阅读说明：本技术 一种解脂耶氏酵母生产脂肪酸乙酯的方法及其发酵培养基 (A kind of method and its fermentation medium of Yarrowia lipolytica production fatty-acid ethyl ester ) 是由 花强 程博谦 韦柳静 高琪 刘顺成 陈骏 于 2018-05-24 设计创作，主要内容包括：本发明公开了一种解脂耶氏酵母生产脂肪酸乙酯的方法及其发酵培养基。该发酵培养基中,所述解脂耶氏酵母为重组解脂耶氏酵母菌株GQY001、GQY004和GQY007中的一种或多种；所述发酵培养基包括碳源和氮源,所述碳源为油酸和/或葵花籽油。利用本发明的发酵培养基生产脂肪酸乙酯可大幅提高脂肪酸乙酯的产量,有利于生物柴油的产业化应用,节能环保,有非常好的应用前景。(The invention discloses the methods and its fermentation medium of a kind of Yarrowia lipolytica production fatty-acid ethyl ester.In the fermentation medium, the Yarrowia lipolytica is one of recombination Yarrowia lipolytica strain GQY001, GQY004 and GQY007 or a variety of；The fermentation medium includes carbon source and nitrogen source, and the carbon source is oleic acid and/or sunflower oil.The yield that fatty-acid ethyl ester can be greatly improved using fermentation medium production fatty-acid ethyl ester of the invention, is conducive to the industrial application of biodiesel, energy conservation and environmental protection has extraordinary application prospect.)

1. a kind of fermentation medium of Yarrowia lipolytica production fatty-acid ethyl ester, which is characterized in that the Yarrowia lipolytica For one of recombination Yarrowia lipolytica strain GQY001, GQY004 and GQY007 or a variety of；The fermentation medium includes Carbon source and nitrogen source, the carbon source are oleic acid and/or sunflower oil.

2. fermentation medium as described in claim 1, which is characterized in that the recombination Yarrowia lipolytica strain is GQY004 Or GQY007, preferably GQY007；

When the carbon source is oleic acid, the recombination Yarrowia lipolytica strain is preferably GQY004；

When the carbon source is sunflower oil, the recombination Yarrowia lipolytica strain is preferably GQY007.

3. the fermentation medium as described in right 1, which is characterized in that the content of the carbon source be 20~80g/L, preferably 20~ 40g/L。

4. fermentation medium as described in claim 1, which is characterized in that the nitrogen source includes that peptone and/or yeast extract Object, the peptone are preferably purchased from OXOID company；The yeast extract is preferably purchased from OXOID company；The fermentation medium Described in the content of nitrogen source be preferably 10~30g/L；

When the nitrogen source includes peptone, the content of the peptone is preferably 15~25g/L, more preferably 20g/L；

When the nitrogen source includes yeast extract, the content of the yeast extract is preferably 8~12g/L, more preferably 10g/L。

5. fermentation medium as claimed in claim 4, which is characterized in that the fermentation medium includes the carbon of 20~40g/L Source, the peptone of 15~25g/L and 8~12g/L yeast extract, the recombination Yarrowia lipolytica strain be GQY001, GQY004 or GQY007.

6. a kind of method of Yarrowia lipolytica production fatty-acid ethyl ester, which is characterized in that it includes the following steps, will be described heavy The seed liquor of group Yarrowia lipolytica strain is inoculated in fermentation medium fermenting and producing as claimed in any one of claims 1 to 5 Fatty-acid ethyl ester.

7. method as claimed in claim 6, which is characterized in that

The amount of the inoculation is the inoculum concentration of the fermentation medium initial OD 600=0.01；

And/or extractant is additionally added after the inoculation, before fermentation；The extractant is preferably dodecane；The use of the extractant Amount preferably 8~12%, more preferably 10%, percentage refers to the percent by volume relative to Preliminary fermentation liquid；

And/or the temperature of the fermentation is 28~32 DEG C, preferably 30 DEG C；

And/or shake culture in the fermentation process；The speed of the concussion is preferably 200~240rpm, more preferably 220rpm；

And/or ethyl alcohol is additionally added in the fermentation process；The ethyl alcohol is preferably primary every addition in 12 hours；The ethyl alcohol Additive amount preferably every time 0.5~1.5%, percentage refers to the percent by volume relative to Preliminary fermentation liquid.

