# Patent

## Title
Method to produce PHBV by recombinant Escherichia coli

## Abstract

The invention discloses a method for production of polyhydroxybutyrate-co-polyhydroxyvalerate (PHBV) by recombinant Escherichia coli harboring plasmid containing both phaCAB and prpE. Different percentage of hydroxyvalerate can be obtained from the recombinant E. coli when cultivated in the medium containing different concentrations of propionic acid. In this patent, we provide a method that integrated all of the genes (i.e. phaCAB, vgb and prpE) required for PHBV production into a single plasmid. The plasmids were then transformed into an E. coli host. Results showed that PHBV can be produced by this recombinant E. coli, and the ration of HV to HB in the co-polymers can be regulated by addition of different concentrations of propionic acid in the medium. The percentage of HV in the co-polymers can be adjusted from about 3% up to more than 35%.

## Background
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to a method to produce poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) by the recombinant Escherichia coli, in particular, overexpression of phaCAB-vgb-prpE gene by the recombinant Escherichia coli to produce PHBV.
2. Description of the Prior Art
Polyhydroxyalkanoates (PHAs) are high molecular weight polymers that can be used as biopolymers, and are biocompatible, absorbable, and biodegradable. PHAs can also be used as biomedical materials, e.g. implants, films or suturing materials. The monomers of PHAs, HA, are linked by ester bonds. A methyl or ethyl group in its C3 position will result in the formation of polyhydroxybutyrate (PHB) or polyhydroxyvalerate (PHV), respectively, whereas polymerization both of PHB and PHV gives rise to poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) with the structural form




PHBV can be used as biodegradable plastics. Due to its extraordinary biocompatibility and thermoplasticity, at present, PHBV is broadly used to produce various types of PHBV films for medical or pharmaceutical use, drug capsules, bone filler, or surgical stitches.
It was known that Ralstonia eutropha can import large amounts of propionate into cells by using propionate permease (PrpP) and can synthesize propionyl CoA from propionate by using propionyl-CoA synthetase (PrpE); hence, the addition of propionate in the bacteria culture medium can induce the formation of 3-ketovaleryl-CoA by facilitating the reaction between propionyl-CoA and acetyl-CoA using preexisted Beta-ketothiolase B (BktB), which consequently resulted in hydroxyvalerate (HV) after the process of reduction catalyzing by the reductase, and said HV can react with hydroxybutyrate (HB) to form PHBV.
Various properties of PHBV including tensile strength, elastic modulus, and crystallinity will decrease accordingly with increased HV amount, while its elasticity and flexibility will notably improved. However, the amount of PHBV synthesized by bacteria in nature is usually very low (less than 5%). In the case of using wild strains in culture medium with propionate to produce PHBV, the growth of most microorganisms will be inhibited which consequently restricts the yield and the availability of the product. Previous studies have demonstrated co-transformed of E. coli with plasmids which one carries phaCAB gene (for PHB synthesis) and the other carries bktB, prpP or prpE gene (relevant genes involved in the metabolism of propionate); yet, culturing of the resulted recombinant E. coli requires two different antibiotics in the growth medium which results in the cost rise and the cost is a major concern in practical production. Furthermore, the numbers and ratios of the two plasmids in E. coli cannot be controlled, and the amount of HV produced by the recombinant E. coli was only 15 ̃20% (mol %) of total PHBV.
The transformed recombinant E. coli containing only one plasmid carries both phaCAB and prpE genes disclosed in present invention. Also, the amount of HV in total PHBV produced by the recombinant E. coli strain disclosed in the present invention is regulated by the concentration of propionate in the culture medium, and is around 35%. Thus, the method to produce PHBV by microorganisms disclosed in the present invention has clearly demonstrated the significant improvements.
SUMMARY OF THE INVENTION
The present invention features a method to produce PHBV by constructing a strain of recombinant Escherichia coli carrying both phaCAB and prpE genes.
In one aspect, the invention is to provide a method to produce PHBV containing different ratios of HV (5% ̃0.35%) by regulating the concentration of propionate in an Escherichia coli culture medium.
In the other aspect, the invention is to provide PHBV containing high percentage of HV.
The present invention discloses a method to produce PHBV by a recombinant Escherichia coli carrying both phaCAB and prpE genes comprising of the following steps:

    
    
        (1) Providing a DNA construct (i.e. a plasmid) carrying phaCAB-vgb-prpE gene;
        (2) providing a Escherichia coli strain;
        (3) transforming of the Escherichia coli with the DNA construct;
        (4) screening and culturing the recombinant Escherichia coli, and
        (5) harvesting the recombinant Escherichia coli and producing PHBV by further purification.

The cloned prpE gene is inserted into the plasmid carrying vgb-phaCAB gene, and forms a DNA construct carrying phaCAB-vgb-prpE gene, in which phaCAB encodes PHB synthase, prpE encodes propionyl-Co A synthetase, and vgb encodes Vitreoscilla Hemoglobin (VHb) which is helpful in production of PHB in E. coli. Next, the transformation of the Escherichia coli with the said DNA construct, in which the Escherichia coli strain is, but is not limited to, DH5α. After screening, the recombinant Escherichia coli is named CT-2E, and the MR medium containing propionate or sodium propionate was inoculated with CT-2E to produce PHBV.

