Method for constructing an engineered bacterium capable of producing fatty acids with methanol as the sole carbon source and applications thereof

Abstract

The application discloses a method for constructing an engineered bacterium capable of producing fatty acids with methanol as the only carbon source. The method comprises knocking out LPL1 genes and IZH3 genes of a high-yield fatty acid engineered strain of Hansenula polymorpha. The method can make the high-yield fatty acid engineered bacterium capable of producing fatty acids with methanol as the only carbon source by knocking out the LPL1 genes and the IZH3 genes.

Description

Technical Field

[0001] This application relates to a method for constructing engineered bacteria capable of producing fatty acids using methanol as the sole carbon source, belonging to the field of microbial genetic engineering and metabolic engineering applications. Background Technology

[0002] Methanol contains both carbon and hydrogen, serving as a hydrogen carrier, while carbon can be used to synthesize various chemicals. Its liquid state facilitates storage and transportation, allowing it to integrate with existing petrochemical infrastructure, making it a promising chemical feedstock. Methanol can be produced from coal, natural gas, and renewable biomass (Energy, 2014, 77, 125). In particular, the "liquid sunshine" route, where CO2 is directly hydrogenated to obtain methanol (Science, 2017, 355, 1296), has been proposed in recent years, ensuring a stable supply of methanol feedstock. Therefore, establishing efficient methanol conversion routes is a feasible path to guarantee future energy and chemical needs. Biosynthetic systems (enzymes and microbial cells) offer high selectivity, green processes, and mild conditions, enabling the synthesis of more complex oxygen-containing chemicals and higher energy-density liquid fuels. Therefore, biological systems and chemical routes complement each other and are expected to become another feasible path for efficient methanol conversion, promoting the clean utilization of coal and carbon resources in my country (Nature Energy, 2018, 3, 925).

[0003] Utilizing microbial cell factories to produce target products such as bulk chemicals, biofuels, and natural products is a sustainable production model. Therefore, constructing cell factories using microorganisms capable of utilizing methanol as a substrate is an important research direction for achieving efficient methanol bioconversion. In nature, there exists a class of "methyltrophic" microorganisms capable of using C1 compounds (methane, methanol, etc.) as carbon sources for growth and metabolism. These mainly include some prokaryotic bacteria and methanol yeasts (such as Pichia pastoris and Hansenula polymorpha). Among them, Hansenula polymorpha possesses many excellent characteristics, including a broad substrate spectrum (glucose, xylose, and methanol, etc.), heat resistance (above 50°C), few byproducts, and ease of high-density fermentation, making it an excellent cell factory chassis cell (FEMS Yeast Research, 2012, 12, 271). However, the lack of efficient genetic manipulation tools severely hinders the application and development of Hansenula polymorpha. The inventors' previous work has built a relatively complete Hansenula polymorpha genetic manipulation platform, which enables convenient and rapid precise genome editing and metabolic regulation, laying a good foundation for building an efficient Hansenula polymorpha cell factory (iScience, 2021, 24, 102168).

[0004] Current research on methyltrophic microorganisms mainly focuses on constructing and enhancing upstream methanol utilization pathways. It is generally believed that if cells can efficiently assimilate methanol, they can achieve efficient synthesis of target products. However, we have found that engineered Hansenula polymorpha strains, which produce high levels of fatty acids, cannot grow in basal media with methanol as the sole carbon source. This phenomenon suggests that in methyltrophic microorganisms, cell growth and product synthesis may compete during methanol utilization. In other words, the methanol metabolic flux may not be able to simultaneously meet the needs of cell growth and product synthesis, but the relevant molecular mechanisms are unclear. Therefore, understanding this competitive relationship and elucidating the related molecular mechanisms will hopefully advance research on natural methyltrophic microbial cell factories, improve the methanol utilization efficiency of natural / synthetic methyltrophic microorganisms, and truly achieve efficient methanol bioconversion to synthesize a variety of high-value-added target products.

[0005] Therefore, this invention aims to obtain a *Hansenula polymorpha* strain capable of efficiently utilizing methanol and producing high levels of fatty acids. In *Hansenula polymorpha* chassis cells of faa1Δ, multi-omics sequencing was performed on the domesticated strain using laboratory adaptive evolution to reveal the molecular mechanisms of lipid metabolism and cell growth competition during methanol assimilation. Two key mutant genes, LPL1 and IZH3, were identified in the domesticated strain. Their double mutation effectively alleviated cytotoxicity caused by fatty acid accumulation and methanol metabolism, and improved the tolerance of wild-type cells to high concentrations of methanol. Finally, global metabolic pathway remodeling in the domesticated strain increased the supply of acetyl-CoA and NADPH, resulting in a fatty acid yield of 15.9 g / L, demonstrating the application potential of *Hansenula polymorpha* as a cell factory. Summary of the Invention

[0006] According to one aspect of this application, a method for constructing an engineered bacterium capable of producing fatty acids using methanol as the sole carbon source is provided. The method involves knocking out the LPL1 and IZH3 genes, enabling the high-yield fatty acid engineered bacterium to produce fatty acids using methanol as the sole carbon source.

[0007] The engineered bacteria that can produce fatty acids using methanol as the sole carbon source refers to the engineered bacteria that can produce fatty acids using methanol as the sole carbon source, that is, can produce fatty acids in a culture medium containing only methanol as the carbon source, but this does not exclude the possibility that they can also produce fatty acids using other carbon sources.

[0008] A method for constructing engineered bacteria capable of producing fatty acids using methanol as the sole carbon source, the method comprising:

[0009] Knock out the LPL1 and IZH3 genes in engineered strains of high-fatty acid-producing Hansenula polymorpha.

[0010] Optionally, the high-fatty acid-producing Hansenula polymorpha engineered strain is obtained by knocking out the FAA1 gene of wild-type Hansenula polymorpha.

[0011] Optionally, the wild-type Hansenula polymorpha is wild-type Hansenula polymorpha NCYC 495leu1.1.

[0012] Optionally, the nucleotide sequence of the LPL1 gene is shown in SEQ ID No. 1;

[0013] The nucleotide sequence of the IZH3 gene is shown in SEQ ID No. 2.

[0014] According to one aspect of this application, an engineered bacterium capable of producing fatty acids using methanol as the sole carbon source is provided, obtained according to the construction method described above.

[0015] According to one aspect of this application, an engineered bacterium capable of producing fatty acids using methanol as the sole carbon source, obtained according to the above-described construction method, is provided, along with its application in the preparation of fatty acids using methanol as the sole carbon source.

[0016] According to one aspect of this application, a method for improving the methanol tolerance of wild-type Hansenula polymorpha is provided, the method comprising:

[0017] Knock out the LPL1 and IZH3 genes in wild-type Hansenula polymorpha.

[0018] Optionally, the nucleotide sequence of the LPL1 gene is shown in SEQ ID No. 1;

[0019] The nucleotide sequence of the IZH3 gene is shown in SEQ ID No. 2.

[0020] Optionally, the wild-type Hansenula polymorpha is wild-type Hansenula polymorpha NCYC 495leu1.1.

[0021] According to one aspect of this application, a method is provided for obtaining a Hansenula yeast strain capable of producing high levels of fatty acids using methanol as the sole carbon source, the method comprising the following steps:

[0022] The high-fatty acid-producing Hansenula polymorpha engineered strain was domesticated as follows:

[0023] The strain was adapted to an initial culture medium containing both glucose and methanol, and then adapted to a culture medium with a gradually decreasing ratio of glucose and methanol until the glucose was reduced to 0, thus obtaining the Hansenula yeast strain capable of producing high levels of fatty acids using methanol as the sole carbon source.

[0024] The high-fatty acid-producing Hansenula polymorpha engineered strain was obtained by knocking out the FAA1 gene of wild-type Hansenula polymorpha.

[0025] The total carbon concentration of glucose and methanol in the culture medium is 0.2–0.5 mol / L.

[0026] Optionally, the total carbon concentration of glucose and methanol in the culture medium is any one of 0.2, 0.3, 0.4, 0.5 mol / L or a range between any two values.

[0027] Optionally, the carbon molar ratio of glucose to methanol in the initial culture medium is x:10-x, where x takes the value of 3 to 7.

