CN117646039A - Method for biosynthesis of isoamyl alcohol by using D-glucose as substrate - Google Patents
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Abstract
The invention provides a method for biosynthesizing isoamyl alcohol by using D-glucose as a substrate. Relates to the field of biochemical engineering, and is characterized in that yarrowia lipolytica Po1g strain is taken as a chassis cell, and the Harris pathway is optimized by screening enzymes with different sources, and the isoamyl alcohol is biosynthesized by using D-glucose in a YPD culture medium. The Harris pathway involves alcohol dehydrogenase (EC 1.1.1. -) and ketoacid decarboxylase (EC 4.1.1.1), and the 2 enzymes are used to construct a co-expression plasmid to construct isoamyl alcohol producing engineering bacteria Po1g/pYLXP' -kivd-yqhd. The result shows that the strain Po1g/pYLXP' -kivd-yqhd can catalyze 2g/L of alpha-ketoisocaproic acid as a substrate to synthesize 51.9mg/L of isoamyl alcohol in an SD-leu culture medium; 0.312mg/L isoamyl alcohol was biosynthesized in YPD medium with 40g/L D-glucose as substrate, 1.53 times the productivity of isoamyl alcohol from Po1g/pYLXP' strain. The invention relates to a method for improving the yield of biosynthesized isoamyl alcohol by optimizing the Harris way of yarrowia lipolytica Po1g strain, and further genetic circuit and fermentation process optimization is expected to greatly improve the yield of isoamyl alcohol.
Description
Technical Field
The invention relates to the field of biochemical engineering, in particular to a method for biosynthesizing isoamyl alcohol by using D-glucose as a substrate.
Background
Isoamyl alcohol, also known as 3-methyl-1-butanol, having the molecular formula C 5 H 12 O, molecular weight 88.148, structural formula (CH 3) 2 CHCH 2 CH 2 OH. The melting point of isoamyl alcohol is-117℃and the boiling point is 131.2 ℃. Isoamyl alcohol is colorless liquid at normal temperature and pressure, has fusel oil taste, is slightly soluble in water, is miscible in diethyl ether, benzene, diethyl ether, chloroform and petroleum ether, is easy to dissolve in acetone, and is soluble in most organic solvents. Isoamyl alcohol is widely used in the fields of liquid fuels, edible flavors, daily essential oils, and the like.
Currently, there are many methods for controlling the flow of liquid. The biosynthesis of isoamyl alcohol is focused on the use of microorganisms such as Corynebacterium glutamicum, escherichia coli and Saccharomyces cerevisiae as chassis cells, and the Ehrlich pathway is used to convert leucine into isoamyl alcohol. However, the related microorganisms have low tolerance to isoamyl alcohol, so that engineering bacteria are difficult to apply to industrial scale production, and yarrowia lipolytica (Yarrowia lipolytica) has strong stress resistance compared with microorganisms such as Saccharomyces cerevisiae and the like, and can adapt to various severe environments such as low temperature, high salt, low pH and the like; and the substrate has a broad spectrum, and can utilize various hydrophobic and hydrophilic carbon sources, such as glucose, glycerol, ethanol, alkanes, waste edible oil and the like. Thus, the strong stress resistance of yarrowia lipolytica confers the potential for high isoamyl alcohol production.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for optimizing Harris pathway of yarrowia lipolytica Po1g strain and synthesizing isoamyl alcohol using D-glucose as substrate, wherein dehydrogenase (EC 1.1.1. -) and ketoacid decarboxylase (EC 4.1.1.1) from different sources are selected to construct coexpression plasmid, and engineering bacteria optimized for Harris pathway are constructed, thereby improving the efficiency of synthesizing isoamyl alcohol using D-glucose.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for synthesizing isoamyl alcohol by using D-glucose, which takes yarrowia lipolytica as a chassis cell to construct engineering bacteria and synthesize the isoamyl alcohol by using the D-glucose.
Alternatively, the yarrowia lipolytica is yarrowia lipolytica Po1g.
