WO2021120548A1 - 基因工程菌及其构建方法、应用，生产nad +的方法 - Google Patents

基因工程菌及其构建方法、应用，生产nad +的方法 Download PDF

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WO2021120548A1
WO2021120548A1 PCT/CN2020/095553 CN2020095553W WO2021120548A1 WO 2021120548 A1 WO2021120548 A1 WO 2021120548A1 CN 2020095553 W CN2020095553 W CN 2020095553W WO 2021120548 A1 WO2021120548 A1 WO 2021120548A1
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nad
strain
genetically engineered
engineered bacteria
genome
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王伟
王康林
付敏杰
金永红
田峰
贾和平
胡志浩
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合肥康诺药物开发有限公司
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Definitions

  • the present invention relates to the field of biotechnology, in particular to a genetically engineered bacteria, its construction method and application, and a method for producing NAD + .
  • Coenzyme I or nicotinamide adenine dinucleotide (NAD + ) is the coenzyme of many dehydrogenases in the body, which connects the tricarboxylic acid cycle and the respiratory chain to realize the transfer of electrons, and it participates in cell material Metabolism, energy synthesis, cell DNA repair, signal transmission and other physiological activities play an irreplaceable role in glycolysis, gluconeogenesis, tricarboxylic acid cycle and respiratory chain. Its structural formula is as follows:
  • NAD + nicotinamide mononucleotide
  • NR nicotinamide ribose
  • Symptom treatment can also be used to prevent liver damage, multiple sclerosis autoimmune neurodegeneration, heart damage caused by heart disease, stroke, brain damage caused by injury and other diseases and repair their sequelae.
  • Biological enzymatic methods such as: starting from nicotinamide, nicotinamide mononucleotide is obtained under the action of nicotinamide phosphoribosyltransferase, and then nicotinamide mononucleotide is obtained under the action of nicotinamide phosphoribose acyltransferase to obtain NAD + ; Some also start with nicotinamide ribose, obtain nicotinamide mononucleotide under the action of nicotinamide ribokinase, and then react to obtain NAD + .
  • the present invention proposes a genetically engineered strain and its construction method, application, and method for producing NAD + .
  • the present invention knocks out the gene encoding adenine deaminase from the strain’s genome, or/ And the genome of the strain integrates the gene encoding the enzyme in the NAD + synthesis pathway, resulting in a genetically engineered strain with high NAD + production.
  • the gene encoding adenine deaminase on the genome of the strain is knocked out, or/and the gene encoding the enzyme in the NAD + synthesis pathway is integrated into the genome of the strain.
  • the strain is Saccharomyces cerevisiae.
  • the enzymes in the NAD+ synthesis pathway are nicotinamidase PNC1, nicotinic acid phosphoribosyl transferase NPT1, nicotinic acid mononucleotide adenosine transferase NMA1, nicotinic acid mononucleotide adenosine transferase At least one of the enzymes NMA2, glutamine-dependent NAD + synthetase QNS1.
  • NMA1 and NMA2 are isoenzymes.
  • the enzyme in the NAD + synthesis pathway is at least one of nicotinic acid phosphoribosyl transferase NPT1 and nicotinic acid mononucleotide adenosyltransferase NMA1.
  • the present invention provides a genetically engineered bacterium, Saccharomyces cerevisiae KH08, whose genome integrates nicotinic acid phosphoribosyl transferase NPT1 and nicotinic acid mononucleotide adenosine in the NAD + synthesis pathway. Transferase NMA1, and the gene encoding adenine deaminase on its genome is knocked out, which can produce high NAD + . Saccharomyces cerevisiae (Saccharomyces cerevisiae) KH08 is deposited in the General Microbiology Center CGMCC of the China Microbial Culture Collection Management Committee, and the deposit number is CGMCC No.19048.
  • Saccharomyces cerevisiae KH08 Saccharomyces cerevisiae KH08 is deposited in the General Microbiology Center CGMCC of the China Microbial Culture Collection Management Committee, and the deposit number is CGMCC No.19048.
  • the present invention also discloses a method for constructing the above-mentioned genetically engineered bacteria, which knocks out the gene encoding adenine deaminase on the genome of the host strain to obtain a strain with high NAD+ yield;
  • the method of "constructing the expression cassette of the gene encoding the enzyme in the NAD + synthesis pathway” is: ligating the encoding gene of the enzyme in the NAD + synthesis metabolic pathway into the expression cassette of the integrated plasmid of the host strain.
  • the above-mentioned integrated plasmid contains components: pMB1 replicon, ampicillin resistance coding gene, G418 resistance selection marker KanMX, ⁇ 1 fragment, ⁇ 2 fragment, GPD promoter, ADH1 terminator, TEF1 promoter and CYC1 terminator.
  • the integrated plasmid is referred to as plasmid pND04 for short.
  • the integration is a delta-site integration method.
  • the ⁇ sequence includes a ⁇ 1 fragment and a ⁇ 2 fragment, wherein the nucleotide sequence of the ⁇ 1 fragment is shown in SEQ ID No. 1, and the nucleotide sequence of the ⁇ 2 fragment is shown in SEQ ID No. 2.
  • the delta sequence is the long terminal repeat sequence on the Ty transposon. They are located on the retrotransposon Ty1 and Ty2 of the chromosomal DNA of Saccharomyces cerevisiae.
  • the delta-site integration is a kind of gene integration using the homology of the delta sequence.
  • the above-mentioned host strain is Saccharomyces cerevisiae, purchased from Yantai Mali Yeast Co., Ltd., and the batch number is YT201902260569. This strain is renamed Saccharomyces cerevisiae KH01.
  • the present invention also proposes the application of the above-mentioned genetically engineered bacteria in the production of NAD + .
  • the present invention also proposes a method for producing NAD + , which uses nicotinamide or/and adenine as a substrate to produce NAD + by using the above-mentioned genetically engineered bacteria.
  • Figure 1 is a schematic diagram of the transformation of NAD + by yeast whole cells.