[0028] Optionally, x can take any one of the values ​​3, 4, 5, 6, and 7, or a range between any two values.

[0029] Optionally, the ratio of glucose to methanol is gradually reduced to a carbon molar ratio of glucose to methanol of x:10-x, where x decreases with a gradient of y, and y takes the value of 0.5 to 1.

[0030] Optionally, the adaptive culture involves acclimatizing a high-fatty acid-producing Hansenula polymorpha engineered strain in a culture medium until it initially yields an OD value of [missing value]. 600 =0.2 inoculation, growth OD at 48h 600 Reaching 5-6.

[0031] Optionally, the method further includes performing genome sequencing and / or transcriptome sequencing analysis on the domesticated high-fatty acid-producing Hansenula polymorpha engineered strain to obtain key gene targets and / or metabolic pathways, and optimizing the key gene targets and / or metabolic pathways.

[0032] Optionally, the method further includes increasing the expression of the MmACL gene in domesticated high-fatty acid-producing Hansenula polymorpha engineered strains.

[0033] Optionally, the method further includes increasing the expression of the ZWF1 and FBP1 genes in domesticated high-fatty acid-producing Hansenula polymorpha engineered strains.

[0034] Optionally, the method further includes increasing the expression of the ZWF1 gene, FBP1 gene, and MmACL gene in domesticated high-fatty acid-producing Hansenula polymorpha engineered strains.

[0035] Optionally, the method further includes increasing the expression of the ZWF1 gene, FBP1 gene, MmACL gene, and RPE gene in the domesticated high-fatty acid-producing Hansenula polymorpha engineered strain.

[0036] Optionally, the method further includes increasing the expression of the ZWF1 gene, FBP1 gene, MmACL gene, and ScIDP2 gene in the domesticated high-fatty acid-producing Hansenula polymorpha engineered strain.

[0037] Optionally, the method further includes increasing the expression of the ZWF1 gene, FBP1 gene, MmACL gene, RPE gene, and ScIDP2 gene in the domesticated high-fatty acid-producing Hansenula polymorpha engineered strain.

[0038] As one implementation scheme, this invention provides a method for improving tolerance to fatty acid and methanol metabolic toxicity and its application. First, previously constructed high-fatty acid-producing Hansenula polymorpha cells were unable to utilize methanol for growth and metabolism, indicating that cell growth and product synthesis severely compete for methanol metabolic flux, leading to cell death. Through laboratory adaptive evolution, a domesticated Hansenula polymorpha strain capable of utilizing methanol and producing high levels of fatty acids was obtained. Second, through genome sequencing analysis, two key mutant genes, LPL1 (specified lipase gene) and IZH3 (Zn metabolism-related membrane protein gene), were identified. Double knockout of these genes enabled the faa1Δ strain to grow and accumulate fatty acids in methanol. Meanwhile, global transcriptome analysis revealed significant upregulation of genes related to the supply of xylulose 5-phosphate (Xu5P), a precursor to methanol metabolism, such as FBP1, RPE, and TAL1. Furthermore, analysis of the percentage of cells stained with propidium iodide (PI) demonstrated that high-fatty acid-producing Hansenula polymorpha primarily underwent cell necrosis mediated by the Rim101 pathway in methanol. Finally, through global metabolic regulation and optimization, fatty acid production reached 15.9 g / L under fed-batch fermentation with methanol as the sole carbon source.

[0039] As one implementation scheme, the present invention provides a polymorphic Hansenula polymorpha strain that produces high levels of fatty acids using methanol as the sole carbon source. The strain was obtained through laboratory adaptive evolution from a Hansenula polymorpha strain in which acyl-CoA synthase (encoded by the gene FAA1) was knocked out.

[0040] The strain fermented for 96 hours in a basic medium containing 10 g / L methanol, and the fatty acid yield reached 480 mg / L. The fatty acid types included C16-1, C16, C18-3, C18-2, C18-1, and C18.

[0041] The strain, after genomic sequencing analysis, contained 15 gene mutations, including a speculative lipase gene (encoded by LPL1) and a Zn metabolism-related membrane protein gene (encoded by IZH3), as well as large chromosomal fragment duplication and deletion.

[0042] The nucleotide sequences of the LPL1 and IZH3 genes are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0043] Transcriptome sequencing analysis of the strain revealed upregulated expression of genes such as 1,6-difructose phosphatase (encoded by gene FBP1), ribulose-5-phosphate-3-epimerase (encoded by gene RPE), and transaldolase (encoded by gene TAL1).

[0044] As one implementation, the present invention provides a method for rescuing high-fatty acid-producing Hansenula mongolica from methanol utilization defects, wherein the LPL1 and IZH3 genes are knocked out in an engineered strain of high-fatty acid-producing Hansenula mongolica (faa1Δ).

[0045] Knocking out the genes LPL1 and IZH3 alleviated the Rim101 pathway-mediated cell death process, involving, but not limited to, RIM101 and RIM13.

[0046] As one implementation, the present invention provides a method for improving the methanol tolerance of wild-type Hansenula polymorpha, wherein the method knocks out the genes LPL1 and IZH3 in wild-type Hansenula polymorpha NCYC 495leu1.1.

[0047] The method can improve the growth rate of Hansenula polymorpha strains in 50 g / L methanol.

[0048] As one implementation, the present invention provides a metabolic engineering strategy to improve the production of fatty acids from methanol in Hansenula polymorpha. The strategy, based on domesticated strains, overexpresses glucose-6-phosphate dehydrogenase (encoded by gene ZWF1), FBP1, ATP:citrate lyase from Mus musculus (encoded by gene MmACL), cytoplasmic isocitrate dehydrogenase from Saccharomyces cerevisiae (encoded by gene ScIDP2), and RPE.

[0049] The metabolic engineering strategy described above for improving the production of fatty acids from methanol in Hansenula polymorpha achieves a fatty acid yield of 15.9 g / L when fed-batch fermentation is carried out in a basal medium with methanol as the sole carbon source.

[0050] The beneficial effects that can be obtained from this application include:

[0051] (1) The method for constructing engineered bacteria provided in this application enables high-fatty acid-producing Hansenula yeast engineered strains to produce fatty acids using methanol as the sole carbon source by knocking out the LPL1 and IZH3 genes, thereby improving the methanol tolerance of wild-type Hansenula yeast.

[0052] (2) The method provided in this application for obtaining a Hansenula yeast strain capable of producing fatty acids with methanol as the sole carbon source is to obtain a strain capable of producing fatty acids with methanol as the sole carbon source through the domestication method provided. Attached Figure Description

[0053] Figure 1 A shows a schematic diagram of fatty acid synthesis by a high-fatty acid-producing strain; Figure 1 B represents the growth of each strain in the glucose fermentation medium. Figure 1C represents the fatty acid production of each strain in glucose fermentation medium. Figure 1 D shows the methanol utilization defect in high-fatty acid-producing Hansenula polymorpha (faa1Δ).

[0054] Figure 2 This study demonstrates laboratory-adapted strains that have acquired methanol utilization and high fatty acid production capabilities through adaptive evolution; among them Figure 2 A is a schematic diagram of the domestication process; Figure 2 B to D represent the growth characteristics of the first to third groups after domestication, respectively. Figure 2 E to G represent the fatty acid yields of the first to third groups after domestication, respectively.

[0055] Figure 3 The genome sequencing analysis identified key mutant genes LPL1 and IZH3; among which... Figure 3 A shows 20 mutations detected; B shows large segments of chromosome duplication and deletion. Figure 3 C represents the growth of HpFA01 after knocking out LPL1 and / or IZH3; Figure 3 D represents the fatty acid production after HpFA01 knocks out LPL1 and / or IZH3; Figure 3 E represents the growth of the wild-type strain after knocking out LPL1 and IZH3.

[0056] Figure 4 Global transcriptome sequencing analysis of differentially expressed genes in domesticated strains; among which Figure 4 A, Figure 4 B indicates that the overall gene expression level of the domesticated strain was significantly downregulated; Figure 4 C indicates that, except for methanol metabolism-related pathways, the other pathways were significantly downregulated.

[0057] Figure 5 The Rim101 pathway-mediated cell necrosis process regulates methanol utilization in high-fatty acid-producing Hansenula polymorpha; among which... Figure 5 A to H represent the cell necrosis rate of each strain; Figure 5 I represents the Rim101 pathway-mediated cell necrosis process.

