Process for producing 1,3-propanediol compound

Abstract

A process of producing an organic compound and/or an intermediary compound as defined herein by feeding carbon dioxide to a culture of a cyanobacterial cell and subjecting said culture to light, wherein said cell is capable of expressing a nucleic acid molecule, wherein the expression of said nucleic acid molecule confer on the cell the ability to convert a glycolytic intermediate into said organic compound and/or into said intermediary compound and wherein said nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient in said culture.

Claims

1. A process of producing 1,3-propanediol compound, comprising: feeding carbon dioxide to a culture of Cyanobacterial cells; and subjecting the culture to light, wherein the cell expresses a nucleic acid molecule and expression of the nucleic acid molecule confers an ability to convert a glycolytic intermediate into 1,3-propanediol, wherein said nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient in said culture, wherein the glycolytic intermediate is hydroxypropionaldehyde, wherein said nucleic acid molecule encodes an enzyme that converts glyceraldehyde-3-phosphate to 1,3-propanediol, wherein said enzyme is glycerol dehydratase, and wherein the glycerol dehydratase has an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 or is encoded by a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 32.

2. The process of claim 1, wherein said enzyme is not sensitive to oxygen inactivation.

3. The process of claim 1, wherein the regulatory system responds to a change in the concentration of the nutrient ammonium in said culture.

4. The process of claim 1, wherein the 1,3-propanediol compound or glycolytic intermediate is separated from the culture.

5. A Cyanobacterial cell that expresses a nucleic acid molecule, wherein expression of the nucleic acid molecule allows the Cyanobacterial cell to convert a glycolytic intermediate into 1,3-propanediol, wherein the nucleic acid molecule is under the control of a regulatory system which responds to a change in the concentration of a nutrient when culturing said Cyanobacterial cell, wherein the glycolytic intermediate is hydroxypropionaldehyde, wherein said nucleic acid molecule encodes an enzyme that converts glyceraldehyde-3-phosphate to 1,3-propanediol, wherein said enzyme is a glycerol dehydratase, and wherein the glycerol dehydratase has an amino acid sequence that is at least 95% identical to SEQ ID NO: 31 or is encoded by a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 32.

6. The Cyanobacterial cell of claim 5, wherein the regulatory system responds to a change in the concentration of the nutrient ammonium.

7. The process of claim 1, wherein the Cyanobacterial cell is obtained from a Synechocystis cell.

8. The process of claim 1, wherein the Cyanobacterial cell is obtained from a Synechocystis PCC 6083 cell.

9. The Cyanobacterial cell of claim 5, wherein the Cyanobacterial cell is obtained from a Synechocystis cell.

10. The Cyanobacterial cell of claim 5, wherein the Cyanobacterial cell is obtained from a Synechocystis PCC 6083 cell.

11. The process of claim 1, wherein the nucleic acid molecule comprised in the cell is integrated into its genome by homologous recombination.

12. The Cyanobacterial cell of claim 5, wherein the nucleic acid molecule comprised in the Cyanobacterial cell is integrated into its genome by homologous recombination.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: The light reaction reproduced from Berg, Tymoczko and Stryer: “Biochemistry” WH Freeman and Co, New York, 2006.

(2) FIG. 2: The Calvin Cycle reproduced from Berg, Tymoczko and Stryer: “Biochemistry” WH Freeman and Co, New York, 1 2006.

(3) FIG. 3: Construction of a recombinant strain: The cassette is introduced by homologous recombination and positioned downstream the SigE controlled promotor of Synechocystis PCC 6803. The black circle shows the suicide plasmid (e.g. pBluescript) that is not able to replicate in Synechocystis PCC 6803. The cassette(s) of interest (denoted as arrow “x” in the figure) will be engineered to be flanked by DNA sequences homologous to a non-coding DNA region (shown as a dotted bar in the figure) in the nrt operon. Via a double crossover event, shown in the figure as dashed crosses, the cassettes of interest are integrated into the Synechocystis genome. Alternatively, the construct can be inserted at a neutral docking site.

