CN115725667A - Artificial dual-bacterium system for producing mcl-PHA (polyhydroxyalkanoate) by utilizing lignocellulose hydrolysate and application thereof - Google Patents
Artificial dual-bacterium system for producing mcl-PHA (polyhydroxyalkanoate) by utilizing lignocellulose hydrolysate and application thereof Download PDFInfo
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Abstract
The invention relates to an artificial double-bacterium system for producing mcl-PHA by utilizing lignocellulose hydrolysate and application thereof, wherein the artificial double-bacterium system consists of engineering bacteria P.putida KT delta ABZF and engineering bacteria E.coli delta 4D. The medium-long-chain polyhydroxyalkanoate is produced by utilizing lignocellulose hydrolysate, and the engineering bacteria E.coli delta.4D mainly utilize xylose in the lignocellulose hydrolysate as a substrate to secrete acetic acid and free fatty acid; the engineering bacterium P.putida KT delta ABZF accumulates biomass by using glucose in lignocellulose hydrolysate and synthesizes medium-long-chain polyhydroxyalkanoate (mcl-PHA) by using acetic acid and free fatty acid. The maximum yield of mcl-PHA obtained by the two bacteria through mixed sugar (glucose and xylose) is 1.64g/L, and the yield is 3.9 times of that obtained by pure culture of the single bacteria. The utilization of complex substrates is realized, and the substrate cost is further reduced.
Description
Technical Field
The invention relates to an artificial double-bacterium system for producing mcl-PHA (microbial alpha-PHA) by utilizing lignocellulose hydrolysate and application thereof, belonging to the field of mixed microbial community research in microbiology.
Background
Due to the energy waste and environmental pollution caused by petroleum-based plastics, there is increasing interest in finding biodegradable alternatives to petroleum-based plastics. Polyhydroxyalkanoates (PHAs) are a class of high molecular polyesters that accumulate in the cytoplasm from bacterial cells in the form of granules [1] Mainly produced from renewable resources and has biodegradability. PHAs can be classified into three types according to the number and chain length of their monomeric carbon atoms: the short-chain polyhydroxyalkanoate (scl-PHA) is composed of monomers with 3-5 carbon atoms, the medium-long-chain polyhydroxyalkanoate (mcl-PHA) is composed of monomers with 6-14 carbon atoms, and the long-chain polyhydroxyalkanoate (lcl-PHA) is self-called by monomers with 15 carbon atoms or more. Wherein, the mcl-PHA has more diversified structures and more flexible physical and mechanical properties [2] And has better biocompatibility and biodegradability. In a word, mcl-PHA can meet the requirements of wide engineering application and has wide application prospect [3-4] 。
However, the current commercial PHA products are mainly short-chain polyhydroxyalkanoates (scl-PHA), and the industrial production of mcl-PHA is currently negligible compared to the production and commercialization of scl-PHA or plastics. The current obstacles limiting the industrial production of mcl-PHA are mainly price cost and production capacity. For example, the cost of microbial production of mcl-PHA requires the provision of expensive precursor materials such as fatty acids, and it is difficult for a single microorganism to simultaneously utilize multiple substrates [5] Resulting in a reduction in productivity. In order to solve these two problems, it is important to construct a microbial cell factory that can utilize an inexpensive carbon source by using metabolic engineering methods. The lignocellulose hydrolysate contains various saccharides such as glucose, xylose, arabinose and the like, is a cheap renewable resource, and the mcl-PHA synthesized by the lignocellulose hydrolysate can be effectively utilizedThe production cost is reduced. Currently, lignocellulose has been applied to PHA synthesis, mainly by converting lignocellulose into fermentable sugars (mainly glucose and xylose) by various treatment methods and then producing PHA by microbial fermentation. Most of the current research is on the microbial production of scl-PHA, whereas the synthesis of mcl-PHA is rare and not high in yield. The reason is that the current research mainly focuses on single microorganism to produce mcl-PHA by utilizing fermentable sugar in lignocellulose, and the single microorganism has low metabolic capability and cannot efficiently utilize complex substrates. For example, davis [6] The mcl-PHA is synthesized from wild ryegrass hydrolysate treated by different methods by using several different pseudomonas by the people, the highest yield is 0.3g/L, and the requirement of industrial production is difficult to meet. In order to overcome the disadvantages of single-strain PHA production, in recent years, some studies have proved that the synthesis of artificial mixed strain system by using metabolic engineering and synthetic biology techniques to produce PHA is one of the solutions. For example, shalin et al [7] The improvement of the yield of the PHB is realized by constructing a co-culture system consisting of Bacillus firmus NII 0830 and Lactobacillus delbrueckii NII 0925. Anbrajan [8] And the like, the scl-PHA is produced by converting acetic acid and butyric acid into substrates by using a double-bacterium system consisting of aeromonas hydrophila and acinetobacter junii, and the yield is 2.64g/L. While the production of mcl-PHA is less studied and of lower yield, e.g.