    
    





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the electrophoresis results of PCR products of Salmonella enterica prpE.
(M: marker; S: Salmonella enterica)
FIG. 2 describes the schematic diagram of the construction steps of pBHB-2-prpE.
FIG. 3 is the electrophoresis results of PCR products of vgb-prpE of pBHB-2-prpE.
(M: marker; S: pBHB-2-prpE; -: pBHB-2)
FIG. 4 is the electrophoresis results of HindIII digestion of vgb-prpE PCR product.
(M: marker; S: pBHB-2-prpE)



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Example 1
Construction of pBHB-2-prpE Plasmids Carrying phaCAB and prpE
Cloning of prpE Gene
Specific primers for prpE gene were designed based on the nucleotide sequence of Salmonella enterica published by National Center for Biotechnology Information (NCBI), and contains HindIII restriction enzyme sites at both ends. Salmonella enterica chromosomal DNA was used as the template for PCR and prpE gene fragment and was obtained by using DNA extraction kit (VIOGENE) after further purification. The prpE PCR product is 1887 bps (FIG. 1), and following ligation with pGEM-T easy vector, the construct was transformed into competent cells. Plates containing X-gal, IPTG, and ampicillin were used for blue-white screening, and the white colonies were selected and further cultured for 12 hours. The plasmid DNA was then extracted, and the extracted plasmid DNA was digested with the restriction enzyme, HindIII, for confirmation.
2. Construction Steps of the Plasmid Carrying prpE and phaCAB Genes.
Excised prpE gene from pGEM-T Easy-prpE using HindIII, extraction of the DNA fragment which is around 2 kb from agarose gel electrophoresis, and digested pBHB-2 plasmid carrying phaCA gene with HindIII and extracted the 8.7 kb DNA fragment from agarose gel electrophoresis. Ligation of the two fragments from the above resulted in the plasmid pBHB-2-prpE (named pBHB-2E), and the detailed schematic diagram is shown in FIG. 2. Transformation of E. coli strain DH5α with pBHB-2E, and stored at −70° C. To confirm whether the prpE, and its upstream genes and transcription start sites were in cis direction, forward primer (vgb-F) for prpE upstream gene, vgb, and reverse primer (prpE-R) for prpE in pBHB-2E were used for PCR reaction. The presence of PCR product of vgb-prpE indicated all the above genes are in cis; otherwise, no PCR product would be found (FIG. 3). The obtained PCR product was further digested with HindIII which resulted in two fragments: 2.0 kb prpE and 0.5 kb vgb (FIG. 4).
Example 2
The Effects of Overexpressing prpE on PHBV Accumulation
To examine whether overexpression of prpE could increase the percentage of hydroxyvalerate (HV) in poly 3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) as expected, E. coli strain XL1-Blue was transformed with pBHB-2E, and renamed as CT-2E. Compared the control E. coli strain (CT-2) transformed with vector without prpE by using Gas Chromatography (GC) for product analysis to further investigate the effects of overexpressing prpE on HV productivity. Transformed E. coli strains were cultured in LB broth for 12 hours and then inoculated into the culture medium in the ratio of 1%. The bacteria liquid was collected and centrifuged 48 hours later, and was subjected to GC analysis after drying. The average of multiple experiment results were calculated using calibration curve and the results are shown in Table 1. According to the results of optical density at 600 nm (OD600) and cell dry weight (CDW) of CT-2E and CT-2, the overexpression of prpE inhibited the growth of CT-2E when compared to CT-2, and the OD600 value, CDW, and PHBV production of CT-2E decreased about 4.62%, 16.28%, and 15.70%, respectively. However, the percentage of HV in PHBV produced increased significantly, about 22.81% of each gram of PHBV produced is HV. The conversion rate of propionate to HV was 72.48%, which indicated that in spite of growth inhibition, overexpression of prpE in E. coli can dramatically increase the usage of propionate and can help to accumulate significant amount of HV.







TABLE 1







Comparison of growth conditions and PHBV


productivity between CT-2 and CT-2E











Strain
CT-2
CT-2E







plasmid
pBHB-2
pBHB-2-prpE



OD600
27.98
26.68



CDW(g/L)
6.8
5.7



PHA/CDW(w/w)
58.71%
43.01%



PHV/PHA(w/w)
4.62%
22.81%



Conversion rate (propionate to
23.91%
72.48%



PHV)










Example 3
The Effects of Propionate at Different Concentrations on PHBV Production
Further exploration, the effects of the added 0 ̃48 mM propionate on PHBV production. CT-2E, pre-cultured for 12 hours with an initial OD600 at 0.05, was sub-cultured in the MR growth medium containing 2% glucose and 1.5% yeast extract (initial pH is 6.5) at 30° C., 200 rpm. As shown in Table 2, the amount of HV increased accordingly with the concentration of propionate; yet, the amount of HV and CDW declined when the concentration of propionate was at 48 mM. The reason of the cell growth arrest might be caused by the high concentration of propionate which subsequently affected the synthesis of PHBV. Based on these results, the optimal concentration of propionate is 32 mM, and under this condition, CDW and the concentration of PHA are 11.25 and 5.15 g/L, respectively; and the amounts of HB and HV are 34.14% and 11.06%, respectively.