[0058] Figure 6 A, Figure 6 B shows the fatty acid production of each strain.

[0059] Figure 7 A represents the biomass of each strain in the fed-batch shake-flask fermentation. Figure 7 B represents the fatty acid yield of each strain.

[0060] Figure 8 This study demonstrates the efficient conversion of HpFAM16u into fatty acids from methanol in fed-batch fermentation in a fermenter. Detailed Implementation

[0061] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials and reagents used can be purchased from biological or chemical companies.

[0062] In this application, the gene modification involves introducing donor DNA containing either "upstream homologous arm of the integration site - integration fragment - downstream homologous arm of the integration site" or "upstream homologous arm of the knockout site - downstream homologous arm of the knockout site" into the host, and using the CRISPR / Cas9 system to achieve gene integration or knockout (iScience, 2021, 24, 102168). The donor DNA and site-targeting sequences involved in this application are shown in Tables 1 and 2.

[0063] Table 1. Donor DNA and gRNA expression vectors involved in the embodiments of this application

[0064] Donor DNA gRNA expression vector Integration site <![CDATA[NS11up-P PMA1 -MmACL-T ADH2 -NS11dw]]> gRNA-NS11 NS11 <![CDATA[NS2up-T CAT1 -ZWF1-P ADH1 +P PMA1 -FBP1-T GAP -NS2dw]]> gRNA-NS2 NS2 <![CDATA[NS3up-P GAP -ScIDP2-T AMO -NS3dw]]> gRNA-NS3 NS3 <![CDATA[P RPE up-P TAL1 -P RPE dw]]> pHpgRNA79 <![CDATA[P RPE ]]>

[0065] The integration site, the 20bp target sequence in the expression vector, and the upstream and downstream homologous arms in Table 1 are referenced in Synth. Syst. Biotechnol., 2021, 6, 63-68. The sequence of the integrated fragment can be found in NCBI based on the provided gene / promoter / terminator names.

[0066] Table 2. Donor DNA and Site-Targeting Sequences Involved in the Embodiments of this Application

[0067] Donor DNA 20bp target sequence Knockout site LPL1up-LPL1dw (SEQ ID No. 5) SEQ ID No. 3 LPL1 IZH3up-IZH3dw (SEQ ID No. 6) SEQ ID No. 4 IZH3

[0068] As one implementation method, the objective of this invention is to obtain a *Hansenula polymorpha* strain capable of efficiently utilizing methanol and producing high levels of fatty acids, and to further explore the molecular mechanisms of lipid metabolism and cell growth competition during methanol assimilation. Furthermore, this molecular mechanism is applied to improve cellular methanol tolerance and fatty acid production. This invention is achieved using the following technical solution:

[0069] In a first aspect, the present invention provides a Hansenula polymorpha strain that efficiently utilizes methanol and produces high levels of fatty acids, which can grow normally in a basal medium with methanol as the sole carbon source and accumulate high concentrations of fatty acids.

[0070] Furthermore, the specific implementation steps of the above technical solution are as follows: Using laboratory adaptive evolution technology, high-fatty acid-producing Hansenula mongolica HpFA01 (genotype MATa,leu1.1,faa1Δ,(P)) is selected. GAP -hCAS9-T AOX1Starting from YPD activated plates, three single colonies were picked and independently subcultured continuously. The initial culture medium was Delft basal medium containing glucose:methanol at a molar ratio of 5:5 (2.5 g / L (NH4)2SO4, 14.4 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, trace elements and vitamins), with OD... 600 =0.2g inoculation, incubated at 37℃ and 220rpm. If the strain can achieve OD after 48h of incubation. 600 If the glucose concentration reaches approximately 5-6, transfer the strain to a new culture medium, specifically Delft basal medium containing a glucose:methanol molar ratio of 4.5:5.5. Otherwise, continue transferring to the original medium. Continue this process, gradually decreasing the glucose content and increasing the methanol content, until the acclimatized strain can grow normally in Delft basal medium with methanol as the sole carbon source, and after 48 hours of culture, the OD value reaches a certain level. 600 When the number of cells reaches approximately 5-6, the acclimatization process is complete. The three groups of acclimatized strains were diluted and plated on YPD plates. Six single colonies from each group were picked and cultured in Delft basal medium (with methanol as the sole carbon source) at 37°C and 220 rpm for 96 hours. Cell growth and fatty acid accumulation were then analyzed.

[0071] Furthermore, to elucidate the molecular mechanism by which the domesticated strains can grow in methanol and synthesize fatty acids, three single colonies from each of the three domesticated strain groups were selected and subjected to genome sequencing analysis along with the starting strain HpFA01. Ten strains were cultured in YPD medium at 37°C and 220 rpm for 16–18 h, and cells were collected. Genome sequencing results identified and characterized two key mutant genes: LPL1 (a hypothesized esterase gene) and IZH3 (a Zn metabolism-related membrane protein gene).

[0072] Furthermore, to investigate the differential gene expression at the transcriptional level in the domesticated strains, a global transcriptomic analysis was performed on the above-mentioned nine domesticated strains and the control strain HpFA01. All ten strains were cultured in YPM (20 g / L peptone, 10 g / L yeast extract, 10 g / L methanol) medium at 37°C and 220 rpm for 24 h. Cells were collected, and total RNA was extracted for transcriptomic analysis. Transcriptomic analysis revealed and identified significantly upregulated expression of genes such as FBP1, RPE, and TAL1.

[0073] A second aspect of the invention provides a method for rescuing methanol utilization defects in high-fatty acid-producing Hansenula polymorpha. LPL1 and IZH3 are seamlessly knocked out.

[0074] Further, the specific implementation steps of the above technical solution are as follows: Starting with the HpFA01 strain, genes LPL1 and IZH3 were seamlessly knocked out. Taking the seamless knockout of gene LPL1 as an example, the genome editing of Hansenula polymorpha in this invention is mainly completed using a self-constructed CRISPR / Cas9 system (iScience, 2021, 24, 102168). First, the sgRNA expression vector pHpgRNA98 targeting gene LPL1 was constructed, with a 20bp target sequence as shown in SEQ ID No. 3. Next, donor DNA molecules were constructed, and sequences of approximately 1000bp upstream and downstream of the coding region of gene LPL1 were amplified respectively. The complete donor DNA fragment was obtained by fusion PCR. The gRNA expression vector pHpgRNA98 and donor DNA were electroporated into strain HpFA01 at a dose of 500ng each and cultured statically at 37℃ on SD plates for 2-3 days. After the transformants were cultured in liquid SD medium, the results were verified by PCR. The strains were then continuously passaged in YPD medium to remove plasmids, and the strains with plasmid loss were stored for later use. The knockout of gene IZH3 (20bp target sequence as shown in SEQ ID No. 4) and other genome editing work described below all followed a similar procedure.

[0075] Furthermore, to verify the growth and fatty acid production of lpl1Δ and izh3Δ strains in methanol medium, the activated recombinant strains were subjected to an initial OD... 600 =0.4% was transferred to Delft basal medium containing 10 g / L methanol and fermented at 37°C and 220 rpm.

[0076] Furthermore, the sequences and functions of LPL1 and IZH3 in Hansenula polymorpha were identified (nucleotide sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, respectively).

[0077] A third aspect of the invention provides a method for improving the methanol tolerance of wild-type Hansenula polymorpha by knocking out the genes LPL1 and IZH3, enabling the strain to tolerate methanol concentrations above 50 g / L. Further, wild-type Hansenula polymorpha 495-3 (genotype MATa,leu1.1,(P...)... GAP -hCAS9-T AOX1 The control strain had both genes LPL1 and IZH3 knocked out, and their growth was compared in Delft basal medium containing 50 g / L methanol. The strain was fermented at 37°C and 220 rpm for 120 h, with OD measured every 24 h. 600 .

[0078] A fourth aspect of this invention provides a metabolic engineering strategy to increase fatty acid production in domesticated bacterial strains. This involves rearranging the central carbon metabolic pathway to increase the supply of precursor acetyl-CoA and cofactor NADPH, and to improve methanol utilization efficiency.