8. the method for claim 7, which is characterized in that described method includes following steps: according at the beginning of fermentation medium The seed liquor of the recombination Yarrowia lipolytica strain is inoculated in the fermentation medium by the inoculum concentration of beginning OD600=0.01 In, the dodecane for being added 8~12% is used as extractant, be placed in the taper culture bottle that ferments in 28~32 DEG C of temperature in 200~ Shake culture under conditions of 240rpm, and every the ethyl alcohol of addition 0.5~1.5% in 12 hours, percentage refers to relative to first Originate the percent by volume of zymotic fluid；

Preferably, described method includes following steps:, will be described according to the inoculum concentration of fermentation medium initial OD 600=0.01 The seed liquor of recombination Yarrowia lipolytica strain is inoculated in the fermentation medium, and the dodecane for being added 10% is used as extraction Agent is placed in fermentation taper culture bottle shake culture under conditions of 30 DEG C of temperature in 220rpm, and added every 12 hours 1% ethyl alcohol, percentage refer to the percent by volume relative to Preliminary fermentation liquid.

9. such as the described in any item methods of claim 6~8, which is characterized in that the kind of the recombination Yarrowia lipolytica strain The preparation of sub- liquid includes the following steps:

(1) the recombination Yarrowia lipolytica strain is inoculated on solid medium and is activated；

(2) colony inoculation that activation obtains in step (1) is obtained into seed liquor in seed culture medium culture；

In step (1), it is preferable that egg of the formula of the solid medium including the glucose of 18~22g/L, 18~22g/L The yeast extract of white peptone and 8~12g/L；It is highly preferred that the glucose of the solid medium being formulated including 20g/L, The peptone of 20g/L and the yeast extract of 10g/L；

In step (2), it is preferable that egg of the formula of the seed culture medium including the glucose of 18~22g/L, 18~22g/L The yeast extract of white peptone and 8~12g/L；It is further preferred that the formula of the seed culture medium includes glucose, the 20g/L of 20g/L Peptone and 10g/L yeast extract.

10. method as claimed in claim 9, which is characterized in that the system of the seed liquor of the recombination Yarrowia lipolytica strain It is standby to include the following steps:

(1) the recombination Yarrowia lipolytica strain is inoculated on solid medium, in 28~32 DEG C of constant incubators, plate Culture 1~3 day, activation；

(2) by activation obtains in step (1) colony inoculation in seed culture medium, in 28~32 DEG C, 200~240rpm of revolving speed Under the conditions of shake culture 24 hours；

In step (1), the temperature of the constant temperature incubation is preferably 30 DEG C；

In step (1), the time of the plate culture is preferably 2 days；

In step (2), the temperature of the shake culture is preferably 30 DEG C；

In step (2), the revolving speed of the shake culture is preferably 220rpm.

Technical field

The present invention relates to the methods and its fermentation medium of a kind of Yarrowia lipolytica production fatty-acid ethyl ester.

Background technique

With being continuously increased for world population, we are faced with petroleum-based energy exhaustion and prevent the environmental requirement of global warming Challenge.If fossil fuel is replaced with bio-fuel, total CO₂Discharge can reduce 60-90%.Two of bio-fuel are common Form is that (biodiesel (biodiesel) refers to animal and plant fat, microbial oil, food and drink for bio-ethanol and biodiesel Waste oil etc. is the liquid fuel of feedstock oil alternative petrifaction diesel made of ester exchange process, with diesel oil intermiscibility pole It is good, and can mix or be used alone with national standard diesel oil).Due to advantage in the environment, bio-fuel be can be significantly reduced Greenhouse gases CO, CO₂And SO_XExhaust gas discharge, bio-fuel causes more and more to pay close attention to.Fatty-acid ethyl ester (FAEE) has The property similar with current gas-oil, it is thus possible to provide significant contribution to the development of the following sustainable fuel.Exploitation is used for Effectively the novel cell factory of production ethyl acetate (FAEE) and method are very important.

Currently, China has become maximum energy-consuming state in the world, China accounts for the 23% of global Energy Consumption amount.2017 " BP world energy sources statistical yearbook (2017) " (Chinese edition) display of publication in July, 2016, global verified oil reserves increased 15000000000 barrels (0.9%) to 1.7 trillion barrels, according to yield level in 2016, this enough world's yield in 50.6.Global stone Oily dosage increases powerful, amplification 1.6%, and daily dosage increases by 1,600,000 barrels, and continuous second year is higher than its ten annuals speedup. India (increasing by 300,000 barrels/day), Europe (increasing by 300,000 barrels/day) and China (increasing by 400,000 barrels/day).In addition, 2016 Nian Kezai The raw energy continues to keep most fast growth rate, renewable energy increases 12% (including wind energy, underground heat, solar energy, biomass Energy, garbage power and bio-fuel, do not include water power), this is that maximum annual increment (increases fifty-five million ton oil to work as since the dawn of human civilization Amount, the reduction amount beyond coal consumption amount).Wherein, biomass fuel increase of production in 2016 2.6% is higher than 2015 0.4%.