Etc. of [9] The method utilizes an artificial double-bacterium system consisting of blue algae and pseudomonas to react CO 2 Converted into mcl-PHA at a yield of 0.156g/L.
Therefore, by applying methods and theories of metabolic engineering and synthetic biology, the artificial double-bacterium system can realize the task which can not be completed by a single microorganism or improve the metabolic function of a multi-cell system [10] And the aim of synthesizing a high value-added product (mcl-PHA) by using a low-cost substrate (lignocellulose) is fulfilled.
Reference documents:
[1]Anderson A J,Dawes E A.Occurrence,metabolism,metabolic role,and industrial uses of bacterial polyhydroxyalkanoates[J].Microbiological Reviews,1990,54(4):450-472.
[2]Gopi S,Kontopoulou M,Ramsay B A,et al.Manipulating the structure of mediumchain-length polyhydroxyalkanoate(MCL-PHA)to enhance thermal properties and crystallization kinetics[J].International Journal of Biological Macromolecules,2018,119:1248-1255.
[3]Wang Q,Luan Y,Cheng X,et al.Engineering of Escherichia coli for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)from glucose[J].Applied Microbiology and Biotechnology,2015,99(6):2593-2602.
[4]Wu F,Misra M,Mohanty A K.Challenges and new opportunities on barrier performance of biodegradable polymers for sustainable packaging[J].Progress in Polymer Science,2021,117.
[5]Morgan-Sagastume F,Hjort M,Cirne D,et al.Integrated production of polyhydroxyalkanoates(PHAs)with municipal wastewater and sludge treatment at pilot scale[J].Bioresource Technology,2015,181:78-89.
[6]Davis R,Kataria R,Cerrone F,et al.Conversion of grass biomass into fermentable sugars and its utilization for medium chain length polyhydroxyalkanoate(mcl-PHA)production by Pseudomonas strains[J].Bioresource Technology,2013,150(4):202-209.
[7]Shalin T,Sindhu R,Pandey A,et al.Production of poly-3-hydroxybutyrate from mixed culture[J].BIOLOGIA,2016,71(7):736-742.
[8]Anburajan P,Kumar A N,Sabapathy P C,et al.Polyhydroxy butyrate production by Acinetobacter junii BP25,Aeromonas hydrophila ATCC 7966,and their co-culture using a feast and famine strategy[J].Bioresource Technology,2019,293.
[9]
H,Hobmeier K,Moos M,et al.Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB[J].Biotechnology for biofuels,2017,10(1):190.
[10]Jones J A,Wang X.Use of bacterial co-cultures for the efficient production of chemicals[J].Current Opinion in Biotechnology,2018,53:33-38.
disclosure of Invention
The invention provides an artificial dual-bacterium system for producing mcl-PHA (polyhydroxyalkanoate) by utilizing lignocellulose hydrolysate and application thereof, and the feasibility is verified, and the specific technology is as follows:
an artificial dual-bacterium system for producing mcl-PHA by using lignocellulose hydrolysate; the artificial double-bacterium system consists of engineering bacterium P.putida KT delta ABZF (p 2-a-J) and engineering bacterium E.coli delta 4D (ACP-SCLAC).
The artificial double-bacterium system utilizes lignocellulose hydrolysate to produce medium-long-chain polyhydroxyalkanoate (mcl-PHA); wherein, the engineering bacteria E.coli delta 4D (ACP-SCLAC) mainly use xylose in lignocellulose hydrolysate as a substrate to secrete acetic acid and Free Fatty Acid (FFA); the engineering bacteria P.putida KT delta ABZF (p 2-a-J) use glucose in lignocellulose hydrolysate to accumulate biomass, and use acetic acid and Free Fatty Acid (FFA) to synthesize medium-long chain polyhydroxyalkanoate (mcl-PHA).
