174(GCSC* No. 6576) E. coli 6576(pSYL107) E. coli MG1047(GCSC* No. 6197) E. coli 6197(pSYL107) E. coli B(GCSC* No. 6572) E. coli 6572(pSYL107) E. coli C-1(GCSC* No. 3121) E. coli 3121(pSYL107) E. coli B(GCSC* No. 2507) E. coli 2507(pSYL107) Available from E. coli Genetic Stock Center, Yale University, New Haven, CT, U. S. A.

Example 6: Production of PHB by recombinant E. coli growing in a minimal medium containing whey Each transformed strain was inoculated into a minimal medium(see: Table 1) containing 35g/L or 70g/L of whey concentration and cultured at 30 0 C for 60 hours while shaking at 250rpm to determine the PHB productivity. The PHB productivity was determined by measuring dry cell weight, PHB concentration and PHB content(percentage), whose results are listed in Table 11.

Table 11: Microbial growth and production of PHB in a minimal medium containing whey *0 a a.

a a.

a *o a.

a a .a* o a Addition of 35g/L of whey Addition of 70g/L of whey Dry cell PHB conc. PHB Dry cell PHB cone. PHB weight content weight content E. coli 4401(pSYL107) 5.05 3.95 78.2 3.95 3.14 79.5 E. coli 6576(pSYL107) 5.1 4 78.4 3.3 2.48 75.2 E. coli 6197(pSYL107) 2.58 0.89 34.5 2.1 0.56 26.7 E. coli 6572(pSYL107) 2.45 1.12 45.7 2.25 0.75 33.3 E. coli 3121(pSYL107) 2.85 1.23 43.2 2.5 0.99 39.6 15 E. coli 2507(pSYL107) 6.3 3 47.6 6.9 3.54 51.3 As shown in Table 11, although each E. coli strain showed different PHB productivity, all 6 transformed E. coli were found to be able to produce PHB from whey. In particular, E.

20 coli 4401(pSYL107) and E. coli 6576(pSYL107) showed the highest productivity of PHB and, all strains except E. coli 2507(pSYL107) showed decreased PHB production, when whey content is increased.

Example 7: Comparison of PHB productivity depending on the whey concentration To determine the effect of whey concentration on PHB productivity, E. coli 4401(pSYL107) and E. coli 6576(pSYL107), which showed the highest productivity of PHB as shown in Example 6, were inoculated into a minimal medium(see: Table 1) containing 10, 20, 30, 40, 50, 60 or 70g/L of whey and cultured at 30 0 C for 60 hours while shaking at 250rpm. The PHB productivity was determined by measuring dry cell weight, PHB concentration and PHB content(percentage), whose results are listed in Table 12.

Table 12: Comparison of PHB productivity depending on the whey concentration

S

S

S

*5

*SS*

S

S

S

a 15 E. coli 4401(pSYL107) E. coli 6576(pSYL107) Whey cone.

Dry cell PHB cone. PHB Dry cell PHB cone. PHB weight content(%) weight(g/L) content(%) 3.63 1.03 28.4 3.23 1.08 33.4 20 2.88 1.49 51.7 6.30 3.56 56.5 5.70 4.47 78.4 6.40 5.20 81.3 40 4.35 3.49 80.2 4.00 3.41 85.3 50 4.50 3.68 81.8 3.85 3.18 82.6 4.15 3.41 82.2 3.80 3.03 79.7 70 3.95 3.14 79.5 3.30 2.48 75.2 As shown in Table 12, two strains showed the maximal PHB productivity when 30g/L of whey was added, and the minimal PHB productivity when 10g/L of whey was added. In case of E. coli 25 4401(pSYL107), PHB concentration and PHB content maintained above 3g/L and 78%, respectively, when whey was added in an amount of more than 30g/L. Furthermore, E. coli 6576(pSYL107) produced PHB at a level of above 3g/L and at a content of above 79%, when whey was added in an amount of 30 to Specifically, PHB concentration at 20g/L of whey amount was relatively high, 3.56g/L, while PHB content at this amount was low, 56.5%.

Therefore, it can be concluded that the recombinant E. coli 4401(pSYL107) and E. coli 6576(pSYL107) may produce much PHB when they were cultured in a minimal medium containing whey.

