ERYTHRITOL PRODUCTION IN CYANOBACTERIA

Abstract

The present invention relates to a process for producing erythritol and to a cyanobacterial cell for the production of erythritol.

Claims

1. A cyanobacterial cell capable of expressing, preferably expressing, at least one functional enzyme selected from the group of enzymes consisting of a phosphatase and a reductase; preferably of an erythrose-phosphatase, an erythritol-phosphatase, and an erythrose reductase; more preferably of an erythrose-4-phosphate reductase and an erythritol-4-phosphate phosphatase, or of an erythrose-4-phosphate phosphatase and an erythrose reductase.

2. A cyanobacterial cell according to claim 1, expressing at least an erythrose-4-phosphate reductase and an erythritol-4-phosphate phosphatase, or an erythrose-4-phosphate phosphatase and an erythrose reductase.

3. A cyanobacterial cell according to claim 1, wherein the at least one functional enzyme is a heterologous enzyme.

4. A cyanobacterial cell according to claim 1, wherein the at least one functional enzyme is selected from the group consisting of an erythrose-4-phosphate phosphatase from Thermo toga maritima, Escherichia coli or Synechocystis PCC6803 and an erythrose-4-phosphate reductase from Saccharomyces cerevisiae, Candida magnolia, Trichoderma reesei, Aspergillus niger or Penicillium chrysogenum.

5. A cyanobacterial cell according to claim 1, wherein the at least one functional enzyme comprises or consists of a polypeptide that has an amino acid sequence with at least 30% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

6. A cyanobacterial cell according to claim 1, wherein the at least one functional enzyme is encoded by a polynucleotide that has a nucleic acid sequence with at least 30% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 15.

7. A cyanobacterial cell according to claim 1, wherein the cyanobacterial cell is a Synechocystis, preferably a Synechocystis PCC 6803.

8. A cyanobacterial cell according to claim 1, wherein a polynucleotide encoding the at least one functional enzyme is under control of a regulatory system which responds to a change in the concentration of a nutrient when culturing said cyanobacterial cell.

9. A process for producing erythritol comprising culturing a cyanobacterial cell according to claim 1, under conditions conducive to the production of erythritol and, optionally, isolating and/or purifying the erythritol from the culture broth.

10. A process according to claim 9, wherein the culture conditions comprise feeding carbon dioxide to the culture and/or subjecting the culture to light.

Description

FIGURE LEGENDS

[0060] FIG. 1.

[0061] Catabolic pathways for formation of erythritol in a cyanobacterium including the enzymes involved.

[0062] FIG. 2.

[0063] Colony PCR to confirm the correct insertion of the plasmid into Synechocystis. Lane 1: DNA ladder; lane 2: negative control; lane 3: Synechocystis strain SAW030 with plasmid conferring erythritol production.

[0064] FIG. 3.

[0065] Growth and level of erythritol production in the supernatant of SAW030 and wild type Synechocystis.

[0066] FIG. 4.

[0067] Toxicity assay for erythritol.

[0068] FIG. 5.

[0069] HPLC data showing the level of erythritol production measured in the supernatant of different Synechocystis erythritol producing strains.

EXAMPLES

[0070] The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

[0071] Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2.sup.nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

Example 1

Cloning Strategy

[0072] We have introduced a specific two enzyme catabolic pathway into a cyanobacterial cell to produce erythritol.

[0073] Two catabolic pathways for the formation of erythritol from erythrose-4-phosphate have been reported in literature (FIG. 1). The pathway of erythritol formation has been best studied in yeast, in which erythrose-4-phosphate (E4P) is first dephosphorylated to d-erythrose, and then reduced to erythritol. In bacteria, erythrose-4-phosphate is described to be reduced to erythritol-4-phosphate first, but the enzymes involved are unknown (Veiga-da-Cunha M, Santos H, Van Schaftingen E: Pathway and regulation of erythritol formation in Leuconostoc oenos. J Bacteriol 1993, 175:3941-3948).

[0074] Phosphatase: of the group of Haloacid Dehalogenase-like phosphatases, with affinity for erythrose-4-phosphate or erythritol-4-phosphate. Such phosphatases usually have a quite broad substrate specificity, but for example YidA (derived from Escherichia coli) has a quite attractive Km for erythrose-4-phosphate.

[0075] Reductase: Closely related to the family of aldose reductases and can usually catalyze the reduction of several aldehydes. Should be able to reduce either erythrose into erythritol or erythrose-4-phosphate into erythritol-4-phosphate.

