CN114164127A - Biological process for improving succinic acid fermentation efficiency - Google Patents
Biological process for improving succinic acid fermentation efficiency Download PDFInfo
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- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 130
- 238000000855 fermentation Methods 0.000 title claims abstract description 95
- 230000004151 fermentation Effects 0.000 title claims abstract description 95
- 239000001384 succinic acid Substances 0.000 title claims abstract description 61
- 230000031018 biological processes and functions Effects 0.000 title abstract description 8
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- 239000012528 membrane Substances 0.000 claims abstract description 16
- 244000005700 microbiome Species 0.000 claims abstract description 15
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- 239000000706 filtrate Substances 0.000 claims abstract description 10
- 241000195597 Chlamydomonas reinhardtii Species 0.000 claims abstract description 8
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 claims abstract description 8
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- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention belongs to the technical field of microorganisms, and discloses a biological process for improving succinic acid fermentation efficiency, which comprises the following steps: the method comprises the steps of inoculating lipolytica saccharomycete seed liquid into a fermentation tank containing a fermentation culture medium for fermentation for 24-48 hours, then inoculating chlamydomonas reinhardtii, adding nutrient solution, continuing to perform fermentation culture for 36 hours, stopping fermentation, coupling the fermentation tank with a ceramic membrane, separating fermentation liquor in the fermentation tank through the ceramic membrane to obtain filtrate and microorganisms, wherein the filtrate is used for subsequently extracting succinic acid, and the microorganisms are used for preparing feed protein. The biological process of the invention improves the yield of succinic acid and reduces the generation of acetic acid as a byproduct.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a biological process for improving succinic acid fermentation efficiency.
Background
Succinic acid (Succinic acid), a scientific name for Succinic acid, is a safe natural organic matter and an important organic synthetic intermediate. Succinic acid as a precursor can be used for producing a plurality of high-added-value derivatives, and is widely applied to the production fields of foods, pharmacy, ion chelating agents, detergents and surfactants. Succinic acid is gradually upgraded to a large amount of chemicals in recent years, and the annual yield of succinic acid worldwide is 3 ten thousand to 5 ten thousand tons. The traditional production of succinic acid is mainly prepared by preparing maleic anhydride from butane and then processing the maleic anhydride by a chemical method, and the catalytic action of heavy metals Pd and Ru is required. With the reduction of petroleum resources and the aggravation of environmental pollution problems, the conversion of petroleum-based chemical production to the biochemical production of succinic acid is necessary, and the efficient and environment-friendly succinic acid fermentation method using renewable resources as raw materials has great application potential.
Compared with the traditional petroleum-based chemical refining method for succinic acid, the production cost of succinic acid by the biological fermentation method is greatly reduced. In addition to fermentation efficiency, post-extraction purification processes downstream of fermentation are the most important factors affecting the cost of succinic acid production. The thallus culture and fermentation environment of the bacteria are close to neutral, and alkali liquor is added continuously to maintain the stability of the pH value in the fermentation process. Therefore, the generated succinate needs to be converted into free succinic acid by acidification treatment, and the cost of the stage accounts for about 60-70% of the production cost. Yeast is tolerant to low pH and can be fermented biologically at low pH to convert the substrate directly to succinate rather than succinate. After fermentation is finished, the fermentation liquor from which the thalli are removed is directly evaporated and crystallized, the downstream processing steps of the product are few, and the production cost can be greatly saved. In addition, yeast cells have advantages over bacteria, can achieve higher biomass, and have excellent and stable performance.
Succinic acid is one of the intermediate metabolites of the central metabolic pathway of the microorganism, the tricarboxylic acid cycle (TCA), and is also a metabolic end product of various facultative anaerobes and strict anaerobes, and can be produced by microorganisms using renewable carbohydrate resources. With the development of biotechnology and microbial molecular genetics, metabolic engineering has been successfully applied to the development of genetically engineered bacteria for high yield of succinic acid as a powerful tool.