The said recombinant E. coli 4401(pSYL107) and E. coli 6576(pSYL107) were named as Escherichia coli/pSYL107 GCSC 4401 and Escherichia coli/pSYL107 GCSC 6576, respectively, and deposited with the Korean Collection for Type Cultures(KCTC) of 52, Oun-Dong, Yusong-Ku, Taejon 305 333, Republic of Korea on July 15, 1997, under the accession numbers of KCTC 0350BP and KCTC 0351BP, respectively.

As clearly illustrated and explained as above, the present 15 invention provides a series of recombinant E. coli strains, DSM 499(pSYL105), DSM 499(pSYL107), DSM 6056(pSYL105),

DSM

6056(pSYL107), KCTC 2223(pSYL105), KCTC 2223(pSYL107), and 4401(pSYL107), 6576(pSYL107), 6197(pSYL107), 6572(pSYL107), 3121(pSYL107), 2507(pSYL107), which can produce PHB by employing lactose or whey as a carbon source. PHB productivity S" can be further increased by the addition of various kinds of complex nitrogen sources in a small amount.

Accordingly, in accordance with the invention, PHB, a promising candidate for biodegradable plastic, can be produced by employing the recombinant E. coli strains in an economical and efficient manner.

23 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A recombinant Escherichia coli selected from the group consisting of E.coli/pSYLl07, GCSC 4401 (KCTC 0350BP) and E.coli/pSYL107 GCSC6576 (KCTC 0351BP), wherein said E.coli produces poly-3-hydroxybutyrate (PHB) from lactose or whey.

2. A recombinant Escherichia coli transformed with a plasmid pSYL105 comprising 3 -lactamase gene, PHB synthesis gene and parB region of plasmid R1 or pSYL107 prepared by inserting a cell division related protein gene (ftsZ) into the plasmid pSYL105.

3. A process for preparing PHB, which comprises the steps of culturing a recombinant E.coli according to claim 1 or claim 2 in a minimal medium containing lactose or whey and recovering PHB from the culture.

4. A process according to claim 3, wherein the minimal medium comprises 13.5g/L of KH 2

PO

4 4g/L of(NH 4 2 HP0 4 1.4g/L of MgSO 4 .7H 2 0, 1.7g/L of citric acid, 0.1g/L of FeSO 4 .7H 2 0, 0.02g/L of CaCl 2 .2H 2 0, 0.022g/L of ZnSO 4 .7H 2 0, 0.005g/L of MnSO 4 .4H 2 0, 0.Olg/L of CuSO 4 .5H 2 0, 0.001g/L of

(NH

6 4 M0 7 0 24 .4H 2 0, 0.002g/L of Na 2

B

4 07.10H 2 0 and 0.Olg/L of S: 25 thiamine.

S: 5. A process according to claim 4, wherein the minimal medium further comprises 0.1 to 10g/L of complex nitrogen source.

6. A process according to claim 5, wherein the complex nitrogen source is selected from a group consisting of tryptone, yeast extract, peptone, casamino acids, cotton H:\Luisa\Keep\specis\48351-97.doc 13/05/99

Claims (3)

1. 7. A process according to any one of claims 3 to 6, wherein the lactose or whey is added to the minimal medium at concentration of 10 to
2. 8. A recombinant Escherichia coli according to claim 1, substantially as hereinbefore described with reference S 10 to any one of the examples. 0*
3. 9. A process according to claim 3, substantially as hereinbefore described with reference to any one of the examples. Dated this 15th day of March 1999 KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY By their Patent Attorneys GRIFFITH HACK 20 Fellows Institute of Patent and Ta M Trade Mark Attorneys of Australia 0 00 *c H:\Bkrot\Xeep\speci\48351-9.doc 15/03/99 Abstract The present invention provides a recombinant Escherichia coli transformed with a recombinant plasmid containing a PHB synthesis gene of Alcaligenes eutrophus, which produces effectively PHB from lactose or whey, and a process for preparing PHB which comprises a step of culturing the recombinant E. coli in a medium containing lactose or whey. The recombinant E. coil is prepared by introducing a plasmid comprising PHB synthesis gene into E. coli which can utilize lactose or whey as a carbon source. In accordance with the present invention, PHB, a promising candidate for biodegradable plastic, can be produced by employing the recombinant E. coli 15 strain in an economical and efficient manner. ee a. a a.* o