WO2015083769A1 - Production method for polyhydroxyalkanoate using only photosynthesis - Google Patents

Abstract

As a result of introducing genes that encode acetoacetyl-CoA synthase in cyanobacteria, the present invention succeeds in producing PHA at a significantly high level of efficiency even when cultivation is performed in an inorganic salt culture solution that does not contain sugar and photosynthesis is performed using CO₂ within air as a carbon source.

Description

Production of polyhydroxyalkanoic acid by photosynthesis alone

The present invention relates to an algae into which acetoacetyl-CoA synthase has been introduced and use of the algae in the production of polyhydroxyalkanoic acid (PHA).

Cyanobacteria are one of the oldest photosynthetic organisms on the planet and have played an important role in the evolution of the oxygen atmosphere we breathe today (Non-patent Document 1). Even today, cyanobacteria play a central role in global carbon recycling, the nitrogen cycle, and most importantly, the maintenance of atmospheric composition (Non-Patent Documents 2 and 3). Cyanobacteria are considered to be ideal producers of various chemicals and biofuels because they use solar energy to fix carbon dioxide to biomass. In the natural environment, fluctuations in nitrogen concentration occur constantly, and microorganisms respond to nutrient starvation by accumulating various carbon and energy storage compounds (Non-patent Document 4). Research on these storage polymers, especially PHA, has attracted considerable interest in recent years in an attempt to address the waste disposal problem caused by petrochemical plastics (Non-Patent Document 5).

Currently, the main biological process utilized for industrial production of PHA is the fermentation of heterotrophic bacteria. Nevertheless, the economic utility of PHA as a general purpose polymer is limited by high production costs due to expensive carbon materials and other necessary materials during the fermentation process. Considerable efforts have been devoted to researching more cost-effective PHA production processes (Non-Patent Document 6). An interesting and promising approach is the use of photosynthetic cyanobacteria as a host for PHA production. Cyanobacteria as a “microbe factory” can directly fix carbon dioxide from the atmosphere to high molecular weight PHA by photosynthesis. In addition to being photoautotrophic, cyanobacteria require minimal nutrients for growth, thus reducing the cost of a growth medium that is complex with the carbon source (Non-Patent Document 7). Thus, the use of cyanobacteria provides a cost-effective and sustainable approach for the production of this environmentally friendly polymer.

The presence of PHA in cyanobacteria was first reported by Carr. In this report, based on acid hydrolysis of poly (3-hydroxybutyrate) (abbreviation: P (3HB)) to crotonic acid and subsequent UV spectrum measurement of the hydrolyzate, PHA was measured in Chloroglea fritschii. Analyzed (Non-Patent Document 8). Since then, many studies have been conducted on Aphanothece sp. (Non-patent document 9), Oscillatoria limosa (Non-patent document 10), and several species of the genus Spirulina (Non-patent documents 11 and 12). And the presence of PHA in several other cyanobacteria, including the thermophilic strain Synechococcus sp. MA19 (Non-Patent Document 13). To date, cyanobacteria have been characterized by the ability to produce PHA containing only 3-hydroxybutyrate (3HB) and / or 3-hydroxyvalerate (3HV) monomers (Non-Patent Documents 9, 10, and 14). . There are many reports on the production of PHA in cyanobacteria, but most of these studies explore the physiological or fermentation aspects of PHA accumulation in cyanobacteria.

The current situation is that the biochemical and molecular basis of PHA synthesis in cyanobacteria is not well understood.

The present invention has been made in view of such circumstances, and an object thereof is to produce polyhydroxyalkanoic acid with high efficiency and low cost by using algae.

Algae can grow without the need for other nutrient sources by photosynthesis using CO ₂ as a carbon source. Among them, cyanobacteria (Cinecocity strain PCC6803) uses genomic information (Kaneko T et al. (1996) DNA Res 3: 109-136) and ease of genetic manipulation by natural transformation ability (Williams JGK ( 1988) Method Enzymol 167: 766-778), suitable for various biotechnology production. Therefore, the present inventor has attempted metabolic alterations in cyanobacteria by increasing the flow of intermediates to PHA biosynthesis and introducing a highly active PHA synthase.

Specifically, in order to produce PHA in cyanobacteria, β-ketothiolase (phaA; derived from Capriavidas), acetoacetyl-CoA reductase (phaB; derived from Capriavidas), and PHA polymerization enzyme (phaC A gene encoding three enzymes (from the genus Chromobacteria). However, PHA production efficiency in the recombinant was low.

Then, next, instead of the β-ketothiolase gene, a gene encoding acetoacetyl-CoA synthase (nphT7; derived from Streptomyces) was introduced to try to modify the metabolic pathway of PHA production (FIG. 1). As a result, it is grown in culture inorganic salts that do not contain sugar, even if the CO ₂ in air was carried out photosynthesis the carbon source, and succeeded in synthesizing a PHA corresponding to 14% of the dry weight of the cyanobacteria . Furthermore, when acetic acid was added as a carbon source, the production efficiency of PHA could be increased to 41% of the dry weight of cyanobacteria (Fig. 2). This value is the world's highest value for PHA production by photosynthesis alone.

