Photo-enzyme catalysis system based on sulfur metabolism of yarrowia lipolytica and application of photo-enzyme catalysis system

Abstract

The invention relates to a photo-enzyme catalysis system based on sulfur metabolism of yarrowia lipolytica and application of the photo-enzyme catalysis system, and belongs to the technical field of genetic engineering and microbial fermentation. The photo-enzyme catalysis system for sulfur metabolism of Yarrowia lipolytica comprises Yarrowia lipolytica, a cadmium binding polypeptide biosynthesis module and a ferredoxin NADP (+) reductase biosynthesis module, the cadmium binding polypeptide biosynthesis module and the ferredoxin NADP (+) reductase biosynthesis module are sequentially connected through subcloning, and the cadmium binding polypeptide biosynthesis module and the ferredoxin NADP (+) reductase biosynthesis module are sequentially connected through subcloning. The yeast cells are located on Yarrowia lipolytica cell chromosomes; the Met25 gene in the recombinant yarrowia lipolytica is knocked out. A light capture and electron transfer system is constructed by recombining a cadmium binding polypeptide gene sequence and a ferredoxin NADP (+) reductase gene in yeast cells, and the oil yield of yarrowia lipolytica is increased.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and microbial fermentation, and in particular to a light-enzyme catalytic system based on sulfur metabolism of Yarrowia lipolytica and application thereof. Background Art

[0002] Several approaches have been explored to enhance NADPH production within cells, but each has its limitations. One approach is to use chemically synthesized quantum dots, such as CdTe. While these quantum dots are very efficient at capturing light energy to generate NADPH, their commercial synthesis increases production costs and reduces economic benefits. In addition, these quantum dots have no specific localization within organelles, which further reduces the efficiency of NADPH production. Another approach is to use rhodopsin-like pigments to capture light energy, but rhodopsin is not as stable and efficient as quantum dots. In addition, the cell's oxidative pentose phosphate pathway can be driven by using xylose, but this approach requires xylose, which increases costs and reduces the overall economic return.

[0003] Yarrowia lipolytica is a GRAS (generally recognized as safe) organism widely used for commercial lipid production. However, the lipid production rate in Yarrowia lipolytica is limited by the insufficient supply of NADPH. NADPH is mainly supplied through the oxidative pentose phosphate pathway, which is essential and leads to a decrease in lipid / glucose conversion. To address this limitation, light, as a renewable energy source, provides a promising way to regenerate NADPH. Quantum dots (QDs), as emerging materials, have the ability to efficiently capture light energy and perform electron transfer, making them ideal for enhancing NADPH production in industrial processes. Therefore, quantum dots (QDs) can be used as a starting point to seek a new way to improve Yarrowia lipolytica. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a light-enzyme catalytic system based on sulfur metabolism of Yarrowia lipolytica and its application. The light-enzyme catalytic system based on sulfur metabolism of Yarrowia lipolytica constructed by the present invention can directly synthesize sulfur-based quantum dots, thereby improving the oil production capacity of Yarrowia lipolytica.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In the first aspect, the present invention provides a photo-enzyme catalytic system based on sulfur metabolism of Yarrowia lipolytica, the system comprising Yarrowia lipolytica, a cadmium-binding polypeptide biosynthesis module and a ferredoxin NADP(+) reductase biosynthesis module, the cadmium-binding polypeptide biosynthesis module and the ferredoxin NADP(+) reductase biosynthesis module being sequentially connected by subcloning and located on the chromosome of the Yarrowia lipolytica cell; the Met25 gene in the recombinant Yarrowia lipolytica is knocked out.

[0007] The present invention knocks out the Met25 gene in the Yarrowia lipolytica cells by genetic engineering, so that the yeast cells can produce hydrogen sulfide, and recombines the cadmium binding polypeptide (CdSBP-SP1) biosynthesis module and the ferredoxin NADP (+) reductase (FNR) biosynthesis module to construct a light capture and electron transfer system. Based on this, the cadmium binding polypeptide (CdSBP-SP1) biosynthesis module can be used in the yeast cells to synthesize cadmium sulfide (CdS) quantum dots with the hydrogen sulfide produced by the Cd ions and the hydrogen sulfide produced by the cadmium binding polypeptide (CdSBP-SP1) to form a light capture system, absorb light energy, and drive the ferredoxin NADP (+) reductase (FNR) biosynthesis module to synthesize FNR to promote NADPH reduction, thereby improving the oil production capacity of Yarrowia lipolytica.

