Method for improving yield of isoamyl alcohol produced by yarrowia lipolytica through mitochondrial zoning

Abstract

The invention relates to a method for improving the yield of isoamyl alcohol in yarrowia lipolytica by utilizing mitochondrial zoning, which comprises the following steps: by taking yarrowia lipolytica Po1f as an original strain, positioning a part of enzymes in an isoamyl alcohol pathway in the zoning of the yarrowia lipolytica by using a mitochondrial positioning tag to form a relatively independent isoamyl alcohol synthesis pathway; meanwhile, precursor substances required by isoamyl alcohol synthesis are enriched, in the same chamber, the conversion efficiency between enzymes is improved, the loss of an action substrate is reduced, and finally, in an SD-Leu-Ura culture medium, the yield of isoamyl alcohol is increased by 88.72% and reaches 0.412 mg / L.

Description

Technical Field

[0001] The invention relates to the field of biochemical engineering, and in particular to a method for increasing the yield of isoamyl alcohol in Yarrowia lipolytica by utilizing protease in mitochondrial compartmentalized expression engineering bacteria. Background Art

[0002] Isoamyl alcohol, also known as 3-methyl-1-butanol, has a molecular formula of C5H 12 O, molecular weight 88.148, structural formula (CH3)2CHCH2CH2OH. The melting point of isoamyl alcohol is -117°C, and the boiling point is 131.2°C. At room temperature and pressure, isoamyl alcohol is a colorless liquid with a fusel oil smell. It is slightly soluble in water, miscible in ether, benzene, ether, chloroform, petroleum ether, easily soluble in acetone, and soluble in most organic solvents. Isoamyl alcohol is widely used in liquid fuels, edible flavors, daily essential oils and other fields.

[0003] Common methods of synthesizing isopentanol include chemical synthesis, photosynthesis and biosynthesis. The main production method in the market is chemical synthesis. However, traditional chemical synthesis has many disadvantages, such as complex production equipment, high temperature, high pressure and catalyst reaction conditions, low raw material utilization, dependence on fossil fuels, and easy to cause environmental pollution. With the development of synthetic biology and genetic engineering technology, people have begun to combine synthetic biology and genetic engineering technology to achieve the synthesis of long-chain alcohols. Biosynthesis has broad application prospects due to its mild reaction conditions, simple process and environmental friendliness.

[0004] Currently, the biosynthesis of isopentanol is concentrated on microorganisms such as Corynebacterium glutamicum, Escherichia coli, and Saccharomyces cerevisiae as chassis cells, using the Ehrlich pathway to convert leucine into isopentanol. However, the tolerance of related microorganisms to isopentanol is low, which makes it difficult to apply engineered bacteria to industrial-scale production. Compared with microorganisms such as Saccharomyces cerevisiae, Yarrowia lipolytica has strong stress resistance and can adapt to a variety of harsh environments. Therefore, the strong stress resistance of Yarrowia lipolytica gives it the potential to produce high levels of isopentanol.

[0005] Existing technologies for synthesizing isopentanol from scratch mainly focus on enhancing the Harris pathway and exogenously introducing ketoacid decarboxylase and alcohol dehydrogenase to achieve the accumulation of isopentanol in Yarrowia lipolytica. However, the isopentanol synthesis pathway in Yarrowia lipolytica is long, and the key enzymes in the pathway are distributed in different organelles. The entire synthesis process will result in low enzyme-to-enzyme conversion efficiency, diffusion loss of substrates before the reaction, and feedback inhibition of the early synthesis pathway by the accumulation of intermediates. These prominent problems have made it difficult to achieve a significant increase in the production of isopentanol in Yarrowia lipolytica. Summary of the invention