Surprisingly, in the strains that showed the highest PHA-producing ability by introducing the acetoacetyl-CoA synthase gene, the expression levels of native PHA biosynthesis-related genes (β-ketothiolase and acetoacetyl-CoA reductase) The expression level of the introduced PHA biosynthesis-related gene was also relatively low compared to the strain into which the β-ketothiolase gene was introduced (FIGS. 3 and 4). On the other hand, the expression of many genes involved in photosynthetic activity was up-regulated compared to these strains (FIG. 5). Therefore, the remarkable effect shown in PHA production is due not only to the forced induction of the flow to PHA production by the expression of the introduced acetoacetyl-CoA synthase gene, but also to an unexpected increase in photosynthetic activity. I think that.

The present invention relates to the algae into which the acetoacetyl-CoA synthase gene has been introduced and the use of the algae in the production of polyhydroxyalkanoic acid. More specifically, the present invention provides the following inventions.

[1] Algae introduced with acetoacetyl-CoA synthase.

[2] The algae according to [1], further introduced with acetoacetyl-CoA reductase and PHA polymerizing enzyme.

[3] The algae according to [1] or [2], wherein the acetoacetyl-CoA synthase is nphT7.

[4] Algae according to [1] or [2], wherein the acetoacetyl-CoA reductase is phaB.

[5] The algae according to [1] or [2], wherein the PHA polymerizing enzyme is phaC.

[6] The algae according to any one of [1] to [5], wherein the algae is cyanobacteria.

[7] A method for enhancing the ability to produce polyhydroxyalkanoic acid in algae, including introducing acetoacetyl-CoA synthase into algae.

[8] A method for producing polyhydroxyalkanoic acid, comprising culturing the algae according to any one of [1] to [6] under conditions capable of photosynthesis.

[9] The production method according to [8], wherein the culture is performed under application of a carbon source.

[10] The production method according to [9], wherein the carbon source is carbon dioxide.

[11] The production method according to [9], wherein the carbon source is acetic acid.

[12] The production method according to [8], wherein the culture is performed under air exchange restriction conditions.

Bioplastics are bio-derived plastics that are beginning to be used in various applications such as beverage containers, car interiors, and personal computers. In addition, a material having degradability (biodegradability) by microorganisms has been developed, and an environmental load reduction effect that is not found in plastics derived from petroleum is expected. However, its productivity is very low, such as several percent or less of the dry weight of the plant or microorganism used, and problems such as growth delay of the plant and microorganism have been reported. Furthermore, the production of bioplastics requires a large amount of sugar and special facilities for culturing microorganisms, and there are problems in terms of cost.

In the present invention, by using an algae into which an acetoacetyl-CoA synthase gene has been introduced, together with an unexpected increase in photosynthetic activity, 14% of the dry weight of cyanobacteria without a solution carbon source, and further as a carbon source By adding a small amount of acetic acid, the world's highest level of PHA production efficiency of 41% was achieved. According to the present invention, it has become possible to dramatically increase the production efficiency of PHA in algae and to greatly reduce the production cost.

It is a figure which shows the metabolic pathway of PHA production which this inventor developed. Synechocystis PCC6803 strain is a graph showing a comparison of accumulation of P (3HB) in _{(pTKP2031V, C Cs A Cn B} Cn, and C _Cs NphT7B _Cn). (A) those bubble _{2-3% CO 2, (B)} 0.4% (w / v) which was shaken supplemented with acetic acid, (C) air exchange limited conditions, 0.4% (w / v) It was replenished with acetic acid and shaken. It is a graph which shows the result of having quantified the expression level of the PHA biosynthesis gene in Synecocystis strain PCC6803. _{(A) pTKP2031V C Cs A Cn} B Cn for _(standard), and C _Cs NphT7B _Cn of (target) results (detection native _{gene), (B) C Cs A} Cn B C against _Cn (standard) _Cs NphT7B _Cn ( Target) results (detect transgene). It is a figure which shows control of the PHA synthesis related gene expression in recombinant Synechocystis. It is a figure which shows the intracellular change in recombinant Synechocystis. _{(a) C Cs A Cn B} Cn for _PTKP2031V, and C _Cs NphT7B _{Cn results, (b) C Cs A Cn} B Cn C Cs NphT7B Cn results for. The black broken line shows the designed route. Genes in which expression variation was observed are shown in italics. Down-regulated genes are indicated with a down arrow beside the gene name. All genes without a downward arrow are genes that are up-regulated. The relationship between the abbreviations in the figure and the corresponding formal names is as follows. AP, allophycocyanin; PC / PEC, phycocyanin / phycoerythrocyanin; Cytb ₆ / f, cytochrome b6 / f complex; PQ, plastoquinone FNR, ferredoxin-NADP (+) reductase; Pc, plastocyanin; PSI, photosystem I; PSII, photosystem II; Ndh, NADH dehydrogenase; Glc-6-P, glucose-6-phosphate; Fru-6-P, fructose-6-phosphate; Fru-1 , 6-bp, fructose-1,6-bisphosphate; Glycerate-1,3-P ₂ , 1,3-bisphosphoglycerate (1,3-biphosphoglycerate); 3- P-Glycerate, 3-phosphoglycerate; Ru-1,5-bisP, ribulose-1,5-biphosphate; PEP, phosphoenolpyruvate.