The invention relates to an artificial double-bacterium system synthesis method for producing mcl-PHA by utilizing lignocellulose hydrolysate, which comprises the following steps: (1) Selecting single colonies of an engineering strain E.coli delta 4D (ACP-SCLAC) and an engineering strain P.putida KT delta ABZF (p 2-a-J) and respectively inoculating the single colonies to an LB liquid culture medium for culture so as to activate the strains; respectively absorbing overnight cultures of the two strains into an M9 liquid culture medium, culturing E.coli delta 4D (ACP-SCLAC) by taking xylose as a carbon source, and culturing P.putida KTdelta ABZF (p 2-a-J) by taking glucose as a carbon source to respectively obtain seed solutions of an engineering strain E.coli delta 4D (ACP-SCLAC) and an engineering strain P.putida KTdelta ABZF (p 2-a-J);
(2) Inoculating the seed liquid of the engineering strain P.putida KT delta ABZF (p 2-a-J) obtained in the step (1) to a fermentation medium containing lignocellulose hydrolysate for fermentation; inoculating E.coli delta 4D (ACP-SCLAC) seed liquid to M9 fermentation medium containing lignocellulose hydrolysate for further fermentation 12 h; seed liquor inoculation of P.putida KT delta ABZF (p 2-a-J) and E.coli delta 4D (ACP-SCLAC) in a volume ratio of 2:1.
the fermentation culture medium inoculated by the two engineering bacteria is an M9 culture medium and a lignocellulose hydrolysate, wherein the ratio of the fermentation culture medium to the lignocellulose hydrolysate is 1:1 volume ratio after mixing 10g/L glucose solution was added to allow more biomass to accumulate in P.putida KT. DELTA. ABZF (p 2-a-J).
The fermentation conditions in the step (2) are as follows: fermentation temperature 30 deg.C, 0.5M H prepared by automatic addition 2 SO 4 And 1M KOH at 7.0, 2vvm into filter sterilized air, and automatically associated Dissolved Oxygen (DO) and stirring rate to maintain DO at 25%.
The method for producing mcl-PHA by using lignocellulose hydrolysate in the artificial double-bacterium system comprises the following steps:
1) Crushing and sieving corn straws, washing the corn straws twice by using distilled water to wash out powdery straws, collecting straw particles of between 20 and 80 meshes, and drying the straw particles;
2) Mixing corn stalk particles with a volume (L) of distilled water of 1:10 (10%, kg/L), then placing the straw water solution into a common glass vessel containing distilled water, adding 1% (v/v) of sulfuric acid (the volume ratio of 0.5M sulfuric acid to the prepared straw water solution is 1); heating the glassware in a high-temperature sterilization pot to 130 ℃, keeping the temperature for 30min, then drying the glassware subjected to the reaction to room temperature, and performing spin-drying dehydration to collect the obtained solution, namely the corn straw pretreatment solution;
3) Freezing and centrifuging the corn stalk pretreatment solution at 9000rpm for 10min to remove solid residues in the solution, and adjusting the pH value to 7.0 by using NaOH to obtain lignocellulose hydrolysate.
The concrete description is as follows:
1. the artificial double-bacterium system consists of engineering bacteria P.putida KT delta ABZF (p 2-a-J) and engineering bacteria E.coli (ACP-SCLAC). Fig. 1 is a metabolic cross-flow diagram of two engineering bacteria, wherein the two bacteria system uses glucose and xylose as carbon sources, and the engineering bacteria e.coli (ACP-SCLAC) use xylose as a substrate to secrete acetic acid and Free Fatty Acid (FFA); the engineering bacterium P.putida KT delta ABZF (p 2-a-J) utilizes glucose as a main growth carbon source and utilizes acetic acid and FFA to synthesize mcl-PHA. Optimization of the ratio of glucose and xylose in the medium and determination of the nitrogen source concentration were then carried out. Wherein, fig. 2A shows that the volume ratio of glucose to xylose is 3:1,1:1 and 1: the yield of mcl-PHA at 3 hours (g/L); FIG. 2B shows the production of mcl-PHA (g/L) at a nitrogen source concentration (g/L) of 1-4. Meanwhile, in order to test the substrate utilization degree of the double bacteria, the concentrations (g/L) of xylose and glucose in the fermentation solution were analyzed by high performance liquid chromatography at different volume ratios of glucose and xylose, as shown in FIG. 3. Wherein, fig. 3A shows that the volume ratio of glucose to xylose is 3:1, the concentration of the two sugars is changed within 64 h; fig. 3B shows that the volume ratio of glucose to xylose is 1: at 3, the concentrations of the two sugars are changed within 64 h; fig. 3C shows that the volume ratio of glucose to xylose was 1: at 1, the concentrations of both sugars were varied over 64 h.