Optionally, the method for screening and synthesizing isoamyl alcohol strains is as follows:
(1) And (5) dehydrogenase screening. The above-mentioned dehydrogenase encoding genes of different origins (YPL 088W, SEQ ID NO: 1), (yiypl 088W, SEQ ID NO: 2), (yqhD, SEQ ID NO: 3), (ScADH 2, SEQ ID NO: 4), (ScADH 7, SEQ ID NO: 5), (ScADH 4, SEQ ID NO: 6), (ScADH 6, SEQ ID NO: 7), (ScADH 1, SEQ ID NO: 8), (ScADH 3, SEQ ID NO: 9), (ScADH 5, SEQ ID NO: 10), (YI2_01, SEQ ID NO: 11), (YIADH 2_02, SEQ ID NO: 12), (YIADH 2_03, SEQ ID NO: 13), (YIADH 2_04, SEQ ID NO: 14), (YIADH 3, SEQ ID NO: 15) was cloned into the SnaBI cleavage site of the vector pYLXP 'using one of the primers see Table 1, and the recombinant plasmid PYP 1g was transformed to obtain the engineering strain Pp 1 g/pXP' -ScP2, pp 1g '-PYP 1, PYP 2' -PYP 3, YPYP 2 '-PYP 3, YP 2' -PYP 3/PYP 1, YP 2 '-PYP 1/PYP 2' -PYP 3, YP 3/PYP 2 '-PYP 1/PYP 2' -PYP 3, YPYP 1/PYP 2 '-PYPYP 2/PYP 3, YP 1/PYP 2' -PYPYP 3/3, YP 3/PYP 2 '-PYPYP 3/PYP 3, YP 3' -PYP 3, YP 3, and YP-PYP 3, XYPYP 3, XYP 3, and PYP 3, XYP 3 XYP, XYP 3 XYP, PX, XYP 3, PX 3 X3 XYP 3 X3, PX 3 X3 3 X3 3, PX, P3 3/PYP-3-3, the 2 strains with highest isoamyl alcohol synthesizing capacity are Po1g/pYLXP '-ScADH2 and Po1g/pYLXP' -yqhD.
(2) Screening of ketoacid decarboxylase. The above-mentioned various sources of ketoacid decarboxylase encoding genes (YLPDC 1, SEQ ID NO: 16), (Kivd, SEQ ID NO: 17), (GoKDC, SEQ ID NO: 18) were ligated between NheI/SalI cleavage sites of the vector pYLXP '-ADH2/pYLXP' -yqhD, respectively, using T4 ligase (Nanjinouzan Biotech Co., ltd.) to obtain 6 recombinant plasmids pYLXP '-Kivd-ADH2, pYLXP' -Kivd-yqhD, pYLXP '-GOKDC-ADH2, pYLXP' -GOKDC-yqhD, pYLXP '-PDC 1-ADH2, pYLXP' -YLXP 1-yqhD (see FIG. 2). The recombinant plasmid is transformed into yarrowia lipolytica Po1g to obtain 6 recombinant yarrowia lipolytica strains: po1g/pYLXP '-Kivd-ADH2, po1g/pYLXP' -Kivd-yqhD, po1g/pYLXP '-GOKDC-ADH2, po1g/pYLXP' -GOKDC-yqhD, po1g/pYLXP '-YLPDC1-ADH2, po1g/pYLXP' -YLPDC1-yqhD.
Alternatively, the expression regulation of the pathway coding genes is controlled by a TEF promoter and an XPR2t terminator.
Alternatively, the conditions for converting α -ketoisocaproic acid to isoamyl alcohol are: the cells were collected during growth to the index by transferring 2% (v/v) of the inoculum size into 25mL of fresh SD-leu C/N80 medium after 2d of activation at 30℃and 200rpm of SD-leu medium. The fermented culture broth was centrifuged at 5000rpm at 4℃to collect the cells. Then, the cells were resuspended in 100mM phosphate buffer, pH6, to give resting cells. And by exogenously adding 2g/L alpha-ketoisocaproic acid, 5mM MgCl 2 The formation of isoamyl alcohol was detected by GC using 1.5mM thiamine diphosphate (ThDP) as substrate.
