CN107475318A - For promoting culture medium, cultural method and the algae strain of cytoalgae secretion amino acid - Google Patents
For promoting culture medium, cultural method and the algae strain of cytoalgae secretion amino acid Download PDFInfo
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- CN107475318A CN107475318A CN201710651573.4A CN201710651573A CN107475318A CN 107475318 A CN107475318 A CN 107475318A CN 201710651573 A CN201710651573 A CN 201710651573A CN 107475318 A CN107475318 A CN 107475318A
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- Prior art keywords
- amino acid
- cytoalgae
- culture medium
- pcc
- cytoalgae pcc
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Abstract
The present invention relates to a kind of culture medium for being used to promote cytoalgae PCC 6803 to secrete amino acid, the culture medium is that with the addition of 3.5 21g L‑1The basal medium of glucose, wherein NaNO3Concentration is in 6 12g L‑1Scope；The cultural method for promoting cytoalgae PCC 6803 to secrete amino acid is further related to, it makes it secrete the condition of amino acid including the use of above-mentioned medium culture cytoalgae PCC 6803；Further relate to secrete algae strains of the cytoalgae PCC 6803 through genetic modification of amino acid.Biological denitrification can be carried out using cytoalgae PCC 6803 using culture medium, cultural method and the algae strain of the present invention, while inorganic nitrogen is converted into organic nitrogen production amino acid.Under the conditions of more excellent, using the cytoalgae PCC 6803 free amino acid daily output that the present invention cultivates up to 52.94 ± 1.50mg L‑1d‑1, sodium nitrate daily consumption reaches 0.51 ± 0.016g L‑1d‑1。
Description
Technical field
The present invention relates to microalgae application field, more specifically it relates to which a kind of be used to promote DNC wireless to secrete amino
The culture medium and cultural method of acid, further relate to secrete algae strains of the cytoalgae PCC 6803 through genetic modification of amino acid.
Background technology
Blue-green algae is that one kind can produce high value added product " cell factory ", genomics, transcription group and metabolism
Group method is had been widely used in blue-green algae genetic modification correlative study.
In today that fossil fuel largely uses, atmosphere polluting problem attention.The gas that combustion of fossil fuel discharges
Nitrogen oxides (NO is included in bodyx), oxysulfide (SOx) etc. pernicious gas.Nitric oxide (NO) is NO in industrial tail gasxMaster
Composition is wanted, it can slowly be oxidized to NO by oxygen in atmosphere2, acid rain, haze are further resulted in, is also damaged the ozone layer, is added
Fast global warming.Therefore, it is necessary to cut down nitrogen oxides from the flue gas of fossil fuel.Traditional denitration means are mainly
, can also be because of ammonia excess generation secondary pollution without any useful products of output by ammonia and reaction of nitrogen oxides generation nitrogen.
Studied and the nitrogen oxides in industrial waste gas be fixed as nitric acid, be further converted to nitrate (cutting edge of a knife or a sword etc., one
Nitrogen oxides (NOx) resource type treating method in kind nitric acid industry tail gas, the patent No. 201310507361；Chang Yanlong etc., one
Oxidative absorption method of the kind by high concentration nitrogen tetraoxide exhaust gas conversion for potash fertilizer, the A of Patent publication No CN 103920385).This
Sample, the tail gas as caused by fossil fuel or chemical reaction, wherein the nitrogen oxides contained can derive as nitrate, it is used as blue-green algae life
Long nitrogen source, depollution is on the one hand removed, on the other hand can be with harvesting biomass and high added value metabolite.Amino acid is the mankind
With the nutrients needed for animal, if blue-green algae can absorb nitrate anion, amino acid is translated into, then its added value is undoubtedly
It can greatly improve.
However, the research that blue-green algae is specifically used to produce amino acid is actually rare, most of researchs concentrate on blue-green algae amino acid generation
Thank the research of path and the research of blue-green algae C, N metabolic regulation.