Up to the present, 2006, Kalscheuer et al. for the first time by one build containing ethyl alcohol production ways Plasmid imports Escherichia coli, as substrate production ethyl alcohol and can be catalyzed transesterification by glucose and oleic acid and produced FAEEs.In subsequent research, use glycerol as carbon source and external source addition oleic acid, by the way that the engineering bacteria is carried out half work The fermenting and producing of industry, makes yield reach 19g/L, this is also the maximum output obtained at present using micro-organisms FAEEs.And 0.5g/L can be reached in the highest for the FAEEs that external source addition fatty acid is produced by saccharomyces cerevisiae, since yield is lower, ferment of making wine The research of mother's production FAEEs can't be applied among industrial production at present still in the experimental study stage.

Therefore, how to improve the yield of fatty-acid ethyl ester is this field urgent problem to be solved.

Summary of the invention

Technical problem to be solved by the present invention lies in overcome to produce fatty-acid ethyl ester by yeast strain in the prior art The not high defect of yield, and provide the method and its fermentation medium of a kind of Yarrowia lipolytica production fatty-acid ethyl ester.This The method of the production fatty-acid ethyl ester of invention can greatly improve the yield of fatty-acid ethyl ester, and the industrialization for being conducive to biodiesel is answered With energy conservation and environmental protection has extraordinary application prospect.

The present invention is achieved through the following technical solutions.

The present invention provides a kind of fermentation medium of Yarrowia lipolytica production fatty-acid ethyl ester, the Yarrowia lipolytica For one of recombination Yarrowia lipolytica strain GQY001, GQY004 and GQY007 or a variety of；The fermentation medium includes Carbon source and nitrogen source, the carbon source are oleic acid and/or sunflower oil.

In the present invention, described recombination Yarrowia lipolytica strain GQY001, GQY004 and GQY007 refer to uracil and bright Propylhomoserin auxotroph Yarrowia lipolytica itself is difficult to synthesize the Yarrowia lipolytica of uracil and leucine, the present invention Middle GQY001, GQY004 and GQY007 are according to note in 107815425 A of Chinese patent CN (specification [0050]-[0070] section) The preparation method of load is made.

In the present invention, the recombination Yarrowia lipolytica strain is preferably GQY004 or GQY007, more preferably GQY007.

In the present invention, when the carbon source is oleic acid, the recombination Yarrowia lipolytica strain is preferably GQY004.

In the present invention, when the carbon source is sunflower oil, the recombination Yarrowia lipolytica strain is preferably GQY007.

In the present invention, the content of the carbon source can be the content of this field routine, preferably 20~80g/L, more preferably 20~40g/L, such as 20,40,60 or 80g/L.

In the present invention, the nitrogen source can be the nitrogen source type of this field routine, such as peptone and/or yeast extract. The peptone can be the peptone of this field routine, preferably be purchased from OXOID company.The yeast extract can be normal for this field The yeast extract of rule is preferably purchased from OXOID company.

In the present invention, the content of the nitrogen source can be the content of this field routine, such as 10~30g/L, then such as 10g/L And 20g/L.

When the nitrogen source includes peptone, the content of the peptone is preferably 15~25g/L, more preferably 20g/L.

When the nitrogen source includes yeast extract, the content of the yeast extract is preferably 8~12g/L, more preferably For 10g/L.

In the present invention, it is preferable that the fermentation medium includes that oleic acid and/or sunflower oil, peptone and yeast extract Object；It is highly preferred that the fermentation medium is made of oleic acid and/or sunflower oil, peptone and yeast extract.

In the present invention, it is preferable that the fermentation medium includes the carbon source of 20~40g/L, the nitrogen source of 10~30g/L；More Preferably, the fermentation medium is made of the carbon source of 20~40g/L, the nitrogen source of 10~30g/L.

