Influence of NADH disturbance on Crabtree effect of Saccharomyces cerevisiae
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摘要: 利用不同拷贝数的载体在酿酒酵母表达NADH氧化酶NOX和可替代氧化酶AOX1，并通过线粒体定位信号减少AOX1对胞质NADH的影响，从而考察NOX和AOX1在分批发酵中对酿酒酵母葡萄糖效应的作用及其对葡萄糖生产衣康酸的影响。研究结果表明，高拷贝载体表达nox和aox1的菌株均有明显的代谢变化，高拷贝表达nox菌株能够氧化胞质NADH，培养基中甘油分泌减少43.94%，衣康酸生产不受影响。而高拷贝表达aox1的菌株存在胞质残留AOX1，会氧化胞质NADH而减少甘油积累。利用线粒体定位信号AAC2和BCS1p将AOX1进一步定位至酿酒酵母线粒体，不再影响甘油合成，120 h时衣康酸产量增加至116.98 mg/L。但是AOX1、AAC2-AOX1和BCS1p-AOX1表达菌株都没有明显减少分批发酵中乙醇的积累。综上所述，在高糖培养基中进行分批发酵，NOX能够减少葡萄糖效应甘油的积累，而AOX1 、AAC2-AOX1和BCS1p-AOX1不能减少葡萄糖效应副产物乙醇的积累。Abstract: Saccharomyces cerevisiae is one of the cell factories in biomanufacturing because of numerous advantages towards industrial fermentations, which include robust growth in low pH, lower temperatures, high tolerance to shear stress, lack of phage contamination, and ease of separation. However, the Crabtree effect of S. cerevisiae made ethanol and glycerol be accumulated due to carbon overflow. For the production of intermediate derivatives of the TCA cycle, such as itaconic acid, the Crabtree effect must be overcome by using a suitable strategy. In this paper, the role of NOX and AOX1 on the Crabtree effect in batch fermentation of S. cerevisiae was investigated by expressing the NADH oxidase NOX and the alternative oxidase AOX1 with plasmids having different copy numbers. It was revealed that both strains expressing NOX and AOX1 in high copy vectors caused significant metabolic changes. The high copy expressing nox strains were able to oxidize cytoplasmic NADH, glycerol secretion in the medium was reduced by 43.94%, and IA concentration was not changed. In contrast, strains with high copy expression of aox1 had cytoplasmic residual AOX1, which oxidized cytoplasmic NADH and reduced glycerol accumulation. Further location of AOX1 to the mitochondria of S. cerevisiae with the mitochondrial location signals AAC2 and BCS1p reduced the effect of AOX1 on glycerol synthesis, and IA production was enhanced to 116.98 mg/L. However, none of the strains expressing AOX1, AAC2-AOX1 and BCS1p-AOX1 significantly alleviated the accumulation of ethanol in batch fermentation. This study helps to improve the production of TCA cycle derivatives from glucose by engineered S. cerevisiae, provides a reference for the production of TCA cycle derivatives from S. cerevisiae in the batch culture at high original glucose concentrations.
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Key words:
- S. cerevisiae /
- glycerol /
- ethanol /
- NOX /
- AOX1 /
- mitochondrial location signals
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图 2 利用替代氧化酶AOX1减少乙醇生产
Figure 2. Reducing ethanol production by overexpressing an alternative oxidase AOX1
Profiles of cell density, ethanol, glycerol, and itaconic acid concentrations in the cultivation of different Saccharomyces cerevisiae strains
图 3 激光共聚焦显微镜观察NOX和AOX1在酿酒酵母中的定位
Figure 3. Location of NOX(A) and AOX1(B) in Saccharomyces cerevisiae
图 4 利用线粒体定位信号将替代氧化酶AOX1定位至线粒体
Figure 4. Further location of AOX1 in mitochondria of Saccharomyces cerevisiae with mitochondrial localization signals
Subcellular localization of AAC2-AOX1-mCherry(A5) and BCS1p-AOX1-mCherry(A7) fusion proteins in Saccharomyces cerevisiae. Strains expressing fusion protein were stained with MitoTracker® Green FM. Scale bar—5 μm.
图 5 AOX1线粒体定位提高衣康酸滴度
Figure 5. Mitochondrial localization of AOX1 increases the titer of itaconic acid
Expression of AAC2-AOX1 (XYY29) and BCS1p-AOX1(XYY31) fusion proteins with high-copy plasmids
表 1 本研究所使用的菌株
Table 1. Strains used in this study.
Strains Genotype Source or reference E.coli DH5a Our lab BY4741 MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0 Professor
Xin-Qing ZhaoXYY2 BY4741, pRS415-PGPD-cadA-TCYC1 [17] XYY21 XYY2, pRS413 This study XYY22 XYY2, pRS423 [17] XYY23 XYY2, pRS413-PGPD2-nox-TCYC1 This study XYY24 XYY2, pRS423-PGPD2-nox-TCYC1 This study XYY25 XYY2, pRS413-PTEF1-aox1-TCYC1 This study XYY26 XYY2, pRS423-PTEF1-aox1-TCYC1 This study XYY29 XYY2, pRS423-PTEF1-aac2-aox1-TCYC1 This study XYY31 XYY2, pRS423-PTEF1-bcs1p-aox1-TCYC1 This study Z1 BY4741, HO△:: PGPD-cadA-TCYC1, pRS423 This study Z2 Z1, GPD2△:: nox This study L1 BY4741, pRS425 This study L2 BY4741, pRS425-PGPD2-mCherry-TCYC1 This study L3 BY4741, pRS425-PGPD2-nox-mCherry-TCYC1 This study A1 BY4741, pRS423 This study A2 BY4741, pRS423-PTEF1-mCherry-TCYC1 This study A3 BY4741, pRS423-PTEF1-aox1-mCherry-TCYC1 This study A4 BY4741, pRS423- PTEF1-aac2-mCherry-TCYC1 This study A5 BY4741, pRS423-PTEF1-aac2-aox1-mCherry-TCYC1 This study A6 BY4741, pRS423-PTEF1-bcs1p-mCherry-TCYC1 This study A7 BY4741, pRS423-PTEF1-bcs1p-aox1-mCherry-TCYC1 This study 
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表 3 本研究所使用的引物
Table 3. Primers used in this study.