(4) FIG. 4: Ammonium controlled production via the NtcA transcriptional regulator. Conditions allowing growth repress production, ammonium depletion promotes production.

(5) FIG. 5: Alcohol resistance. After five days, 100 μl of each culture was diluted and transferred to solid medium prepared from the BG-11 medium. Solid cultures were grown in an incubator at 30° C. under continuous illumination (70 μEinstein/m.sup.2/s) using two TL tubes without any addition (control) or with butanol, respectively ethanol added at various concentrations. After a week, single colonies were observed and counted. The amount of colonies was compared to the control sample.

(6) FIG. 6: A preferred design of expression cassettes is given in FIG. 6.

EXAMPLES

Example 1: Biochemical Background of the Cyanobacteria of the Invention

(7) The energy, in the form of ATP, as well as the reductive power in the form of NADPH, that are both needed to drive the subsequent highly endergonic dark reactions of photosynthesis, are catalyzed by the two photosystems of oxygenic photosynthesis, PS-II and PS-I, arranged according to the well-known Z-scheme, plus the membrane-bound ATPase (FIG. 1).

(8) In phototrophic organisms like Cyanobacteria, CO.sub.2 is fixed in the so-called Calvin cycle. This is a cyclic series of reductive steps that result in the net conversion of CO.sub.2 into C3 compounds, such as glyceraldehyde-3-phosphate, phosphoglycerate and pyruvate (FIG. 2).

(9) This pathway is essentially endergonic and in Nature driven by sunlight. It consumes CO.sub.2 and water and yields C3 compounds (e.g. pyruvate) and oxygen:
CO.sub.2+H.sub.2O+Solar energy.fwdarw.C3 compounds+O.sub.2  (1)

(10) This reaction cannot proceed spontaneously: It is driven by the consumption of the ATP and NADPH, generated in the ‘light’ reactions of photosynthesis. Subsequently, the C3 compounds are used in Nature (i.e. in phototrophic organisms like plants and Cyanobacteria) as the building blocks to make new cells and/or plants. This requires additional amounts of reducing power (as NADPH) and energy conserved during the light capturing reactions (as ATP) and also allows the organisms to proliferate (grow and survive).

(11) Nature also sustains an entirely different mode of (microbial) life: Numerous bacterial and fungal species are able to conserve sufficient energy (as ATP) to proliferate by fermentation, in which they use so-called substrate level phosphorylation to generate their energy. This respiration-independent mode of energy conservation relies on metabolic pathways that result in redox neutral dissimilation of the energy source. The most abundant pathways have evolved with sugars (e.g. glucose) as energy source and therefore all have glycolysis in common:
Glucose.fwdarw.glyceraldehyde-P.fwdarw.pyruvate+reducing power  (2)

(12) Redox neutrality is maintained by the generalized reaction:
pyruvate+reducing power.fwdarw.fermentation products  (3)

(13) Thus, it will contain the functional biochemistry to reduce the above-mentioned intermediates to the end product and will have as its biocatalytic input and output the combination of (1) and (3), respectively:
CO.sub.2+H.sub.2O+Solar energy.fwdarw.organic product+O.sub.2

Example 2: Description of the Expression System Used

(14) Genetic Cassettes

(15) The identity of an organic product formed (and excreted into the environment) in the process of the invention depends on the species-specific gene cassettes (i.e. nucleic acid molecules represented by nucleotide sequences) that encode the respective biochemical pathway (see table 1). Preferred enzymes encoded by nucleic acid molecules are substantially oxygen insensitive.