[0079] Furthermore, the implementation steps of the above technical solution are as follows:

[0080] (1) Increase the supply of precursor acetyl-CoA

[0081] Starting with the domesticated strain HpFAM01 (strain number 10 from the third domestication group), the URA3 gene was knocked out as a gene editing selection marker, and the KU80 gene was knocked out to increase homologous recombination efficiency, resulting in strain HpFAM03. Overexpression of ATP:citrate lyase (encoded by the gene MmACL) from Musmusculus was performed to increase intracellular acetyl-CoA supply. The specific procedure was consistent with the second aspect of this invention described above. The constructed donor DNA was obtained by fusion PCR, including approximately 1000 bp homologous arms on each side and the expression cassette P. PMA1 -MmACL-T ADH2 The integration site is the genomic neutral site NS11 (Synth.Syst.Biotechnol.,2021,6,63-68.), therefore the constructed sgRNA expression vector is gRNA-NS11, achieving the integration of the gene MmACL.

[0082] (2) Increase the supply of cofactor NADPH

[0083] Based on the above-mentioned strains, glucose-6-phosphate dehydrogenase (encoded by gene ZWF1), 1,6-difructose phosphatase (encoded by gene FBP1), and cytoplasmic isocitrate dehydrogenase derived from Saccharomyces cerevisiae (encoded by gene ScIDP2) were further overexpressed. The specific procedure is consistent with the second aspect of the present invention described above, wherein the constructed donor DNA, the required sgRNA expression vector, and the integration site are shown in Table 1.

[0084] (3) Improve methanol utilization efficiency

[0085] To improve the methanol utilization efficiency of the strain, it was considered to increase the supply of the methanol utilization precursor xylulose 5-phosphate by overexpressing ribulose-5-phosphate-3-epimerase (encoded by the gene RPE). A strongly methanol-inducible promoter P was used. TAL1 Replace its original promoter P RPE This achieves overexpression of the gene RPE. The specific process is consistent with the second aspect of the present invention described above, wherein the constructed donor DNA, the required sgRNA expression vector, and the integration site are shown in Table 1.

[0086] Furthermore, to improve the fermentation stability of the strain, the final modified strain was supplemented with the URA3 and KU80 genes to obtain the prototrophic strain HpFAM16u. Batch fed-batch fermentation was performed with an initial loading volume of 300 mL in a 1.0 L fermenter, using the initial OD... 600 Inoculation was performed at a ratio of 1. When the methanol in the fermenter was depleted, 600 g / L methanol and 5× Delft medium were added at a feed-feed ratio of 2:1. The remaining methanol level was monitored in real time during fermentation, and the feed-feed rate was adjusted accordingly. The aeration rate was 18–48 sL / h, the stirring rate was 400–800 rpm, the dissolved oxygen was controlled at 30%, and the pH was adjusted to 5.6 with 2M KOH and 1M HCl.

[0087] In this embodiment, wild-type Hansenula polymorpha NCYC 495leu1.1 can be purchased from the China General Microbiological Culture Collection Center (CGMCC), strain number 2.2412;

[0088] Strain 495-3 has been documented in iScience, 2021, 24, 102168, and is available to the public from the applicant. This biological material can only be used for the verification of the patent content and cannot be used for other purposes.

[0089] Example 1: Defects in methanol utilization by high-fatty acid-producing Hansenula polymorpha

[0090] (1) Construction of high fatty acid-producing strains

[0091] Figure 1 A shows a schematic diagram of fatty acid synthesis by a high-fatty acid-producing strain.

[0092] Based on the wild-type Hansenula polymorpha NCYC 495leu1.1, a Hansenula polymorpha CRISPR / Cas9 genome editing system was constructed to obtain strain 495-3 (genotype MATa,leu1.1,(P)). GAP -hCAS9-T AOX1 (iScience, 2021, 24, 102168) Strain HpFA01 was obtained by knocking out acyl-CoA synthase (encoded by FAA1) of the starting strain 495-3 alone, strain HpFA02 was obtained by knocking out acyl-CoA oxidase (encoded by POX1) alone, and strain HpFA03 was obtained by knocking out both FAA1 and POX1 genes at the same time.

[0093] (2) Strains culture and fatty acid fermentation

[0094] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract;

[0095] Glucose fermentation medium (basic components of glucose): (NH4)2SO4 2.5 g / L, KH2PO4 14.4 g / L, MgSO4·7H2O 0.5 g / L, add approximately 900 mL ddH2O, adjust pH to 5.6, bring volume to 950 mL, and sterilize at 115℃ for 30 min. After sterilization, add 1 mL Vitamin solution, 2 mL Trace metal solution, and 50 mL 400 g / L glucose solution. Add leucine (60 mg / L) and uracil (20 mg / L) before use.

[0096] Methanol fermentation medium (basal components containing 10 g methanol / L): (NH4)2SO4 2.5 g / L, KH2PO4 14.4 g / L, MgSO4·7H2O 0.5 g / L, add approximately 900 mL ddH2O, adjust pH to 5.6, bring volume to 1 L, and sterilize at 115℃ for 30 min. After sterilization, add 1 mL Vitamin solution, 2 mL Trace metal solution, and 12.6 mL pure methanol. Add leucine (60 mg / L) and uracil (20 mg / L) before use.

[0097] The formulations for the Trace-metal solution are shown in Table 3; the formulations for the Vitamin solution are shown in Table 4.

[0098] Table 3. Formulation of Trace-metal solution

[0099]

[0100] Table 4. Formulation of Vitamin Solution

[0101]

[0102] After bacterial activation, strains 495-3, HpFA01(faa1Δ), HpFA02(pox1Δ), and HpFA03(faa1Δpox1Δ) were streaked onto YPD solid medium and incubated statically at 37°C for 2 days. Single colonies were picked and added to 3 / 15 mL of YPD medium (i.e., 3 mL of medium was added to a 15 mL culture tube) and cultured at 37°C with shaking at 220 rpm for 24 hours. Inoculation was performed according to the initial OD... 600 =0.2% inoculated into the fermentation medium, with a liquid volume of 20 mL / 100 mL Erlenmeyer flask, and fermented at 37℃ and 220 rpm. Samples were taken at fixed points (every 12 h) or at the endpoint (60 h) for biomass (expressed as absorbance at 600 nm) and yield analysis.

[0103] Experimental results showed that wild-type Hansenula polymorpha and strains with FAA1 and POX1 knocked out, either alone or in combination, were cultured in basal media containing glucose or methanol. In glucose fermentation medium, the strains grew normally, and the faa1Δ strain was able to accumulate large amounts of fatty acids (e.g., ...). Figure 1 (As shown in BC). In methanol fermentation medium, wild-type strains and strains with POX1 knockout alone grew normally but did not accumulate fatty acids. However, strains with FAA1 knockout exhibited methanol utilization defects (e.g., Figure 1 (As shown in D). Therefore, it is believed that in Hansenula polymorpha, fatty acid synthesis and cell growth compete for methanol metabolic flux, ultimately leading to cell death.

[0104] Example 2: Laboratory Adaptive Evolution

[0105] Three single clones of strain HpFA01 were independently and sequentially passaged. The initial culture medium was a basal medium containing glucose and methanol in a 5:5 carbon-to-molar ratio, with a total carbon-to-molar concentration of 0.3 mol / L. The cultures were initially cultured in this initial medium with OD... 600 =0.2 inoculation, when the strain can achieve OD at 48h 600 Once the concentration reaches 5-6, adjust the carbon molar ratio of glucose to methanol to 4.5:5.5, and then transfer to the initial OD200. 600 =0.2 inoculation, when the strain can achieve OD at 48h 600 To achieve a carbon molar ratio of 5-6, the ratio of glucose to methanol was adjusted in increments of 0.5, and this process was repeated until a carbon molar ratio of 1:9 was reached. The strain was then cultured in a medium with a glucose-methanol carbon molar ratio of 0:10 until it could grow in a medium with methanol as the sole carbon source and achieve an OD value of [missing value] after 48 hours of fermentation. 600 Once the score reaches 5-6, the domestication process is complete (see diagram). Figure 2 A).