The bacteria for producing succinic acid comprise two main types of succinic acid natural producing bacteria and metabolic engineering recombinant bacteria. The natural succinic acid-producing strains are isolated from rumen, and include Actinobacillus succinogenes, Anaerobiospirillum succinogenes, and Entamoxifen succinogenes. They are gram-negative bacteria, mesophilic and CO-philic, which are both facultative anaerobic or strictly anaerobic2And producing succinic acid by using a TCA reduction branch. The actinobacillus succinogenes is a good-performance easily-obtained strain, can be fermented by using various carbon sources, can tolerate high-concentration succinic acid and glucose, and can have tolerance to the glucose and the succinic acid as high as 158g/L and 104 g/L. The anaerobiospirillum succinogenes can naturally produce succinic acid with higher concentration, but has poor tolerance to high-concentration glucose and succinate, strict anaerobic environment is required, the nutrition condition is complex, and the production cost is higher. The mannheim succinic acid-producing bacterium can also produce succinic acid using various carbon sources, has a complete TCA cycle, and is capable of aerobic growth, but its succinic acid-producing ability is weaker than that of the above two natural host bacteria.
In nature, these naturally succinic acid-producing microorganisms are present in places where amino acids and vitamins are abundant, they are mostly auxotrophic strains, many also conditionally pathogenic bacteria, the strains grow poorly and require expensive media that are rich in nutrients. Furthermore, the genetic information and research tools of these succinic acid-producing bacteria are not sufficiently mature. To solve these problems, bacteria such as Escherichia coli and Corynebacterium glutamicum which have a clear genetic background and do not naturally produce succinic acid have been widely used in the research of producing succinic acid by metabolic engineering.
Escherichia coli belongs to facultative anaerobes. The production of succinic acid under aerobic conditions requires inactivation of Succinate Dehydrogenase (SDH), blocking the downstream conversion of succinate, an intermediate metabolite of the TCA cycle, and also allows the accumulation of succinate by opening the glyoxylate shunt. The succinic acid fermentation under anaerobic conditions is carried out by a TCA reduction branch and a partial glyoxylate branch. In addition, Escherichia coli may overflow acetic acid during fermentation, and the main acetic acid production pathway needs to be blocked by metabolic engineering means. In addition to E.coli, C.glutamicum is also a good, safe and easily controllable production host which can be subjected to aerobic-anaerobic two-stage fermentation. Therefore, the engineering bacteria such as escherichia coli, corynebacterium glutamicum and the like are used as succinic acid production strains, and the yield of succinic acid reach higher levels. However, since bacteria are not resistant to acidic environment, continuous alkali liquor feeding is required for maintaining pH neutrality in cell growth and fermentation processes, so that the generated succinate needs to be converted into free succinic acid by acidification treatment and then crystallization extraction is carried out in an extraction stage after fermentation.
The production of succinic acid in yeast was originally intended to improve the flavor of wine produced by Saccharomyces cerevisiae, and studies have been mainly focused on succinate dehydrogenase, fumarase, and the like in the TCA cycle. Succinate accumulation can be realized by knocking out a gene encoding succinate dehydrogenase to block the TCA cycle of mitochondria; meanwhile, the corresponding coding genes of succinate dehydrogenase and isocitrate dehydrogenase are knocked out, so that succinate can be accumulated by utilizing the oxidative TCA cycle and the glyoxylate pathway together. Considering that the theoretical yield of succinic acid produced by the TCA reduction branch is twice that of the TCA oxidation branch, the institute of Process engineering, the department of Chinese academy of sciences, on the basis of knocking out the pyruvate decarboxylase, glycerol-3-phosphate dehydrogenase and fumarase genes of Saccharomyces cerevisiae, the Shichen laboratory expresses the enzymes (pyruvate carboxylase PYC2p, malate dehydrogenase MDH3p, Escherichia coli-derived fumarase EcFumCp and fumarase FRDS1p) reacted in each step of the TCA reduction branch in the cytoplasm. At low pH (pH controlled at 3.8) and sufficient CO was added2In the process, 12.97g/L succinic acid can be obtained by fermenting the recombinant strain in a 3L fermentation tank for 120h, and the yield is 0.21mol/mol glucose. Although Saccharomyces cerevisiae is tolerant to environmental stresses such as low pH, lower yields and yields limit the development of Saccharomyces cerevisiae as a cell factory for succinic acid production.