[0008] As a preferred embodiment of the first aspect, the yeast cell is Yarrowia lipolytica Po1fk-AD.

[0009] As a preferred embodiment of the first aspect, the cadmium binding polypeptide (CdSBP-SP1) biosynthetic module is a nucleotide sequence that can encode an amino acid sequence as shown in SEQ ID No:23.

[0010] As a preferred embodiment of the first aspect, the ferredoxin NADP(+) reductase (FNR) biosynthesis module is a nucleotide sequence that can encode an amino acid sequence as shown in SEQ ID No:25.

[0011] As a preferred embodiment of the first aspect, the nucleotide sequence of the cadmium binding polypeptide (CdSBP-SP1) biosynthetic module is shown in SEQ ID No:1.

[0012] As a preferred embodiment of the first aspect, the nucleotide sequence of the ferredoxin NADP(+) reductase (FNR) biosynthesis module is shown in SEQ ID No:2.

[0013] As a preferred embodiment of the first aspect, the cadmium binding polypeptide biosynthesis module and the ferredoxin NADP(+) reductase biosynthesis module connected in sequence are plasmid pUrlp-Met25KO-XP-CdSBP-SP1-24-FNR.

[0014] As a preferred embodiment of the first aspect, the cadmium binding polypeptide (CdSBP-SP1) biosynthesis module also includes a peroxisome targeting signal peptide encoding module; the ferredoxin NADP(+) reductase (FNR) biosynthesis module also includes a peroxisome targeting signal peptide encoding module.

[0015] The present invention constructs a targeted signal peptide encoding module based on the recombinant light capture and electron transfer system of yeast cells, and the signal peptide can be pulled to the peroxisome in Yarrowia lipolytica to achieve protein localization. The present invention adds a signal peptide to the C-terminus of the cadmium binding polypeptide CdSBP-SP1 and the FNR enzyme, which can pull the cadmium sulfide quantum dots to the peroxisome and shorten the path of electron transfer. In addition, the signal peptide can locate the cadmium sulfide quantum dots to the surface of the peroxisome, and can effectively remove the toxicity of hydrogen peroxide generated by the quantum dot photocatalysis process.

[0016] As a preferred embodiment of the first aspect, as a preferred embodiment of the first aspect, the cadmium binding polypeptide (CdSBP-SP1) biosynthetic module is a nucleotide sequence that can encode an amino acid sequence as shown in SEQ ID No:24.

[0017] As a preferred embodiment of the first aspect, the ferredoxin NADP(+) reductase (FNR) biosynthesis module is a nucleotide sequence that can encode an amino acid sequence as shown in SEQ ID No:26.

[0018] As a preferred embodiment of the first aspect, the nucleotide sequence of the cadmium binding polypeptide (CdSBP-SP1) biosynthetic module is shown in SEQ ID No:3.

[0019] As a preferred embodiment of the first aspect, the nucleotide sequence of the ferredoxin NADP(+) reductase (FNR) biosynthesis module is shown in SEQ ID No:4.

[0020] As a preferred embodiment of the first aspect, the connected cadmium binding polypeptide biosynthesis module and ferredoxin NADP(+) reductase biosynthesis module are plasmids pUrlp-Met25KO-XP-CdSBP-SP1(SKL)-24-FNR(SKL).

[0021] In a second aspect, the present invention further provides use of the recombinant Yarrowia lipolytica described in the first aspect in producing oils and fats.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention utilizes the sulfur metabolism of Yarrowia lipolytica, and constructs a light capture and electron transfer system by recombining the cadmium binding polypeptide CdSBP-SP1 and the FNR gene in yeast cells. The light capture system in the present invention is a sulfur-based quantum dot, such as CdS, which can directly capture light energy, regenerate NADPH through the FNR gene electron transfer system, and increase the lipid yield in Yarrowia lipolytica. The present invention also constructs a peroxisome targeting signal peptide, which is composed of three amino acids (SKL) at the C-terminus of the protein. This signal peptide can be pulled to the peroxisome in Yarrowia lipolytica to achieve protein localization. The present invention adds a signal peptide to the C-terminus of the cadmium binding polypeptide CdSBP-SP1 and FNR to shorten the path of electron transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram of the plasmid pUrlp;