In a preferred embodiment of the invention, the fermentation medium includes the oleic acid and/or sunflower seeds of 20~40g/L Oil, the peptone of 15~25g/L and 8~12g/L yeast extract, the recombination Yarrowia lipolytica strain be GQY001, GQY004 or GQY007.

In a preferred embodiment of the invention, the fermentation medium is grouped as by the group of following quality volume: 20~ The oleic acid and/or sunflower oil of 40g/L, the peptone of 15~25g/L and the yeast extract of 8~12g/L, the recombination solve rouge Ye Shi yeast strain is GQY001, GQY004 or GQY007.

In an of the invention preferred embodiment, the fermentation medium include 20g/L oleic acid and/or sunflower oil, The peptone of 20g/L and the yeast extract of 10g/L, the recombination Yarrowia lipolytica strain be GQY001, GQY004 or GQY007。

In a preferred embodiment of the invention, the fermentation medium is grouped as by the group of following quality volume: 20g/L Oleic acid and/or sunflower oil, the peptone of 20g/L and 10g/L yeast extract, the recombination Yarrowia lipolytica strain For GQY001, GQY004 or GQY007.

The present invention, the preparation method of the fermentation medium can be the method for this field routine, as long as by each component with water, Preferably distilled water dissolves, then with high-temperature sterilization.The condition of the high-temperature sterilization can be the condition of this field routine, example Such as 115 DEG C of sterilizing 25min.

The present invention also provides a kind of methods of Yarrowia lipolytica production fatty-acid ethyl ester comprising following steps: will The seed liquor of the recombination Yarrowia lipolytica strain is inoculated in aforesaid fermentation culture medium fermenting and producing fatty-acid ethyl ester.

When wherein, with aforementioned fermentation medium fermented and cultured recombination Yarrowia lipolytica strain production fatty-acid ethyl ester, make With conventional method, i.e., the seed liquor for recombinating Yarrowia lipolytica strain is inoculated in prior culture media, then carried out normal Rule fermentation.

The amount of the inoculation is preferably the inoculum concentration of the fermentation medium initial OD 600=0.01.

Extractant can also be added after the inoculation, before fermentation.The extractant can extract dissolution for this field routine The reagent of fatty-acid ethyl ester, preferably dodecane.The dosage of the extractant can be this field routine dosage, preferably 8~ 12%, more preferably 10%, percentage refers to the percent by volume relative to Preliminary fermentation liquid.

The temperature of the fermentation can be the temperature of this field routine, preferably 28~32 DEG C, more preferably 30 DEG C.

The fermentation process can routinely carry out shake culture by this field.The speed of the concussion can be this field routine Speed, preferably 200~240rpm, more preferably 220rpm.

Ethyl alcohol can also be added in the fermentation process.The ethyl alcohol can be primary every addition in 12 hours.The ethyl alcohol adds Dosage can be every time 0.5~1.5%, such as 1%, and percentage refers to the percent by volume relative to Preliminary fermentation liquid.

Preferably, described method includes following steps:, will according to the inoculum concentration of fermentation medium initial OD 600=0.01 The seed liquor of the recombination Yarrowia lipolytica strain is inoculated in the fermentation medium, and the dodecane for being added 8~12% is made For extractant, shake culture under conditions of 28~32 DEG C of temperature in 200~240rpm is placed in fermentation taper culture bottle, and Every the ethyl alcohol of addition 0.5~1.5% in 12 hours, percentage refers to the percent by volume relative to Preliminary fermentation liquid.

It is highly preferred that described method includes following steps: according to the inoculum concentration of fermentation medium initial OD 600=0.01, The seed liquor of the recombination Yarrowia lipolytica strain is inoculated in the fermentation medium, 10% dodecane conduct is added Extractant was placed in fermentation taper culture bottle shake culture under conditions of 30 DEG C of temperature in 220rpm, and every 12 hours The ethyl alcohol of addition 1%, percentage refer to the percent by volume relative to Preliminary fermentation liquid.

As known to those skilled in the art, rouge is being produced with the seed liquor fermented and cultured of the recombination Yarrowia lipolytica strain Before fat acetoacetic ester, the seed liquor of recombination Yarrowia lipolytica strain can be prepared according to this field routine operation, it is general to wrap Include following steps:

(1) the recombination Yarrowia lipolytica strain is inoculated on solid medium and is activated；

(2) colony inoculation that activation obtains in step (1) is obtained into seed liquor in seed culture medium culture.