The present invention provides algae into which acetoacetyl-CoA synthase has been introduced. In the present invention, “algae” means an organism having photosynthetic ability among organisms living in the hydrosphere. Examples of the algae used in the present invention include cyanobacteria and marine algae. However, the growth rate is fast, the genetic recombination technique is established, the genome sequence is decoded, and the gene Cyanobacteria are preferable for the reason that expression vectors are maintained. Specific examples of cyanobacteria include the genus Synechocystis, the genus Microcystis (for example, Microcystis aeruginosa), the genus Arthrospira (for example, Arthrospira platensis), Cyanothece genus cyanobacteria, Alcaligenes genus cyanobacterium (eg, Alcaligenes eurtophus), Anabaena genus cyanobacterium, Synechococcus genus cyanobacterium, Thermosynechococcus (Thermosynechococcus) (E.g., Thermosynechococcus elongats), Gloeobacter genus cyanobacterium (e.g., Gloeobacter violaceus), Acaryochris (Acaryoch) loris) cyanobacterium (for example, Acaryochloris marina), Nostoc genus cyanobacterium (for example, Nostock punctiforme), Trichodesmium genus cyanobacterium, Prochloron Examples include the genus cyanobacteria and the genus Prochlorococcus genus cyanobacteria.

The “acetoacetyl CoA synthase” used in the present invention is an enzyme that catalyzes a reaction for producing acetoacetyl CoA by irreversible condensation of acetyl CoA and malonyl CoA. By introducing the enzyme into algae, the flow to PHA production can be forcibly induced (FIG. 1). The acetoacetyl CoA synthetase introduced into the algae is not particularly limited as long as it has the above enzyme activity, but nphT7 is preferable. The nucleotide sequence and amino acid sequence of typical nphT7 derived from the genus Streptomyces are shown in SEQ ID NOs: 1 and 2, respectively.

In the present invention, acetoacetyl CoA reductase and PHA polymerizing enzyme can be further introduced into algae. The “acetoacetyl CoA reductase” in the present invention is an enzyme that catalyzes a reaction for producing 3-hydroxybutyl CoA from acetoacetyl CoA. The acetoacetyl CoA reductase introduced into the algae is not particularly limited as long as it has the above enzyme activity, but phaB is preferable. The base sequence and amino acid sequence of typical phaB derived from the genus Capriavidas are shown in SEQ ID NOs: 3 and 4, respectively, and the base sequence and amino acid sequence of phaB from a typical Pseudomonas genus are shown in SEQ ID NOs: 5 and 6, respectively. Further, the “PHA polymerization enzyme” in the present invention is an enzyme that catalyzes a reaction for producing PHA from 3-hydroxybutyl CoA. The PHA polymerase introduced into the algae is not particularly limited as long as it has the above enzyme activity, but phaC is preferable. The base sequence and amino acid sequence of phaC derived from a typical genus Chromobacterium are shown in SEQ ID NOs: 7 and 8, respectively.

It should be noted that the amino acid sequence of the above-mentioned enzyme introduced into algae may be mutated in nature, or may be artificially mutated. As long as it has the above enzyme activity, an enzyme in which one or more amino acids (for example, within 30 amino acids, within 10 amino acids, within 5 amino acids, within 3 amino acids) are mutated (substitution, deletion, insertion, addition) is also used in the present invention can do.

In the present invention, the phrase “introduced” into the algae means that the enzyme is introduced into the algae as a gene and includes the case where the enzyme is introduced into the algae as a protein. The gene encoding the enzyme can be inserted into an appropriate vector and introduced into algae by a general method such as natural transformation, electroporation, or conjugation. As a promoter for expressing a gene, for example, a light-inducible psbAII promoter can be used. When introducing the enzyme into algae in the form of a protein, for example, a microinjection method or a method of introducing it in combination with a signal peptide can be used.

Thus, in the algae into which the above enzyme has been introduced, the production capacity of PHA is enhanced. Therefore, the present invention provides a method for enhancing the ability to produce PHA in algae, which comprises introducing the enzyme into the algae. In addition, PHA can be produced with high efficiency by culturing the algae with enhanced PHA production ability under conditions capable of photosynthesis. Therefore, the present invention also provides a method for producing PHA, which comprises culturing the algae into which the enzyme is introduced under conditions capable of photosynthesis.