In 2L fermentation liquor, the optimal culture conditions of an artificial double-bacterium system, namely P.putida KT delta ABZF (p 2-a-J), are obtained, and the inoculation is carried out 12h after the E.coli (ACP-SCLAC) inoculation, wherein the inoculation ratio is 2:1, the ratio of glucose to xylose in the fermentation medium is 1:1, the nitrogen source concentration was 3g/L, the culture temperature was 30 ℃ and the pH was 7.0. The artificial dual-bacterium system can rapidly consume glucose under the three mixed sugar ratios, and a small amount of xylose remains after 64 hours, which proves that the artificial dual-bacterium system can efficiently utilize two kinds of sugar. Wherein, the artificial double-bacterium system is cultured in a fermentation medium with the total sugar concentration of 20g/L and the glucose and the xylose of 10g/L respectively under the conditions to produce mcl-PHA, and the highest mcl-PHA yield is 1.64g/L.
2. Based on the optimized artificial double-bacterium system, the artificial double-bacterium system synthesizes mcl-PHA with lignocellulose hydrolysate as a substrate. Optimization of the fermentation medium composition was then performed. In 2L of fermentation culture solution, the optimal fermentation culture medium for obtaining the artificial dual-bacterium system comprises the following components in a volume ratio of lignocellulose hydrolysate to M9 culture medium of 1:1, and additionally 10g/L glucose is added. FIG. 4 shows the mcl-PHA production of the dual-bacterial system in different fermentation media containing lignocellulose hydrolysate, and it can be seen that the highest mcl-PHA yield of 1.02g/L was obtained when the optimal media components (glucose concentration of 10.50g/L, xylose concentration of 10.21 g/L) and the other optimal conditions (P.putida KT. Delta. ABZF (p 2-a-J) were inoculated 12h after E.coli (ACP-SCLAC) inoculation, the inoculation ratio was 2, the nitrogen source concentration was 3g/L, the culture temperature was 30 ℃, and the pH was 7.0).
The invention has the advantages that:
the invention provides an artificial dual-bacteria system for producing mcl-PHA (alpha-PHA) by utilizing lignocellulose hydrolysate and application thereof, wherein the artificial dual-bacteria system is constructed by utilizing engineering P.putida KT delta ABZF (p 2-a-J) and E.coli (ACP-SCLAC), tasks such as substrate synthesis, PHA synthesis and the like are distributed to different strains for execution, the different strains are subjected to targeted design, transformation and reconstruction, the dual-bacteria form a mutually beneficial symbiotic relation based on 'nutrition supply' and 'detoxification', the metabolic function of the system is improved, the overload of single-bacteria metabolism is avoided, and the mcl-PHA can be produced by utilizing the lignocellulose hydrolysate. As shown in FIG. 2, the maximum yield of mcl-PHA obtained by the double bacteria through mixed sugar (glucose and xylose) is 1.64g/L, and the yield is 3.9 times of that of pure culture of single bacteria; as shown in FIG. 4, the dual-bacteria system can utilize lignocellulose hydrolysate to produce mcl-PHA, so that the utilization of complex substrates is realized, and the substrate cost is further reduced, the maximum mcl-PHA yield of the artificial dual-bacteria system is 1.02g/L, and the method has competitive advantages compared with other researches for synthesizing PHA by utilizing renewable resources.
