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中表达还原型烟酰胺腺嘌呤二核苷酸（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。结果表明，高拷贝载体表达nox、aox1基因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，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。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 has been regarded as one of the cell factories in biomanufacturing because of numerous advantages towards industrial fermentations, including 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 Saccharomyces cerevisiae leads to accumulation of ethanol and glycerol due to carbon overflow. For the production of intermediate derivatives of the TCA (Tricarboxylic acid cycle) cycle, such as itaconic acid, the Crabtree effect must be eliminated by using a suitable strategy. In this paper, the role of NOX and AOX1 on the Crabtree effect in batch fermentation of Saccharomyces cerevisiae was investigated by expressing the NADH (Nicotinamide adenine dinucleotide) 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 Saccharomyces cerevisiae with the mitochondrial location signals AAC2 and BCS1p weakened 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 Saccharomyces cerevisiae, providing a reference for the production of TCA cycle derivatives from Saccharomyces cerevisiae in the batch culture at high original glucose concentrations.
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Key words:
- Saccharomyces 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
图 3 NOX(a)和AOX1(b)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
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 本文所使用的菌株
Table 1. Strains used in this paper
Strain Genotype Source or reference E.coli DH5α — Our lab BY4741 MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0 Refer [18] XYY2 BY4741, pRS415-PGPD-cadA-TCYC1 Refer [17] XYY21 XYY2, pRS413 This paper XYY22 XYY2, pRS423 Refer [17] XYY23 XYY2, pRS413-PGPD2-nox-TCYC1 This paper XYY24 XYY2, pRS423-PGPD2-nox-TCYC1 This paper XYY25 XYY2, pRS413-PTEF1-aox1-TCYC1 This paper XYY26 XYY2, pRS423-PTEF1-aox1-TCYC1 This paper XYY29 XYY2, pRS423-PTEF1-aac2-aox1-TCYC1 This paper XYY31 XYY2, pRS423-PTEF1-bcs1p-aox1-TCYC1 This paper Z1 BY4741, HO△:: PGPD-cadA-TCYC1, pRS423 This paper Z2 Z1, GPD2△:: nox This paper L1 BY4741, pRS425 This paper L2 BY4741, pRS425-PGPD2-mCherry-TCYC1 This paper L3 BY4741, pRS425-PGPD2-nox-mCherry-TCYC1 This paper A1 BY4741, pRS423 This paper A2 BY4741, pRS423-PTEF1-mCherry-TCYC1 This paper A3 BY4741, pRS423-PTEF1-aox1-mCherry-TCYC1 This paper A4 BY4741, pRS423- PTEF1-aac2-mCherry-TCYC1 This paper A5 BY4741, pRS423-PTEF1-aac2-aox1-mCherry-TCYC1 This paper A6 BY4741, pRS423-PTEF1-bcs1p-mCherry-TCYC1 This paper A7 BY4741, pRS423-PTEF1-bcs1p-aox1-mCherry-TCYC1 This paper 
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表 3 本文所使用的引物
Table 3. Primers used in this paper
Name Sequence Name Sequence zw-1F cggccgctctagaactagtggatccgcaatgtttcgttggttataacattag zw-13F atcaccaagctttgcaaagatggccatggtgagcaagggcgaggaggataac zw-1R ctacaacgattttactcataagctttgataaggaaggggagcgaagg zw-13R cgaggtcgacggtatcgataagcttctacttgtacagctcgtccatgccg zw-2F tcgctccccttccttatcaaagcttatgagtaaaatcgttgtagtcggtg zw-14F atcaccaagctttgcaaagatggccatgagtaaaatcgttgtagtcggtgc zw-2R cgaggtcgacggtatcgataagcttttatttttcagccgtaagggcagcc zw-14R tatcctcctcgcccttgctcaccattttttcagccgtaagggcagccattg zw-3F ggctgcccttacggctgaaaaataaaagcttatcgataccgtcgacctcg zw-15F ctaaagggaacaaaagctggagctcatagcttcaaaatgtttctactcct zw-3R cgaattcctgcagcccgggggatccgcaaattaaagccttcgagcgtccc zw-15R cgaggtcgacggtatcgataagcttctacttgtacagctcgtccatgcc zw-4F ctaaagggaacaaaagctggagctccagcaatgtttcgttggttataacattag zw-16F caaaccaagctttgcaaagatggccatgtatccgacctcaggatgcgcga zw-4R cgaggtcgacggtatcgataagcttttatttttcagccgtaagggcagcc zw-16R tatcctcctcgcccttgctcaccattatcacctcatcccgttcccagccag zw-5F cggccgctctagaactagtggatccatagcttcaaaatgtttctactcctt zw-17F caaaccaagctttgcaaagatggccatgtcttccaacgcccaagtcaaaac zw-5R cgaattcctgcagcccgggggatccgcaaattaaagccttcgagcgtccc zw-17R tatcctcctcgcccttgctcaccattttgaacttcttaccaaacaagatc zw-6F ctaaagggaacaaaagctggagctcatagcttcaaaatgtttctactcc zw-18F zw-17F zw-6R gtaatggcagtgctgttttaactagttttgtaattaaaacttagattagattg zw-18R tatcctcctcgcccttgctcaccattatcacctcatcccgttcccagccag zw-7F gttttaattacaaaactagttaaaacagcactgccattactaatacacctc zw-19F caaaccaagctttgcaaagatggccatgtcggataagccgattgacatac zw-7R taatgatgatgatgatgatggatccgttttgtttaagctgatgcaattttttg zw-19R tatcctcctcgcccttgctcaccattccattgtcatgttgtatgtagtttg zw-8F tctaagttttaattacaaaactagtatgtcttccaacgcccaagtcaaaa zw-20F zw-19F zw-8R tcgcgcatcctgaggtcggatacattttgaacttcttaccaaacaagatc zw-20R zw-18R zw-9F gatcttgtttggtaagaagttcaaaatgtatccgacctcaggatgcgcg zw-21F cctgtgtgacatttatgacggttttagagctagaaatagcaag zw-9R taatgatgatgatgatgatggatcctcatatcacctcatcccgttcccag zw-21R cgtcataaatgtcacacagggatcatttatctttcactgcgga zw-10F tctaagttttaattacaaaactagtatgtcggataagccgattgacatac zw-22F catccaaaatattaaattttacttttattacatacaactttttaaactaatatacacattagtttatcattatcaatactcgcca zw-10R tcgcgcatcctgaggtcggatacattccattgtcatgttgtatgtagtttg zw-22R caactattagctctaaatccatatcctcataagcagcaatcaattctatctatactttaaagcaaattaaagccttcgagcgtccc zw-11F caaactacatacaacatgacaatggaatgtatccgacctcaggatgcgcgag zw-23F cgcactatctggtgcaaactgttttagagctagaaatagcaag zw-11R taatgatgatgatgatgatggatcctatcacctcatcccgttcccagccag zw-23R agtttgcaccagatagtgcggatcatttatctttcactgcgga zw-12F ctaaagggaacaaaagctggagctcgcaatgtttcgttggttataacatt zw-24F gtattttggtagattcaattctctttccctttccttttccttcgctccccttccttatcaatgcttgctgtcatgagtaaaatcgttgtagtcgg zw-12R ccatggccatctttgcaaagcttggtgataaggaaggggagcgaaggaa zw-24R gtataatgataaattggttgggggaaaaagaggcaacaggaaagatcagagggggagggggggggagagtgtttatttttcagccgtaagggcagcc
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