[0106] Experimental results showed that although the three groups of acclimatization processes were not entirely the same, taking anywhere from 35 to 65 days, all ultimately yielded mutant strains capable of growing normally in methanol and synthesizing fatty acids. Figure 2 Five to six single clones were selected from each domestication group and subjected to fatty acid fermentation in a basal medium containing 10 g / L methanol under the same conditions as in Example 1. The growth and fatty acid synthesis of the domesticated strains were observed. All domesticated strains exhibited similar growth characteristics and fatty acid synthesis. The final OD value after 96 hours of culture was [not specified]. 600 Reaching around 8 ( Figure 2 BD), fatty acid production is between 280 and 450 mg / L. Figure 2(EG). Among them, strains 3-10 have the best growth status and can maintain a final fatty acid yield of about 400 mg / L, which can be used as the starting strain for subsequent high fatty acid production (also known as strain HpFAM01).

[0107] Example 3: Reverse metabolic engineering based on genomics and transcriptomics

[0108] (1) Genome sequencing analysis

[0109] To investigate the molecular mechanism by which domesticated strains synthesize fatty acids from methanol, we performed genome sequencing analysis. First, three domesticated strains (1-1, 1-2, 1-14, 2-2, 2-6, 2-16, 3-10, 3-12, 3-18) and the faa1Δ starting strain (strain HpFA01) were selected from each domestication group, totaling 10 strains. Cells were cultured in YPD medium for 16–18 hours, and the genomes were extracted for sequencing analysis. The faa1Δ starting strain underwent third-generation genome sequencing and assembly, which effectively avoided false positives in subsequent analyses and enabled analysis of large chromosome doubling and deletion. Using the faa1Δ starting strain as a reference genome, the remaining nine domesticated strains underwent resequencing analysis.

[0110] The results are as follows Figure 3 As shown in Figure A, 20 mutations (SNPs and InDel) were detected, involving 15 genes. Among them, a hypothetical lipase (encoded by the LPL1 gene) showed mutations in all three groups of acclimatization studies. In the first group, the mutations were S205P and F185L, while in the second and third groups, they were frameshift mutations caused by a 1bp insertion. In addition, a common mutation site was found in the second and third groups: the gene IZH3, where amino acid position 38 was mutated from E to a stop codon, leading to premature gene termination. IZH3 encodes a membrane protein related to Zn metabolism. These genes are key sites for further validation of reverse metabolic engineering targets. Large chromosomal fragment duplication and deletion (…) Figure 3 B) also involves a large number of genes, and these mutations may be an important reason why the domesticated strains are able to utilize methanol and produce high levels of fatty acids.

[0111] (2) Validation of key genomic targets

[0112] Based on the genome sequencing results, key sites were validated and analyzed, and the fermentation conditions were the same as in Example 1. Knockout of the LPL1 gene alone in strain HpFA01 failed to induce growth of the strain in methanol. Figure 3C). Therefore, it is speculated that other synergistic genes may play a key role. Given that Groups II and III share a common mutant gene, IZH3 (E38*), in addition to LPL1, we performed single and combined knockout of IZH3 in the starting strain. We found that combined knockout of LPL1 and IZH3 resulted in high-fatty acid-producing *Hansenula polymorpha* growing in basal medium with methanol as the sole carbon source, achieving a maximum growth OD of [insert growth rate here]. 600 The concentration reached approximately 3. However, the IZH3 strain alone failed to grow, indicating that LPL1 and IZH3 exerted a synergistic effect. Ultimately, the double-knockout strain accumulated 40.9 mg / L of fatty acids after 120 hours of fermentation. Figure 3 D).

[0113] To verify the potential role of genes LPL1 and IZH3 in enhancing cellular methanol tolerance, both LPL1 and IZH3 were double-knocked out in wild-type *Hansenula polymorpha* strain 495-3. The results showed that the double-knockout strain exhibited a significantly higher growth rate than the control strain in a basal medium containing 50 g / L methanol. Figure 3 E) indicates that the double knockout strain has a higher methanol tolerance, which also proves the application advantages of genes LPL1 and IZH3 in improving the methanol tolerance of cells.

[0114] (3) Transcriptome sequencing analysis

[0115] To analyze the differences in gene expression at the transcriptional level between the domesticated and originating strains, the nine domesticated and originating strains were cultured in YPM (10 g / L methanol, 20 g / L peptone, 10 g / L yeast extract) for 24 h. Cells were then collected, and RNA was extracted for transcriptome sequencing analysis. The overall differences in gene expression are shown below. Figure 4 As shown, the overall gene expression level of the domesticated strain was significantly downregulated. Figure 4 The downregulation of numerous genes (A, B), including those related to amino acid metabolism, can help cells reduce meaningless protein synthesis, thus enhancing the strain's resistance to cytotoxicity caused by fatty acid accumulation and methanol metabolism. Specifically, analysis of differential expression of genes in the central carbon metabolism pathway revealed that, except for pathways related to methanol metabolism, other pathways were also significantly downregulated. Figure 4 C). Upregulation of expression of certain genes (FBP1, RPE, TAL1) in the non-oxidative phase of the gluconeogenesis and pentose phosphate pathways may be related to enhanced supply of the methanol metabolic precursor Xu5P. These gene targets can lay the foundation for further improving the methanol utilization efficiency and fatty acid production of the strain.

[0116] Example 4: Cell Necrosis Regulation of High-Fat Lipid-Producing Strains' Methanol Utilization

[0117] To investigate the intracellular changes that enabled the domesticated strain to grow normally and produce high levels of fatty acids using methanol as the sole carbon source, cells were cultured in YPM medium and initially inoculated with OD. 600 The concentration was set at 0.2, and cells were cultured at 37°C and 220 rpm for 24 h. Cells were then collected. The cells were centrifuged at 1000 g for 5 min and washed twice with PBS buffer. The cells were resuspended in PBS containing 5 μg / mL propidium iodide (PI) and incubated at room temperature for 10 min. Fluorescence was detected by flow cytometry at 488 / 620 nm, and the cell necrosis rate was calculated.

[0118] Cells that can be stained by PI are generally called necrotic cells. The above experiments show that... Figure 5 It can be seen that the cell necrosis rate of faa1Δ (strain HpFA01) exceeded 90%, compared to the wild-type (strain 495-3, corresponding to Figure 5 Wild-type strain (F) and domesticated strain (HpFAM01, corresponding to F) Figure 5 The mortality rate of the domesticated strains in group B was less than 6%, indicating that high-fatty acid-producing Hansenula polymorpha cells underwent cell necrosis-mediated death in methanol-medium medium. Simultaneously, knocking out genes LPL1 and IZH3, individually or in combination, significantly reduced the cell necrosis rate, and the two may have a synergistic effect. To further demonstrate this conclusion, the key genes RIM101 and RIM13 in the Rim101 pathway, the cell necrosis regulatory pathway of strain HpFA01, were knocked out individually. The results showed that knocking out both significantly reduced the cell necrosis rate. Therefore, when high-fatty acid-producing Hansenula polymorpha utilizes methanol, fatty acids increase the intracellular diacylglycerol (DAG) content, and DAG has been reported to induce cell necrosis. On the other hand, methanol, and its oxidation product formaldehyde, can induce DNA-protein cross-linking, leading to cell death. These two factors exhibit a dose-dependent synergistic effect, jointly inducing cell necrosis mediated by the Rim101 pathway and ultimately resulting in cell death. lpl1Δ can alleviate DAG-induced cell necrosis, while izh3Δ can weaken DNA-protein cross-linking. Together, they reduce the cell necrosis rate, enabling cells to grow and accumulate fatty acids.