Drawings
FIG. 1 is a schematic diagram of the relationship between two bacteria
FIG. 2 optimization of glucose and xylose ratio and nitrogen source (ammonium chloride) concentration in mixed sugar medium by two bacteria
FIG. 3 shows the utilization of mixed sugar by two bacteria at different sugar mixing ratios
FIG. 4 mcl-PHA production by two bacteria in different lignocellulosic hydrolysate media
Detailed Description
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. The dual-bacteria relationship is designed as shown in figure 1, and the dual-bacteria system consists of engineering bacteria P.putida KT delta ABZF (p 2-a-J) and engineering bacteria E.coli (ACP-SCLAC). The double-bacterium system takes lignocellulose hydrolysate (mainly containing glucose, xylose and the like) as a substrate, the engineering bacterium E.coli (ACP-SCLAC) prefers to utilize xylose to secrete Free Fatty Acid (FFA) and acetic acid, and the engineering bacterium P.putida KT delta ABZF (p 2-a-J) utilizes glucose as a growth carbon source and utilizes acetic acid and FFA to synthesize mcl-PHA. The dual-bacteria system utilizes an intermediate metabolite acetic acid to construct a dual-bacteria detoxification symbiotic relationship, namely the engineering bacteria P.pudida KT delta ABZF (p 2-a-J) utilize acetic acid secreted by the engineering bacteria E.coli (ACP-SCLAC) as a carbon source, and simultaneously relieve the inhibition of the acetic acid on the engineering bacteria E.coli (ACP-SCLAC).
An artificial dual-bacterium system for producing mcl-PHA by utilizing lignocellulose hydrolysate, which is prepared by the following steps:
(1) Determination of the ratio of glucose and xylose in a Mixed sugar Medium
M9 inorganic salt medium in fermentation medium (g/L): na (Na) 2 HPO 4 ·7H 2 O 12.8，KH 2 PO 4 3，NaCl 0.5，MgSO 4 0.24, 1mL/L of trace element solution, and pH 7.0; glucose and xylose were additionally added at a total sugar concentration of 20g/L, with a glucose to xylose ratio of 3:1 or 1:1 or 1:3; the nitrogen source (ammonium chloride) concentration was designed to be 1g/L.
(2) Determination of nitrogen source concentration in sugar-mixed culture medium
Determining that the ratio of glucose to xylose in the culture medium is 1: after 1, the nitrogen source (ammonium chloride) concentration was designed to be 1 to 4g/L under the above conditions.
(3) Determination of culture medium components in lignocellulose hydrolysate fermentation culture medium
The fermentation medium in the step (1) is an M9 inorganic salt medium (g/L): na (Na) 2 HPO 4 ·7H 2 O 12.8，KH 2 PO 4 3，NaCl 0.5，MgSO 4 0.24, 1mL/L of trace element solution and pH 7.0; the ratio of lignocellulose hydrolysate to M9 medium in the medium was designed to be 1:1 (M1-1) or 9:1 (M9-1), no additional glucose was added; the ratio of lignocellulose hydrolysate to M9 medium was designed to be 1:1 (M1-1 +) or 9:1 (M9-1 +), with the addition of 10g/L glucose; the nitrogen source (ammonium chloride) concentration was designed to be 3g/L.
The application method for producing mcl-PHA in cellulose hydrolysate by using the artificial dual-thallus system is characterized by comprising the following steps:
1) Crushing and sieving corn straws, washing the corn straws twice by using distilled water to wash out powdery straws, collecting straw particles of between 20 and 80 meshes, and drying the straw particles;
2) Mixing corn stalk particles with a volume (L) of distilled water of 1:10 (10%, w/v), then placing the straw aqueous solution into a common glass ware containing distilled water, adding 1% (v/v) of sulfuric acid (the volume ratio of 0.5M sulfuric acid to the prepared straw aqueous solution is 1); heating the glassware in a high-temperature sterilization pot to 130 ℃, keeping the temperature for 30min, then drying the glassware subjected to the reaction to room temperature, and performing spin-drying dehydration to collect the obtained solution, namely the corn straw pretreatment solution;
3) Freezing and centrifuging the corn stalk pretreatment solution at 9000rpm for 10min to remove solid residues in the solution, and adjusting the pH value to 7.0 by using NaOH to obtain lignocellulose hydrolysate.
After fermentation for 64h, the mcl-PHA content in the fermentation liquor is determined to be 1.02g/L, which indicates that the constructed artificial dual-bacterium system can utilize lignocellulose resources to produce mcl-PHA.
The invention is described in further detail below with reference to the following figures and specific examples:
1. culture medium formula and solution formula
LB medium (g/L): peptone 10.0, naCl 10.0, yeast extract 5.0, pH 7.0.