[0006] In view of this, the present invention locates the enzymes in the isopentanol synthesis pathway in the mitochondria through mitochondrial localization protein tags, so that the isopentanol that should be synthesized in the cytoplasm can also be synthesized in the mitochondrial compartment, thereby improving the conversion efficiency of the substrate in the metabolic pathway, increasing the storage space of the final product, and reducing feedback inhibition in the metabolic pathway. Ultimately, this method achieves an isopentanol yield of 0.412 mg / L in Yarrowia lipolytica.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The object of the present invention is to provide a method for increasing the production of isopentanol in Yarrowia lipolytica by compartmentalization, characterized in that the method comprises expressing endogenous α-isopropylmalate dehydrogenase (Leu1, YALI1_B02345g, Protein ID: CAB99455.1), β-isopropylmalate dehydrogenase (Leu2, YALI1_C00464g, Protein ID: AAF70314.1) α-isopropylmalate synthase (Leu4, YALI0_B07447g, Protein ID: CAG82839.1) and heterologous α-ketoacid decarboxylase (Kivd, EC 4.1.1.1, Protein ID: CAG34226.1) and alcohol dehydrogenase (yqhD, EC 1.1.1.2, Protein ID: CAG82839.1) in Yarrowia lipolytica, respectively. ID: NP_417484.1), and the first 26 amino acid sequences of Saccharomyces cerevisiae cytochrome 4 (COX4) were used as mitochondrial localization tags to localize some enzymes in the isopentanol biosynthesis pathway to the mitochondria for expression.

[0009] Furthermore, the mitochondrial localization signal peptide MLS, the first 26 amino acid sequence of Saccharomyces cerevisiae cytochrome 4 (COX4), is fused and expressed at the N-terminus of α-isopropylmalate dehydrogenase (Leu1, SEQ ID NO.1), β-isopropylmalate dehydrogenase (Leu2, SEQ ID NO.2), α-isopropylmalate synthase (Leu4, SEQ ID NO.3), α-ketoacid decarboxylase (Kivd, SEQ ID NO.4), and alcohol dehydrogenase (yqhD, SEQ ID NO.5) to form a new fusion protein.

[0010] Furthermore, the yeast host is Yarrowia lipolytica Po1f.

[0011] Furthermore, the expression regulation of the pathway encoding genes is controlled by the TEF promoter and the XPR2t terminator.

[0012] Furthermore, the Yarrowia lipolytica engineered bacteria uses the mitochondrial localization signal peptide MLS to express α-ketoacid decarboxylase and alcohol dehydrogenase and α-isopropylmalate dehydrogenase, β-isopropylmalate dehydrogenase and α-isopropylmalate synthase in the Harris pathway in the mitochondria of the yeast host.

[0013] By means of the above scheme, the present invention has at least the following advantages:

[0014] The present invention uses Yarrowia lipolytica as a starting strain. Through our invention, α-ketoacid decarboxylase and alcohol dehydrogenase in the isoamyl alcohol synthesis pathway and α-isopropylmalate dehydrogenase, β-isopropylmalate dehydrogenase and α-isopropylmalate synthase in the Harris pathway are located in the mitochondrial compartment of Yarrowia lipolytica using mitochondrial localization tags, forming a relatively independent isoamyl alcohol synthesis pathway, while also increasing the storage space for precursor substances required for isoamyl alcohol synthesis and reducing feedback effects. In the same compartment, the conversion efficiency between enzymes is improved, the loss of substrates is reduced, and ultimately the isoamyl alcohol yield is increased to 0.412 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 . Schematic diagram of the compartmentalization of mitochondria in the production of isopentanol in Yarrowia lipolytica.

[0016] Figure 2 .Growth curve of engineered bacteria.

[0017] Figure 3 .Sugar consumption of engineered bacteria.

[0018] Figure 4 .Gas chromatography quantitative analysis results of isopentanol produced by engineered bacteria. Specific implementation methods

[0019] Involved detection methods:

[0020] 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 (30m×0.25mm×0.20μm), the detector type is FID, the injection port temperature is 230℃, the transfer line temperature is 280℃, the injection volume is 1μL, the split ratio is 20:1, the carrier gas flow rate is 0.5mL / min, the programmed temperature is to reach 50℃ and hold for 5min. The temperature is increased to 160℃ at 10℃ / min and held for 2min. The temperature is increased to 220℃ at 60℃ / min and held for 10min. The post-column temperature is 150℃. The peak time of isoamyl alcohol is 3.163min.