TABLE-US-00002 TABLE 2 Characteristics of phosphatases D-erythrose-4-P Vmax Km Kcat (umol/min/mg SEQ ID gene donor organism (mM) (s-1) protein) NO: ref TM1254 Thermotoga maritima 0.152 2.63 5, 6 Kunetsova MSB8 et al, 2005 YidA Escherichia coli 0.019 19 1, 2 Kunetsova et al, 2006 sll1524 Synechocystis PCC6803 3, 4

TABLE-US-00003 TABLE 3 Characteristics of reductases D-erythrose NADPH SEQ ID Km Kcat Kcat gene donor organism NO: (mM) (s-1) Km (s-1) ref ErCm Candida magnolia 9, 10 8.5 7.6 0.016 48 (Lee et al, JH110 2010) Gcy1p Saccharomyces 7, 8 3.4 Ookura et al, cerevisiae 2007 GLD1 Hypocrea jecorina 11, 12 0.016-0.134 530-36.5 Jovanovi et (Trichoderma al, 2013 reesei) ALR1 Aspergillus niger 13, 14 0.139 25 Jovanovi et al, 2013 Pc20g15580 Penicillium 15, 16 ? ? chrysogenum

Example 2

Biochemical Background of a Cyanobacterial Cell According to the Present Invention

[0076] The genes encoding the phosphatase TM 1254, derived from Thermotoga maritima MSB8 (Kuznetsova et al., 2005), and the erythrose reductase Gcylp, derived from Saccharomyces cerevisiae (Ookura and Kasumi, 2007) were codon-optimized for expression in Synechocystis and obtained through chemical synthesis. These genes were each cloned with a trc promoter into a RSF 1010-based conjugative plasmid pVZ. Introduction of the phosphatase-encoding gene, in combination with a gene encoding one of the erythrose reductases (via a conjugative plasmid) should allow the transconjugant Synechocystis strain to produce erythritol from erythrose-4-phosphate. These strains were tested by colony PCR to confirm the presence of the plasmid (FIG. 2). FIG. 2 depicts SAW030 in the third lane with a band of 2200 bp, representing the phosphatase and reductase, whereas the second lane shows wildtype Synechocystis as a negative control.

Example 3

Production of Erythritol by a Cyanobacterial Cell

[0077] Mutant cultures obtained in example 2 were selected for presence of the plasmid by growth on agar plates containing 20 g/ml of kanamycine. A selected mutant was named Synechocystis SAW030. This SAW030 mutant was inoculated in BG-11 medium supplemented with 10 mM TES-buffer-NaOH (pH=8.0) and 20 ug/ml kanamycine and grown to stationary phase within several days (OD of 1.5). An aliquot of the initial culture was used to inoculate 100 ml BG-11 supplemented with 10 mM TES-buffer-NaOH (pH=8.0) and with 10 g/ml kanamycine to an OD of 0.1. The culture was incubated at low light intensity (40 E), 30 C. and shaking at 120 rpm. After every few days, an 800 l sample was taken for measurement of optical density (A730) and determination of erythritol concentration in the supernatant. With the help of standard concentrations of erythritol, the concentration of erythritol in the culture was determined via an HPLC method (FIG. 3). In conclusion, erythritol production increases in time (at least up to 35 days) to a concentration of at least 550 uM (60 mg/L) in the extracellular medium.

Example 4

Resistance to Erythritol of Synechocystis PCC6803

[0078] Synechocystis PCC6803 was inoculated in 10 ml BG-11 supplemented with 10 mM TES-buffer-NaOH (pH=8.0) and with 10 g/ml kanamycine at an OD of 0.2. The culture was incubated at low light intensity (40 E), 30 C. and shaking at 120 rpm. It was clearly shown (FIG. 4) that up to a concentration of 10 gr/L erythritol, cultures are not affected with respect to growth-rate.

Example 5

Biochemical Background and Production of Erythritol of Several Additional Cyanobacterial Cells According to the Present Invention

[0079] The genes encoding phosphatases TM 1254 and YidA, and the erythrose reductases Gcy1p, GLD1, ALR1 and Pc20g15580 were codon-optimized for expression in Synechocystis and obtained by chemical synthesis. These genes were each cloned with a trc promoter into a vector containing homologous regions targeting for genome integration at the slr0168 gene. Introduction of the phosphatase-encoding gene, in combination with a gene encoding one of the erythrose reductases (via natural transformation and homologous recombination) allows the transformant Synechocystis strains to produce erythritol from erythrose-4-phosphate. Mutant cultures were selected for by growth on agar plates containing 20 g/ml of kanamycine. The resulting strains were tested by colony PCR to confirm the presence of the desired genes in the genome. From the strains obtained, strains comprising TM1254 and GLD1, YidA and GLD1, and TM1254 and Gcy1p were selected for further analysis.

[0080] These strains were inoculated in BG-11 medium supplemented with 25 mM CAPSO-buffer-NaOH (pH=9.0) and 20 ug/ml kanamycine and grown to stationary phase within several days (OD of 1.5). An aliquot of the initial culture was used to inoculate 100 ml BG-11 supplemented with 25 mM CAPSO-buffer-NaOH (pH=9.0) and 20 ug/ml kanamycine to an OD of 0.1. The culture was incubated at low light intensity (40 E), 30 C. and shaking at 120 rpm. After 23-30 days of culture, an 800 l sample was taken for measurement of optical density (A730) and for determination of erythritol concentration in the supernatant. Using standard concentrations of erythritol, the concentration of erythritol in the culture was determined using HPLC (FIG. 5). In conclusion, erythritol production was detected in the extracellular medium of each of the tested strains; strain TM1254/GLD1 produced 0.05 mM erythritol (retention 15.75 min.), strain YidA/GLD1 produced 0.09 mM erythritol (retention 15.75 min.), and strain TM1254/Gcylp produced 0.1 mM erythritol (retention 15.72 min.). These results are clearly in the same magnitude as strain SAW030 in example 3.

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