Hall etc. is from anabena (Anabaena sp.) 29151, Synechococcus (Synechococcus sp.) 602, Synechococcus
AN Tx20 and the screening of cytoalgae (Synechocystis sp.) 29108, which are separated to, can excessively synthesize and secrete tryptophan, phenylpropyl alcohol
Propylhomoserin, tyrosine, methionine and arginic mutant strain (Hall et al., 1980.Approach to recognition
of regulatory mutants of cyanobacteria.Journal of Bacteriology 143:981-988)。
Wild type cytoalgae 29108 is extracellular to be nearly no detectable phenylalanine, and tyrosine content is also less than 1 μM of (0.16mg L-1), but
Two metabolic regulation mutant strains 4FP30 and 4FP31 of its anti-4- fluorophenylalanine extracellular tryptophan, tyrosine and phenylpropyl alcohol ammonia
Acid content significantly raises, and in the two mutant strains, the extracellular content of tyrosine respectively reaches 13 μM of (2.12mg L-1) and
8μM(1.3mg L-1)。
Wolters etc. is using chemical mutagen ethylmethane sulfonate (ethyl methanesulfonate, EMS) to wild
Type Synechococcus PCC 7942 carries out mutagenesis, screens the algae strain of anti-D-Arg, therefrom screen two plants of main production arginine and
Mutant strain APM31 and APM40 (Wolters et al., the 1991.Deregulation of arginine of citrulling
biosynthesis in Synechococcus sp.PCC 7942.Applied Microbiology and
Biotechnology 35:56-59).This report claims, and the extracellular arginine contents of APM31 and extracellular amino acid content are respectively
99mg L-1With 320mg L-1, the extracellular arginine contents of APM40 and extracellular amino acid content are respectively 265mg L-1And 428mg
L-1.For further studies have shown that in the two algae strains, the content of extracellular citrulling is respectively 154mg L-1With 124mg L-1。
After deducting uncommon citrulling, the total content of APM31 and the extracellular common amino acids of APM40 is actually respectively 166mg L-1
With 304mg L-1.Divided by the yield of total cultivated days 14d, APM31 and APM40 extracellular common amino acid is respectively 11.86mg
L-1d-1With 21.71mg L-1d-1。
Cytoalgae (Synechocystis sp.) PCC 6803, the Chroococcales belonged in Cyanophyta, Chroococcaceae, collection
Born of the same parents' Trentepohlia, is separated to by R.Kunisawa.PCC is Pasteur DSMZ (the Paster Cuture of France
Collection abbreviation).It has natural genetic transformation system, is the mould of blue-green algae and photosynthesis molecular biology research
One of formula biology.There has been no the report on producing amino acid with cytoalgae PCC 6803 at present.
The content of the invention
Inventor in process of scientific research, it was unexpectedly found that, when to the culture medium for cultivating cytoalgae PCC 6803
After being improved, frustule can be made to extracellular a large amount of secretion amino acid；And it also found, to its arginine catabolism
After two genes in approach are knocked out, arginine and total amino acid even more greatly improve to extracellular secretion.
Found based on more than, the invention provides applications of the cytoalgae PCC 6803 in biosynthesis amino acid.
Present invention also offers a kind of culture medium for promoting cytoalgae PCC 6803 to secrete amino acid, the culture medium is to add
3.5-21g L are added-1The basal medium of glucose.Basal medium described in the present invention refers to can be to cytoalgae PCC
6803 provide the culture medium of necessary nutrient matter, include and are applied to culture cytoalgae various known to blue-green algae culture field
PCC 6803 all culture mediums.The BG11 talked about in embodiment is only for example son and used, in order to which experiment can
Cytoalgae PCC 6803 is promoted to secrete the concentration of glucose of amino acid.
By the glucose that debita spissitudo is added in basal medium so that cytoalgae PCC 6803 is largely to extracellular point
Amino acid is secreted, either the wild types of culture cytoalgae PCC 6803 are still cultivated and participate in amino acid metabolism, transhipment has correlation gene
Mutant strain is such.Therefore, the culture medium can be used for producing amino acid.
In one embodiment, 6-12g L are also included in the culture medium-1NaNO3。
In a preferred embodiment, 12g L are included in the culture medium-1NaNO3。
Why NaNO is used3As nitrogen source, it is because usually containing nitrogen oxides in industrial flue gas, nitre can be fixed as
Acid group.In the present invention, not only grown, also a large amount of synthesis, divided using the nitrate anion in culture medium, cytoalgae PCC 6803
Amino acid is secreted, thus also a large amount of secretion amino acid while denitration is carried out.These extracellular amino acid can be separated as people or
The nutrients of poultry, improve the added value of microalgae denitration.