[0024] Figure 2 Schematic diagram of the plasmid vector pYLXP template;

[0025] Figure 3 is a schematic diagram of the plasmid vector pYL24 template;

[0026] Figure 4 Schematic diagram of hydrogen sulfide gas detection results of the initial yeast (left picture) and the recombinant yeast strain 1 (right picture);

[0027] Figure 5 This is a schematic diagram of the detection results of hydrogen sulfide gas in the tail gas of the fermentation tank of the recombinant yeast strain 2;

[0028] Figure 6 This is a schematic diagram of the observation results of the fermentation broth of the recombinant yeast strain 3 under a confocal microscope;

[0029] Figure 7 It is a schematic diagram comparing the results of lipid production of the initial yeast and recombinant yeast 2 and 3;

[0030] Figure 8 This is the significant difference analysis of the final oil production results of the initial yeast, recombinant yeast 2, and 3;

[0031] Fig. 9 Schematic diagram comparing the lipid yield results of the initial yeast and recombinant yeasts 2 and 3. DETAILED DESCRIPTION

[0032] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0033] In this specific embodiment, the PCR amplification system is: total volume 50 μL; DNA template, 1 μL; forward primer (10 μM) and backward primer (10 μM) 2 μL each; dNTP (10 mM), 1 μL; 2×Phanta Max Buffer, 25 μL; Max Super-Fidelity polymerase, 1 μL; and finally, double distilled water is used to make up 50 μL.

[0034] In this specific embodiment, the PCR amplification procedure is as follows: amplification conditions are 95°C pre-denaturation for 4 min (1 cycle); 95°C denaturation for 15 sec, annealing at 60°C for 15 sec, and extension at 72°C for 1 min (34 cycles); and extension at 72°C for 5 min (1 cycle).

[0035] The initial strain used in this specific embodiment is Yarrowia lipolytica Po1fk-AD, which is obtained by the following method: using Yarrowia lipolytica Po1f as the starting strain, knocking out the coding gene ku70 responsible for non-homologous recombination in the strain to obtain Po1fΔku70 (MatA, Δku70::hisG, leu2 270, ura3-302, xpr2-322, axp1-2), abbreviated as Po1fk (knockout method refers to Kretzschmar, A. et al. Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining. Curr. Genet. 59, 63-72 (2013).); inserting genes ACC1 and DGA1 into the aforementioned Po1fk strain, the function of ACC1 and DGA1 is to promote the lipid production of yeast (construction method reference: Wand, K. et al. Engineering the Lipid and Fatty Acid Metabolism in Yarrowia lipolytica for Sustainable Production of High Oleic Oils.ACS Synth.Biol.11,1542-1554(2022).) to obtain the initial strain Po1fk-AD used in this specific embodiment; among them, Yarrowia lipolytica Po1f was purchased from the American Type Culture Collection and numbered ATCC MYA-2613.

[0036] The reagent used for seamless cloning in this specific implementation is the Seamless Cloning Kit produced by Beyotime. The product has a detailed cloning introduction. Cloning and sequencing can obtain linear fragments of the corresponding promoter and gene, and PCR expansion can be used for subcloning. The subcloning uses Beyotime's T4 ligase, and the subclones are digested for enzyme verification. The positive clones can be digested with AvrII to obtain linearized fragments for lipolytic yeast transfection. The PCR enzyme used is Max Super-Fidelity polymerase from Nanjing Novozyme Biotechnology Co., Ltd.

[0037] The pYL24, pYLXP' and pUrlp vectors used in this specific embodiment are all from the Synthetic Biology and Intelligent Control Laboratory of Guangdong Technion-Israel Institute of Technology.

[0038] Example 1

[0039] 1. Construction of recombinant yeast 1: Po1fk-AD-Met25KO

[0040] 1 Construction of plasmid for knocking out Met25 gene: pUrlp-Met25KO

[0041] 1.1 Amplify the target fragment: amplify the Met25up nucleotide sequence shown in SEQ ID No: 5; amplify the Met25down nucleotide sequence shown in SEQ ID No: 6.