The cultivation of the algae into which the enzyme has been introduced can be carried out in a medium of an inorganic salt containing no sugar, thereby reducing the cultivation cost. As the medium to be used, for example, BG-11 medium or the like can be used. When the algae are grown for a certain period of time and then transplanted and cultured in a medium with a limited nutrient source (for example, a BG-11 medium lacking sodium nitrate), the production efficiency of PHA can be further increased. As conditions for the pH of the medium, the culture temperature, and the illumination, general conditions such as pH 8.0, 30 ° C., and 100 μmol photons m ⁻² s ⁻¹ can be used. In culture, it is preferable to perform shaking or bubble with air.

In the present invention, PHA production efficiency can be dramatically increased by culturing under the application of a carbon source. Carbon dioxide or acetic acid can be used as the carbon source. When carbon dioxide is applied, the bubbled air may be enriched with carbon dioxide. In this case, the concentration of carbon dioxide is preferably 2 to 3% (v / v). Moreover, what is necessary is just to add acetic acid to a culture medium, when applying acetic acid. The concentration of acetic acid to be added is preferably 0.4% (w / v) or more.

In the present invention, the production efficiency of PHA can be remarkably increased by setting the air exchange restriction condition. The air exchange restriction condition can be created, for example, by closing the mouth of the incubator with a cotton plug and covering with an aluminum foil.

The recovery of PHA from the algae thus cultured can be performed, for example, by an extraction method using methanol or ethanol, or a method using a crushing device such as Beadsbeader.

It should be noted that whether or not the ability of PHA production in cultured algae was enhanced depends on the control (algae into which the enzyme has not been introduced, for example, a vector that does not contain the enzyme gene has been introduced). It is possible to evaluate whether or not the production amount of PHA is increased as compared with (algae). For example, as described in this example, the amount of PHA produced in algae can be quantified by subjecting the dried cells to chloroform extraction, decomposing the extract with methanol, and removing the organic phase containing hydroxyacylmethyl ester. It can be performed by subjecting to gas chromatography mass spectrometry.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[Materials and methods]
(1) Chemicals and reagents Unless otherwise stated, all chemicals were purchased from Nacalai Tesque (Tokyo, Japan) or Wako Pure Chemical (Tokyo, Japan). KOD plus highly accurate DNA polymerase was purchased from Toyobo (Tokyo, Japan). Restriction enzymes and DNA ligation kits were purchased from Takara (Shiga, Japan).

(2) Living organisms and culture conditions All Synechocystis strain PCC6803 (Williams JGK (1988) Method Enzymol 167: 766-778) is 20 mM HEPES-KOH (at 30 ° C. under continuous illumination of 100 μmol photons m ⁻² s ⁻¹ ). The cells were cultured in BG-11 medium (Rippka R et al. (1979) Journal of General Microbiology 111: 1-61) buffered with pH 8.0. Liquid cultures were shaken (100 rpm) or bubbled with air enriched with 2-3% (v / v) CO ₂ . Escherichia coli DH5α used for plasmid cloning was grown in LB medium at 37 degrees with shaking (180 rpm). Kanamycin (50 μg / mL) or ampicillin (100 μg / mL) was added for selection and maintenance of the plasmid. To promote PHA biosynthesis in cyanobacteria, a two-stage culture was performed. Cultures were first grown in BG-11 medium until late logarithmic growth phase, then harvested, washed and transferred to BG-11 medium lacking sodium nitrate. Phosphorus deficiency was performed by culturing cells in BG-11 without potassium phosphate. To study the effects of carbon supplementation on PHA accumulation, different carbons [0.2% (w / v) and 0.4% (w / v) fructose and / or acetic acid] were added. The conditions for restricting air exchange in culture were performed by closing the mouth of the incubator with a spigot and covering with an aluminum foil (Non-patent Document 7). The cyanobacterial culture was cultured for 7 days, 10 days, or 14 days under the above culture conditions, collected by centrifugation (8000 g, 10 minutes), and then freeze-dried.

(3) Plasmid construction and transformation of Synechocystis The construct for the transformation of Synechocystis is derived from pTKP2031V. pTKP2031V was designed for homologous recombination with the kanamycin resistance cassette into the genome between sites slr2030 and slr2031 (Satoh S et al. (2001) J Biol Chem 276: 4293-4297). Expression of all cyanobacteria constructs was under the control of the psbAII promoter. A gene cluster containing β-ketothiolase (phaA _Cn ) and acetoacetyl CoA reductase (phaB _Cn ) was transformed into C. nekatol H16 using primers phaAB _Cn (F; NdeI) and phaAB _Cn (R; HpaI) (Table 1). Amplified from chromosomal DNA.

The underline in the table indicates the restriction enzyme digestion site.