[0119] Example 5: Improving fatty acid synthesis in Hansenula polymorpha using methanol as a substrate

[0120] (1) Strategy selection and combination

[0121] Although genes such as FBP1, RPE, and TAL1 do not promote methanol utilization in high-fatty acid-producing cells, enhancing Xu5P supply may increase fatty acid production in domesticated strains. In addition, enhancing the supply of acetyl-CoA and NADPH, precursors required for fatty acid synthesis, is also expected to further improve fatty acid synthesis in domesticated strains. To increase intracellular NADPH supply, FBP1 was expressed in the HpFAM03 strain (HpFAM03 was developed from the domesticated strain HpFAM01, with the URA3 gene knocked out as a gene editing selection marker and the KU80 gene knocked out to increase homologous recombination efficiency) to increase the metabolic flux from gluconeogenesis to PPP, and coupled with the ZWF1 gene (i.e., the DNA fragment T integrated at the NS2 site). CAT1 -ZWF1-P ADH1 -P PMA1 -FBP1-T GAP This resulted in a 30% increase in fatty acid production. Simultaneously, to increase intracellular acetyl-CoA supply, the HpFAM03 strain expressed ATP:citrate lyase derived from Musmusculus (encoded by the gene MmACL) (i.e., the DNA fragment P is integrated at the NS11 site). PMA1 -MmACL-T ADH2 In strain HpFAM03, a pyruvate dehydrogenase complex (PDH) mutant derived from Escherichia coli was expressed (i.e., a DNA fragment P was integrated at the 4NS5 site). GAP -EcLpdA * -T FBA -T PDB -EcAceE-P TEF1 -P ADH1 -EcAceF-T AMO Both EcAceE and EcAceF can be obtained from the NCBI database, and EcLpdA... * The sequence can be found in Biochemistry 32, 2737-2740 (1993). The β-oxidation process was modified in the HpFAM03 strain (i.e., PEX10 gene knockout, pex10Δ). Results showed that, under the same conditions as in Example 1, fatty acid fermentation was carried out in a basal medium containing 10 g / L methanol for 96 h. Only overexpression of MmACL increased fatty acid yield by 10%, and FBP1-ZWF1 increased fatty acid yield by 31.1%. Other strategies had no significant effect, and pex10Δ even caused cell growth failure. This result indicates that single-pathway modification has no significant effect on the methanol-to-fatty acid process; a global metabolic rearrangement is needed to increase fatty acid synthesis precursors and cofactors. Figure 6 A, In the diagram, Control is strain HpFAM03)

[0122] Furthermore, simultaneous expression of FBP1-ZWF1 and MmACL (integration site and integration fragment as described above) in the HpFAM03 strain resulted in HpFAM09, which significantly increased fatty acid production by nearly 60%. Building on this, to promote methanol utilization and increase Xu5P supply, ribulose-5-phosphate-3-epimerase (encoded by the gene RPE) was further expressed (i.e., in P... RPE The site integrates DNA fragment P TAL1 HpFAM13 was obtained, which resulted in a further 30% increase in fatty acid yield, bringing the fatty acid yield to 680 mg / L under the condition of 10 g / L methanol supplementation. Figure 6 B). Figure 6 The fermentation conditions in B are the same as in Example 1, except that 10 g / L of methanol is added after 48 hours, and the fermentation ends after 120 hours.

[0123] (2) Shake flask batch fermentation

[0124] To avoid the impact of auxotrophs on cell growth, the auxotroph genes URA3 and KU80 were reintroduced into HpFAM03, HpFAM09, and HpFAM13, resulting in prototrophic strains (HpFAM02u, HpFAM09u, and HpFAM15u, respectively). These strains were then subjected to shake-flask-level fed-batch fatty acid fermentation. Initial culture volumes were 50 / 250 mL, in a basal medium containing 10 g / L methanol, with an initial inoculum size of OD0.05. 600 =0.4. No feeding was performed for the first 42 hours. Starting from 42 hours, 0.6 mL of methanol (10 g / L) and 0.4 mL of 5×Deflt nutrients were added every 24 hours. The pH was not adjusted for the first 86 hours according to pH test paper. From 86 hours onwards, the pH was adjusted to approximately 5 every 24 hours using 2M KOH (pH test paper). The 5×Deflt nutrient solution consisted of (NH4)2SO4 12.5 g / L, KH2PO4 72 g / L, MgSO4·7H2O 2.5 g / L, 5 mL / L Vitamin solution, and 10 mL / L Trace metal solution.

[0125] However, it was found that the increase in fatty acid production of HpFAM15u was not as significant as in the aforementioned batch fermentation during shake-flask fed-batch fermentation. This is speculated to be due to insufficient NADPH supply during the long fermentation process. Therefore, HpFAM09u and HpFAM15u further expressed cytoplasmic isocitrate dehydrogenase derived from Saccharomyces cerevisiae (encoded by the gene ScIDP2) (i.e., the DNA fragment P is integrated at the NS3 site). GAP -ScIDP2-T AMOStrains HpFAM14u and HpFAM16u were obtained. The results of fed-batch fermentation of these strains in shake flasks are as follows: Figure 7 As shown. Figure 7 A shows the growth status. The control strain grew the slowest. In the early stage, M14u and M16u grew slightly faster than M15u. In the later stage of fermentation, M15u grew better, reaching the highest OD. 600 The OD values ​​exceeded 50, while the final OD values ​​of the control strain, M14u, and M16u were basically the same; regarding fatty acid accumulation, from Figure 7 As can be seen from B, after 168 hours of fermentation, the fatty acid production of the three engineered strains was significantly higher than that of the control strain, and the production of M14u and M16u was also significantly higher than that of M15u. Among them, M16u reached the highest at 8.3 g / L, which was 2.8 times that of the control strain.

[0126] (3) Batch fed fermentation in fermenters

[0127] Based on the results of shake-flask fed-batch fermentation, the best strain, HpFAM16u, was selected for fed-batch fermentation experiments in a fermenter. The fermentation process was conducted in a DasGip parallel bioreactor system (Eppendorf). The initial fermentation medium was a basal medium containing 10 g / L methanol, with a 1.0 L fermenter containing 0.3 L of liquid, and an initial inoculum size of OD0.05. 600 =1. When the methanol in the fermenter is depleted, begin adding 600 g / L methanol and 5× Delft medium at a 2:1 feed ratio. Monitor the remaining methanol levels in real time during fermentation and adjust the feed rate accordingly. Aeration rate is 18–48 sL / h, stirring speed is 400–800 rpm, dissolved oxygen is controlled at 30%, and pH is adjusted to 5.6 using 2M KOH and 1M HCl.

[0128] Fermentation results as follows Figure 8 As shown, after 25 hours, methanol consumption was depleted, and feeding began. The methanol consumption rate fluctuated initially, but stabilized at 0.6 g / h from 72 hours until the end of the fermentation process. Cells grew rapidly, and fermentation ended after 183 hours, with the final OD value... 600 The yield reached 137.6 g / L, and the fatty acid yield reached 15.9 g / L, demonstrating the application potential of Hansenula yeast as a cell factory for the efficient conversion of methanol into fatty acids and other products.