[0021] Example 1: Construction of an expression vector containing a mitochondrial localization signal peptide at the N-terminus

[0022] i: High-fidelity enzyme 2-round amplification of MLS sequence (primers: pYLXP'2m F1 / pYLXP'2mF2 / pYLXP'2m R), generally requires two rounds of PCR. In the first round, add a small amount of primers, 0.5pmol of each primer in 10uL system, 15 cycles to obtain the full-length template; in the second round, take 1uL PCR product as template, add 20pmol of full-length upstream and downstream primers (primers: pYLXP'2m F1 / pYLXP'2mR), 25 cycles to obtain the full-length MLS. 2% gel recovery of MLS target fragment 118bp.

[0023] ii: pYLXP' was digested with SnaBI and purified by column. The recovered fragment was 6382 bp in length.

[0024] iii: One-step cloning was used to obtain the recombinant plasmid pYLXP'm, which was then transformed into Escherichia coli JM109.

[0025] Example 2: Construction of a recombinant plasmid for mitochondrial compartmental expression of the leucine synthesis pathway

[0026] i: Using the Lactococcus Lactis genome as a template, primers pYLXP'm-kivd F / R were used to amplify Kivd(m); using the Escherichia coli JM109 genome as a template, primers pYLXP'm-yqhD F / R were used to amplify the gene fragment yqhD(m). Using the Yarrowia lipolytica Po1f genome as a template, primers pYLXP'm-YlLeu4F / R, pYLXP'm-YlLeu1 F / R, pYLXP'm-YlLeu2 F / R were used to amplify genes YlLeu4(m), YlLeu1(m), and YlLeu2(m).

[0027] ii: Using pYLXP'm as a vector, integrate YlLeu4(m), YlLeu1(m), YlLeu2(m), Kivd(m) and yqhD(m) into the SnaBI restriction site of the vector to obtain recombinant plasmids: pYLXP'-YlLeu1(m), pYLXP'-YlLeu2(m), pYLXP'-YlLeu4(m), pYLXP'-Kivd(m), pYLXP'-yqhD(m). The integration is pYLXP'-Kivd(m)-yqhD(m)-YlLeu1(m)-YlLeu4(m)-YlLeu2(m) recombinant plasmid.

[0028] iii: Transform the recombinant plasmid into Yarrowia lipolytica Po1f to obtain the engineered strain Po1f / pYLXP'-Kivd(m)-yqhD(m)-YlLeu1(m)-YlLeu4(m)-YlLeu2(m).

[0029] Example 3: Construction of a recombinant plasmid for cytoplasmic expression of the leucine biosynthesis pathway

[0030] i: Using the Lactococcus Lactis genome as a template, primers Kivd F / R were used to amplify Kivd; using the Escherichia coli JM109 genome as a template, primers yqhD F / R were used to amplify the gene fragment yqhD. Using the Yarrowia lipolytica Po1f genome as a template, primers YlLeu4F / R and YlLeu1 F / R were used to amplify genes YlLeu4 and YlLeu1.

[0031] ii: The Kivd, yqhD, YlLeu4, and YlLeu1 gene fragments were cloned into the SnaBI restriction site of the vector pYLXP' using a one-step cloning kit (Nanjing Novozymes Biotech Co., Ltd.) to obtain plasmids pYLXP'-Kivd, pYLXP'-yqhD, pYLXP'-YlLeu4, and pYLXP'-YlLeu1.

[0032] iii: Integrate plasmids pYLXP'-Kivd, pYLXP'-yqhD, pYLXP'-YlLeu4, and pYLXP'-YlLeu1 into the recombinant plasmid pYLXP'-Kivd-yqhD-YlLeu1-YlLeu4.