  • the present invention integrates the encoding genes of enzymes in the NAD + synthesis pathway into the genome of the strain, especially nicotinic acid phosphoribosyl transferase NPT1 and nicotinic acid ribose monophosphate adenylyltransferase NMA1, which can greatly increase yeast transformation production
  • nicotinic acid phosphoribosyl transferase NPT1 and nicotinic acid ribose monophosphate adenylyltransferase NMA1 which can greatly increase yeast transformation production
  • the ability of NAD + after yeast fermentation is mature, by directly adding nicotinamide or/and adenine to the fermentation broth for whole cell transformation, the NAD + content accumulated in the yeast cell can be qualitatively increased;
  • adenine When using adenine as a substrate, adenine will produce a large amount of hypoxanthine by-products during the production of NAD + , which makes the purification of NAD + difficult. Therefore, by knocking out adenine deaminase from the strain genome
  • the coding gene of the present invention completely loses or weakens the activity of adenine deaminase, can reduce the amount of substrate adenine, and can alleviate the impact of hypoxanthine by-products on subsequent purification.
  • the genetically engineered bacteria of the present invention is compared with In wild-type yeast, when the same amount of NAD + is produced, the amount of adenine is reduced by at least 3 times, and the accumulation of hypoxanthine is reduced by at least 6 times;
  • Colony color milky white; growth temperature: 28-30°C; optimum pH: 5.0-6.0; colony morphology: the surface is smooth, moist, viscous, easy to provoke, uniform texture; reproduction mode: budding reproduction.
  • Figure 1 is a schematic diagram of yeast whole cell transformation into NAD + .
  • glycogen is glycogen reserve
  • Saccharomyces cerevisiae is Saccharomyces cerevisiae cell
  • Nm is nicotinamide
  • Na is niacin
  • Ade is adenine
  • PRPP is 5-phosphoribose- 1-pyrophosphate
  • AMP is 5-adenosine monophosphate
  • ATP is 5-adenosine triphosphate
  • NaMN is nicotinic acid ribose monophosphate
  • NaAD is nicotinic acid adenine dinucleotide
  • NAD is nicotinamide adenine dinucleotide
  • NPT1 is nicotinic acid phosphoribosyltransferase
  • NMA1 is nicotinic acid ribose monophosphate adenylyltransferase 1
  • NMA2 is nic
  • Figure 2 is a nucleic acid gel map of the Eco105I digestion verification of plasmid pND08, in which 1, 2, 3, and 4 are pND08 plasmids.
  • Fig. 3 is a nucleic acid gel image verified by PCR of Saccharomyces cerevisiae KH07 strain, where 1 is the genome of Saccharomyces cerevisiae KH01 strain, 2, 3, and 4 are the genome of Saccharomyces cerevisiae KH07 strain, and M is Marker.
  • Figure 4 shows the HPLC chromatogram of NAD+.
  • Figure 5 shows the HPLC chromatograms of niacin, hypoxanthine, adenine and nicotinamide.
  • NPT1, NMA1, and AAH1 used in the following examples all represent genes, and NPT1, NMA1, and AAH1 all represent enzymes.
  • the source information of the kits, reagents and plasmids used in the examples is as follows: agarose gel DNA recovery kit (upgraded spin column type) (Shanghai Jierui Bioengineering Co., Ltd., catalog number GK2043-200); GBclonart seamless cloning reagent Kit (Suzhou Shenzhou Gene Co., Ltd., Item No. GB2001-48); Yeast Genomic DNA Extraction Kit (Beijing Kangwei Century Biotechnology Co., Ltd., Item No. CW0569); Plasmid pUC57 (Wuhan Miaoling Biotechnology Co., Ltd., Item No.
  • Plasmid pNL01 contains GPD promoter and TEF1 promoter;
  • PCR amplification was performed with primers pND05-F1 and pND05-R1 to obtain a 1811bp dual-promoter fragment; the agarose gel DNA recovery kit was used to gel-recover the dual-promoter fragment, and then combined with SmaI enzyme The recovered pND04 plasmid was cut for seamless cloning (using GBclonart seamless cloning kit), and the plasmid pND05 was constructed.
  • PCR amplification was performed with primers pND06-F1 and pND06-R1 to obtain a 1654bp KanMX fragment; the KanMX fragment was gel-recovered with an agarose gel DNA recovery kit, and then the recovered pND05 was digested with SdaI The plasmid was cloned seamlessly (using GBclonart seamless cloning kit), and the plasmid pND06 was constructed.
  • PCR amplification was performed with primers pEZTEF1-F1 and pEZTEF1-R1 to obtain a 6391bp vector fragment 1;
  • PCR amplification was performed with primers pEZGPD-F1 and pEZGPD-R1 to obtain 7678bp vector fragment 2;
  • FIG. 2 is the nucleic acid gel map of the Eco105I restriction enzyme digestion verification of plasmid pND08. Among them, 1, 2, 3, 4 are pND08 plasmids, and marker is Thermo Scientific GeneRuler DNA Ladder Mix (Product No. SM0333); Figure 2 shows that the plasmid pND08 can be digested with Eco105I to obtain two bands of 1309bp and 7574bp, and the plasmid pND08 is verified correctly.
  • the adenine deaminase expressed by the AAH1 gene can degrade adenine into hypoxanthine with lower solubility, which affects the subsequent purification work and causes the waste of adenine. Therefore, knocking out the AAH1 gene can reduce the amount of adenine. It can also reduce the amount of by-product hypoxanthine to achieve the purpose of reducing costs.