In a specific embodiment, the basal medium is BG11.
Present invention also offers a kind of cultural method for promoting cytoalgae PCC 6803 to secrete amino acid, including the use of above-mentioned
Culture medium raises together the step of culture cytoalgae PCC 6803 makes it secrete amino acid under light illumination.Mixed breeding is to instigate cytoalgae etc. one
Algae both carried out autophyting growth further through existing organic carbon source in intake culture medium in incubation by photosynthesis slightly,
Such as glucose etc., carry out heterotrophic growth training method.
In one embodiment, the cytoalgae PCC 6803 is wild type algae strain, or for participate in amino acid metabolism,
The algae strain that one or more genes of transhipment are knocked, the gene for participating in amino acid metabolism, transporting include：slr0782、
Sll1336, cphA and bgtB.
In one embodiment, slr0782 genes are knocked in the cytoalgae PCC 6803.
In a further preferred embodiment, in addition to slr0782, in the cytoalgae PCC 6803 sll1336, cphA and
One or more of bgtB is knocked.
In a preferred embodiment, slr0782 and sll1336 is knocked in the cytoalgae PCC 6803.
In a preferred embodiment, slr0782, sll1336 and cphA are knocked in the cytoalgae PCC 6803.
In a preferred embodiment, slr0782, sll1336, cphA and bgtB quilt in the cytoalgae PCC 6803
Knock out.
In one embodiment, the amino acid is any of following amino acid or multiple combinations：
Glycine, alanine, leucine, isoleucine, threonine, valine, serine, proline, aspartic acid, day
Winter acid amides, glutamic acid, glutamine, lysine, arginine, histidine, tyrosine, tryptophan, phenylalanine, methionine,
Cysteine/cystine.
Present invention also offers the algae strains of cytoalgae PCC 6803 of secretion amino acid, and it is participation amino acid metabolism, transhipment
The algae strain that is knocked of one or more genes, it is described participate in amino acid metabolism, the gene of transhipment includes：slr0782、
Sll1336, cphA and bgtB.
In a preferred embodiment, the slr0782 in the algae strain is knocked.
In a further preferred embodiment, the slr0782 in the algae strain and sll1336 are knocked.
In a further preferred embodiment, slr0782, sll1336 and cphA in the algae strain are knocked.
In a further preferred embodiment, slr0782, sll1336, cphA and bgtB in the algae strain are knocked.
Gene slr0782, sll1336, cphA, bgtB involved in the present invention is the data that blue-green algae field is commonly used
Storehouse cyanobase (http://genome.microbedb.jp/cyanobase/) in name to gene.
Brief description of the drawings
Fig. 1 is plasmid pHB5646 structure chart；
Fig. 2 is plasmid pHB5647 structure chart；
Fig. 3 is plasmid pHB5649 structure chart；
Fig. 4 is plasmid pHB5648 structure chart；
Fig. 5 is wild type and mutant strain in 30 DEG C, the μ E m of light intensity 30-2s-1, mixed breeding (BG11+1g L-1Glucose), stand
The comparison of upgrowth situation under condition of culture；
Fig. 6 is in L containing 12g-1NaNO3、1g L-1The arginine yield of wild type and mutant strain in the nutrient solution of glucose
Statistical chart, wherein the mutant strain for knocking out slr0782 genes is referred to as Δ H, knock out two mutant strains of slr0782, sll1336 gene
Referred to as Δ MH, three mutant strains for knocking out slr0782, sll1336, cphA gene are referred to as Δ CMH, knock out slr0782, sll1336,
CphA, bgtB four mutant strains are referred to as Δ BCMH (following figure is identical with this)；
Fig. 7 is in L containing 12g-1NaNO3、1g L-1The amino acid production of wild type and mutant strain in the nutrient solution of glucose
Statistical chart；
Fig. 8 is in L containing 12g-1NaNO3、7g L-1The amino acid production of wild type and mutant strain in the nutrient solution of glucose
Statistical chart；
Fig. 9 is in L containing 12g-1NaNO3、21g L-1The amino acid production of wild type and mutant strain in the nutrient solution of glucose
Statistical chart.