The obtained PCR product was digested with NdeI and HpaI and inserted into pTKP2031V digested with NdeI and HpaI to obtain pTKP2031V-phaAB _Cn . PHA synthase (phaC _Cs ) was prepared from chromosomal DNA of Chromobacterium sp. USM2 using primers phaC _Cs (F; SfuI) and phaC _Cs (R; AatI). This PCR fragment was digested with SfuI and AatI and subcloned into an appropriate restriction enzyme site of pTKP2031V-phaAB _Cn by ligation to obtain pTKP2031V-phaC _Cs A _Cn B _Cn . pTKP2031V-phaC _Cs nphT7phaB _Cn was constructed by replacing phaA _Cn with nphT7 in pTKP2031V-phaC _Cs A _Cn B _Cn . The nphT7 gene derived from Streptomyces sp. 190 was prepared using the template pHis_nphT7 (Okamura E et al. (2010) Proc Natl Acad, which was prepared in advance using primers nphT7 (F; SfuI) and nphT7 (R; AatI). Sci USA 107: 11265-11270). The transformation of Synechocystis was performed according to a previous report (Osanai T et al. (2011) J Biol Chem 286: 30962-30971). Briefly, 100-200 μL of logarithmic growth culture was mixed with the plasmid solution to a final concentration of 1-2.5 μg / mL. The mixture was spread on a nitrocellulose membrane filter on a BG-11 plate and incubated overnight at 30 ° C. under continuous white light (75-100 μmol photons / m ² s). The membrane filter was transferred onto a new BG-11 plate containing 50 μg / mL kanamycin. Kanamycin resistant clones were isolated and replated three times. The presence and isolation of heterologous genes in cyanobacteria genome was confirmed by PCR analysis and sequencing.

(4) Quantitative analysis of PHA About 25-30 mg of lyophilized cyanobacteria cells were washed with methanol and dried at 65 ° C. overnight. Subsequently, the dried cells were extracted with chloroform at 65 ° C. for 48 hours. The chloroform extract was subjected to methanol decomposition with a solution consisting of 85% (v / v) methanol and 15% (v / v) concentrated sulfuric acid at 100 ° C. for 140 minutes (Braunegg G et al. (1978) Eur J Appl Microbiol Biotechnol 6: 29-37). The organic phase containing hydroxyacylmethyl ester was subjected to gas chromatography mass spectrometry using an Agilent 7890A GC / 5975 MSD system equipped with an HP-5 column (Agilent, USA).

(5) RNA preparation Total RNA was extracted from cells using Trizol reagent (Invitrogen, USA) in combination with PureLink RNA Mini Kit (Invitrogen, USA) according to the manufacturer's protocol. A small amount of DNA remaining in the RNA sample was removed by digestion with DNase I (Takara, Japan). The quantity and quality of RNA samples were analyzed using a Bioanalyzer 2100 (Agilent, USA).

(6) Real-time PCR analysis For cDNA synthesis, 250 ng of RNA was used and QuantiTect Reverse Transcription Kit (Qiagen, USA) was used. For real-time PCR quantification, Thunderbird SYBR qPCR Mix (Toyobo, Japan) and gene-specific primers were used, and Mx3000P QPCR system (Agilent, USA) was used. Cycling conditions are as follows. 40 cycles of 10 minutes at 95 ° C, 15 seconds at 95 ° C, and 1 minute at 60 ° C. To ensure reaction specificity, a melting curve analysis (60 ° C-95 ° C) was performed after each amplification. Transcription levels were quantified based on determination of quantification cycle (Ct). The transcription level of the gene of interest was normalized to the level of the housekeeping gene (16S rRNA) used in this study. Synechocystis strain PCC6803 strain C _Cs NphT7B _Cn and C _Cs A _Cn B _Cn (target) expression level for pTKP2031V (reference material) and C _Cs A _Cn B _Cn (reference material) C _Cs NphT7B _Cn (target) In order to compare the expression level of the gene of interest, comparative quantification was performed.

(7) RNA-seq library preparation, Illumina sequencing, and data analysis For each sample, 2 μg of total RNA was subjected to ribosomal RNA removal using Ribo-Zero rRNA removal kit (Epicentre, USA). A cDNA library for RNA-seq was constructed from total RNA from which rRNA was removed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit (Illumina, USA) according to the manufacturer's instructions. Briefly, the preparation of a cDNA library includes the following steps. RNA fragmentation, cDNA synthesis, 3 ′ end adenylation, adapter ligation, and enrichment of cDNA templates. Library quantification was performed using a Bioanalyzer 2100 (Agilent, USA) and a 4-6 pM template was used for cluster generation. The library was sequenced on a Miseq (Illumina, USA) instrument with a 2x250 paired end protocol. The sequence data is provided to NCBI Gene Expression Omnibus (GEO) as accession number GSE50688. RNA-seq data analysis was performed using CLC Genomics Workbench 6 software (CLC bio, Denmark). Sequence reads were pre-processed to trim low quality reads and screen for reads shorter than 20 bp. The sorted sequence reads were mapped to the Synechocystis PCC 6803 genome (NC_000911), allowing up to two mismatches. Reference genome sequences and annotations were downloaded from NCBI (23 May 2013). Sequence reads mapped to non-coding RNA and reads that did not map to a unique position were excluded from further analysis. Transcript level is RPKM (Mortazavi A et al. (2008) Nat Meth 5), which normalizes the number of gene reads by the length of the gene and the total number of reads mapped in the sample. : 621-628). Statistical analysis was performed, and a gene with a FDR (False Discovery Rate) p-value correction of <0.05 was determined as a differentially controlled gene (Baggerly KA et al. (2003) Bioinformatics 19: 1477-1483).