(3) Engineering bacteria with obviously enhanced isoamyl alcohol synthesis efficiency: po1g/pYLXP' -Kivd-yqhD recombinant strain. The conditions for producing isoamyl alcohol by using D-glucose are as follows: the cells were cultured at 30℃and 200rpm in SD-leu medium for 2d activation, transferred to 25mL of YPD (C/N80, 40g/L D-glucose) medium at 2% (v/v) and reacted at 30℃for 3d, and the production of isoamyl alcohol was detected by GC.
Optionally, the concentration of the substrate D-glucose is 40g/L.
Alternatively, the conversion conditions are 30℃for 3d.
Compared with the prior art, the invention has the following beneficial effects:
1: the Harris pathway of yarrowia lipolytica Po1g strain is optimized through screening various dehydrogenases and ketoacid decarboxylases commonly used in research, and guidance is provided for the research and development of yarrowia lipolytica Po1g strain in isoamyl alcohol production.
2. The engineering bacterium Po1g/pYLXP' -kivd-yqhd can catalyze 2g/L of alpha-ketoisocaproic acid in an SD-leu culture medium to be used as a substrate for biosynthesis of 51.9mg/L isoamyl alcohol; 0.312mg/L isoamyl alcohol was biosynthesized in YPD medium with 40g/L D-glucose as substrate, 1.53 times the productivity of isoamyl alcohol from Po1g/pYLXP' strain.
Drawings
FIG. 1. Schematic diagram of the synthetic pathway for the production of isoamyl alcohol using D-glucose as substrate. The pathway consists of leucine metabolism, dehydrogenase and ketoacid decarboxylase.
FIG. 2A schematic diagram of the selected Harris pathway assembly. The 3 ketoacid decarboxylases and the 2 dehydrogenases were combined on pYLXP ' plasmids, respectively, pYLXP ' -Kivd-ADH2, pYLXP ' -Kivd-yqhD, pYLXP ' -GOKDC-ADH2, pYLXP ' -GOKDC-yqhD, pYLXP ' -YLPDC1-ADH2, pYLXP ' -YLPDC1-yqhD recombinant plasmid map, respectively, by T4 ligation. The expression of the genes on the recombinant plasmid is controlled by pTEF promoter and XPR2t terminator, and the genes are connected in series in the form of monocistronic to form the conversion path from alpha-ketoisocaproic acid to isoamyl alcohol.
FIG. 3 effects of different dehydrogenases on the conversion efficiency of isovaleraldehyde. The dehydrogenases from different sources are screened by measuring the yield of isoamyl alcohol from isovaleraldehyde catalyzed by yarrowia lipolytica Po1g engineering bacteria.
FIG. 4 Harris pathway functional validation of yarrowia lipolytica Po1g engineering bacteria. (a) Gas chromatograms of alpha-ketoisocaproic acid as substrate to isoamyl alcohol. (b) Quantitative analysis results of the conversion of alpha-ketoisocaproic acid as substrate into isoamyl alcohol.
FIG. 5 results of a gas chromatography quantitative analysis of yarrowia lipolytica Po1g engineering bacteria for the production of isoamyl alcohol using D-glucose.
Detailed description of the preferred embodiments
The invention provides an optimized method for synthesizing isoamyl alcohol from head by taking D-glucose as a substrate by using a Harris pathway, wherein the optimized method for synthesizing isoamyl alcohol by taking D-glucose as the substrate can be used for producing isoamyl alcohol by constructing yarrowia lipolytica Po1g engineering bacteria.