In addition to Saccharomyces cerevisiae, yarrowia lipolytica is another species of yeast that has been extensively studied in recent years. It belongs to non-traditional yeast, is strictly aerobic, has two-state property, and utilizes complete TCA cycle and electron transfer chain to maintain growth. Lipolytic yeast have many specific physiological, metabolic and genetic properties. It can be used on a wide range of substrates including glucose, glycerol, ethanol, fatty acids, lipids, n-alkanes, etc. It can grow in the pH value range of 3.0-7.5, and can still be fermented when the pH value is lower than 3.0. Through natural screening or further metabolic engineering modification, the lipolysis yeast can accumulate a large amount of organic acids such as citric acid, isocitric acid, alpha-ketoglutaric acid and the like, so the lipolysis yeast gradually develops into an important industrial strain.
Yovkova et al finely controls the metabolic flow of lipolysis yeast cells producing alpha-ketoglutarate by metabolic engineering and reduces the accumulation of byproduct pyruvate. Recently, it has been reported in the literature that recombinant bacteria capable of accumulating succinic acid can be obtained by mutating the succinate dehydrogenase-encoding gene of a lipolytic yeast or replacing the promoter thereof. Yuzbasheev et al constructed a recombinant strain of lipolytic yeast lacking succinate dehydrogenase activity by a gene knockout strategy, and then utilized N-methyl-N' -nitro-N-nitrosoguanidine for chemical mutagenesis and screening, and the obtained mutant strain was fermented in a complete medium for 7 days to produce 17g/L succinic acid. Subsequently, the subject group obtained a mutant strain with enhanced bacterial growth and succinic acid biosynthesis by using an directed evolution method, and 40.5g/L succinic acid was synthesized by fed-batch fermentation in a fermentation tank for 36 hours. The fermentation process does not need to control the pH value, and the yield of the domesticated mutant strain are greatly improved. The above studies initially demonstrated the potential of understanding lipid yeast as a cell factory for succinic acid production.
However, some problems still need to be solved, the recombinant strain Y.lipolytica generates more by-product acetic acid during succinic acid fermentation, the growth of cells is influenced, the biomass of the strain is low, and the fermentation period is long. Acetic acid can be partially dissociated into CH3COO in the neutral environment of the cell-And H+The pH value in the membrane is reduced, so that the pH difference inside and outside the membrane is reduced, the proton driving force is weakened, the energy for promoting ADP phosphorylation to generate ATP is insufficient, and the normal metabolism and physiological activity of cells are disturbed.
Disclosure of Invention
The invention aims to provide a biological process for improving the succinic acid fermentation efficiency.
The invention is realized by the following technical scheme.
A bioprocess for increasing succinic acid fermentation efficiency comprising the steps of:
inoculating the lipolytica yeast seed liquid into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 6-8% for fermentation, wherein the temperature is 28-29 ℃, the rotation speed is 400-plus 500rpm, the ventilation volume is 0.5-0.6vvm, the fermentation time is 24-48h, then inoculating chlamydomonas reinhardtii according to the inoculation amount of 6-8%, meanwhile, nutrient solution accounting for 5-10% of the volume of the fermentation liquid is added, continuing to ferment and culture for 36h, stopping fermentation, coupling the fermentation tank with a ceramic membrane, separating the fermentation liquid in the fermentation tank by the ceramic membrane to obtain filtrate and microorganism, wherein the filtrate is used for subsequently extracting succinic acid, and the microorganism is used for preparing feed protein; in the whole fermentation process, defoaming is carried out by feeding foam enemy.