  • PCR amplification was performed with primers AAH1-F1 and AAH1-R1, and then agarose gel DNA recovery kit was used to run the gel to recover the 1588bp AAH1 knockout fragment I;
  • Saccharomyces cerevisiae competent cells from Saccharomyces cerevisiae KH01 (this is the host strain, the host strain is Saccharomyces cerevisiae), purchased from Yantai Mali Yeast Co., Ltd., the batch number is YT201902260569, this strain is renamed as Saccharomyces cerevisiae KH01) glycerol cryopreservation tube, take a ring of bacteria liquid, streak activation on YPD plate (YPD medium composition: yeast powder 10g/L, peptone 20g/L, glucose 20g/L), and place it in a 30°C incubator , Culture for 3 days; then pick a single colony from the YPD plate and inoculate it into a 4mL YPD test tube, incubate at 30°C and 250rpm for 16h, transfer the test tube bacterial solution to a 30mL YPD shake flask at 2% of the inocul
  • Electrotransformation Take 1 ⁇ g of AAH1 knock-out fragment I and add it to 100 ⁇ L of Saccharomyces cerevisiae competent cells, place on ice for a while, transfer to a 2mm electroporation cup in an ice bath, after 1.5KV electric shock, resuspend in 1mL YPD medium and transfer Put it in the EP tube and incubate at 30°C for 1-3h to obtain the transformation solution. Take 200 ⁇ L and 300 ⁇ L of the transformation solution respectively and spread on the YPD plate containing 500mg/L G418 antibiotic, and place it in a 30°C incubator for 3- The transformant was obtained in 5 days, and the transformant was selected and streaked for purification.
  • the genome was extracted with a yeast genome extraction kit.
  • the primers were used to perform PCR on AAH1-500F/AAH1-500R to verify that the knockout was correct.
  • This transformant strain was named Saccharomyces cerevisiae KH07SG (Contains G418 resistance);
  • Example 2 in 1.2 prepare competent cells of Saccharomyces cerevisiae KH07SG strain; According to the method in Example 2 in 1.3, transform 500ng of pSH65 plasmid into competent cells of Saccharomyces cerevisiae KH07SG strain to obtain a transformation solution. The solution was spread on YPD containing 30mg/L zeo (bleomycin) or YPD containing 15mg/L Phleo (phleomycin). After culturing in a 30°C incubator for 3 days, the transformants were picked to inoculate YPG ( Replace 20g/L glucose with 20g/L galactose.
  • zeo zeo
  • YPD containing 15mg/L Phleo phleomycin
  • Test tube culture at 30°C and 250rpm for 2-3h. After the bacterial solution is diluted, take 100 ⁇ L each of the bacterial solution of different dilutions and apply separately YPD plate, cultured in a 30°C incubator for 3 days, pick a single colony and spot the YPD plate and the YPD plate containing 500mg/L G418 antibiotic respectively, and screen the strains with elimination of resistance (that is, grow on the YPD plate, at 500mg/L containing 500mg/L). G418 antibiotic YPD strain on the plate), this strain was named Saccharomyces cerevisiae KH07S.
  • PCR amplification was performed with primers AAH1-F2 and AAH1-R2, and then agarose gel DNA recovery kit was used to run the gel to recover the 1589bp AAH1 knockout fragment II;
  • Figure 3 shows that Saccharomyces cerevisiae KH01 strain has a target band of 2031bp, while Saccharomyces cerevisiae KH07 strain has two bands of 1011bp and 1151bp, and Saccharomyces cerevisiae KH07 strain AAH1 has two bands. Knockout and elimination are correct.
  • the method of producing NAD+ by shaking flask fermentation is: pick a ring of bacteria liquid from the glycerin cryostat tube of Saccharomyces cerevisiae to streak the YPD plate, place it in an incubator at 30°C for 2-3 days; use an inoculation ring to pick up the activated wine Yeast monoclonal to contain 50mL fermentation medium (Fermentation medium formula: glucose 50g/L, casein extract 15g/L, yeast extract 15g/L, NaCl 5g/L, KH 2 PO 4 1g/L, K 2 HPO 4 1g/L, MgSO 4 ⁇ 7H 2 O 0.3g/L, pH 5.4) 500mL shake flask, placed at 30 °C, 250 rpm conditions, after 72 hours of incubation, add adenine and nicotinamide to make adenine final The concentration is 3g/L, the final concentration of nicotinamide is 6g/L, continue to incubate at 30°C
  • Fermentation extract Take 20mL of the above fermentation broth in a 50mL centrifuge tube, collect the bacteria by centrifugation, add 5mL 0.2% formic acid water to the bacteria, vortex to mix, in a 95°C water bath, stir at 1000rpm for 5min, and quickly transfer to ice Put it on an ice bath, stir at 1000 rpm for 5-10 minutes, centrifuge at 7500 rpm at 4°C for 5 minutes, and take the supernatant to obtain the fermentation extract;
  • Detection Detect the content of adenine, hypoxanthine, and NAD + in the fermentation extract by high performance liquid chromatography HPLC.
  • the detection method is: draw 1 mL of NAD + extract, filter it with a 0.22 ⁇ m filter, and then sample for detection;
  • Figure 5 shows the HPLC chromatograms of niacin, hypoxanthine, adenine and nicotinamide;
  • Figure 4 shows that the retention time of NAD + is 4.811 min;
  • Figure 5 shows that the retention time of niacin is 2.952 min, the retention time of hypoxanthine is 3.515 min, the retention time of adenine is 5.858 min, and the retention time of nicotinamide is 6.669 min.
  • the 6178bp integration fragment was transformed into the cells of Saccharomyces cerevisiae KH01 to obtain transformants.
  • the transformants were streaked and purified, and then the NAD + was produced by fermentation in the shake flask described in 5.1 of Example 2 Methods
  • the transformants were screened and the dominant transformants with high NAD + yield were obtained, and the transformant strain was named as Saccharomyces cerevisiae KH06 after elimination according to the two steps in Example 2;
  • the 6178bp integration fragment was transformed into the cells of Saccharomyces cerevisiae KH07 to obtain transformants.
  • the transformants were taken and purified by streaking, and then the transformation was screened by the method of producing NAD + by shaking flask fermentation in 5.1 of Example 2 The transformant with high yield of NAD + was obtained.