[0042] The Met25up fragment was amplified using primers with sequences as shown in SEQ ID No: 16 and SEQ ID No: 17 and obtained by yeast colony PCR. Similarly, the Met25down fragment was amplified using primers as shown in SEQ ID No: 18 and SEQ ID No: 19.

[0043] 1.2 Plasmid construction

[0044] according to Figure 1, the pUrlp vector is a circular plasmid containing leucine Leu2 and uracil Ura3 tags. The expression of the uracil Ura3 gene is driven by the TEF promoter and regulated by the XPR2 terminator. In addition, LoxP sites are provided before the TEF promoter and after the XPR2 terminator, so that the Ura3 screening marker gene can be recovered using the Cre-LoxP system. The pUrlp plasmid vector can also be inserted into the homologous arms upstream and downstream of the site-specific integration site, which can achieve site-specific insertion and recombination of genes in chromosomes. pUrlp is cut with AvrII and NheI, and the Met25up nucleotide sequence shown in SEQ ID No:5 can be inserted into the AvrII restriction site, and the Met25down nucleotide sequence shown in SEQ ID No:6 can be inserted into the NheI restriction site. The transformed plasmid is pUrlp-Met25KO.

[0045] 2. Construction of recombinant strain 1

[0046] The Met25 gene in the initial strain Yarrowia lipolytica Po1fk-AD was replaced with the Ura uracil screening gene by homologous recombination technology to obtain a recombinant strain 1 in which the Met25 gene was knocked out, as follows:

[0047] The plasmid pUrlp-Met25KO was transferred into Yarrowia lipolytica Po1fk-AD, and the Met25 gene in the strain was replaced with the Ura uracil selection gene, thereby obtaining the recombinant yeast 1 with the Met25 gene knocked out: Po1fk-AD-Met25KO. The subsequent modified strains 2 and 3 both inserted the target gene at this position, so as to simultaneously knock out the Met25 gene and insert the gene to be overexpressed.

[0048] Strain culture method: The initial strain Yarrowia lipolytica Po1fk-AD (named as Po1fk-AD in the present invention) was cultured in YPD medium for 12 hours, and then 300 μL was added to 3 mL of fresh YPD medium and cultured for 5 hours. Centrifuge at 1000 rpm for 5 minutes, collect the cells, discard the supernatant, and wash the cells with sterilized ddH2O.

[0049] Transformation method: centrifuge at 1000rpm for 5min, collect the cells, and discard the supernatant; add 1mL of 100mM lithium acetate to the cells, gently blow to make the cells evenly suspended, place at room temperature for 5min, and then centrifuge at 1000rpm for 5min, collect the cells, and prepare yeast competent cells. The transformation mixture system includes 240μL PEG (50% W / V), 36μL 1.0M lithium acetate, 10μL ss-DNA, and the transformation fragment is the linearized plasmid pUrlp-Met25KO, and finally fill it up to 360μL with sterilized ddH2O. Add each item in the above order to the competent cells of Yarrowia lipolytica, blow it evenly with a pipette, place it in a 42℃ water bath for 30min, centrifuge it at 2000rpm for 2min, discard the supernatant, add 1mL YPD liquid culture medium, and culture it at 30℃200rpm for 2h.

[0050] Screening method: The transformed recombinant strains were screened on CSM-Ura plates, the positive strains were verified by colony PCR, and the ones with the correct bands were fermented in shake flasks. Specifically: centrifuge at 2000rpm for 5 minutes at room temperature, discard the supernatant, wash twice with sterile water, and finally resuspend the cells with 100μL sterile water, and apply the plate lacking UR A for screening; the screening culture conditions were 30℃ and cultured for more than 48h. The transformants were picked for overnight culture, the genome was extracted, and PCR was verified. The correct clones were obtained with the correct target fragments, and the correct cloned strains were named: Po1fk-AD-Met25KO.

