Methods and genetically engineered micro-organisms for the combined production of PDO, BDO and PHP by fermentation

1. A method for combined production of 1,3-propanediol (PDO), 2,3-butanediol (BDO), and Polyhydroxypropionic acid (PHP) by fermentation, comprising culturing a genetically engineered micro-organism characterized in that: compared with corresponding wild-type starting micro-organism, the D-lactate dehydrogenase gene in the genetically engineered micro-organism is deleted or functionally inactivated, and the genetically engineered micro-organism comprises a heterogenous polynucleotide encoding the Coenzyme A-dependent Aldehyde dehydrogenase and a heterogenous polynucleotide encoding the Polyhydroxyalkanoate synthase, under conditions suitable for the genetically engineered micro-organism to grow and produce PDO, BDO and PHP, and isolating the produced PDO, BDO and PHP.

2. A method for combined production of 1,3-propanediol (PDO), 2,3-butanediol (BDO), and Polyhydroxypropionic acid (PHP) by fermentation of a genetically engineered bacterium, comprising the steps of:

(a) constructing a genetically engineered bacterial strain capable of producing PDO, BDO and PHP by deleting the D-lactate dehydrogenase gene from a wild-type PDO-producing bacterium and introducing polynucleotides encoding the Coenzyme A-dependent Aldehyde dehydrogenase and the Polyhydroxyalkanoate synthase, respectively, into said bacterium;

(b) fermenting the strain by means of fed-batch fermentation; and

(c) isolating PDO, BDO and PHP from the broth.

3. The method of claim 2 , wherein step (a) comprising:

(i) deleting D-lactate dehydrogenase gene, comprising:

isolating and purifying genomic DNA sample from a wild-type PDO-producing bacterium of Klebsiella, Citrobacter , or Enterobacter;

carrying out gene amplification using the purified genomic DNA sample as template and primers designed based on the gene sequence of D-lactate dehydrogenase;

purifying the amplified D-lactate dehydrogenase gene fragment and inserting it into a cloning vector;

selecting positive cloning vector and digesting, recovering and inserting the gene fragment into a suicidal vector carrying a resistance gene, and then transfecting competent cells of E. coli SM10 with the suicidal vector; and

conducting parents exchanging experiment between the E. coli cells containing the recombinant plasmid and wild-type host cells, selecting a strain in which the D-lactate dehydrogenase gene has been deleted, using the resistance gene of the suicidal vector as the selection marker; and

(ii) introducing polynucleotides encoding the Coenzyme A-dependent Aldehyde dehydrogenase and the Polyhydroxyalkanoate synthase, comprising:

cloning the gene of Coenzyme A-dependent Aldehyde dehydrogenase by using the genomic DNA sample from the wild-type PDO-producing bacterium of Klebsiella, Citrobacter , or Enterobacter as template, digesting and then inserting the gene into an expression vector to construct a first recombinant expression vector;

inserting the gene of Polyhydroxyalkanoate synthase derived from Ralstonia or Cupriavidus into the first recombinant expression vector to construct a second recombinant expression vector; and

transforming the second recombinant expression vector the competent cells of the genetically engineered bacterial strain constructed in step (i) in which the gene of D-lactate dehydrogenase has been deleted, identifying and isolating the positive clones as the desired strains.

4. The method of claim 2 , wherein step (b) comprises:

culturing the cells of the genetically engineered strain in a solid medium for 16-24 h,

inoculating the cells into a seed culture medium for culture at 30-37° C. under aerobic conditions,

inoculating the culture, at a ratio of 1%-5%, into a fermentation medium with glycerol as the substrate for fermentation at 30-37° C., and

conducting fed-batch fermentation for 40-60 h at a pH of 5.0-8.0 with glycerol and alkali solution mixed at a ratio of 1:0.05-1.0 as the feeding substrate.

5. The method of claim 2 , wherein step (c) comprises:

subjecting the broth to microfiltration and ultrafiltration, subjecting the broth filtrate to desalination via electrodialysis, and isolating PDO and BDO from the desalinated broth filtrate via concentration and rectification; and

extracting PHP from the cellular remnant separated by filtration.

6. The method of claim 2 , wherein the wild-type PDO-producing bacterium is from Klebsiella, Citrobacter, Enterobacter or Serratia.

7. The method of claim 2 , wherein step (b) is carried out under aerobic conditions.

8. The method of claim 4 , wherein the substrate used in the fermentation is glycerol, glycerol broth, crude glycerol by-product of biodiesel industry or crude glycerol by-product of soap industry.

9. The method of claim 3 , wherein the D-lactate dehydrogenase gene fragment is inserted into the cloning vector pMD18-T; wherein cloning the gene of Coenzyme A-dependent Aldehyde dehydrogenase by using the genomic DNA sample from the wild-type PDO-producing bacterium of Klebsiella, Citrobacter , or Enterobacter as template, digesting and then inserting the gene into an expression vector pDK6 to construct a first recombinant expression vector PAD_pDK6; wherein inserting the gene of Polyhydroxyalkanoate synthase derived from Ralstonia or Cupriavidus into the first recombinant expression vector PAD_pDK6, to construct a second recombinant expression vector PAD PhaC_pDK6; and wherein the second recombinant expression vector PAD_PhaC_pDK6 is transformed into the competent cells of the genetically engineered bacterial strain constructed in step (i).