[0129] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution. sequence list <110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences <120> Construction method and application of engineered bacteria that can produce fatty acids using methanol as the sole carbon source <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1521 <212> DNA <213> Artificial Sequence <400> 1 atggaatcta agaactgtca tcttgttgtt ctttgtcacg gtctttgggg agtgtctgag 60 cacttcgcct atattgaggc ccagctgaaa aatcatgctt ctctaagctc tagcaaagac 120 cagctaatag tctatactac aaagaccaac gagaggttca aaacctacga cggtatcgac 180 ctctgcggta ctcgcgtcgc agaggagatt cttgctgaag ccctaagatt gcaggaacag 240 ggattgactg ttagtaaatt ttctgttgtt ggctattctt taggaggttt aattgcacgg 300 tatgccatcg gtgttctgca ctatcgcggt tttttctgca atatcgagcc tgttaatttc 360 actagcttct gttcgccgca tgttggagtc ctcactcccg gccagagcgt cagtatcaag 420 attttcaact ggttggtgcc tgtgctactg ggcaaaagcg gccaccagct ttttctcaaa 480 gactcgtcta cagtgcctct tttgaaactg atgtcgcttc cccataccgt attctaccgc 540 ggcctcgcaa agttcaaaaa catctcgctg tattcgaaca tccgctccga catccggaca 600 tcctggtggt gttctggaat cagttacatc aatccttttg acatcttgga caagaatcct 660 aacgtcaaaa ttgaccagga cggatatatt cactttgaga acggctccaa gtttagcctg 720 aagttcattg aagactacaa accggtcgtt ctggatgcgg ataagccaat cacgttcaca 780 ggcctagtcg acgtctcgtc caaagcaaaa ctcaaccaaa tggttcagga ggacctccca 840 gccgacaacg acgtcccggc acagtcgaaa actgccaatt tcttccagag aaaattcaag 900 tgggtcatca tgctgtttcg gttcggtgta tacctgccaa tgtgggtcac gtggtttgtg 960 ctgcacaact ggttgcaagt ctttaattcc actctgcgag tgatcaagga gtcgtcgaaa 1020 ctggccgaat ttctggagct ttgcgaggag cctgagcctg cacggccgcc gctcaccaaa 1080 cggctcagcg aagccagcat tgacctgcac cgcctggagc atgggctgca tgaccaagga 1140 gatgagttca tcgactctgt ctttgatgcc atcacctcga cgaacgacat caaccgcagc 1200 atttttgagg cacgtgaaaa cggcccgctc tcgctcacca ccacaatcaa caagatcggc 1260 gagttcgacc tttctgcaga gccagataca ccggatgcac tccggtaccg gcagattctg 1320 agtcctttta aattggacct ttccgaccat caaaaggaca ttatcagtca tctcaataag 1380 ctaccatggc gcaaatttcc gatctacatc accaagacaa acgccacgca cgcggccgcc 1440 attgtccgac acgacgatcc tgcttttgag gagggtaagg tcgtggtgaa gcatttcgtt 1500 gaggaagtgt tccagcaata g 1521 <210> 2 <211> 1458 <212> DNA <213> Artificial Sequence <400> 2 atgtcttcca ctggcattga ggtgcacggg tcggacctgt ttgatgagca cgaccagaag 60 ctcatgcagc ggatcaagtc gcagtccgag ctgaacagcg acgcgtcgct ggaagagcgt 120 gtgctggagc ggtttgaccg gtttctggcc aagctggagt cgcggatcga ggatttcgaa 180 aagtacttta ccaactccgc ccacaaatcg aatcggaccc actacacatt ctttgaggcg 240 ttgaagtcca tcaaggccgc cgtgattcag aaccagcagc acaatctgca ggcgttcgga 300 cagatcttgg acaggtatta tggcagtctg ctggacgaga aaaaagagta cgactcgacg 360 gacatgtacg caaaaatatc gaccgggctg cagtttctgg acgagaaaat tgacctgttt 420 gagaagacct tcaacatccc gctaagcccg caagaacacc ttggaaaatt gaaggagaaa 480 ctgtacaatt tcgataaggc cgtggctgcc ggctcgaagc gacttcttca tttctacgag 540 ctgcctttcc aatggcgaga aaacaagtac attatctacg gctaccggtt caactcgggc 600 catctctcgg ctctgaagag cgttttccag ctccacaacg aaactgccaa tatttggaca 660 cacatattgg gatctctgct actcctgtac attatgctca aacactatcc ggcaaccgag 720 atttatgccc gttccagctt tggcgacaag ctggtgacca actgcttctt ccttgcctcc 780 atcaagtgtc tcatgtcgtc tgtcgtctgg cacacctacg actgcatctc gcaactcaag 840 ctgcgcaaaa gattcgcgtg tatcgactac acaggtatca cagtgttgat cacggcgtcc 900 atcatcacca ccgaacacat cgcgctcaag gagttccctg ccgcagagct cggcttcatc 960 tcgttttcta ccatcgccgg catcgtcggg gtgctcttca cgtggtccga gttcttcgac 1020 aagccagagt cgcggcccgt cagaatcctc tttttcatct cgctctcggc actcggcgtc 1080 gccgccttct gctccagtgc tgtgctcaag ggcctggaat actcgttcca tctgtacgcc 1140 ccgctgttga aaagctttgt gtggtacctg accggagtcc agttttacgg cacattggtg 1200 cccgaaagat ggagaaacga cgtcattgtc gacaagctgg acatctgcga cgaggcaatc 1260 agcgagctag agaaatcgga caaactggac gaatacctca acaaaacccc ggacgaaact 1320 ccgatgcgca agagctggtt ttccatgtgg tgggtcgact acgtcttcag ctcgcaccat 1380 atctggcata tatttgtgtt gctgggcgtt ctgggccact actcggctat tttggaaatg 1440 ttttctctcg ttaactag 1458 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gagaggttca aaacctacga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 acgaccagaa gctcatgcag 20 <210> 5 <211> 2027 <212> DNA <213> Artificial Sequence <400> 5 ctttgctggg ctgtcaaatg agtccaggtg atcggtcaga atgggcaagt tctctggccc 60 gccgattgga ggattcaggg cactctcggg aactaggaga ttctctataa tttctggtgg 120 agtgtaagtc gtgtaatcat aaacaaagaa tgcagacaga agaagaacaa ttgttccgct 180 aacaacacct gatgtgagga aaaacagctt cagcactctt ccgacagcgg tattcgtcca 240 aaaacttgat ttcggccgct tgacaggcgg taacgaaccg acctcgttcg aacgaggaaa 300 acccgtttgg aattgtctag agagcaaggc agtcgccagc agcggttttt tcaaatagac 360 tggagcagag ctggccaccg gcttggggca accccagata cttggtttag gccgcaaagc 420 cagtccaaga gtcgtttttg gcaggccaac ctttattatt gatgggaaca tactaaaggt 480 gctcttctaa gaaaagtgta ggcggccaaa ttacaaccaa atttaatagc caaacacaat 540 tgaataaaaa caaactgtgt gtttgaccta tagggatttt ataatgccaa gtgtaaggaa 600 cagacctgac cgaaaagtgc caatgaaaaa aagattttag atgttttcaa ccaccaatat 660 ccgatcggag ttgttggcaa tagggggtgc acgtttgctt ttttcataga tccatagtta 720 ccgtgcaacc aacctgatcc attgcgttcc ggccggcatt aaacggtggg cgccctttct 780 aggaatccga cgtgcatatt acatatttga ggggtgcaa agttcataac tcacatctgt 840 gcaccaaagt tatggcccgt ctcggtcgac cctgtttact cacctctgca ccgcgtagaa 900 ttttatggaa atccacatct cacaatcagt tattgtcttg cgacaatctc ttatctctgc 960 gtcgtgtgga ttataaaaga ccctgtaaat tccctagccc gccacgcatt cagcatttat 1020 agagagtaaa tctatcagca actccttgaa tgaaaattct tcaatgctca attcgcctcg 1080 ctcagctcgg caatgatggc ctccagcaca ccgccaaatc tgccgaccag cccgacaaaa 1140 ctgtctacca acacgttttt gtcgcccgac tccagcacac caggcgctac ttccaacgca 1200 agaccgtcta tcctgcttgc caggctcatt ttttcatgct cctctgtatc tctggcccgc 1260 tcctcgaaag acccctgcgg ccgcgccgat tctgccgcgt ctctctccgg cgtggccccc 1320 agctccatgt tacccttctc ggtgaccgga gtcatcgcca gcggcgccgt cgaagcgaga 1380 ttgatgttca caccaaactg attgagcgtg tctggcgacg ctgctgccgt cttgtcgtcc 1440 atcaactgtg agtgcaacat cggcacaggc atctggagcc gcatctgcga gcgcgcacgc 1500 gacaacgacc ccggctcgga cttcatctcc tggtactcgt tgttgctcag cgcgggagca 1560 gtctgtgggc caggaggaac gttctgcgac ggtatcttac tgttgatcgg ctgcaacagc 1620 ggctgctgcg aggccgacga gggaagcggc ttcatgtgtg gcagccgttg cggtgatggc 1680 acgaggccgg cctcctttag ccgctgcggc gagggcgtcc tgttgcccag actcgatagt 1740 cctgtgctgg aagctcctat gcgccgaacg gcaggctcct cagcctgcac gggggttgac 1800 ggcgactcct gacgcgacac ggttgacgcc cttggccgtt tttcctgtgc agccgtctct 1860 ggaagcgtgt ccagactggg gatctctata ggcacattgg ggaccgcgga cacctctgcg 1920 acgggcgacg acggcgtctc atcggactcg tcctgctcaa acagctttag cagcgatttt 1980 ggggcggtgg tgctagtcgc ggcggccgag ttagctgtgt ttcgcag 2027 <210> 6 <211> 2023 <212> DNA <213> Artificial Sequence <400> 6 tcggacaatc tgctggaaca ggagatcggc cggcgcatct ggtggtgcat ctatgtgttt 60 gactgtggcc agaccatcac gtttggtcgt ccgctgggga tcccctgtgc gggtatcgac 120 gcacggctgc cgttgaatat cagcgacaca gagctgactg cggcgtctga gtgcttgccc 180 ggcgaggtgc aggaaccgac aatctacacg agcgtgcggc tgcagagtct gttccacctg 240 ctcaccaact gcatctacga gcgcatcacc tccgacccat tcccgtccgc caaagagctg 300 ctcgagtggg acacgcggtt catagggcgg tggaagagcc tggtcccgga ctacttccag 360 caggacaagg aggttttgca gccgttcacg cttgcacacc acgttctgca ttggagacgg 420 aaaaacttgc agatcatcat gtacaggaca agtctgctga aaaaagtgtt ccgccgtcca 480 gacgatgagc cgctatgcca ggaggaagag gaggccgcgg aaaagtgtct gcgcaagtgc 540 tcctcgacga tccagagcat ggcagcgttg tggcagcgca aagaaaccac cagcaggatg 600 gaggcgtggt acacggtttt cttccttgtt cccgctttgc tcatgcctct tgtgtgtctg 660 cggaatgacc cgctatctgc gcaggcacag caatggaaac aggacatttt gaccgccgag 720 caggttctcc agagactgct gcacatctgt ccgatagcta ccaaaatcct ggagctcagc 780 aagtctctgg gggccgatta cgttgcctct gctgacgcgg cagctgactt gcttgccagt 840 gcgggaacgg acgagagccc gatgtcgcag ctcacacacc tgcattcggt gctgtggccg 900 ttgtcgtttg acatcgagca gcagtttctg tagcaggatt acgtaattta cgataaaaat 960 tttatggtgc ttagcccata caaagtaatt tttggtgaat atttttgttt gcgatactct 1020 catcatcaaa caatgatgca ttgtgctgtc acgactggtc cacgtcgatc ggtcttgagg 1080 acgagtgtgt agagtagtag tttgagtacg actcgggttc gttgtgcggc ggcgggcttg 1140 actgcgtgtg ggtttccaga cgttcgtgcg ggcgcagcag gggagccgtt tcgccaggct 1200 ccggcgcctg cttgccaaca gagcacacca aacaacacga cgccccgatt gctagtagca 1260 acatgccaat cacgacgccg atcagcagag ccacaaacgg caagtgcagg tggatcgggc 1320 ccttcgagtc gctgagccag tccgggttaa tcgaccggtc cacaagcaag atgcctgtgg 1380 cgtagcagct gttaatcgcg agcccttggc gcaaaatgcg cacctcgtgg tcgttaccct 1440 cgcgtacgcg caacatcgag atgcccgacg taagctcgcg acacgagaga tgcacgtttt 1500 ccagcagcgt cggaatggtg tcgttgcact cgataatttt ttgcatgcct ccggcaggca 1560 gaatgtgcaa ccgaacaaat ttctcgagtc tggcgaggtt gtgtagctct gacgtgaagt 1620 tctccagttt catcgagcgt gagctcggca caatgaacga ataatcctcg tagagaagat 1680 gatcaagctg caaaagttta agcaccttga ggaactcgtc gccgtcgaca gtggaaagca 1740 gctctggcac gccaatctgc acggtggcag gaaacacgag actgtctatt ggatgggcca 1800 ctcctttgtc aaagagcacc tcggactcaa tcgacacatc cactgctgta ccgtttatgt 1860 agagtttatc tgaatctgtc tctatctcaa gcggtttgtc cagcaacgta gtcagggtag 1920 ctgagtcctg gaagtcgtcg tagatcaggc cgtttaccac aaggtttccc aaaatctcca 1980 gcatgacgtc gcggttcgcg tagagatagt ccagcacgag atc 2023