Further, the air conditioner is provided with a fan,
the preparation method of the lipolytic yeast seed liquid comprises the following steps: inoculating the lipolysis saccharomycetes into an YPG culture medium for culturing to obtain a seed solution, and then carrying out ultrasonic treatment, wherein the ultrasonic frequency is controlled to be 25KHz, the power is 6W, the ultrasonic interval is 10s, the ultrasonic time is 2s, and the total ultrasonic time is 120-180 s.
Further, the air conditioner is provided with a fan,
the fermentation medium comprises the following components: 80g/L of glycerol, 30g/L of corn steep liquor, 5g/L of magnesium carbonate, 2g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.1g/L of ferrous sulfate heptahydrate, 0.01g/L of biotin and 6.5 of pH value.
Further, the air conditioner is provided with a fan,
the nutrient solution comprises the following components: glycerol 200g/L, inositol 10 g/L.
Further, the air conditioner is provided with a fan,
the molecular weight cut-off of the ceramic membrane is 10000-20000 Da.
The invention also claims succinic acid prepared by any one of the biological processes.
Compared with the prior art, the starting point and the obtained beneficial effects of the invention mainly comprise but are not limited to the following aspects:
the lipolysis yeast is non-traditional yeast, has good robustness, tolerance to high-concentration organic acid and low pH and good potential for producing succinic acid, and secretes a large amount of acetic acid due to the fact that the TCA cycle of Y.lipolytica delta sdh5 is blocked, which causes the discordance of glycolysis and TCA cycle flow; the production of acetic acid not only consumes substrate but also has toxic effects on cells, which is not favorable for the realization of high yield, high productivity and high conversion rate.
The acetic acid is gradually generated by the lipolysis yeast in the fermentation, and the lipolysis yeast generates larger toxicity for the middle and later period fermentation along with the accumulation of the acetic acid.
After the fermentation system of the lipolytic yeast is completely established, a proper amount of inositol is added, so that the CO can be enhanced2The fixed reaction weakens the cycle of glyoxylic acid, efficiently produces alpha-ketoglutaric acid and further generates succinic acid; the glycerol can be used as a carbon source for co-decomposing the lipid yeast and can also improve the cell membrane permeability of the lipid-decomposing yeast.
The ultrasonic pretreatment of the lipolytic yeast seed liquid with low intensity can activate the strain, shorten the fermentation culture time of the strain and improve the yield of succinic acid.
Drawings
FIG. 1 Effect of different factors on succinic acid production.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the present application will be clearly and completely described below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A bioprocess for increasing succinic acid fermentation efficiency comprising the steps of:
inoculating lipolytic yeast PGC01003 (mutant strain of lipolytic yeast with sdh5 knocked out, see literature of Robust detailed acid production from raw microbial utilization engineered Yarrowia lipolytica.Biotechnology for Biofuses.2016) into YPG culture medium (with the components of 10g/L yeast powder, 20g/L peptone and 20g/L glycerol) for culture to obtain seed solution, and then performing ultrasonic treatment with ultrasonic frequency of 25KHz, power of 6W, ultrasonic interval of 10s, ultrasonic time of 2s and total ultrasonic time of 120 s;
subjecting the seed liquid (concentration of 1 × 10) to ultrasonic treatment8cfu/mL) is inoculated into a fermentation tank containing a fermentation medium according to the inoculation amount of 6 percent for fermentation, the temperature is 28 ℃, the rotating speed is 500rpm, the ventilation volume is 0.6vvm, the fermentation time is 36 hours, and then chlamydomonas reinhardtii (the concentration is 1 multiplied by 10) is inoculated according to the inoculation amount of 8 percent5cfu/mL), adding nutrient solution accounting for 10% of the volume of the fermentation liquid, continuing to perform fermentation culture for 36h, stopping fermentation, coupling the fermentation tank with a ceramic membrane, separating the fermentation liquid in the fermentation tank by the ceramic membrane to obtain filtrate and microorganisms, wherein the filtrate is used for subsequently extracting succinic acid, and the microorganisms are used for preparing feed protein; defoaming by feeding foam enemy in the whole fermentation process; the molecular weight cut-off of the ceramic membrane is 20000 Da;
the fermentation medium comprises the following components: 80g/L of glycerol, 30g/L of corn steep liquor, 5g/L of magnesium carbonate, 2g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.1g/L of ferrous sulfate heptahydrate, 0.01g/L of biotin and 6.5 of pH value;
the nutrient solution comprises the following components: glycerol 200g/L, inositol 10 g/L.