  • the transformant strain was eliminated according to the step 2 in Example 2, it was named Saccharomyces cerevisiae KH08, and it was preserved in the Chinese Microbial Culture Collection Management Committee. Center CGMCC, the deposit number is CGMCC No.19048;

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Abstract

提供一种基因工程菌及其制备方法，该工程菌菌株基因组上的腺嘌呤脱氨酶的编码基因被敲除，或/和菌株基因组上整合有NAD +合成代谢途径中的酶的编码基因，其中所述NAD +合成代谢途径中的酶可为烟酰胺酶PNC1、烟酰酸磷酸核糖转移酶NPT1、烟酰酸单核苷酸腺苷转移酶NMA1、烟酰酸单核苷酸腺苷转移酶NMA2、谷氨酰胺依赖性NAD +合成酶QNS1中的至少一种。还提供了该基因工程菌在生产NAD +中的应用。

Description

基因工程菌及其构建方法、应用，生产NAD +的方法 技术领域
本发明涉及生物技术领域，尤其涉及一种基因工程菌及其构建方法、应用，生产NAD +的方法。
背景技术
辅酶I，即烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide，NAD +)，是体内很多脱氢酶的辅酶，连接三羧酸循环和呼吸链，用来实现电子的传递，它参与细胞物质代谢、能量合成、细胞DNA修复、传递信号等多种生理活动，在糖酵解，糖异生，三羧酸循环和呼吸链中发挥着不可替代的作用，其结构式如下所示：
Figure PCTCN2020095553-appb-000001
从19世纪40年代，NAD +首次被发现开始，研究者对NAD +的探索越来越偏重于血管再生、基因修复、抗衰老、减少出生缺陷等方面，让NAD +在抗衰老和医药领域展现出活力。由于NAD +分子量比较大，不利于人体的吸收，补充其前体烟酰胺单核苷酸(NMN)或者烟酰胺核糖(NR)可能是最科学有效的补充NAD +的方式，但是NMN和NR不能穿过血脑屏障，而NAD +却可以，这使得NAD +在治疗成瘾和其他大脑失衡疾病方面具有不可替代的作用，可用于慢性疾病、重量控制、情绪障碍、酒精和毒瘾等疾病和症状的治疗，还可用于防止肝 损伤、多发性硬化自身免疫性神经退行性变、心脏病、中风造成的心脏损伤、受伤造成的脑损伤等病症及其后遗症的修复。
NAD +的合成有方法有：从头合成路径，数种以烟酰胺单核苷酸、烟酰胺核糖、烟酰胺或烟酸为起始物的补救路径等；但从头合成路径，NAD +产量有限，所以NAD +的工业化生产多是从NAD +前体烟酰胺单核苷酸、烟酰胺核糖、烟酰胺或烟酸的补救路径开始，采用生物酶法或者全细胞转化法来生产NAD +。生物酶法例如：从烟酰胺开始，在烟酰胺磷酸核糖转移酶作用下得到烟酰胺单核苷酸，然后烟酰胺单核苷酸在烟酰胺磷酸核糖腺苷酰基转移酶作用下得到NAD +；也有从烟酰胺核糖开始，在烟酰胺核糖激酶作用下得到烟酰胺单核苷酸，进而反应得到NAD +。但该路径由于需要制备多种酶液，工艺繁琐，因此工业化成本相对较高；另有直接从化学合成的烟酰胺单核苷酸开始，经生物酶催化后生产NAD +，但该方法由于化学合成烟酰胺单核苷酸，所以成本高且存在手性化合物的问题。已有报道利用酵母(Sakai,T.,et al.Accumulation of Nicotinamide Adenine Dinucleotidein Baker's Yeast by Secondary Culture.Agr.Biol.Chem.,37(5),1049-1056,1973)、产氨棒杆菌(Elhariry,H.M.,et al.S434F in NrdE Generates the Thermosensitive Phenotype ofCorynebacterium ammoniagenes CH31and EnhancesThermolability by Increasing the SurfaceHydrophobicity of theNrdE(Ts)Protein.Appl.Environ.Microbiol.71(9),5582-5586,200)、芽孢杆菌等进行全细胞转化烟酰胺和腺嘌呤等物质生产NAD +，但产量低，导致生产成本高。
发明内容
基于背景技术存在的技术问题，本发明提出了一种基因工程菌及其构建方法、应用，生产NAD +的方法，本发明将菌株基因组上的腺嘌呤脱氨酶的编码基 因敲除，或/和菌株基因组上整合有NAD +合成途径中的酶的编码基因，得到了NAD +产量高的基因工程菌。
本发明提出的一种基因工程菌，菌株基因组上的腺嘌呤脱氨酶的编码基因被敲除，或/和菌株基因组上整合有NAD +合成途径中的酶的编码基因。
上述腺嘌呤脱氨酶的编码基因被敲除后，会使得腺嘌呤脱氨酶的活性完全丧失或减弱。
优选地，所述菌株为酿酒酵母(Saccharomyces cerevisiae)。
优选地，所述NAD +合成途径中的酶为烟酰胺酶PNC1、烟酰酸磷酸核糖转移酶NPT1、烟酰酸单核苷酸腺苷转移酶NMA1、烟酰酸单核苷酸腺苷转移酶NMA2、谷氨酰胺依赖性NAD +合成酶QNS1中的至少一种。
上述NMA1和NMA2是为同工酶。
优选地，所述NAD +合成途径中的酶为烟酰酸磷酸核糖转移酶NPT1、烟酰酸单核苷酸腺苷转移酶NMA1中的至少一种。
优选地，本发明提供了一种基因工程菌为酿酒酵母(Saccharomyces cerevisiae)KH08，其基因组上整合有NAD +合成途径中的烟酰酸磷酸核糖转移酶NPT1和烟酰酸单核苷酸腺苷转移酶NMA1，且其基因组上腺嘌呤脱氨酶的编码基因被敲除，能高产NAD +。酿酒酵母(Saccharomyces cerevisiae)KH08保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19048。