The method for producing isoamyl alcohol using D-glucose as a substrate and the application thereof provided in the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Detailed description of the preferred embodiments
Example 1: screening of alcohol dehydrogenase
The dehydrogenase-encoding genes of different origins (YPL 088W, SEQ ID NO: 1), (YIPL 088W, SEQ ID NO: 2), (yqhD, SEQ ID NO: 3), (ScADH 2, SEQ ID NO: 4), (ScADH 7, SEQ ID NO: 5), (ScADH 4, SEQ ID NO: 6), (ScADH 6, SEQ ID NO: 7), (ScADH 1, SEQ ID NO: 8), (ScADH 3, SEQ ID NO: 9), (ScADH 5, SEQ ID NO: 10), (YIADH 2_01, SEQ ID NO: 11), (YIADH 2_02, SEQ ID NO: 12), (YIADH 2_03, SEQ ID NO: 13), (YIADH 2_04, SEQ ID NO: 14), (YIADH 3 SEQ ID NO: 15) were amplified by PCR techniques (see Table 1 for primers). The dehydrogenases were assembled onto the expression vector plasmid pYLXP' using ClonExp one-step cloning kit (Nanjinouzan Biotechnology Co., ltd.) to obtain recombinant plasmids, respectively. The recombinant plasmid was transformed into E.coli JM109, and colony PCR was performed using a universal primer pair (TEF_F2/XPR2_seq) to verify the recombinant results. And the plasmid was subjected to Sanger sequencing to confirm successful construction of the recombinant plasmid. And (3) transforming the recombinant plasmid into 1g of yarrowia lipolytica Po to obtain 16 engineering bacteria. Expression of all genes was controlled by the pTEF-strong promoter and XPR2t terminator. Yarrowia lipolytica strains harboring the pYLXP' plasmid were used as a blank and strains harboring other recombinant plasmids were experimental groups.
The cells were activated by culturing at 30℃and 200rpm for 2 days, inoculated in 25mL of SD-leu (C/N=80) medium at 2% (v/v), and collected by centrifugation (4℃at 5000rpm for 8 min) at 200rpm for 2 days. Then, the cells were resuspended in 100mM phosphate buffer (pH=6), and 2g/L of isovaleraldehyde was added as a substrate, and the cells were transformed at 30℃and 200rpm for 4 hours, and isoamyl alcohol was extracted by using an equal volume of ethyl acetate, and the amount of isoamyl alcohol produced was detected by a gas chromatograph to screen the conversion efficiency of isovaleraldehyde after expression of dehydrogenases of different origins in yarrowia lipolytica Po1g.
The method for analyzing isoamyl alcohol by gas chromatography is as follows: the gas chromatograph model is Beijing Agilent gas chromatograph, the chromatographic column model is DB-1 (30 m multiplied by 0.25mm multiplied by 0.20 mu m), the detector type FID, the sample inlet temperature is 230 ℃, the transmission line temperature is 280 ℃, the sample injection amount is 1 mu L, the split ratio is 20:1, the carrier gas flow rate is 0.5mL/min, the programmed temperature rise is up to 50 ℃, and the temperature is kept for 5min. Heating to 160 ℃ at 10 ℃ per minute, and keeping for 2 minutes. Heating to 220 ℃ at 60 ℃ per minute, and keeping for 10 minutes. The post-column temperature was 150 ℃.
The gas chromatography detection result shows that the conversion value of Po1g/pYLXP' -yqhD to isoamyl alcohol in engineering bacteria is highest and reaches 952mg/L; po1g/pYLXP' -ScADH2 times, the isoamyl alcohol conversion value can reach 862mg/L (as shown in FIG. 3).
Thus, the pYLXP '-yqhD, pYLXP' -SCADH2 was used to construct the subsequent Harris synthesis pathway.
TABLE 1 primers used in the present invention
Example 2: harris pathway assembly and functional verification
The various sources of the genes encoding the ketoacid decarboxylases (YLPDC 1, SEQ ID NO: 16), (Kivd, SEQ ID NO: 17), (GoKDC, SEQ ID NO: 18) were amplified by PCR techniques (primers see Table 1). Respectively using ClonExpress one-step cloning kit (NanjinoxPraise biosciences limited) assemble the coding gene of the ketoacid decarboxylase onto the expression vector plasmid pYLXP 'to obtain 3 recombinant plasmids pYLXP' -YLPDC1, pYLXP '-Kivd, pYLXP' -GoKDC. The plasmid pYLXP' -Kivd was cleaved with NheI/SalI and the 9091bp fragment was recovered in gel. The plasmids pYLXP '-ADH2 and pYLXP' -yqhD were cut with AvrII/SalI, fragments of 2096bp and 2213bp were recovered by