[result]
(1) Enhanced PHA production in recombinant cyanobacteria In the well-studied Capriavidus necator biosynthetic pathway, P (3HB) synthesis occurs in a three-step reaction, and acetyl-CoA acetoidase by β-ketothiolase It begins with condensation to acetyl-CoA (Tsuge T (2002) Journal of Bioscience and Bioengineering 94: 579-584). It is postulated that the acetyl-CoA pool in cyanobacteria is insufficient to previously drive a thermodynamically unfavorable condensation reaction under photosynthesis conditions (Lan EI, Liao JC (2012) Proc Natl Acad Sci USA 109: 6018-6023). Instead of relying solely on the condensation mediated by native β-ketothiolase to generate acetoacetyl-CoA, Streptomyces sp. CL190-derived acetoacetyl-CoA synthase (nphT7 _Ss ) was converted to P (3HB ) Incorporated into the route design.

The nphT7 _Ss gene catalyzes the irreversible condensation of acetyl-CoA and malonyl-CoA to produce acetoacetyl-CoA and drives the reaction to PHA production. The generation of carbon dioxide from the condensation reaction efficiently pushes the reaction towards the production of acetoacetyl CoA (Okamura E et al. (2010) Proc Natl Acad Sci USA 107: 11265-11270). USM2 (phaC _Cs ) (Bhubalan K et al. (2011) Appl Environ Microbiol 77: 2926-2933), a highly active PHA synthase derived from Chromobacterium sp., Was co-expressed with nphT7 _Ss in Synechocystis The photosynthetic production of P (3HB) was improved. From the viewpoint of simultaneous effects of nutrient limitation, carbon supplementation, and air exchange limitation on PHA accumulation (Non-patent Document 7, Sharma L, Mallick N (2005) Bioresour Technol 96: 1304-1310), these culture conditions, Biosynthetic experiments in the presence of nitrogen or phosphorus deficiency, air exchange limitations, and / or carbon sources (CO ₂ , acetic acid, and / or fructose) were designed.

For the design of the PHA production pathway in Synechocystis, the vector plasmid pTKP2031V was used to introduce a heterologous gene via homologous recombination between the sites of slr2030 and slr2031 (Satoh S et al. (2001) J Biol Chem 276 : 4293-4297). Synechocystis was transformed with a plasmid carrying the phaC _Cs gene, nphT7 _Ss gene, and C. nekatol acetoacetyl CoA reductase (phaB _Cn ) gene under the control of the light-inducible psbAII promoter. A successful transformant strain, C _Cs NphT7B _Cn , was analyzed for PHA production under a two-stage culture system consisting of a continuous cell growth stage and a PHA accumulation stage. The C _Cs NphT7B _Cn strain was facilitated by direct photosynthesis of PHA from CO ₂ and reached a maximum 14 wt% P (3HB) capacity on day 7 of culture (FIG. 1A). In comparison, phaC _Cs, C. Nekatoru β- ketothiolase (phaA _Cn), and C _Cs A _Cn B _Cn in P under the same culture conditions (3HB) capacity to express phaB _Cn (7wt%) reduction observed in It was. Only the pTKP2031V strain in which the kanamycin resistance cassette was integrated in the genome showed the lowest P (3HB) productivity (5 wt%). However, prolonged culture up to day 14 had no significant effect on the ability of cyanobacteria to accumulate P (3HB) under photoautotrophic conditions. At higher cell densities, competition for carbon dioxide and light may limit P (3HB) accumulation.

In order to increase P (3HB) production, an external carbon source [0.4% (w / v) acetic acid] was fed to the cyanobacterial culture during the PHA accumulation phase. The C _Cs NphT7B _Cn strain recorded a maximum PHA volume of 29 wt% on the 10th day of culture (FIG. 1B). The increase in the P (3HB) pool by the addition of the carbon source supports the initial findings regarding the effects of external carbon source supplementation on PHA production (Non-Patent Document 7, Sharma L, Mallick N (2005) Bioresour Technol 96: 1304-1310). In the effect of air exchange restriction, a significant increase (up to 41 wt%) of P (3HB) was observed in the C _Cs NphT7B _Cn strain (FIG. 1C). These observations imply that P (3HB) accumulation capacity in Synechocystis is affected by the supply of carbon source and air exchange. Interestingly, it was found that an increase in CO ₂ supply (5%) to the light-independent culture of Synechocystis increased the PHA capacity in the C _Cs NphT7B _Cn strain to 16 wt% (Table 2). Simultaneous addition of acetic acid and fructose to nitrogen and phosphorus deficient cultures of pTKP2031V strain showed a decrease in P (3HB) accumulation compared to light-independent conditions (5% CO ₂ ). Under the same conditions, there was no significant change in the P (3HB) capacity in the C _Cs NphT7B _Cn strain and the C _Cs A _Cn B _Cn strain. The order of PHA-producing ability of recombinant Synechocystis on day 7 under the culture conditions tested in this study is C _Cs NphT7B _Cn > C _Cs A _Cn B _Cn > pTKP2031V.