[0033] iiii: Transform the recombinant plasmid into Yarrowia lipolytica Po1f to obtain the engineered strain Po1f / pYLXP'-Kivd-yqhD-YlLeu1-YlLeu4.

[0034] Example 4: Construction of a recombinant plasmid for mitochondrial and cytoplasmic localization fusion expression of the leucine biosynthesis pathway

[0035] i: Integrate the plasmids pYLXP'-Kivd, pYLXP'-yqhD, pYLXP'-YlLeu1(m), pYLXP'-YlLeu4(m), and pYLXP'-YlLeu2(m) into pYLXP'-Kivd-yqhD-YlLeu1(m)-YlLeu4(m)-YlLeu2(m).

[0036] ii: Transform the recombinant plasmid into Yarrowia lipolytica Po1f to obtain the engineered strain Po1f / pYLXP'-Kivd-yqhD-YlLeu1(m)-YlLeu4(m)-YlLeu2(m).

[0037] Example 5: Fermentation culture of successfully constructed Yarrowia lipolytica engineered bacteria

[0038] i: Cultivate in SD-leu medium at 30°C and 200 rpm for 2 days for activation, inoculate 2% (v / v) into 25 mL of SD-leu (C / N=80) medium, culture at 30°C and 200 rpm for 5 days, and take samples every 24 hours. Extract isopentanol from the samples using a solid phase microextraction instrument, and detect the amount of isopentanol generated using a gas chromatograph.

[0039] ii: According to gas phase analysis, the isopentanol production was the highest at 96h, with the mitochondrial-localized engineered bacteria reaching 0.412 mg / L, an increase of 88.72% compared with the control and 31.89% compared with the cytoplasmic-localized engineered bacteria.

[0040] The above description is only an optional implementation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for increasing the production of isoamyl alcohol in Yarrowia lipolytica by compartmentalization, characterized in that: The invention comprises expressing endogenous α-isopropylmalate dehydrogenase (Leu1, YALI1_B02345g, Protein ID: CAB99455.1), β-isopropylmalate dehydrogenase (Leu2, YALI1_C00464g, Protein ID: AAF70314.1), α-isopropylmalate synthase (Leu4, YALI0_B07447g, Protein ID: CAG82839.1) and heterologous α-ketoacid decarboxylase (Kivd, EC 4.1.1.1, Protein ID: CAG82839.1) in Yarrowia lipolytica. The first 26 amino acids of Saccharomyces cerevisiae cytochrome 4 (COX4) were used as mitochondrial localization tags to localize some enzymes in the isopentanol biosynthesis pathway to the mitochondria for expression.

2. The method according to claim 1, characterized in that The mitochondrial localization signal peptide MLS, the first 26 amino acid sequences of Saccharomyces cerevisiae cytochrome 4 (COX4), are fused and expressed at the N-termini of proteins of α-isopropylmalate dehydrogenase (Leu1, SEQID NO.1), β-isopropylmalate dehydrogenase (Leu2, SEQ ID NO.2), α-isopropylmalate synthase (Leu4, SEQID NO.3), α-ketoacid decarboxylase (Kivd, SEQ ID NO.4), and alcohol dehydrogenase (yqhD, SEQ ID NO.5) to form a new fusion protein.

3. According to claim 1 and claim 2, using Yarrowia lipolytica Po1f as the expression host, transforming the recombinant plasmid into Yarrowia lipolytica Po1f to obtain an engineered strain containing all pathway genes. Characterized in that: The Yarrowia lipolytica engineered bacteria uses the mitochondrial localization signal peptide MLS to express α-ketoacid decarboxylase and alcohol dehydrogenase and α-isopropylmalate dehydrogenase, β-isopropylmalate dehydrogenase and α-isopropylmalate synthase in the Harris pathway in the mitochondria of the yeast host.

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