[0051] 2. Construction of recombinant yeast 2: Po1fk-AD-Met25KO-FC

[0052] 1. Plasmid construction

[0053] 1.1 Obtain optimized target sequence:

[0054] Based on the optimized cadmium binding polypeptide sequence (CdSBP-SP1) and ferredoxin NADP(+) reductase sequence (FNR) of Yarrowia lipolytica, the nucleotide sequence of the obtained target sequence is as follows:

[0055] CdSBP-SP1 nucleotide sequence: SEQ ID No: 1;

[0056] FNR nucleotide sequence: SEQ ID No: 2;

[0057] 1.2 Plasmid construction

[0058] according to Figure 2The pYLXP vector is a circular plasmid containing a leucine Leu2 tag for screening in Yarrowia lipolytica. The vector also includes a strong promoter TEF and a terminator XPR2, which play an important role in the expression of the target gene in Yarrowia lipolytica. Figure 2 After digestion with SnaBI and KpnI, the PCR amplification product of CdSBP-SP1 was added for seamless cloning to obtain the plasmid: pYLXP , -CdSBP-SP1.

[0059] according to Figure 2 , pYL24 vector is a circular plasmid containing a leucine Leu2 tag for screening in Yarrowia lipolytica. The vector also includes a strong promoter TDH and a terminator MIG1, which play an important role in the expression of the target gene in Yarrowia lipolytica. Figure 3 After being digested with XbaI and KpnI, the PCR amplification product of FNR was added for seamless cloning to obtain the plasmid: pYL24-FNR.

[0060] The above two constructed plasmids were transformed into Escherichia coli competent cells respectively, and then the conventional plasmid extraction steps were performed. The extracted plasmids were digested to obtain the target fragments CdSBP-SP1 sequence and FNR sequence. The gene CdSBP-SP1 fragment and the FNR fragment were then connected by subcloning, and the two genes were inserted into the plasmid pUrlp-Met25KO for subcloning to obtain the plasmid pUrlp-Met25KO-XP-CdSBP-SP1-24-FNR.

[0061] 2. Construction of recombinant strain 2

[0062] The above plasmid was extracted and linearized with AvrII to recover the fragment, and the linearized plasmid was transfected into Yarrowia lipolytica. Specifically:

[0063] Strain culture method: After culturing the initial strain Yarrowia lipolytica Po1fk-AD in YPD medium for 12 hours, 300 μL was taken and added to 3 mL of fresh YPD medium and cultured for 5 hours. Centrifuge at 1000 rpm for 5 minutes, collect the cells, discard the supernatant, and wash the cells with sterilized ddH2O.

[0064] Transformation method: centrifuge at 1000rpm for 5min, collect the cells, and discard the supernatant; then add 1mL of 100mM lithium acetate to the cells, gently blow to make the cells evenly suspended, place at room temperature for 5min, and then centrifuge at 1000rpm for 5min, collect the cells, and prepare yeast competent cells. The transformation mixture system includes 240μL PEG (50% W / V), 36μL 1.0M lithium acetate, 10μL ss-DNA, and the transformation fragment is the linearized plasmid pUrlp-Met25KO-XP-CdSBP-SP1-24-FNR, and finally fill it up to 360μL with sterilized ddH2O. Add each item to the competent cells of Yarrowia lipolytica in the above order, blow evenly with a pipette, place in a 42℃ water bath for 30min, centrifuge at 2000rpm for 2min, discard the supernatant, add 1mL YPD liquid culture medium, and culture at 30℃ 200rpm for 2h.

[0065] Screening method: The recombinant strains were screened on CSM-Ura plates, the positive strains were verified by colony PCR, and the ones with the correct bands were fermented in shake flasks. The details are as follows: centrifuge at room temperature for 5 minutes at 2000rpm, discard the supernatant, wash twice with sterile water, and finally resuspend the cells with 100μL sterile water, and apply the plate lacking URA for screening. The screening culture conditions are 30℃ and culture for more than 48h. The transformants were picked for overnight culture, the genome was extracted, and PCR was verified. The correct clones were obtained with the correct target fragments, and the correct cloned strains were named: Po1fk-AD-Met25KO-FC.

[0066] 3. Construction of recombinant yeast 3: Po1fk-AD-Met25KO-FC (SKL)

[0067] 1. Plasmid construction

[0068] 1.1 Obtaining an optimized target sequence with a peroxisome targeting signal peptide (SKL):

[0069] Based on the optimized cadmium binding polypeptide sequence (CdSBP-SP1) and ferredoxin NADP(+) reductase sequence (FNR), a peroxisome targeting signal peptide (SKL) was added to obtain the nucleotide sequence of the target sequence as follows:

[0070] CdSBP-SP1 (SKL) nucleotide sequence: SEQ ID No: 3;

[0071] FNR (SKL) nucleotide sequence: SEQ ID No: 4;

[0072] 1.2 Plasmid construction

[0073] The plasmid vector pYLXP template was digested with SnaBI and KpnI, and the PCR amplification product of CdSBP-SP1 (SKL) was added for seamless cloning to obtain the plasmid: pYLXP , -CdSBP-SP1-SKL.