gel, and the recombinant plasmids pYLXP '-Kivd-ADH2 and pYLXP' -Kivd-yqhD were obtained by T4 ligation. The plasmid pYLXP ' -GOKDC is digested by ClaI/AvrII, after 8927bp fragments are recovered by glue, the plasmids pYLXP ' -ADH2 and pYLXP ' -yqhD are digested by ClaI/NheI, fragments of about 2035bp and 2422bp are recovered by glue, and 4 recombinant plasmids pYLXP ' -GOKDC-ADH2 and pYLXP ' -GOKDC-yqhD are obtained by T4 ligation. The plasmid pYLXP ' -YLPDC1 is digested by NheI/ClaI, a fragment of about 2950bp is recovered by gel, plasmids pYLXP ' -ADH2 and pYLXP ' -yqhD are digested by ClaI/AvrII, fragments of 8282bp and 8399bp are recovered by gel, and the T4 is connected to obtain 2 recombinant plasmids pYLXP ' -YLPDC1-ADH2 and pYLXP ' -YLPDC1-yqhD. And (3) transforming the recombinant plasmid into yarrowia lipolytica strain Po1g to obtain 6 recombinant yarrowia lipolytica strains. The cells were activated by culturing at 30℃and 200rpm for 2 days, inoculated in 25mL of SD-leu (C/N=80) medium at 2% (v/v), and collected by centrifugation (5000 rpm, 8 min) at 200rpm for 2 days at 30 ℃. The cells were then resuspended using 100mM phosphate buffer (pH=6) and 2g/L alpha-ketoisocaproic acid, 5mM MgCl were added 2 1.5mM thiamine diphosphate (ThDP) is used as a substrate, the substrate is converted for 4 hours at 30 ℃ and 200rpm, isoamyl alcohol is extracted by using equal volume of ethyl acetate, and the yield of the conversion of alpha-ketoisohexanoic acid into isoamyl alcohol is detected by using a gas chromatograph, so that the optimal recombinant Harris pathway in yarrowia lipolytica Po1g engineering bacteria is screened.
The gas chromatography analysis result shows that the peak time of isoamyl alcohol is 3.163min. The highest amount of isoamyl alcohol produced by Po1g/pYLXP' -kivd-yqhD in engineering bacteria reaches 51.9mg/L (shown in figure 4).
Example 3: analysis and identification of de novo isoamyl alcohol synthesis using glucose as substrate
The engineering bacteria Po1g/pYLXP '-Kivd-yqhD and Po1g/pYLXP' are inoculated in SD-leu liquid culture medium, and are cultured for 2d to activate under the conditions of 30 ℃ and 200rpm, and are transferred into 25mL YPD (C/N80, 40g/L D-glucose) culture medium with the inoculum size of 2% (v/v), reacted for 3d at 30 ℃, the isoamyl alcohol is extracted by using equal volume of ethyl acetate, and the production amount of the isoamyl alcohol is detected by using a gas chromatograph.
The gas chromatographic analysis result shows that the yield of isoamyl alcohol of the engineering bacteria Po1g/pYLXP '-yqhD, which is reacted for 3D at 30 ℃ in YPD culture medium by using 40g/L of D-glucose as a substrate, is 0.312mg/L, and is 1.53 times that of the strain Po1g/pYLXP' (as shown in figure 5).
The foregoing is merely an alternative embodiment of the invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the principles of the invention, and such modifications and variations should also be considered as being within the scope of the invention.
Claims (9)
1. A method for biosynthesizing isoamyl alcohol by using glucose as substrate. The method is characterized in that alpha-ketoisohexanoic acid is used as a substrate, yarrowia lipolytica is used as a chassis cell, and dehydrogenases (EC 1.1.1. -) and ketoacid decarboxylases (EC 4.1.1.1) from different sources are screened to optimize the Harris pathway, namely the efficiency of catalyzing the conversion of alpha-ketoisohexanoic acid into isoamyl alcohol, so as to obtain engineering bacteria for synthesizing the isoamyl alcohol by taking glucose as the substrate (the reaction process is shown in figure 1).
2. The dehydrogenase (EC 1.1.1. -) according to claim 1, specifically comprising: (1) alcohol dehydrogenases of different origins (ADH, EC 1.1.1.1); (2) Saccharomyces cerevisiae BY 4741-derived aldehyde-ketone reductase-encoding gene (YPL 088W, SEQ ID NO: 1); (3) Yarrowia lipolytica Po1g of a source aldehyde-ketone reductase-encoding gene (YlYPL 088W, SEQ ID NO: 2); (4) Escherichia coli JM109 NADPH-dependent aldehyde reductase-encoding gene of 109 origin (yqhD, EC 1.1.1.2,SEQ ID NO:3).