Figure 10 is in NaNO containing various concentrations3、7g L-1The amino acid of wild type and mutant strain produces in the nutrient solution of glucose
Measure statistical chart.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
1. algae strain and condition of culture
The condition of culture of cytoalgae PCC 6803 is 30 DEG C, 30 μ E m-2s-1Quiescent culture under the conditions of continuous light (early daily,
Late each shake is once).This assay medium is culture mediums of the BG11 after certain change, by NaNO3Concentration is adjusted to 1.5-
24g L-1, and add 1-21g L-1Glucose.By determining OD in incubation730To monitor its growth, treat that frustule reaches
To logarithmic phase (OD730≈ 0.8) when take it to do every experiment.The solid plate of algae strain passage adds 1.4% agar, 1g L-1
Glucose, 8mM TES (pH 8.2) and 0.3%Na2S2O3.When culture medium adds antibiotic, spectinomycin (Sp), to block that mould
Plain (Km), streptomysin (Sm) concentration are all 25mg L-1, erythromycin (Em) and chloramphenicol (Cm) are 10mg L-1, while use more
Amount of each antibiotic concentration more than halves use during kind antibiotic.
2. the structure of gene knockout mutant strain and gene overexpression mutant strain
In cyanobase (http://genome.microbedb.jp/cyanobase/) find cytoalgae PCC 6803
In 4 genes sequence：Slr0782 (arginine dehydrogenase gene), sll1336 (arginine deiminase gene), cphA are (blue
Algae granule synthase gene), bgtB (basic amino acid absorption and transport body gene).
The plasmid for knocking out this four genes is built first.According to the both sides primers pair of gene to be knocked out,
Enter performing PCR as template using the genomic DNAs of wild type cytoalgae PCC 6803 to expand to obtain aforementioned four genetic fragment, connect respectively
Plasmid pHB5641, pHB5642, pHB5643, pHB5644 are obtained after on to carrier T.Inserted at pHB5642 ApaI sites
About 1.2Kb Kmr(kalamycin resistance) genetic fragment obtains plasmid pHB5646 (Fig. 1), for knocking out slr0782 genes；
About 1Kb Em is inserted at pHB5641 EcoRV sitesr(Erythromycinresistant) genetic fragment obtains plasmid pHB5647 (Fig. 2), uses
To knock out sll1336 genes；About 2.2Kb Sm is inserted at pHB5643 HpaI sitesr(streptomycin resistance) genetic fragment is entered
Plasmid pHB5649 (Fig. 3) is obtained after row connection, for knocking out cphA genes；About 2Kb is inserted at pHB5644 BalI sites
Spr(Spectinomycin resistance) genetic fragment obtains plasmid pHB5648 (Fig. 4), for knocking out bgtB genes.
Above-mentioned plasmid is transferred in cytoalgae PCC 6803 successively, respectively obtains the mutant strain for knocking out slr0782 genes
Δ H, two mutant strain Δ MH of slr0782, sll1336 gene are knocked out, knock out slr0782, sll1336, cphA gene and obtain three
Mutant strain Δ CMH, basic amino acid absorption and transport body gene bgtB is finally knocked out in three mutant strains and obtains four mutant strain Δs
BCMH.The transformant that all orientations knock out, screening and detection through excessively taking turns, finally obtains the mutant strain being kept completely separate.
3. the growth curve of mutant strain
To above-mentioned 4 mutant strains in 30 DEG C, 30 μ E m-2s-1Lower BG11 culture mediums add glucose mixed breeding culture, depict
Growth curve, influence of the detection gene knockout to growth, as a result as shown in figure 5, the growth rate of each knockout mutant strain does not have not only
Have and slow down, also slightly improved than wild type on the contrary.