本发明还公开了上述基因工程菌的构建方法，将宿主菌株基因组上的腺嘌呤脱氨酶的编码基因敲除获得NAD +产量高的菌株；
或者分别构建NAD +合成途径中的酶的编码基因的表达框，接着将所述酶的 编码基因的表达框整合于宿主菌株基因组上构建获得NAD +产量高的菌株；
或者分别构建NAD +合成途径中的酶的编码基因的表达框，接着将所述酶的编码基因的表达框整合于敲除腺嘌呤脱氨酶的编码基因的宿主菌株基因组上构建获得NAD +产量高的菌株。
上述“构建NAD +合成途径中的酶的编码基因的表达框”的方法为：将NAD +合成代谢途径中的酶的编码基因连接至宿主菌株的整合质粒的表达框内。
上述整合质粒包含的组件有：pMB1复制子、氨苄青霉素抗性的编码基因、G418抗性筛选标记KanMX、δ1片段、δ2片段、GPD启动子、ADH1终止子、TEF1启动子和CYC1终止子，将整合质粒简称为质粒pND04。
优选地，所述整合为δ-位点整合法。
优选地，δ序列包含δ1片段和δ2片段，其中，δ1片段的核苷酸序列如SEQ ID No.1所示，δ2片段的核苷酸序列如SEQ ID No.2所示。
δ序列是Ty转座子上的长末端重复序列，它们位于酿酒酵母染色体DNA的逆转录转座子Ty1和Ty2上，δ-位点整合就是利用δ序列的同源性进行基因整合的一种方式，文献Semkiv,M.V.,et al.Increased ethanol accumulation from glucose via reduction of ATP level in a recombinant strain of Saccharomyces cerevisiaeoverexpressing alkaline phosphatase.BMC Biotechnol 42(14),2014中记载有关于δ序列的表述。将需要过表达的基因整合到酿酒酵母基因组上，可以增加发酵过程中基因的稳定性。
上述宿主菌株为酿酒酵母(Saccharomyces cerevisiae)，从烟台马利酵母有限公司购买得到，批号为YT201902260569，将此菌株重新命名为酿酒酵母KH01。
本发明还提出了上述基因工程菌在生产NAD +中的应用。
本发明还提出了一种生产NAD +的方法，以烟酰胺或/和腺嘌呤作为底物利用上述基因工程菌生产NAD +。
上述以烟酰胺和腺嘌呤作为底物利用上述基因工程菌生产NAD +的流程图参见图1，图1为酵母菌全细胞转化NAD +的示意图。
有益效果：
1.本发明将NAD +合成途径中的酶的编码基因整合到菌株基因组上，特别是烟酸磷酸核糖转移酶NPT1和烟酸核糖单磷酸腺苷酰基转移酶NMA1，可以极大地增加酵母转化生成NAD +的能力，酵母发酵成熟后通过直接添加烟酰胺或/和腺嘌呤至发酵液中进行全细胞转化，可使酵母胞内积累的NAD +含量有质的提升；
2.以腺嘌呤为底物时，在生产NAD +时，腺嘌呤会产生大量次黄嘌呤副产物，给NAD +的纯化增加了困难，因此，通过在菌株基因组上敲除腺嘌呤脱氨酶的编码基因，使得腺嘌呤脱氨酶的活性完全丧失或者减弱，可以减少底物腺嘌呤的用量，同时可以缓解次黄嘌呤副产物对后续纯化造成的影响，本发明的基因工程菌相比于野生型酵母，生产同样量的NAD +时，腺嘌呤的用量降低了至少3倍，次黄嘌呤的积累量减少了至少6倍；
3.将NAD +合成途径中的酶的编码基因整合到敲除腺嘌呤脱氨酶的编码基因的宿主菌株基因组上，即可以提高NAD +产量，又可以减少底物腺嘌呤的用量，同时可以缓解次黄嘌呤副产物对后续纯化造成的影响，降低成本；与野生型酵母相比，NAD +的产量提高80％以上。
生物保藏说明
酿酒酵母(Saccharomyces cerevisiae)KH08，于2019年11月28日，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，地址为中国，北京，保藏编号为CGMCC No.19048，其形态学及生理化学特性如下：
菌落颜色：乳白色；生长温度：28-30℃；最适pH：5.0-6.0；菌落形态：表面光滑、湿润、粘稠，容易挑起，质地均匀；生殖方式：出芽生殖。
附图说明
图1为酵母菌全细胞转化NAD +的示意图，其中，glycogen为糖原储备，Saccharomyces cerevisiae为酿酒酵母细胞，Nm为烟酰胺、Na为烟酸、Ade为腺嘌呤、PRPP为5-磷酸核糖-1-焦磷酸、AMP为5-单磷酸腺苷、ATP为5-三磷酸腺苷、NaMN为烟酸核糖单磷酸、NaAD为烟酸腺嘌呤二核苷酸、NAD为烟酰胺腺嘌呤二核苷酸、NPT1为烟酸磷酸核糖转移酶、NMA1为烟酸核糖单磷酸腺苷酰基转移酶1、NMA2为烟酸核糖单磷酸腺苷酰基转移酶2。
图2为质粒pND08的Eco105I酶切验证的核酸胶图，其中，1、2、3、4为pND08质粒。
图3为酿酒酵母KH07菌株PCR验证的核酸胶图，其中，1为酿酒酵母KH01菌株的基因组，2、3、4为酿酒酵母KH07菌株的基因组，M为Marker。
图4为NAD +的HPLC色谱图。
图5为烟酸、次黄嘌呤、腺嘌呤和烟酰胺的HPLC色谱图。
具体实施方式
下面，通过具体实施例对本发明的技术方案进行详细说明。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所使用的NPT1、NMA1、AAH1均表示基因，NPT1、NMA1、AAH1均表示酶。