(16) TABLE-US-00001 TABLE 1 Examples of preferred donor organisms for the nucleic acid molecules or genes to be introduced into a Cyanobacterium with the pathway they catalyze. For e.g. the production of ethanol and propanediol various alternative donor organisms can be suggested. donor genes pathway Sarcina ventriculi pyruvate decarboxylase, Pyruvate .fwdarw. acetaldehyde Lactobacillus brevis alcohol dehydrogenase Acetaldehyde .fwdarw. ethanol Clostridium thiolase pyruvate .fwdarw. acetoacetylCoA acetobutilicum hydroxybutyrylCoA acetoacetylCoA .fwdarw. butyrylCoA dehydrogenase butyrylCoA .fwdarw. butyraldehyde crotonase butyraldehyde .fwdarw. 2-butanol butyryl-CoA dehydrogenase Butanol dehydrogenase Pseudomonas syringiae ethylene forming enzyme 2-ketoglutarate .fwdarw. ethylene Lactococcus lactis lactate dehydrogenase pyruvate .fwdarw. D-lactate Lactococcus lactis acetolactate synthase pyruvate .fwdarw. 2-acetolactate Klebsiella pneumoniae acetolactate decarboxylase 2-acetolactate .fwdarw. acetoin diacetyl reductase diacetyl .fwdarw. acetoin acetoin reductase acetoin .fwdarw. 2,3 butanediol glycerol dehydratase 2,3 butanediol .fwdarw. 2-butanone 1,3 propanediol 2-butanone .fwdarw. 2-butanol dehydrogenase Clostridium thiolase acetylCoA .fwdarw.acetoacetylCoA acetobutilicum ac.acetylCoA transferase acetoacetylCoA .fwdarw. acetoacetate acetoacetate decarboxylase acetoacetate .fwdarw. acetone Clostridium thiolase acetylCoA .fwdarw. acetoacetylCoA acetobutilicum ac.acetylCoA transferase acetoacetylCoA .fwdarw. acetoacetate Klebsiella pneumoniae acetoacetate decarboxylase acetoacetate .fwdarw. acetone propanol dehydrogenase acetone .fwdarw. propanol Synechocystis PCC glycerol-3-P dehydrogenase GAP .fwdarw. glycerol-P 6083 glycerol-3-P Phosphatase glycerol-P .fwdarw. glycerol K. pneumoniae glycerol dehydratase glycerol .fwdarw. OHpropionaldehyde oxidoreductase OHprop.aldehyde .fwdarw. 1,3-propanediol

(17) Homologous Integration and Ammonium Controlled Expression

(18) The genes/cassettes, necessary for the different pathways and respective organic products in Synechocystis, are preferably introduced to Synechocystis via chromosomal integration. This will be achieved by homologous recombination which allows to precisely define the chromosomal site of insertion. Appropriate plasmids for this purpose known to be applicable in Synechocystis sp PCC 6830 are pBluescript (Stratagene, USA) or pGEM-T (Promega, USA). A strategy with respect to the construct is exemplified in FIG. 3 but alternative (neutral) docking sites for integration will be considered.

(19) We will make use of the fact that expression of a number of glycolytic genes of Synechocystis are under control of a group 2 sigma-factor, σ.sup.E. In turn, expression of the gene encoding this factor, SigE, is switched on by the transcriptional regulator NtcA.sup.1,3. This switch is, amongst other unidentified signals, dependent on the extracellular nitrogen availability via the intracellular α-oxoglutarate/glutamate ratio: under conditions of ammonium depletion of the medium to less than 1 mM.sup.2, NtcA binds to α-oxoglutarate and the resulting NtcA-α-oxoglutarate complex has a high binding affinity for and positive control on the SigE promotor. Thus, a gene cassette under SigE control will be expressed upon ammonium depletion. As a consequence, during ammonium excess conditions, the carbon flux will be directed towards biosynthesis whereas in the stationary phase this flux will be directed to production (see FIG. 4). For Synechococcus the external ammonium threshold for the switch to σ.sup.E synthesis was found to be submillimolar range.sup.2.

Example 3: Alcohol Resistance

(20) Synechocystis PCC 6803 strain was grown on BG-11 medium (Stanier R Y, et al. Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol. Rev. (1971) 35:171-205) in an orbital shaker at 30° C. under continuous illumination using two TL tubes, which provided average light intensity of approximately 70 μE.Math.m.sup.−2.Math.s.sup.−1. To quantify the influence of alcohols on the net growth rate, cells were grown without any addition (control) or with butanol, respectively ethanol added at various concentrations.