Example 2
A bioprocess for increasing succinic acid fermentation efficiency comprising the steps of:
inoculating lipolytic yeast PGC01003 into YPG culture medium, culturing to obtain seed liquid, and performing ultrasonic treatment with ultrasonic frequency of 25KHz, power of 6W, ultrasonic interval of 10s, ultrasonic time of 2s, and total ultrasonic time of 180 s;
subjecting the seed liquid (concentration of 1 × 10) to ultrasonic treatment8cfu/mL) was inoculated into a fermentation tank containing a fermentation medium at an inoculum size of 8% for fermentation,the temperature is 29 ℃, the rotating speed is 400rpm, the ventilation capacity is 0.5vvm, the fermentation time is 24h, and then chlamydomonas reinhardtii (the concentration is 1 multiplied by 10) is inoculated according to the inoculation amount of 6 percent5cfu/mL), adding nutrient solution accounting for 6% of the volume of the fermentation liquid, continuing to perform fermentation culture for 36h, stopping fermentation, coupling the fermentation tank with a ceramic membrane, separating the fermentation liquid in the fermentation tank by the ceramic membrane to obtain filtrate and microorganisms, wherein the filtrate is used for subsequently extracting succinic acid, and the microorganisms are used for preparing feed protein; defoaming by feeding foam enemy in the whole fermentation process; the molecular weight cut-off of the ceramic membrane is 10000 Da;
the fermentation medium comprises the following components: 80g/L of glycerol, 30g/L of corn steep liquor, 5g/L of magnesium carbonate, 2g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.1g/L of ferrous sulfate heptahydrate, 0.01g/L of biotin and 6.5 of pH value;
the nutrient solution comprises the following components: glycerol 200g/L, inositol 10 g/L.
Example 3
The influence of different factors on the succinic acid yield and the acetic acid yield in the biological process of the invention is as follows:
succinic acid production and acetic acid production were measured at different time points (0,12,24,36,48,60,72, 84).
The groups are set according to different factor treatments:
the invention comprises the following steps: example 1;
control 1: the same procedure as in example 1 was repeated except that Chlamydomonas reinhardtii was not added;
control 2: the rest of the process was the same as example 1 without adding a nutrient solution;
control 3: the rest of the procedure was the same as in example 1, except that the ultrasonic treatment was not carried out.
As shown in figure 1, the succinic acid content is stably increased along with the increase of the fermentation time, wherein succinic acid is hardly produced in 12 hours before fermentation, the acid production efficiency is rapidly improved after 24 hours, but the lipolysis yeast can gradually produce acetic acid in the fermentation, and the toxicity to the middle and later-period fermentation of the lipolysis yeast is relatively high along with the accumulation of the acetic acid, and at 36 hours, the acetic acid in the fermentation broth can be used as a carbon source to perform non-light action and the glycerol is relatively difficult to be used as the carbon source by adding Chlamydomonas reinhardtii, so that the fermentation production of the lipolysis yeast is optimizedAn acid environment; adding appropriate amount of inositol to enhance CO2Fixing reaction to weaken glyoxylate cycle, ensuring that tricarboxylic acid cycle is not interrupted and continuously supplying alpha-ketoglutaric acid continuously, and further generating succinic acid; in the early stage of fermentation, the low-intensity ultrasonic pretreatment is carried out on the seed liquid, so that the strain can be activated, and the yield of succinic acid is increased; compared with a comparison 1, the invention can improve 18.7g/L by adding Chlamydomonas reinhardtii, greatly improve acid production efficiency, reduce the content of acetic acid as a byproduct by more than 90% (see table 1), improve 18.1g/L by comparing with a comparison 2 without adding a nutrient solution, and improve 9.8g/L by comparing with a comparison 3 without adopting ultrasonic treatment.