实施例中使用的试剂盒、试剂和质粒来源信息如下：琼脂糖凝胶DNA回收试剂盒(升级版离心柱型)(上海捷瑞生物工程有限公司，货号GK2043-200)；GBclonart无缝克隆试剂盒(苏州神洲基因有限公司，货号GB2001-48)；酵母基因组DNA提取试剂盒(北京康为世纪生物科技有限公司，货号CW0569)；质粒pUC57(武汉淼灵生物科技有限公司，货号P0087)；pUG6质粒(武汉淼灵生物科技有限公司，货号P0104)；pSH65质粒(武汉淼灵生物科技有限公司，货号P1352)；SmaI、SdaI等限制性内切酶(赛默飞世尔科技(中国)有限公司)。
实施例1酿酒酵母中δ整合表达质粒的构建
1含δ序列的整合质粒的构建
1.1以酿酒酵母基因组为模板，分别用表1中所列引物对PCR扩增得到235bp片段I和261bp片段II，其中片段I包含154bp的δ1片段，δ1片段的序列如SEQ ID No.1所示，片段II包含180bp的δ2片段，δ2片段的序列如SEQ ID No.2所示；
1.2以质粒pUC57为模板，用表1中所列引物对PCR扩增得到2682bp的pUC57片段；
1.3使用琼脂糖凝胶DNA回收试剂盒对pUC57片段、片段I和片段II进行胶回收处理，然后使用GBclonart无缝克隆试剂盒进行无缝克隆，构建得到质粒pND04。
表1步骤1.1和1.2中所用引物的序列
Figure PCTCN2020095553-appb-000002
2含双启动子的整合质粒的构建
取质粒pNL01，其核苷酸序列如SEQ ID No.3所示，质粒pNL01包含GPD启动子、TEF1启动子；
以质粒pNL01为模板，用引物pND05-F1和pND05-R1进行PCR扩增得到1811bp的双启动子片段；使用琼脂糖凝胶DNA回收试剂盒对双启动子片段进行胶回收处理，然后与SmaI酶切回收的pND04质粒进行无缝克隆(使用GBclonart无缝克隆试剂盒)，构建得到质粒pND05。
表2引物pND05-F1和pND05-R1的序列
引物名称 序列(5′→3′)
pND05-F1 TGTAATAGGATCAACCTGCAGGCCCGTTAGCATATCTACAATTGGGTGAAATG
pND05-R1 TCATTTTATATGTTTATATTCACCCCCATGGGTTGGCCGATTCATTAATGCAG
3含酵母筛选标记的整合质粒的构建
以pUG6质粒为模板，用引物pND06-F1和pND06-R1进行PCR扩增得到 1654bp的KanMX片段；用琼脂糖凝胶DNA回收试剂盒对KanMX片段进行胶回收处理，然后与SdaI酶切回收的pND05质粒进行无缝克隆(使用GBclonart无缝克隆试剂盒)，构建得到质粒pND06。
表3引物pND06-F1和pND06-R1的序列
引物名称 序列(5′→3′)
pND06-F1 TAATGTAATAGGATCAACCTGCAGGTTAATTAACTGCAGGTCGACAACCCTTAATATAAC
pND06-R1 TTGTAGATATGCTAACGGGCCTGCAATTTAAATCACTAGTGGATCTGATATCACCTAATAAC
4含目的基因的整合质粒的构建
4.1以质粒pND06为模板，用引物pEZTEF1-F1和pEZTEF1-R1进行PCR扩增得到6391bp的载体片段1；
4.2以酿酒酵母基因组为模板，用引物NPT1-F1和NPT1-R1进行PCR扩增得到1347bp的NPT1片段；
4.3使用琼脂糖凝胶DNA回收试剂盒对载体片段1、NPT1片段进行胶回收处理，然后使用GBclonart无缝克隆试剂盒进行无缝克隆，构建得到质粒pND07；
4.4以质粒pND07为模板，用引物pEZGPD-F1和pEZGPD-R1进行PCR扩增得到7678bp载体片段2；
4.5以酿酒酵母基因组为模板，用引物NMA1-F1和NMA1-R1进行PCR扩增得到NMA1片段；
4.6使用琼脂糖凝胶DNA回收试剂盒对载体片段2、NMA1片段进行胶回收处理，然后使用GBclonart无缝克隆试剂盒进行无缝克隆，构建得到质粒pND08，其核苷酸序列如SEQ ID No.4所示；
4.7对质粒pND08进行酶切验证，结果参见图2，图2为质粒pND08的 Eco105I酶切验证的核酸胶图，其中，1、2、3、4为pND08质粒，marker为Thermo Scientific GeneRuler DNA Ladder Mix(货号SM0333)；由图2可以看出质粒pND08用Eco105I酶切后可以得到1309bp和7574bp两条带，质粒pND08验证正确。
表4 4.1、4.2、4.4和4.5中所用引物的序列
引物名称 引物序列(5′→3′)
pEZTEF1-F1 GAATTCTGCAGATATCCATCACACTG
pEZTEF1-R1 TTTGTAATTAAAACTTAGATTAGATTGCTATGC
NPT1-F1 ATCTAATCTAAGTTTTAATTACAAAGGATCCATGTCAGAACCAGTGATAAAGTCTC
NPT1-R1 AGTGTGATGGATATCTGCAGAATTCTTAGGTCCATCTGTGCGCTTC
pEZGPD-F1 ACCCGGGGCGAATTTCTTATG
pEZGPD-R1 TTTGTTTGTTTATGTGTGTTTATTCGAAACTAAG
NMA1-F1 GAATAAACACACATAAACAAACAAAATGGATCCCACAAGAGCTCC
NMA1-R1 AAATCATAAGAAATTCGCCCCGGGTTCATTCTTTGTTTCCAAGAACTTGCTTAAC
实施例2酿酒酵母(二倍体)中AAH1基因的敲除
AAH1基因表达的腺嘌呤脱氨酶能将腺嘌呤降解成溶解性更低的次黄嘌呤，影响后续纯化工作，且造成腺嘌呤的浪费，所以将AAH1基因敲除，即可以减少腺嘌呤用量，又可以减少副产物次黄嘌呤的量，达到减少成本的目的。
二倍体酵母中有两个AAH1等位基因，只敲除其中一个，让其自身发生置换的效率太低，所以需要分别对两个等位基因进行敲除。在分别对两个等位基因进行敲除时，要设计好两次敲除所用的同源臂，避免两次敲除发生在同一位置。具体操作如下所述：
1第一个AAH1基因的敲除