(21) After 5 days, 100 μl of each culture was appropriately diluted and transferred to solid medium prepared from BG-11 and supplemented with 0.3% sodium thiosulfate, 10 mM N-tris[hydroxymethyl]-2-aminoethanesulfonic acid (TES) pH 8.2, 5 mM glucose and 1.5% bactoagar. Solid cultures were grown in an incubator 30° C. under continuous illumination. After a week, single colonies were observed and counted. The amount of colonies was compared to the control sample.

(22) From the results shown below in FIG. 5, it is concluded that the net specific growth rate decreases linearly with increasing alcohol concentration and that the growth rate is reduced by 50% at concentrations of approximately 0.17 M butanol respectively 0.29 M ethanol. Therefore, it is to be expected that the two phases production process of the invention is much more efficient than a single phase production process.

(23) TABLE-US-00002 TABLE 3 list of all primers used HOMOLOGY REGION 1 Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI Reverse AAATCTCGAGACCAGGACATCCGACTTGC XhoI HOMOLOGY REGION 2 Forward CACGACTAGTGTGACCGGGTCATTTTTTTGCTATTTATTCC SpeI Reverse AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAG XbaI Product: Lactic acid Forward ldh CAATCTCGAGATGGCTGATAAACAACGTAAG XhoI Reverse ldh CAATGAATTCTTAGTTTTTAACTGCAGAAGCAAATTC EcoRI Product: Ethanol Forward pdc ATAACTCGAGGACAATAGGTGCTTTAATCAC XhoI Reverse pdc CGACGATATCAGGTGTAAAATACCATTTATTAAAATAG EcoRV Forward adh CATTGATATCATGTCTAACCGTTTGGATGG EcoRV Reverse adh CATACTGCAGCTATTGAGCAGTGTAGCCAC PstI Product: 1,3-Propanediol Forward gpd AAATCTCGAGTCAGTGGAGACAATAGTCG XhoI Reverse gpd AAATATCGATATGCGTAATTTCCCAGAAATC ClaI Forward dhak CATAAAGCTTATGAAATTCTATACTTCAACGACAG HindIII Reverse dhak AAATGATATCTTACCAGGCGAAAGCTC EcoRV Forward Gl dehydr AAATATCGATTTATTCAATGGTGTCAGGCTG ClaI Reverse Gl dehydr CCAAAAGCTTATGAAAAGATCAAAACGATTTG HindIII Forward oxidoreductase GGGTGATATCTTAAGGTAAAGTAAAGTCAACCCAC EcoRV Reverse oxidoreductase AAATGAATTCATGTTAAACGGCCTGAAAC EcoRI Lactic acid-II set of primers Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI HomologyI Reverse GTTGTTTATCAGCCATACCAGGACATCCGACTTG HomologyI Reverse for CTGCGTGCAATCCATCTTGTTCAATCATTTAGTTTTTAACTGCAGAAGCAAATTC ldh Reverse for GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG KAN Reverse for AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI HomologyII Ethanol-II set of primers Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI HomologyI Reverse GATTAAAGCACCTATTGTCACCAGGACATCCGACTTG HomologyII Reverse pdc CTACCTTACCATCCAAACGGTTAGACATAGGTGTAAAATACCATTTATTAAAATAG Reverse adh CAATCCATCTTGTTCAATCATCTATTGAGCAGTGTAGCCACCGTC Reverse KAN GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG Reverse AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI HomologyII 1,3-Propanediol Forward AAATGGTACCGAACTGAGATTAGCCCCGGAC KpnI HomologyI Reverse TATTGTCTCCACTGAACCAGGACATCCGACTTG HomologyI Reverse dhg CTGTCGTTGAAGTATAGAATTTCATATGCGTAATTTCCCAGAAATCCAAAATACG Reverse dha k GGTTCAGCCTGACACCATTGAATAATTACCAGGCGAAAGCTCCAGTTGGAGC Reverse GTGGTTGACTTTACTTTACCTTAAATGAAAAGATCAAAACGATTTGCAGTACTGG glycerol dehydrts Reverse CAATCCATCTTGTTCAATCATATGTTAAACGGCCTGAAACC oxidored Reverse KAN GCAAAAAAATGACCCGGTCACTCAGAAGAACTCGTCAAGAAGG Reverse AAATTCTAGATAACTGCGGTAGCACTAAAGCCGCTGCCTTAC XbaI HomologyII Ethylene Forward Efe TAAAGTCGACAAGGAGACTAGCATGACCAAC SalI Reverse Efe TAAAGAATTCTTAGGAGCCGGTGG EcoRI 2-Butanol (Clostridium) forward thl AAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG reverse thl TTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG forward 3bdh AAGGAGATTCCAATGAAAAAGGTATGTGTTATAG reverse 3bdh TTATTTTGAATAATCGTAGAAACCTTTTCCTG forward crt- AAGGAGATTCCAATGTCAAAAGAGATTTATGAATCAG etf reverse crt-etf CTACAATTTTTTTACCAAATTCAAAAACATTCC forward ald AAGGAGATTCCAATGGATTTTAATTTAACAAGAG reverse ald TTATCTAAAAATTTTCCTGAAATAACTAATTTTCTGAACTTC forward bdh AAGGAGATTCCAATGCTAAGTTTTGATTATTCAATAC reverse bdh TTAATATGATTTTTTAAATATCTCAAGAAGCATCCTCTG 2-Butanol (L. lactis and K. pneumoniae) Foreward L. AAGGAGACTACTATGTCTGAGAAACAATTTGGGGC lactis als Reverse L. TCAGTAAAATTCTTCTGGCAAT lactis als Foreward L. AAGGAGACTACTATGTCAGAAATCACACAACTTTTTCA lactis aldB Reverse L. TCATTCAGCTACATCAATATCTTTTTTCAAAGC lactis aldB Foreward L. AAGGAGACTACTATGTCTAAAGTTGCAGCAGTTACTGG lactis butA Reverse L. TTAATGAAATTGCATTCCACCATC lactis butA Foreward L. AAGGAGACTACTGTGGCTTGGTGTGGAATCTGT lactis butB Reverse L. TTATAGACCTTTTCCAGTTGGTG lactis butB Foreward K. AAGGAGACTACTATGAAAAGATCAAAACGATTTGCAG pneumoniae dhaB Reverse K. TCAGAATGCCTGGCGGAAAAT pneumoniae dhaB Foreward K. AAGGAGACTACTATGAGCTATCGTATGTTTGATTATCTGG pneumoniae dhaT Reverse K. TCAGAATGCCTGGCGGAAAAT pneumoniae dhaT Acetone Foreward thl AAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG Reverse thl TTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG Foreward AAGGAGGCGGCGATGAACTCTAAAATAATTAG ctfAB Reverse TTATGCAGGCTCCTTTACTATATAAT ctfAB Foreward adc AAGGAGGCGGCGATGTTAAAGGATGAAGTA Reverse adc CCCTTACTTAAGATAATCATATATAACTTCAGC Propanol Foreward thl AAGGAGATTCCAATGAGAGATGTAGTAATAGTAAG Reverse thl TTAGTCTCTTTCAACTACGAGAGCTGTTCCCTG Foreward AAGGAGGCGGCGATGAACTCTAAAATAATTAG ctfAB Reverse TTATGCAGGCTCCTTTACTATATAAT ctfAB Foreward adc AAGGAGGCGGCGATGTTAAAGGATGAAGTA Reverse adc CCCTTACTTAAGATAATCATATATAACTTCAGC Foreward K. AAGGAGAATTCCAATGCATACCTTTTCTCTGC pneumoniae aad Reverse K. TCATTGCAGGTTCTCCAGCAGTTGC pneumoniae aad

REFERENCES

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