Claims

1. A method for constructing engineered bacteria capable of producing fatty acids using methanol as the sole carbon source, characterized in that, The construction method includes: Knockout of high-fatty acid-producing Hansenula polymorpha engineered strains LPL1 Genes and IZH3 Gene; The high-fatty acid-producing Hansenula polymorpha engineered strain was obtained by knocking out the FAA1 gene of wild-type Hansenula polymorpha. The wild-type Hansenula polymorpha is wild-type Hansenula polymorpha NCYC 495 leu1.1; The nucleotide sequence of the LPL1 gene is shown in SEQ ID No. 1; The nucleotide sequence of the IZH3 gene is shown in SEQ ID No.

2.

2. The engineered bacteria capable of producing fatty acids using methanol as the sole carbon source obtained by the construction method according to claim 1.

3. The engineered bacteria capable of producing fatty acids using methanol as the sole carbon source obtained by the construction method according to claim 1, and the engineered bacteria capable of producing fatty acids using methanol as the sole carbon source according to claim 2, in the preparation of fatty acids using methanol as the carbon source.

4. A method for improving the methanol tolerance of wild-type Hansenula polymorpha, characterized in that, The method includes: Knockout of wild-type Hansenula polymorpha LPL1 Genes and IZH3 Gene; The LPL1 The nucleotide sequence of the gene is shown in SEQ ID No. 1; The IZH3 The nucleotide sequence of the gene is shown in SEQ ID No. 2; The wild-type Hansenula polymorpha is wild-type Hansenula polymorpha NCYC 495 leu1.

1.

5. A method for obtaining a Hansenula polymorpha strain capable of producing high levels of fatty acids using methanol as the sole carbon source, characterized in that, The method includes the following steps: The high-fatty acid-producing Hansenula polymorpha engineered strain was domesticated as follows: The strain was adapted to an initial culture medium containing both glucose and methanol, and then adapted to a culture medium with a gradually decreasing ratio of glucose and methanol until the glucose was reduced to 0, thus obtaining the Hansenula yeast strain capable of producing high levels of fatty acids using methanol as the sole carbon source. The high-fatty acid-producing Hansenula polymorpha engineered strain was obtained by knocking out wild-type Hansenula polymorpha. FAA1 Genes obtained; The wild-type Hansenula polymorpha is wild-type Hansenula polymorpha NCYC 495 leu1.1; The total carbon concentration of glucose and methanol in the culture medium is 0.2–0.5 mol / L; The ratio of glucose to methanol gradually decreases to a carbon molar ratio of glucose to methanol of x:10-x, where x decreases with a gradient of y, and y takes the value of 0.5 to 1.

6. The method according to claim 5, characterized in that, The initial culture medium contains glucose and methanol in a carbon molar ratio of x:10-x, where x ranges from 3 to 7. The adaptive culture involves acclimatizing a high-fatty acid-producing Hansenula polymorpha engineered strain in a culture medium until it initially reaches an OD value of [missing value]. 600 =0.2 inoculation, OD growth at 48 h 600 Reaching 5-6.

7. The method according to claim 5, characterized in that, It also includes performing genome sequencing and / or transcriptome sequencing analysis on domesticated high-fatty acid-producing Hansenula polymorpha engineered strains to obtain key gene targets and / or metabolic pathways, and optimizing the key gene targets and / or metabolic pathways.

8. The method according to claim 5, characterized in that, The method also includes improving the quality of high-fatty acid-producing Hansenula polymorpha engineered strains. MmACL Gene expression.

9. The method according to claim 5, characterized in that, The method also includes improving the quality of high-fatty acid-producing Hansenula polymorpha engineered strains. ZWF1 Genes and FBP1 Gene expression.

10. The method according to claim 5, characterized in that, The method also includes improving the quality of high-fatty acid-producing Hansenula polymorpha engineered strains. ZWF1 Gene, FBP1 Genes and MmACL Gene expression.

11. The method according to claim 5, characterized in that, The method also includes improving the quality of high-fatty acid-producing Hansenula polymorpha engineered strains. ZWF1 Gene, FBP1 Gene, MmACL Genes and RPE Gene expression.

12. The method according to claim 5, characterized in that, The method also includes improving the quality of high-fatty acid-producing Hansenula polymorpha engineered strains. ZWF1 Gene, FBP1 Gene, MmACL Genes and ScIDP2 Gene expression.

13. The method according to claim 5, characterized in that, The method also includes improving the quality of high-fatty acid-producing Hansenula polymorpha engineered strains. ZWF1 Gene, FBP1 Gene, MmACL Gene, RPE Genes and ScIDP2 Gene expression.

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