[0074] The plasmid vector pYL24 template was digested with XbaI and KpnI, and the PCR amplification product of FNR (SKL) was added for seamless cloning to obtain the plasmid: pYL24-FNR (SKL).

[0075] The two constructed plasmids were transformed into competent Escherichia coli cells, followed by conventional plasmid extraction steps. The extracted plasmids were digested to obtain the target fragments CdSBP-SP1 (SKL) sequence and FNR (SKL) sequence. The gene CdSBP-SP1 (SKL) fragment and the FNR (SKL) fragment were then connected by subcloning. The two genes were inserted into the plasmid pUrlp-Met25KO for subcloning to obtain the plasmid pUrl p-Met25KO-XP-CdSBP-SP1 (SKL)-24-FNR (SKL).

[0076] 2. Construction of recombinant strain 3

[0077] The above plasmid was extracted and linearized with AvrII to recover the fragment, and the linearized plasmid was transfected into Yarrowia lipolytica. Specifically:

[0078] Strain culture method: After culturing the initial strain Yarrowia lipolytica Po1fk-AD in YPD medium for 12 hours, 300 μL was taken and added to 3 mL of fresh YPD medium and cultured for 5 hours. Centrifuge at 1000 rpm for 5 minutes, collect the cells, discard the supernatant, and wash the cells with sterilized ddH2O.

[0079] Transformation method: Centrifuge at 1000rpm for 5min, collect the cells, and discard the supernatant. Then add 1mL of 100mM lithium acetate to the cells, gently blow to evenly suspend the cells, place at room temperature for 5min, and then centrifuge at 1000rpm for 5min to collect the cells and prepare yeast competent cells. The transformation mixture system includes 240μL PEG (50% W / V), 36μL 1.0M lithium acetate, 10μL ss-DNA, and the transformation fragment is the linearized plasmid pUrlp-Met25KO-XP-CdSBP-SP1(SKL)-24-FNR(SKL), and finally fill to 360μL with sterilized ddH2O. Add each item to the newly prepared Yarrowia lipolytica competent cells in the above order, pipette and mix evenly, place in a 42°C water bath for 30 min, centrifuge at 2000 rpm for 2 min, discard the supernatant and add 1 mL YPD liquid culture medium, and culture at 30°C and 200 rpm for 2 h.

[0080] Screening method: The recombinant strains were screened on CSM-Ura plates, and the positive strains were verified by colony PCR. The ones with the correct bands were fermented in shake flasks. The details are as follows: centrifuge at room temperature for 5 minutes at 2000rpm, discard the supernatant, wash twice with sterile water, and finally resuspend the cells with 100μL sterile water, and apply the plate lacking URA for screening. The screening culture conditions are 30℃ and culture for more than 48h. The transformants were picked for overnight culture, the genome was extracted, and PCR was verified. The correct clones were obtained by extracting the genome and PCR verification. The correct clones were named Po1fk-AD-Met25del-FC (SKL).

[0081] The primers used in this example are shown in Table 1:

[0082] Table 1: Primer information used in this example

[0083]

[0084]

[0085] Example 2 Fermentation tank fermentation

[0086] The recombinant yeast strains 1, 2, and 3 of Example 1 were cultured in yeast CSM-leu liquid medium (purchased from Shanghai Qiyuan Biotechnology Co., Ltd., item number QYK-17854). For seed shake flask culture, the culture medium contained 80 g / L glucose (purchased from Shanghai Shenggong A501991-0500), 0.7 g / L completely synthetic medium without leucine (Sunrise Catalog#: 1005-100), 1.7 g / L yeast nitrogen source without amino acids and ammonium sulfate (Sunrise Catalog#: 1501-250), and 1.1 g / L ammonium sulfate (purchased from Shanghai Shenggong A620060-0500). Yeast was cultured at 250 rpm and 30°C. For the bioreactor, glucose was fed at a constant rate (15 g / day). After 2 days of fermentation, the peristaltic pump supplemented cadmium acetate (Sigma755087-5G) until 1 mM cadmium acetate was reached on the 6th day, and blue light with a wavelength of 410 nm and a power of 2 W was provided. Strain 2 and strain 3 were compared with an illumination group and a non-illumination group, and then the fermentation broth was tested.