3. The alcohol dehydrogenase (ADH, EC 1.1.1.1) according to claim 1, claim 2. Mainly comprises the following steps: (1) ADH encoding genes derived from Saccharomyces cerevisiae BY4741 (ScADH 2, SEQ ID NO: 4), (ScADH 7, SEQ ID NO: 5), (ScADH 4, SEQ ID NO: 6), (ScADH 6, SEQ ID NO: 7), (ScADH 1, SEQ ID NO: 8), (ScADH 3, SEQ ID NO: 9) and (ScADH 5, SEQ ID NO: 10); (2) An ADH encoding gene derived from Yarrowia lipolytica Po g (YIdH2_01, SEQ ID NO: 11), (YIdH2_02, SEQ ID NO: 12), (YIdH2_03, SEQ ID NO: 13), (YIdH2_04, SEQ ID NO: 14), (YIdH 3, SEQ ID NO: 15).
4. The ketoacid decarboxylase of different origin (EC 4.1.1.1) according to claim 1. The main sources include: (1) Yarrowia lipolytica Po1g of a pyruvate decarboxylase encoding gene (YLPDC 1, SEQ ID NO: 16); (2) Lactococcus Lactis (Kivd, SEQ ID NO: 17); (3) Derived from Gluconobacter oxydans (GoKDC, SEQ ID NO: 18).
5. The optimized Harris pathway according to claim 1, wherein plasmid pYLXP' is used as an expression vector for the transformation pathway, and a recombinant plasmid containing all pathway genes is constructed.
6. According to claim 1 and claim 5, the recombinant plasmid is transformed into yarrowia lipolytica Po1g by taking yarrowia lipolytica Po1g as an expression host, so as to obtain the engineering strain containing all the path genes.
7. The optimized Harris pathway according to claim 1 and the recombinant engineering strain according to claim 6, characterized in that the transformation conditions used are as follows: (1) The cells were cultured at 30℃and 200rpm for 2d activation, transferred to 25mL of fresh SD-leu (C/N=80) medium at an inoculum size of 2% (v/v), and collected by centrifugation for 2-3 d. (2) Centrifuging at 4deg.C and 5000rpm for 8min to collect thallus, and re-suspending thallus with 100mM phosphate buffer solution with pH of 6 to obtain resting cell. (3) 2g/L of alpha-ketoisocaproic acid or isovaleraldehyde is respectively added as a substrate, and the mixture is converted for 4 hours at 30 ℃ and 200 rpm; (4) detecting the formation of isoamyl alcohol using a gas chromatograph.
8. Isoamyl alcohol synthesis method according to claim 1 and recombinant engineering strain according to claim 5, characterized in that the transformation conditions used are as follows: (1) Culturing at 30deg.C and 200rpm for 2d for activation, transferring into 25mL YPD (C/N=80) medium containing 40g/L glucose with 2% (v/v) inoculation amount, and reacting at 30deg.C for 3d; (2) the formation of isoamyl alcohol was detected using a gas chromatograph.
9. The isoamyl alcohol synthesis method according to claim 1, claim 7, and claim 8. The method is characterized in that the screening process is as follows: (1) Screening engineering bacteria containing alcohol dehydrogenase coding genes from different sources, wherein the result can catalyze the isovaleraldehyde to synthesize the isoamyl alcohol with the highest enzyme activity of Po1g/pYLXP' -yqhD; (2) Screening strains containing alcohol dehydrogenase encoding genes (yqhD and GoKDC) and serially connected with ketoacid decarboxylase encoding genes from different sources, wherein the strain with the highest activity for catalyzing alpha-ketoisohexanoic acid to synthesize isoamyl alcohol is Po1g/pYLXP' -Kivd-yqhD; (3) The engineering bacteria Po1g/pYLXP '-yqhD takes 40g/L of D-glucose as a substrate in YPD culture medium, the isoamyl alcohol yield of 3D reaction at 30 ℃ is 0.312mg/L, and the yield is 1.53 times of that of the Po1g/pYLXP' strain.
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