1.1以pUG6质粒为模板，用引物AAH1-F1和AAH1-R1进行PCR扩增，然后用琼脂糖凝胶DNA回收试剂盒跑胶回收得到1588bp的AAH1敲除片段I；
1.2制备酿酒酵母感受态细胞：从酿酒酵母KH01(此为宿主菌株，宿主菌株为酿酒酵母(Saccharomyces cerevisiae)，从烟台马利酵母有限公司购买得到，批号为YT201902260569，将此菌株重新命名为酿酒酵母KH01)甘油冻存管中取一环菌液，在YPD平板(YPD培养基的成分为：酵母粉10g/L，蛋白胨20g/L，葡萄糖20g/L)划线活化，置于30℃培养箱，培养3天；然后从YPD平板上挑取单菌落接种到4mL YPD试管，30℃、250rpm条件下培养16h，将试管菌液按2％的接种量转接至30mL YPD摇瓶中，30℃、250rpm条件下培养至OD 600＝0.8-1.2，收集菌体，用冷的无菌水洗涤两次，再用冷的1M山梨醇洗涤两次后，最后用1M山梨醇将菌体重悬至300μL的体系，得到酿酒酵母感受态细胞，按100μL分装成三份后，放置冰中，备用；
1.3电转化：取AAH1敲除片段I 1μg加入至100μL酿酒酵母感受态细胞中，冰中放置片刻，转移至冰浴的2mm电转杯中，1.5KV电击后，用1mL YPD培养基重悬后转移至EP管中，于30℃摇床温育1-3h得到转化液，分别取200μL、300μL转化液涂布于含500mg/L G418抗生素的YPD平板上，置于30℃培养箱中培养3-5天得到转化子，挑取转化子划线纯化，用酵母基因组提取试剂盒提取基因组，用引物对AAH1-500F/AAH1-500R进行PCR验证敲除正确，将此转化子菌株命名为酿酒酵母KH07SG(含有G418抗性)；
表5第一个AAH1基因的敲除所用到的引物
Figure PCTCN2020095553-appb-000003
Figure PCTCN2020095553-appb-000004
2 KH07SG菌株消抗
根据实施例2中1.2的方法，制备酿酒酵母KH07SG菌株的感受态细胞；根据实施例2中1.3的方法，将500ng的pSH65质粒转化至酿酒酵母KH07SG菌株的感受态细胞中得到转化液，将转化液涂布于含30mg/L zeo(博来霉素)的YPD或者含15mg/L Phleo(腐草霉素)的YPD平板，于30℃培养箱中培养3天后，挑取转化子接种YPG(以20g/L半乳糖替代20g/L葡萄糖，其他与YPD培养基相同)试管，30℃、250rpm条件下，培养2-3h，菌液稀释后，取不同稀释程度的菌液各100μL分别涂布YPD平板，于30℃培养箱中培养3天，取单菌落分别点YPD平板和含500mg/L G418抗生素的YPD平板，筛选抗性消除的菌株(即在YPD平板上生长，在含500mg/L G418抗生素的YPD平板上不长的菌株)，将此菌株命名为酿酒酵母KH07S。
3第二个AAH1基因的敲除(在酿酒酵母KH07S中敲除第二个AAH1基因)
3.1以pUG6质粒为模板，用引物AAH1-F2和AAH1-R2进行PCR扩增，然后用琼脂糖凝胶DNA回收试剂盒跑胶回收得到1589bp的AAH1敲除片段II；
3.2按照实施例2中1.2的步骤制备KH07S菌株的感受态细胞；
3.3按照实施例2中1.3的步骤将AAH1敲除片段II转化至KH07S菌株细胞中得到转化子，取转化子划线纯化并提取基因组，用引物对AAH1-500F/AAH1-500R进行PCR验证敲除正确，将此转化子菌株命名为酿酒 酵母KH07G(含有G418抗性)；
表6引物AAH1-F2和AAH1-R2的序列
Figure PCTCN2020095553-appb-000005
4按照实施例2中2的步骤，将酿酒酵母KH07G菌株消抗，获得正确消抗的菌株，命名为酿酒酵母KH07，用表5中的引物对AAH1-500F/AAH1-500R进行PCR确认酿酒酵母KH07菌株，结果参见图3，图3为酿酒酵母KH07菌株PCR验证的核酸胶图，其中，1为酿酒酵母KH01菌株的基因组，2、3、4为酿酒酵母KH07菌株的基因组，M为Marker，为Thermo Scientific GeneRuler 1kb Plus DNA Ladder(货号：SM1332)；由图3可知，酿酒酵母KH01菌株有2031bp的目的条带，而酿酒酵母KH07菌株有1011bp和1151bp两条带，酿酒酵母KH07菌株AAH1基因双敲除及消抗正确。
5试验
5.1取酿酒酵母菌株KH01、KH07S和KH07用相同的摇瓶发酵生产NAD +的方法来验证基因敲除的效果；
摇瓶发酵生产NAD +的方法为：从酿酒酵母甘油冻存管挑取一环菌液划线YPD平板，置于30℃培养箱，培养2-3天；用接种环挑取活化后的酿酒酵母单克隆到含50mL发酵培养基(发酵培养基配方：葡萄糖50g/L、酪蛋白提取物15g/L、酵母提取物15g/L、NaCl 5g/L、KH 2PO 4 1g/L、K 2HPO 4 1g/L、MgSO 4·7H 2O 0.3g/L，pH5.4)的500mL摇瓶中，置于30℃、250rpm条件下， 培养72h后添加腺嘌呤和烟酰胺，使腺嘌呤终浓度为3g/L，烟酰胺终浓度为6g/L，继续于30℃、250rpm条件下培养72h后停止发酵得到发酵液；
发酵提取液：取上述发酵液20mL于50mL离心管中，离心收集菌体，向菌体中加入5mL 0.2％甲酸水，涡旋混匀，于95℃水浴中，1000rpm搅拌5min，迅速转移至冰上冰浴，1000rpm搅拌5-10min，在于4℃，7500rpm离心5min，取上清即为发酵提取液；
检测：用高效液相色谱HPLC法检测发酵提取液中腺嘌呤、次黄嘌呤、NAD +的含量，检测方法为：吸取1mL NAD +提取液，用0.22μm滤膜过滤后进样检测；