Wherein, in step (1), it is preferable that the solid medium formula including 18~22g/L glucose, 18~ The peptone of 22g/L and the yeast extract of 8~12g/L；It is highly preferred that the formula of the solid medium includes 20g/L's The yeast extract of glucose, the peptone of 20g/L and 10g/L.

Wherein, in step (1), it is preferable that the formula of the solid medium is grouped as by the group of following quality volume: 18 The yeast extract of the glucose of~22g/L, the peptone of 18~22g/L and 8~12g/L；It is highly preferred that the solid culture The formula of base is grouped as by the group of following quality volume: the yeast of the glucose of 20g/L, the peptone of 20g/L and 10g/L extracts Object.

Wherein, in step (2), it is preferable that the seed culture medium formula including 18~22g/L glucose, 18~ The peptone of 22g/L and the yeast extract of 8~12g/L；It is highly preferred that the formula of the seed culture medium includes 20g/L's The yeast extract of glucose, the peptone of 20g/L and 10g/L.

Wherein, in step (2), it is preferable that the formula of the seed culture medium is grouped as by the group of following quality volume: 18 The yeast extract of the glucose of~22g/L, the peptone of 18~22g/L and 8~12g/L；It is highly preferred that the seed culture The formula of base is grouped as by the group of following quality volume: the yeast of the glucose of 20g/L, the peptone of 20g/L and 10g/L extracts Object.

Preferably, the preparation of the seed liquor of the recombination Yarrowia lipolytica strain includes the following steps:

(1) the recombination Yarrowia lipolytica strain is inoculated on solid medium, in 28~32 DEG C of constant incubators, Plate culture 1~3 day, activation；

(2) by activation obtains in step (1) colony inoculation in seed culture medium, in 28~32 DEG C, revolving speed 200~ Shake culture 24 hours under conditions of 240rpm.

Wherein, in step (1), the temperature of the constant temperature incubation is preferably 30 DEG C.

Wherein, in step (1), the time of the plate culture is preferably 2 days.

Wherein, in step (2), the temperature of the shake culture is preferably 30 DEG C.

Wherein, in step (2), the revolving speed of the shake culture is preferably 220rpm.

Those skilled in the art know, further preferably include the step of harvest fatty-acid ethyl ester after fermenting and producing fatty-acid ethyl ester Suddenly.The method that the harvest fatty-acid ethyl ester uses generally is fermented after 72h, takes fermentation liquid upper layer, organic phase is taken after centrifugation i.e. .

Wherein, the means that this field routine can be used in the content of the fatty-acid ethyl ester are detected, such as gas-chromatography Method.

The step of those skilled in the art know, may also include harvest thallus after fermenting and producing fatty-acid ethyl ester, it is described The method that fatty-acid ethyl ester uses is harvested generally to be centrifuged bacterium solution, is dry.

Wherein, the condition of the centrifugation is preferably 12,000rpm centrifugation 5min.

Wherein, the condition of the drying is preferably and dries in 105 DEG C to constant weight.

Wherein it is preferred to be washed with deionized 2-3 times after the centrifugation.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is that:

(1) present invention in recombination Yarrowia lipolytica strain GQY001, GQY004 and GQY007 using different carbon source into Row culture, comparison yield it is found that when wherein using oleic acid as carbon source, the yield of fatty-acid ethyl ester up to 9.2~11.8g/L, It is to use glucose as many as 10 times of carbon source；When wherein using sunflower oil as carbon source, the yield of fatty-acid ethyl ester can Up to nearly 13.5~19.1g/L, as many as 20 times of glucose as carbon source are reached as high as.In addition, fatty-acid ethyl ester produced Component is concentrated mainly on C18:1 and C18:2, and unsaturated fatty acid ethyl ester proportion is more to can reduce the molten of fatty-acid ethyl ester Point is convenient for industrial application, and fatty acid carbon chain is longer, and fatty-acid ethyl ester production capacity is higher under equimolar.

(2) present invention use reproducible vegetable oil as carbon source can mass production biodiesel fatty acid ethyl ester, this general It is reduced using the cost of microbial method production biodiesel fatty acid ethyl ester, can be applied to commercially produce, be conducive to biology The industrial application of diesel oil, energy conservation and environmental protection have extraordinary application prospect.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

In following embodiments, Yarrowia lipolytica strain GQY001, GQY004 and GQY007 is recombinated according to Chinese patent CN The preparation method recorded in 107815425 A (patent No. 201610818867.7) is made.

In following embodiments, peptone and yeast extract are purchased from OXOID company.