4. dissociate arginic detection in culture
Because the gene of insertion inactivation relates generally to arginine metabolism, inventor is first to wild type and 4 mutant strain inspections
The arginic situation of generation is surveyed.In preliminary experiment, algae strain is checked in NaNO3Content is 1.5g L-1、3g L-1、6g L-1、12g L-1、24g L-1BG11 in carry out mixture growth (1g L-1Glucose) situation, find 24g L-1NaNO3It can suppress
It grows, 12g L-1NaNO3And algae strain can be supported preferably to grow below.Because an object of the present invention is consumed by NOx
Convert the NO obtained3 -, we employ higher NaNO3Concentration, i.e. 12g L-1Carry out subsequent experimental.In L containing 12g-1NaNO3
And add 1g L-1The BG11 culture algae strains of glucose, the mixed breeding culture 10d under illumination condition.As a result as shown in fig. 6, at this
Under part, Δ MH arginine yield highest (0.48 ± 0.06mg L-1OD730 -1), it is predominantly located at intracellular；Δ CMH arginine production
Measure as 0.11 ± 0.05mg L-1OD730 -1, also it is predominantly located at intracellular；Δ BCMH algae strains arginine yield is 0.38 ± 0.01mg L-1OD730 -1, Major Secretory is to extracellular.Δ BCMH has knocked out one compared to Δ CMH more and has absorbed arginic transporter gene bgtB,
This shows that bgtB has played effect in arginic secretion process.Under normal circumstances, being secreted into extracellular arginine can pass through
Absorption and transport body weight, which is newly transported intracellular back and is metabolized for frustule, to be used, and when the transporter gene is knocked, transport leading to for absorption
Road is cut off, and extracellular arginine just can not transport intracellular back so that extracellular arginine increase.
5. concentration of glucose is on total free amino acid yield and secretes significantly affecting for feature
Inventor further have detected algae strain in 1g L-1Glucose, 12g L-1NaNO3Under the conditions of cultivate 10 days total free ammonias
Base acid yield, as a result as shown in fig. 7, being still two mutant strain Δ MH yield highest (8.28 ± 0.13mg L-1OD730 -1), and
And extracellular (5.38 ± 0.05mg L are secreted into mostly-1OD730 -1)。
In 7g L-1Glucose, 12g L-1NaNO3Under conditions of same culture 10 days, the total free amino acid production of each algae strain
Amount is as shown in Figure 8.Now, not only the amino acid production of mutant strain greatly improves, and the yield of wild type is also significantly increased.This
Outside, raising of the raising of these algae strain total amino acid yield from extracellular amino acid production.Wherein, Δ MH total free amine group
Acid yield reaches 79.72 ± 1.34mg L-1OD730 -1, extracellular amino acid production reaches 78.84 ± 1.52mg L-1OD730 -1, it is remote high
In 1g L-1Amino acid production during glucose, extracellular amino acid have a daily output of 52.94 ± 1.50mg L-1d-1, higher than Wolters
Et al. (1991) Synechococcus PCC 7942 mutant strain APM31 and APM40 (extracellular ammonia has been screened using the method for mutagenesis
The base acid daily output is respectively 11.86mg L-1d-1With 21.71mg L-1d-1).On this condition, Δ MH nitrate daily consumption reaches
To 0.51 ± 0.016g L-1d-1.Δ H algaes strain is repeating larger (the total free amino acid 80.49-107.6mg L of experiment large deviations- 1OD730 -1), but a mutant strain Δ H and two mutant strain Δ MH free amino acid yield are in all algae strains from the point of view of average value
Higher.Δ CMH, Δ BCMH amino acid yield are less than the above two, but also reach 57.9 ± 0.78mg L-1OD730 -1With
58.37±2.75mg L-1OD730 -1.Therefore, either preferable Δ H and Δ MH, or Δ CMH, Δ BCMH are even wild
Type (20.55 ± 4.41mg of total free amino acid L-1OD730 -1), potential production amino acid algae strain can be all used as, and for biology
Denitration.7g L-1Glucose, 12g L-1NaNO3Under conditions of cultivate 10 days after 21 kinds of ammonia in wild type and mutant cultures
The situation of base acid yield is as shown in table 1- tables 3.
Table 1 is in L containing 12g-1NaNO3、7g L-1The ammonia of wild type and mutant strain per OD value cells in the nutrient solution of glucose
Base acid yield
* in HPLC detections, both amino acid cannot distinguish between.