M9 inorganic salt medium (g/L): na (Na) 2 HPO 4 ·7H 2 O 12.8，KH 2 PO 4 3，NH 4 Cl 2，NaCl 0.5，MgSO 4 0.24, 1mL/L of trace element solution and pH 7.0. The carbon source is added after converting glucose and xylose according to the required amount.
Microelement formula (g/L1M HCl): feSO 4 ·7H 2 O 2.78，MnCl 2 ·4H 2 O 1.98，CoCl 2 ·6H 2 O 2.38，CaCl 2 ·2H 2 O 1.47，CuCl 2 ·2H 2 O 0.17，ZnSO 4 ·7H 2 O 0.29。
Glucose and xylose mother liquor: accurately weighing 100g of glucose and xylose, respectively dissolving in a common glass container filled with 200mL of distilled water (the final concentration of sugar is 500 g/L), sterilizing with high pressure steam at 115 deg.C for 30min, cooling, and storing at 4 deg.C.
1M sodium citrate buffer: 210g of citric acid hydrate was added to 750mL of water, 50-60g of NaOH was added to pH =4.3, and the volume was made to 1L. pH =4.8 when this buffer was diluted to 0.05M.
2. Gene sequence related to engineering bacteria
The patent relates to two engineering bacteria, namely E.coli (ACP-SCLAC) and P.putida KT delta ABZF (p 2-a-J). The invention name of a construction method in E.coli (ACP-SCLAC) is that the E.coli is constructed by disclosing a genetic engineering bacterium which prefers to efficiently secrete acetic acid and FFA by using xylose according to a method for improving the acetic acid and FFA secretion capability of escherichia coli and making escherichia coli prefer to use xylose, wherein the Chinese patent application number of the E.coli (ACP-SCLAC) applied by 2018-8-9 of Tianjin university is 201810900753.6. The related genes are ACP gene and SCLAC gene, which respectively code ricinoyl carrier protein thiolase and laccase, and the nucleotide sequences are shown in SEQ ID No.1 and SEQ ID No. 2.
The other engineering bacterium P.putida KT delta ABZF (p 2-a-J) related by the invention is constructed according to a construction method of a genetic engineering bacterium for improving the acetic acid assimilation capability, which is disclosed as a method for improving the assimilation capability of Pseudomonas putida KT2440 and applied to 2018-8-9 of Tianjin university with the Chinese patent application number of 201810900753.6. The engineered bacterium involves six genes fadA, fadB, phaZ, yqeF, phaJ and acs, which respectively encode 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, PHA depolymerase, acyltransferase, enoyl-CoA hydratase and acetyl-CoA synthetase. The nucleotide sequence is shown by SEQ ID No.3-8 correspondingly.
Example 1 Artificial double-strain System of engineering bacteria E.coli (ACP-SCLAC) and P.putida KTA delta ABZF (p 2-a-J) for producing mcl-PHA by mixed sugar in laboratory
(1) Single colonies of the engineering bacteria E.coli (ACP-SCLAC) and the engineering bacteria P.putida KT delta ABZF (p 2-a-J) are respectively inoculated into 5ml of LB liquid culture medium, and are placed on a constant temperature shaking table at 30 ℃ for overnight culture under the condition of 220rpm so as to activate the bacteria. 1mL of each of the overnight cultures of the two strains was aspirated into 100mL of M9 liquid medium, and E.coli (ACP-SCLAC) cultured with xylose as a carbon source and P.putida KT. DELTA. ABZF (p 2-a-J) cultured with glucose as a carbon source.
(2) And (2) mixing the seed solution of the engineering bacteria E.coli (ACP-SCLAC) and P.putida KT delta ABZF (p 2-a-J) obtained in the step (1) according to the ratio of 2:1 in sequence, p.putida KT Δ ABZF (p 2-a-J) was inoculated 12h after e.coli (ACP-SCLAC) inoculation.
(3) The fermentation medium in the step (2) is an M9 inorganic salt medium (g/L): na (Na) 2 HPO 4 ·7H 2 O 12.8，KH 2 PO 4 3，NaCl 0.5，MgSO 4 0.24, 1mL/L of trace element solution and pH 7.0. Wherein the total sugar concentration is 20g/L, the ratio of glucose to xylose is 3:1 or 1:1 or 1:3. the concentration of the nitrogen source (ammonium chloride) is 1-4g/L.