(2) Expression levels of PHA synthesis-related genes Expression levels of Synechocystis native PHA biosynthesis genes composed of phaC _Ss , phaA _Ss and phaB _Ss were monitored by real-time PCR analysis (FIGS. 2A and 2B). Surprisingly, quantifying the expression levels of phaA _Ss and phaB _Ss in Synechocystis C _Cs NphT7B _Cn strain showed about 2-fold lower expression relative to the TKP2031V strain. However, in the investigated recombinant Synechocystis (pTKP2013V, C _Cs A _Cn B _Cn , and C _Cs NphT7B _Cn ), there was no significant difference in the expression level of the native phaC _Ss gene. The expression levels of phaC _Cs and phaB _Cn introduced into the genome on the same operon were at least 3 times lower in the C _Cs NphT7B _Cn strain than in the C _Cs A _Cn B _Cn strain. Despite the higher PHA accumulation level in C _Cs NphT7B _Cn, the expression levels of most PHA biosynthesis-related genes in this strain were relatively low compared to C _Cs A _Cn B _Cn and pTKP2013V.

(3) Analysis of Synechocystis Transcriptional Response under Photoautotrophic PHA Accumulation Conditions To obtain knowledge about PHA accumulation in cyanobacteria, transcripts of recombinant Synechocystis with different PHA-producing ability were analyzed. An RNA-seq library was prepared from cells cultured for 7 days in nitrogen-deficient BG-11 under photoautotrophic conditions. Sequencing was performed using an Illumina platform that yielded a total of 93 million reads (average of 15.5 million reads per sample) for 6 samples. Scatter plots between two biological replicates for each recombinant feline city sample showed a correlation coefficient between 0.96-0.98, indicating the reproducibility of the sequence data. The expression level of each gene was quantified as RPKM (reads per kilobase of exon model per million mapped reads).

RNA-seq data provides detailed information on genes that are regulated in response to photoautotrophic PHA accumulation conditions in recombinant Synechocystis strains pTKP2013V, C _Cs A _Cn B _Cn, and C _Cs NphT7B _Cn . In general, genes that showed higher expression in Synechocystis were primarily involved in photosynthesis, electron transport chains, protein metabolic processes, and nucleic acid metabolism. In particular, transcription levels of genes involved in the activities of photosystem I (psaB, psaA, psaF, and psaL) and photosystem II (psbA3, psbA2, psbX, psbY, psbU, psbK, and psbD2) were the most abundant. . The gene expression was compared in the recombinant strains (C _Cs A _Cn B _Cn and C _Cs NphT7B _Cn ) in which PHA production was more efficient than the reference strain (pTKP2013V). Genes up-regulated in C _Cs A _Cn B _Cn and C _Cs NphT7B _Cn strains significantly enhanced photosynthesis, transport, and intercellular communication. In contrast, down-regulated genes have been found to be largely involved in cofactors and vitamins, protein metabolic processes, and DNA binding. The photosystem I reaction center subunit XII gene (psaM) detected only in cyanobacteria was strongly up-regulated in both C _Cs A _Cn B _Cn and C _Cs NphT7B _Cn . PsaJ, another photosystem I reaction center subunit gene, was found to be up-regulated more than 10 times. Both of these subunits are required for the formation of functional photosystem I (Xu W et al. (2001) Biochim Biophys Acta 1507: 32-40). In addition to genes encoding photosystem I subunits, genes involved in photosystem II were also significantly upregulated. PsbX and PsbK, which are considered essential for the stability of Photosystem II, were induced more than 5 times in both C _Cs A _Cn B _Cn and C _Cs NphT7B _Cn . Upregulation of PetG and PetL, which is a cytochrome B6-f complex subunit and important for the stability or association of the complex, was also observed (Schwenkert S et al. (2007) Plant Physiol 144: 1924-1935). Two genes involved in porphyrin and chlorophyll metabolism, magnesium-protoporphyrin IX monomethyl ester cyclase (sll1874) and protoheme IX fanesyltransferase (sll1899), were up-regulated in both strains. Overall, genes encoding proteins involved in several aspects, such as photosynthetic activity, such as metabolism of photosystems I and II, cytochromes, and chlorophyll, are up-regulated in recombinant Synechocystis that actively synthesizes PHA. It was.