Name Sequence zw-1F cggccgctctagaactagtggatccgcaatgtttcgttggttataacattag zw-1R ctacaacgattttactcataagctttgataaggaaggggagcgaagg zw-2F tcgctccccttccttatcaaagcttatgagtaaaatcgttgtagtcggtg zw-2R cgaggtcgacggtatcgataagcttttatttttcagccgtaagggcagcc zw-3F ggctgcccttacggctgaaaaataaaagcttatcgataccgtcgacctcg zw-3R cgaattcctgcagcccgggggatccgcaaattaaagccttcgagcgtccc zw-4F ctaaagggaacaaaagctggagctccagcaatgtttcgttggttataacattag zw-4R cgaggtcgacggtatcgataagcttttatttttcagccgtaagggcagcc zw-5F cggccgctctagaactagtggatccatagcttcaaaatgtttctactcctt zw-5R cgaattcctgcagcccgggggatccgcaaattaaagccttcgagcgtccc zw-6F ctaaagggaacaaaagctggagctcatagcttcaaaatgtttctactcc zw-6R gtaatggcagtgctgttttaactagttttgtaattaaaacttagattagattg zw-7F gttttaattacaaaactagttaaaacagcactgccattactaatacacctc zw-7R taatgatgatgatgatgatggatccgttttgtttaagctgatgcaattttttg zw-8F tctaagttttaattacaaaactagtatgtcttccaacgcccaagtcaaaa zw-8R tcgcgcatcctgaggtcggatacattttgaacttcttaccaaacaagatc zw-9F gatcttgtttggtaagaagttcaaaatgtatccgacctcaggatgcgcg zw-9R taatgatgatgatgatgatggatcctcatatcacctcatcccgttcccag zw-10F tctaagttttaattacaaaactagtatgtcggataagccgattgacatac zw-10R tcgcgcatcctgaggtcggatacattccattgtcatgttgtatgtagtttg zw-11F caaactacatacaacatgacaatggaatgtatccgacctcaggatgcgcgag zw-11R taatgatgatgatgatgatggatcctatcacctcatcccgttcccagccag zw-12F ctaaagggaacaaaagctggagctcgcaatgtttcgttggttataacatt zw-12R ccatggccatctttgcaaagcttggtgataaggaaggggagcgaaggaa zw-13F atcaccaagctttgcaaagatggccatggtgagcaagggcgaggaggataac zw-13R cgaggtcgacggtatcgataagcttctacttgtacagctcgtccatgccg zw-14F atcaccaagctttgcaaagatggccatgagtaaaatcgttgtagtcggtgc zw-14R tatcctcctcgcccttgctcaccattttttcagccgtaagggcagccattg zw-15F ctaaagggaacaaaagctggagctcatagcttcaaaatgtttctactcct zw-15R cgaggtcgacggtatcgataagcttctacttgtacagctcgtccatgcc zw-16F caaaccaagctttgcaaagatggccatgtatccgacctcaggatgcgcga zw-16R tatcctcctcgcccttgctcaccattatcacctcatcccgttcccagccag zw-17F caaaccaagctttgcaaagatggccatgtcttccaacgcccaagtcaaaac zw-17R tatcctcctcgcccttgctcaccattttgaacttcttaccaaacaagatc zw-18F zw-17F zw-18R tatcctcctcgcccttgctcaccattatcacctcatcccgttcccagccag zw-19F caaaccaagctttgcaaagatggccatgtcggataagccgattgacatac zw-19R tatcctcctcgcccttgctcaccattccattgtcatgttgtatgtagtttg zw-20F zw-19F zw-20R zw-18R zw-21F cctgtgtgacatttatgacggttttagagctagaaatagcaag zw-21R cgtcataaatgtcacacagggatcatttatctttcactgcgga zw-22F catccaaaatattaaattttacttttattacatacaactttttaaactaatatacacattagtttatcattatcaatactcgcca zw-22R caactattagctctaaatccatatcctcataagcagcaatcaattctatctatactttaaagcaaattaaagccttcgagcgtccc zw-23F cgcactatctggtgcaaactgttttagagctagaaatagcaag zw-23R agtttgcaccagatagtgcggatcatttatctttcactgcgga zw-24F gtattttggtagattcaattctctttccctttccttttccttcgctccccttccttatcaatgcttgctgtcatgagtaaaatcgttgtagtcgg zw-24R gtataatgataaattggttgggggaaaaagaggcaacaggaaagatcagagggggagggggggggagagtgtttatttttcagccgtaagggcagcc
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