(4) And (2) fermenting by adopting a 5L fermentation tank, wherein the liquid carrying capacity is 2L, the fermentation temperature is controlled to be 30 ℃, filtered and sterilized air is supplied at the speed of 2vvm, the Dissolved Oxygen (DO) is controlled to be 25% by automatically controlling the rotating speed, and the pH is controlled to be 7.0 by automatically adding 0.5M H2SO4 and 1M KOH. The fermenter culture experiment was run for 64h, 2 replicates were run, and the ratio of glucose and xylose was 1: when the concentration of ammonium chloride is 3g/L at 1 hour, the highest mcl-PHA yield is 1.64g/L, which indicates that the constructed artificial double-bacterium system can effectively utilize glucose xylose mixed sugar in a laboratory as a carbon source to produce mcl-PHA.
Example 2 engineering bacteria E.coli (ACP-SCLAC) and P.putida KTDELTA ABZF (p 2-a-J) Artificial double-bacteria System for producing mcl-PHA by mixing and fermenting cellulose hydrolysate
(1) Crushing and sieving corn straws, washing the corn straws twice by using distilled water to wash out powdery straws, collecting straw particles of 20-80 meshes, and drying the straw particles; mixing corn stalk particles with the volume (L) of distilled water, wherein the mass (kg) of the corn stalk particles is 1:10 (10%, w/v), putting the straw water solution into a common glass vessel containing distilled water, adding 1% (v/v) of sulfuric acid (the volume ratio of 0.5M sulfuric acid to the prepared straw water solution is 1; heating the glassware in a high-temperature sterilization pot to 130 ℃, keeping the temperature for 30min, then drying the glassware subjected to the reaction to room temperature, and performing spin-drying dehydration to collect the obtained solution, namely the corn straw pretreatment solution; and finally, freezing and centrifuging the corn straw pretreatment liquid at 9000rpm for 10min to remove solid residues in the liquid, and adjusting the pH value to 7.0 by using NaOH to obtain the lignocellulose hydrolysate. Analyzing the sugar content (g/L) in the pretreatment solution by using high performance liquid chromatography, wherein the glucose concentration in the M1-1 culture medium is 0.45 +/-0.02, and the xylose concentration is 9.44 +/-1.06; the concentration of glucose in the M1-1+ culture medium is 10.50 +/-1.58, and the concentration of xylose is 10.21 +/-0.19; the concentration of glucose in the M9-1 culture medium is 2.03 +/-0.14, and the concentration of xylose is 22.56 +/-1.24; the concentration of glucose in the M9-1+ culture medium is 10.31 +/-0.95, and the concentration of xylose is 21.80 +/-0.48.
(2) Single colonies of the engineering bacteria E.coli (ACP-SCLAC) and the engineering bacteria P.putida KT delta ABZF (p 2-a-J) are respectively inoculated into 5ml of LB liquid culture medium, and are placed on a constant temperature shaking table at 30 ℃ and are cultured overnight at 220rpm so as to activate the bacteria. 1mL of each of the overnight cultures of the two strains was aspirated into 100mL of M9 liquid medium, and E.coli (ACP-SCLAC) cultured with xylose as a carbon source and P.putida KT. DELTA. ABZF (p 2-a-J) cultured with glucose as a carbon source. (3) Mixing the recombinant strain E.coli delta p-m-a-e obtained in the step (2) and the recombinant strain P.putida-acs seed solution according to the weight ratio of 1:1, and inoculating the P.putida-acs 24h after inoculating the E.coli delta p-m-a-e.
(3) And (3) mixing the seed liquid of the engineering bacteria E.coli (ACP-SCLAC) and the seed liquid of the engineering bacteria P.putida KT delta ABZF (p 2-a-J) obtained in the step (2) according to the ratio of 2:1 in sequence, p.putida KT Δ ABZF (p 2-a-J) was inoculated 12h after e.coli (ACP-SCLAC) inoculation.
(4) The fermenter parameter control in step (3) was the same as in example 1. After fermentation for 64h, the yield of mcl-PHA produced by fermentation with M1-1+ (the ratio of lignocellulose hydrolysate to M9 medium is 1.
While the invention has been described in further detail in connection with specific embodiments thereof, it will be apparent to those skilled in the art that various modifications and adaptations can be made therein without departing from the principles of the invention.