The invention also compares the fermentation acid production rate and the acetic acid content at the 72h time point, which is shown in table 1.
TABLE 1
| Group of | The fermentation acid production rate is g/L.h | Acetic acid g/L |
| The invention | 0.95 | 0.26 |
| Control 1 | 0.69 | 2.94 |
| Control 2 | 0.70 | 0.25 |
| Control 3 | 0.82 | 0.37 |
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (6)
1. A bioprocess for increasing succinic acid fermentation efficiency comprising the steps of:
inoculating the lipolytica yeast seed liquid into a fermentation tank containing a fermentation culture medium according to the inoculation amount of 6-8% for fermentation, wherein the temperature is 28-29 ℃, the rotation speed is 400-plus 500rpm, the ventilation volume is 0.5-0.6vvm, the fermentation time is 24-48h, then inoculating chlamydomonas reinhardtii according to the inoculation amount of 6-8%, meanwhile, adding nutrient solution accounting for 5-10% of the volume of the fermentation liquid, continuing to ferment and culture for 36h, stopping fermentation, coupling the fermentation tank with a ceramic membrane, separating the fermentation liquid in the fermentation tank by the ceramic membrane to obtain filtrate and microorganism, wherein the filtrate is used for extracting succinic acid, and the microorganism is used for preparing feed protein; in the whole fermentation process, defoaming is carried out by feeding foam enemy.
2. The bioprocess of claim 1, wherein the preparation method of the lipolytic yeast seed liquid is as follows: inoculating the lipolysis saccharomycetes into an YPG culture medium for culturing to obtain a seed solution, and then carrying out ultrasonic treatment, wherein the ultrasonic frequency is controlled to be 25KHz, the power is 6W, the ultrasonic interval is 10s, the ultrasonic time is 2s, and the total ultrasonic time is 120-180 s.
3. The bioprocess of claim 1 wherein the components of the fermentation medium are: 80g/L of glycerol, 30g/L of corn steep liquor, 5g/L of magnesium carbonate, 2g/L of ammonium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 0.1g/L of ferrous sulfate heptahydrate, 0.01g/L of biotin and 6.5 of pH value.
4. The bioprocess of claim 1 wherein the nutrient solution comprises the components of: glycerol 200g/L and inositol 10 g/L.
5. The bioprocess of claim 1 wherein the molecular weight cut-off of the ceramic membrane is 10000-.
6. Succinic acid produced by a bioprocess according to any one of claims 1-5.
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| CN105838632A (en) * | 2016-05-19 | 2016-08-10 | 江南大学 | Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof |
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| CN107916275A (en) * | 2017-12-06 | 2018-04-17 | 山东大学 | A kind of method using the aerobic ambroin acid of Yarrowia lipolytica strain with reduction TCA approach |
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| CN105838632A (en) * | 2016-05-19 | 2016-08-10 | 江南大学 | Saccharomyces cerevisiae gene engineering bacteria for producing succinic acid and application thereof |
| CN106544284A (en) * | 2016-11-01 | 2017-03-29 | 临沂大学 | A kind of restructuring Yarrowia lipolytica engineered strain and its construction method and application |
| CN107916275A (en) * | 2017-12-06 | 2018-04-17 | 山东大学 | A kind of method using the aerobic ambroin acid of Yarrowia lipolytica strain with reduction TCA approach |
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