TABLE 2







The effects of propionate at different concentrations on cell dry weight and production of PHBV.















Propionate
PHBV
PHB
PHV
Cell dry
HV
HB
PHBV



Concentration
percentage
percentage
percentage
weight
Concentration
Concentration
Concentration
HV/PHBV


(mM)
(%)
(%)
(%)
(g/L)
(g/L)
(g/L)
(g/L)
(%)


















0
55.86
55.86
0.00
9.25
0.00
5.17
5.17
0.00


8
49.41
48.20
1.21
11.38
0.14
5.48
5.62
2.46


16
45.19
40.56
4.62
12.50
0.58
5.07
5.65
10.23


24
45.63
37.03
8.60
11.38
0.98
4.21
5.19
18.85


32
45.74
34.14
11.60
11.25
1.30
3.84
5.15
25.36


48
31.95
20.92
11.03
8.5
0.94
1.78
2.72
34.51









Example 4
The Effects of Different Concentrations of Sodium Propionate on Production of PHBV
Explore the effects of sodium propionate at different concentrations on recombinant strain, CT-2E. The concentrations of sodium propionate are between 0-48 mM, and according to Table 3, the amount of HV increased along with the concentrations of sodium propionate. On the other hand, cell growth arrest was observed while the concentration of sodium propionate was increased; moreover, the final cell dry weight was between 9.25 ̃10.38 g/L. According to the results, the amount of HV increased up to 5% when the concentration of sodium propionate reached 32 mM. Furthermore, PHBV production in the presence of propionate and sodium propionate was compared, and the results are shown in Table 3, addition of propionate can result in higher HV concentrations.







TABLE 3







The effects of sodium propionate at different concentrations on cell dry weight and PHA production















sodium propionate
PHBV
HB
HV
Cell dry
HV
HB
PHBV



Concentration
percentage
percentage
percentage
weight
Concentration
Concentration
Concentration
HV/PHBV


(mM)
(%)
(%)
(%)
(g/L)
(g/L)
(g/L)
(g/L)
(%)


















0
39.22
39.22
0.00
10
0.00
3.92
3.92
0.00


16
43.35
39.90
3.45
10.38
0.36
4.14
4.50
7.96


32
44.67
34.16
10.51
10
1.05
3.42
4.47
23.54


48
45.29
29.85
15.45
9.25
1.43
2.76
4.19
34.10

## Claims

1. A method for producing poly 3-hydroxybutyrate-co-3-hydroxyvalerate by construction of a recombinant Escherichia coli carrying phaCAB of Ralstonia eutropha, wherein phaCAB is polyhydroxyalkanoates biosynthesis operon and propionyl-CoA synthase (prpE) genes of Salmonella enterica comprising the steps of:
(a) Providing a DNA construct carrying phaCAB-vgb-prpE genes, wherein vgb is Vitreoscilla globin gene from Vitreoscilla stercoraria; 
(b) Providing an Escherichia coli strain;
(c) Transforming the DNA construct into the Escherichia coli strain;
(d) Screening and culturing the recombinant Escherichia coli; 
(e) Harvesting the recombinant Escherichia coli and producing poly 3-hydroxybutyrate-co-3-hydroxyvalerate by further purification;
the prpE DNA fragment is ligated into the plasmid carrying phaCAB gene in cis, wherein the phaCAB gene encodes polyhydroxyvalerate synthase having the nucleotide sequence of SEQ ID NO: 1, and forms a DNA construct carrying phaCAB-vgb-prpE genes, which is subsequently transformed into the Escherichia coli; following screening, the recombinant Escherichia coli is cultured in the medium for expression of poly 3-hydroxybutyrate-co-3-hydroxyvalerate.

2. The method of claim 1, wherein the Escherichia coli strain is DH5α.

3. The method of claim 1, wherein vgb gene in the phaCAB-vgb-prpE DNA construct encodes Vitreoscilla Hemoglobin.

4. The method of claim 1, wherein prpE in the phaCAB-vgb-prpE DNA construct encodes propionyl coenzyme A synthetase.

5. The method of claim 1, wherein phaCAB, vgb and prpE genes are all cloned into the same plasmid.

6. The method of claim 1 or claim 5, wherein the plasmid carrying phaCAB, vgb, and prpE genes also carries a ampicillin-resistance gene.

7. The method of claim 1, wherein the culture medium used is minimum requirements medium (MR medium) comprising different concentrations of propionate or sodium propionate.

8. The method of claim 7, wherein the MR medium is consisting of 2% glucose and 1.5% yeast extract.