[0087] Experimental example

[0088] 1. Hydrogen sulfide detection

[0089] The initial identification of hydrogen sulfide is done by testing filter paper soaked in lead nitrate. Strains that produce hydrogen sulfide will dye the filter paper black. The generation rate of candidate strains is then tested using a hydrogen sulfide gas probe. Specifically:

[0090] The preliminary identification of hydrogen sulfide is carried out using filter paper soaked in lead nitrate. The filter paper is soaked in 1g / l lead nitrate solution for 30 minutes, taken out and attached to the mouth of the shaking bottle. The filter paper will show color in about ten minutes. The black color is lead sulfide precipitation, which is a positive strain. Later in the fermentation tank, the exhaust gas can be passed through the gas probe for real-time detection. The gas probe model is S_M4C-H2S-100, purchased from Shanghai Songbai Gas Sensor Company.

[0091] The results are as follows Figure 4 As shown, the filter paper test of the initial yeast did not change color, while the recombinant yeast 1 ( Figure 4 The filter paper test on the right side shows black. Figure 5 As shown, a large amount of hydrogen sulfide is also produced in the tail gas of the fermentation tank of recombinant yeast 2. It can be seen that the strain with Met25 knocked out in the present invention can produce hydrogen sulfide.

[0092] 2. Quantum dot detection

[0093] CdS quantum dots were observed under a confocal microscope, with an excitation wavelength of 405nm and a receiving spectrum of 420-490nm. Specifically:

[0094] Laser wavelength: 405nm, power 1.0%, pinhole 1.21AU, detection wavelength 420-490nm, GaASP 700V, digital gain 1.0.

[0095] T-PMT channel: laser wavelength 639nm, power 0.3%, pinhole 1.00AU, detection wavelength 300-900nm, GaASP300V, digital gain 1.0.

[0096] The results are as follows Figure 6 As shown, when the fermentation broth of the recombinant strain 3 was observed under a confocal microscope, the quantum dot fluorescence could be clearly observed and precisely located in a specific organelle, while this phenomenon was not observed in the recombinant strain 1.

[0097] 3. Oil and glucose content detection

[0098] Some samples were taken every 24 hours for testing, glucose was detected by high performance liquid chromatography, and lipids were detected by gas chromatography, specifically: For lipid analysis, 4 OD units of cell pellets were centrifuged at 14,000 rpm for 3 minutes, and after the pellet was dried, 500 μL of 0.5N sodium-methanol solution and 100 μL of 2g / L C13 standard were added. After vortex mixing at 1200 rpm for 2 hours, 40 μL of 98% H2SO4 and 400 μL of n-hexane were added. After vortex mixing at 1200 rpm for 10 minutes, centrifuged at 14,000 rpm for 2 minutes, and the organic phase was taken for GC-FID analysis. GC method reference (Tai, M. & Stephanopoulos, G. Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab. Eng. 15, 1-9 (2013)): The oven conditions are as follows: 150 ° C (1 minute), heating to 230 ° C in 10 minutes, and keeping at 230 ° C for 2 minutes. The split ratio is 10: 1. The total lipids analyzed in this paper include C16, C16, C16, C18, C18: 1, and C18: 2.

[0099] For glucose analysis, the cell culture medium was centrifuged at 14,000 rpm for 3 minutes, and the supernatant was filtered and analyzed by HPLC. The mobile phase was 5 mM H2SO4, and the RID detector was used at a flow rate of 0.4 mL / min and a temperature of 50°C. The glucose standard concentrations were 0 g / L, 1.25 g / L, 2.5 g / L, 5 g / L, 10 g / L, and 20 g / L

[0100] Recombinant strain 1 does not have a light-harvesting and electron-transfer system, and its performance is no different from that of strains 2 and 3 under dark conditions, so its final yield is replaced by the dark fermentation results of strains 2 and 3. The strain with Met25 knockout can eliminate the toxicity of cadmium ions and form CdS@SP1 nanoparticles under the wrapping of SP1. The nanoparticles are responsible for light energy harvesting, and FNR is responsible for converting light energy into NADPH. The results are shown in Figure 2. Figure 7-9 As shown, the oil production and oil yield of the recombinant yeast 1 were lower than those of the recombinant strains 2 and 3. It can be seen that the recombinant yeast containing the cadmium binding polypeptide CdSBP-SP1 and the FNR gene constructed by the present invention can increase the oil production and successfully increase the lipid production in the bioreactor (Yarrowia lipolytica) from 3.1 g / L to 7.1 g / L (Table 2).