On the other hand, the transcription level of the gene encoding the protein involved in protein metabolism (transcription, translation, amino acid synthesis, etc.) was decreased in the recombinant Synechocystis strains C _Cs A _Cn B _Cn and C _Cs NphT7B _Cn . Genes encoding these proteins [DNA mismatch protein (MutL) methionine sulfoxide reductase B (sll1680), prohibitin (slr1106), exozyme S synthetic protein B (ExsB), 3-dehydroquinate dehydrator (AroQ) and hydrogenase (HypA)] This decrease in transcription levels may be related to decreased growth of Synechocystis under nitrogen deficient conditions. Cells respond to nutrient-restricted conditions by a decrease in metabolic activity that occurs concomitant with PHA accumulation. Genes involved in cofactor and vitamin metabolism [lipopeptide antibiotics iturin a biosynthetic protein (slr0495), cobalamin synthase (CobS), 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), cobalt precorin Decreased expression levels of 6x reductase (CobK), riboflavin biosynthetic protein (RibG), repolitol transferase (LipB), and o-succinylbenzoate synthase (sll0409)] were observed.

A comparison between gene expression in recombinant Synechocystis strains C _Cs NphT7B _Cn and C _Cs A _Cn B _Cn was performed to gain substantial insight into the overall response of cyanobacteria to adapt to excessive PHA accumulation It was. This analysis indicated that the C _Cs NphT7B _Cn strain utilizes a combination of induced stress response, photosynthesis, energy metabolism, and transport during the PHA accumulation phase. In particular, proteins involved in several aspects of photosynthetic activity, such as uroporfinogen decarboxylase (HemE), ferredoxin component (slr1205), protochlorophyllidoductase subunit (BchB), protohome IX fanesyltransferase (CtaB ), Genes encoding photosystem II reaction center protein N (PsbN), and iron stress chlorophyll II binding protein (IsiA) are up-regulated in C _Cs NphT7B _Cn strain compared to C _Cs A _Cn B _Cn strain It was.

The enhanced photosynthetic activity suggests that the carbon fixation capacity has been enhanced to accommodate increased diversion of carbon to the polymer. Polyhydroxyalkanoic acids are bacterial storage compounds that are synthesized in response to physiological stress conditions (Anderson AJ, Dawes EA (1990) Microbiol Rev 54: 450-472). In recent studies, stress-related genes in cyanobacteria, including cochaperonin (groES), holiday junction resolvase (ruvC), molecular chaperone (groEL), superoxide dismutase (sodB), and heat shock protein 90 (htpG), Slightly up-regulated. Although PHA accumulation is thought to confer survival and stress tolerance in a changing environment (Pham TH et al. (2004) Microbiology 150: 3405-3413), stress conditions are reactions that support PHA production. May be stimulating. In addition, the transcriptional level of the overall nitrogen regulator NtcA was detected at increased levels. NtcA is known to regulate the expression of many genes involved in nitrogen metabolism (Bradley RL, Reddy KJ (1997) J Bacteriol 179: 4407-4410), and the induction of the gene encoding this protein is It may be related to nitrogen-deficient culture conditions applied to increase PHA synthesis in Synechocystis. Conversely, down-regulation of genes involved in DNA binding, transport, translation, and DNA repair was observed in the C _Cs NphT7B _Cn strain.

As described above, according to the present invention, PHA, which is one of the bioplastics, can be produced with high efficiency and low cost. Therefore, the present invention can greatly contribute to the production of various chemicals and biofuels using PHA.

SEQ ID NOs: 9-26
<223> Artificially synthesized primer sequences

Claims (12)

1. Algae introduced with acetoacetyl-CoA synthase.
2. Furthermore, the algae of Claim 1 into which the acetoacetyl CoA reductase and PHA polymerizing enzyme were introduce | transduced.
3. The algae according to claim 1 or 2, wherein the acetoacetyl-CoA synthase is nphT7.
4. The algae according to claim 1 or 2, wherein the acetoacetyl-CoA reductase is phaB.
5. The algae according to claim 1 or 2, wherein the PHA polymerizing enzyme is phaC.
6. The algae according to any one of claims 1 to 5, wherein the algae is cyanobacteria.
7. A method for enhancing the ability to produce polyhydroxyalkanoic acid in algae, which comprises introducing acetoacetyl-CoA synthase into the algae.
8. A method for producing polyhydroxyalkanoic acid, comprising culturing the algae according to any one of claims 1 to 6 under conditions capable of photosynthesis.
9. The production method according to claim 8, wherein the culture is performed under application of a carbon source.
10. The production method according to claim 9, wherein the carbon source is carbon dioxide.
11. The production method according to claim 9, wherein the carbon source is acetic acid.
12. The production method according to claim 8, wherein the culture is performed under air exchange restriction conditions.

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