HPLC的检测条件为：色谱柱为waters C18(4.6×150mm，5μm)，紫外检测器检测，波长＝260nm；流速＝1.0mL/min，进样量＝5μL，柱温＝30℃，流动相A为甲醇，流动相B为10mM乙酸铵水溶液(pH＝5.0)，梯度洗脱，洗脱程序如表7所示；典型色谱图参见图4和图5，图4为NAD +的HPLC色谱图，图5为烟酸、次黄嘌呤、腺嘌呤和烟酰胺的HPLC色谱图；由图4可以看出，NAD +的保留时间为4.811min；由图5可以看出，烟酸的保留时间为2.952min，次黄嘌呤的保留时间为3.515min，腺嘌呤5.858min，烟酰胺的保留时间为6.669min。
表7
时间(min) 流动相A(v/v％) 流动相B(v/v％)
0.00-0.01 2 98
0.01-7.00 7 93
7.00-8.00 80 20
8.00-9.00 80 20
9.00-9.10 2 98
9.10-13.00 2 98
结果：发酵提取液中，菌株KH01、KH07S和KH07的细胞内腺嘌呤和次黄嘌呤的含量如表8所示：
表8
酿酒酵母菌株 腺嘌呤(mg/L) 次黄嘌呤(mg/L)
KH01 6.11 127.23
KH07S 9.94 117.34
KH07 379.87 20.46
由表8可以看出，AAH1基因双敲除能明显减少腺嘌呤到次黄嘌呤的降解，次黄嘌呤的积累量减少了至少6倍。
实施例3酿酒酵母KH01整合NPT1和NMA1基因
取实施例1制得的质粒pND08，用MssI单酶双切，再用琼脂糖凝胶DNA回收试剂盒跑胶回收得到6178bp整合片段；
按照实施例2中1.2和1.3的步骤，将6178bp整合片段转化至酿酒酵母KH01的细胞中得到转化子，取转化子划线纯化，然后用实施例2的5.1中的摇瓶发酵生产NAD +的方法筛选转化子，得到NAD +产量高的优势转化子，将此转化子菌株按照实施例2中的2步骤消抗后命名为酿酒酵母KH06；
试验：取酿酒酵母KH06菌株，按照实施例3的5.1中的摇瓶发酵生产NAD +的方法生产NAD +，并以酿酒酵母KH01为对照，结果如表10所示。
表10不同菌株的NAD +产量
菌株 NAD +(g/kg DCW)
KH01 11.3
KH06 20.5
由表10可以看出，超表达烟酸磷酸核糖转移酶的基因NPT1和烟酸核糖单磷酸腺苷酰基转移酶的基因NMA1的酿酒酵母KH06菌株比酿酒酵母KH01菌株的NAD +产量提高了80％以上。
实施例4酿酒酵母KH07整合NPT1和NMA1基因
按照实施例3中的步骤，将6178bp整合片段转化至酿酒酵母KH07的细胞中得到转化子，取转化子划线纯化，然后用实施例2的5.1中的摇瓶发酵生产NAD +的方法筛选转化子，得到NAD +产量高的优势转化子，将此转化子菌株按照实施例2中2的步骤消抗后，命名为酿酒酵母(Saccharomyces cerevisiae)KH08，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19048；
试验：按照实施例2的5.1中的摇瓶发酵生产NAD +的方法，取酿酒酵母KH08单菌落接种于含50mL发酵培养基的500mL摇瓶中，于30℃、250rpm条件下，培养72h后添加腺嘌呤和烟酰胺，使腺嘌呤终浓度为0.8g/L，烟酰胺终浓度为6g/L，继续于30℃、250rpm条件下培养72h后停止发酵得到发酵液。以酿酒酵母KH07菌株为对照，发酵结果如表11所示。
表11不同菌株的NAD +产量
菌株 NAD +(g/kg DCW)
KH07 12.0
KH08 21.5
由表11可以看出，超表达烟酸磷酸核糖转移酶的基因NPT1和烟酸核糖单磷酸腺苷酰基转移酶的基因NMA1的酿酒酵母KH08菌株比酿酒酵母KH07菌株的NAD +产量提高约80％。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种基因工程菌，其特征在于，菌株基因组上的腺嘌呤脱氨酶的编码基因被敲除，或/和菌株基因组上整合有NAD +合成途径中的酶的编码基因。
  2. 根据权利要求1所述基因工程菌，其特征在于，所述菌株为酿酒酵母。
  3. 根据权利要求1或2所述基因工程菌，其特征在于，所述NAD +合成途径中的酶为烟酰胺酶PNC1、烟酰酸磷酸核糖转移酶NPT1、烟酰酸单核苷酸腺苷转移酶NMA1、烟酰酸单核苷酸腺苷转移酶NMA2、谷氨酰胺依赖性NAD +合成酶QNS1中的至少一种。
  4. 根据权利要求1-3任一项所述基因工程菌，其特征在于，所述NAD +合成途径中的酶为烟酰酸磷酸核糖转移酶NPT1、烟酰酸单核苷酸腺苷转移酶NMA1中的至少一种。
  5. 根据权利要求1-4任一项所述基因工程菌，其特征在于，所述基因工程菌保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19048。
  6. 一种如权利要求1-5任一项所述基因工程菌的构建方法，其特征在于，将宿主菌株基因组上的腺嘌呤脱氨酶的编码基因敲除获得NAD +产量高的菌株；
    或者分别构建NAD +合成途径中的酶的编码基因的表达框，接着将所述酶的编码基因的表达框整合于宿主菌株基因组上构建获得NAD +产量高的菌株；
    或者分别构建NAD +合成途径中的酶的编码基因的表达框，接着将所述酶的编码基因的表达框整合于敲除腺嘌呤脱氨酶的编码基因的宿主菌株基因组上构建获得NAD +产量高的菌株。
  7. 根据权利要求6所述基因工程菌的构建方法，其特征在于，所述整合为δ-位点整合法。
  8. 根据权利要求6或7所述基因工程菌的构建方法，其特征在于，δ序列包含δ1片段和δ2片段，其中，δ1片段的核苷酸序列如SEQ ID No.1所示，δ2片段的核苷酸序列如SEQ ID No.2所示。
  9. 一种如权利要求1-4任一项所述基因工程菌在生产NAD +中的应用。
  10. 一种生产NAD +的方法，其特征在于，以烟酰胺或/和腺嘌呤作为底物利用权利要求1-4任一项所述基因工程菌生产NAD +。
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