[0101] Table 2: Analysis results of high-quality yield of different strains

[0102]

[0103] The present invention utilizes the sulfur metabolism of Yarrowia lipolytica to directly synthesize sulfur-based quantum dots, such as CdS, by recombining the cadmium-binding polypeptide CdSBP-SP1 and FNR genes in yeast cells, which is convenient and economical. By using a localization signal peptide (SKL) to encapsulate the quantum dots, they can be precisely located in specific organelles, thereby improving catalytic efficiency and NADPH regeneration capacity. In addition, the production of hydrogen sulfide in yeast can also detoxify metal ions in the culture medium, making it possible to use waste glycerol for lipid production, making the fermentation process more environmentally friendly. The use of light as a renewable energy source further improves economic benefits, and quantum dots have higher stability and efficiency than pigments.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A light-enzyme catalytic system based on sulfur metabolism of Yarrowia lipolytica, characterized in that: The system comprises Yarrowia lipolytica, a cadmium binding polypeptide biosynthesis module and a ferredoxin NADP(+) reductase biosynthesis module, wherein the cadmium binding polypeptide biosynthesis module and the ferredoxin NADP(+) reductase biosynthesis module are sequentially connected through subcloning and are located on the chromosome of the Yarrowia lipolytica cell; and the Met25 gene in the recombinant Yarrowia lipolytica is knocked out.

2. The light-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to claim 1, characterized in that: The cadmium binding polypeptide biosynthesis module is a nucleotide sequence that can encode an amino acid sequence as shown in SEQ ID No: 23; the ferredoxin NADP (+) reductase biosynthesis module is a nucleotide sequence that can encode an amino acid as shown in SEQ ID No:

25.

3. The light-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to claim 2, characterized in that: The nucleotide sequence of the cadmium binding polypeptide biosynthesis module is shown in SEQ ID No: 1; the nucleotide sequence of the ferredoxin NADP (+) reductase biosynthesis module is shown in SEQ ID No:

2.

4. The light-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to claim 1, characterized in that: The subclones sequentially connect the cadmium binding polypeptide biosynthesis module and the ferredoxin NADP (+) reductase biosynthesis module to form a plasmid pUrlp-Met25KO-XP-CdSBP-SP1-24-FNR.

5. The light-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to claim 1, characterized in that: The cadmium binding polypeptide biosynthesis module also includes a peroxisome targeting signal peptide encoding module; the ferredoxin NADP (+) reductase biosynthesis module also includes a peroxisome targeting signal peptide encoding module.

6. The light-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to claim 5, characterized in that: The cadmium binding polypeptide biosynthesis module is a nucleotide sequence that can encode an amino acid sequence as shown in SEQ ID No: 24; the ferredoxin NADP (+) reductase biosynthesis module is a nucleotide sequence that can encode an amino acid sequence as shown in SEQ ID No:

26.

7. The light-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to claim 6, characterized in that: The nucleotide sequence of the cadmium binding polypeptide biosynthesis module is shown in SEQ ID No:3; the nucleotide sequence of the ferredoxin NADP(+) reductase biosynthesis module is shown in SEQ ID No:

4.

8. The light-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to claim 5, characterized in that: The subclones sequentially connect a cadmium binding polypeptide biosynthesis module including a peroxisome targeting signal peptide encoding module and a ferredoxin NADP (+) reductase biosynthesis module to form a plasmid pUrlp-Met25KO-XP-CdSBP-SP1 (SKL) -24-FNR (SKL).

9. Use of the photo-enzyme catalytic system for sulfur metabolism of Yarrowia lipolytica according to any one of claims 1 to 8 in the production of oils and fats.

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