Claims (7)
1. An artificial dual-bacterium system for producing mcl-PHA by using lignocellulose hydrolysate; the artificial double-bacterium system is characterized by consisting of an engineering bacterium P.putida KT delta ABZF (p 2-a-J) and an engineering bacterium E.coli delta 4D (ACP-SCLAC).
2. An artificial dual bacteria system for the production of mcl-PHA as claimed in claim 1; the method is characterized in that: the artificial double-bacterium system utilizes lignocellulose hydrolysate to produce medium-long-chain polyhydroxyalkanoate (mcl-PHA); wherein, the engineering bacteria E.coli delta 4D (ACP-SCLAC) mainly use xylose in lignocellulose hydrolysate as a substrate to secrete acetic acid and Free Fatty Acid (FFA); the engineering bacterium P.putida KT delta ABZF (p 2-a-J) utilizes glucose in lignocellulose hydrolysate to accumulate biomass, and utilizes acetic acid and Free Fatty Acid (FFA) to synthesize medium-long chain polyhydroxyalkanoate (mcl-PHA).
3. The method for synthesizing an artificial dual-bacteria system for producing mcl-PHA from lignocellulose hydrolysate as recited in claim 1 or 2, comprising the steps of:
(1) Selecting single colonies of an engineering strain E.coli delta 4D (ACP-SCLAC) and an engineering strain P.putida KT delta ABZF (p 2-a-J) to respectively inoculate the single colonies into an LB liquid culture medium for culture so as to activate thalli; respectively sucking overnight cultures of the two strains into an M9 liquid culture medium, culturing E.coli delta 4D (ACP-SCLAC) by taking xylose as a carbon source, and culturing P.putida KT delta ABZF (p 2-a-J) by taking glucose as a carbon source to respectively obtain seed solutions of an engineering strain E.coli delta 4D (ACP-SCLAC) and an engineering strain P.putida KT delta ABZF (p 2-a-J);
(2) Inoculating the seed liquid of the engineering strain P.putida KT delta ABZF (p 2-a-J) obtained in the step (1) to a fermentation medium containing lignocellulose hydrolysate for fermentation; inoculating E.coli delta 4D (ACP-SCLAC) seed liquid to M9 fermentation medium containing lignocellulose hydrolysate for continuous fermentation 12h after inoculation; seed liquid inoculation volume ratio of P.putida KT delta ABZF (p 2-a-J) and E.coli delta 4D (ACP-SCLAC) is 2:1.
4. the method as set forth in claim 3, wherein the fermentation medium inoculated by the two engineering bacteria is M9 medium and the lignocellulose hydrolysate is mixed in a ratio of 1:1 volume ratio after mixing 10g/L glucose solution was added to allow more biomass to accumulate in P.putida KT. DELTA. ABZF (p 2-a-J).
5. The method as set forth in claim 3, wherein the fermentation conditions in step (2) are: the fermentation temperature was 30 ℃, the pH was controlled to 7.0 using 0.5m H2SO4 and 1M KOH in an auto-addition configuration, air was admitted at 2vvm rate after filtration sterilization, and the Dissolved Oxygen (DO) value and the stirring rate were automatically related to maintain DO at 25%.
6. The method of producing mcl-PHA using the lignocellulosic hydrolysate in the artificial dual bacterial system of claim 1, comprising the steps of:
(1) Crushing and sieving corn straws, washing the corn straws by using distilled water to wash off powdery straws, collecting straw particles of 20-80 meshes, and drying the straw particles;
(2) Preparing a straw water solution by taking the mass of corn straw particles and the volume of distilled water as 1/10 (kg/L), then placing the straw water solution into a common glass vessel containing distilled water, adding 1% of sulfuric acid for pretreatment, and fully stirring and uniformly mixing; heating the glassware in a high-temperature sterilization pot to 130 ℃, keeping the temperature for 30min, then drying the glassware subjected to the reaction to room temperature, and performing spin-drying dehydration to collect the obtained solution, namely the corn straw pretreatment solution;
(3) Freezing and centrifuging the corn stalk pretreatment solution at 9000rpm for 10min to remove solid residues in the solution, and adjusting the pH value to 7.0 by using NaOH to obtain lignocellulose hydrolysate.
7. The method as claimed in claim 6, wherein the volume ratio of the aqueous solution of the prepared straw added with the sulfuric acid with 1% of the sulfuric acid amount being 0.5M is 1:100.
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