Table 2 is in L containing 12g-1NaNO3、7g L-1The born of the same parents of wild type and mutant strain per OD value cells in the nutrient solution of glucose
Outer amino acid production
Table 3 is in L containing 12g-1NaNO3、7g L-1The born of the same parents of wild type and mutant strain per OD value cells in the nutrient solution of glucose
Interior amino acid production
In 21g L-1Glucose, 12g L-1NaNO3Under conditions of cultivate 10 days, each algae strain total free amino acid yield as scheme
Shown in 9.Total free amino acid of Δ MH mutant strains and extracellular amino acid production ratio are in 7g L under the conditions of this-1Have under glucose condition
Declined, but still highest, respectively 41.26 ± 4.74 and 38.63 ± 4.54mg L-1OD730 -1, significantly larger than in 1g L-1Portugal
Yield under the conditions of grape sugar.Inventor has also been attempted in 3.5g L-1Glucose, 12g L-1NaNO3Under conditions of culture Δ MH dash forward
Mutant, its total amino acid yield can reach 31.33 ± 0.13mg L after 10 days-1OD730 -1, wherein extracellular amino acid production reaches
To 27.65 ± 1.48mg L-1OD730 -1.Therefore, glucose concentration range is in 3.5-21g L-1Shi Douneng significantly improves amino acid
Yield, but with 7g L-1Glucose is optimal.
6.NaNO3Influence of the concentration to Δ MH total free amino acid yield
Inventor is tested in 7g L by taking two higher mutant strain Δ MH of free amine group acid yield as an example-1Glucose condition
Lower different NaNO3Influence of the concentration to amino acid production, as a result as shown in Figure 10, it was demonstrated that 12g L-1Concentration is optimal, but in 6g L-1
When also have high yield.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (9)
1. applications of the cytoalgae PCC 6803 in biosynthesis amino acid.
2. a kind of culture medium for being used to promote cytoalgae PCC 6803 to secrete amino acid, it is characterised in that the culture medium is to add
3.5-21g L are added-1The basal medium of glucose.
3. culture medium according to claim 2, it is characterised in that 6-12g L are also included in the culture medium-1NaNO3。
4. a kind of cultural method for promoting cytoalgae PCC 6803 to secrete amino acid, it is characterised in that including the use of claim 2
Or the culture medium described in 3 raises together the step of culture cytoalgae PCC 6803 makes it secrete amino acid under light illumination.
5. cultural method according to claim 4, it is characterised in that the cytoalgae PCC 6803 is wild type algae strain,
Or the algae strain to participate in amino acid metabolism, one or more genes of transhipment are knocked, the participation amino acid metabolism, transhipment
Gene include：slr0782、sll1336、cphA、bgtB.
6. cultural method according to claim 5, it is characterised in that slr0782 genes in the cytoalgae PCC 6803
It is knocked.
7. cultural method according to claim 6, it is characterised in that sll1336, cphA in the cytoalgae PCC 6803
It is knocked with one or more of bgtB.
8. according to the cultural method any one of claim 4-7, it is characterised in that the amino acid is following amino acid
Any of or multiple combinations：Glycine, alanine, leucine, isoleucine, threonine, valine, serine, dried meat ammonia
Acid, aspartic acid, asparagine, glutamic acid, glutamine, lysine, arginine, histidine, tyrosine, tryptophan, phenylpropyl alcohol
Propylhomoserin, methionine, cysteine/cystine.
9. a kind of algae strains of cytoalgae PCC 6803 for secreting amino acid, it is characterised in that for participation amino acid metabolism, transport one
The algae strain that individual or multiple genes are knocked, the gene for participating in amino acid metabolism, transporting include：slr0782、sll1336、
CphA and bgtB.
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| CN103314101A (en) * | 2010-11-03 | 2013-09-18 | 加利福尼亚大学董事会 | Biofuel and chemical production by recombinant microorganisms via fermentation of proteinacious biomass |
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| CN103314101A (en) * | 2010-11-03 | 2013-09-18 | 加利福尼亚大学董事会 | Biofuel and chemical production by recombinant microorganisms via fermentation of proteinacious biomass |
| US20160060643A1 (en) * | 2014-09-03 | 2016-03-03 | Arizona Board Of Regents On Behalf Of Arizona State University | Premethylation of dna for high efficiency transformation of cyanobacteria |
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