Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway.
Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway.
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DOI：
10.1016/j.synbio.2022.02.007
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发表时间：
2022-06
影响因子：
4.8
通讯作者：
Pfleger BF
中科院分区：
文献类型：
--
作者：
Gambacorta FV;Wagner ER;Jacobson TB;Tremaine M;Muehlbauer LK;McGee MA;Baerwald JJ;Wrobel RL;Wolters JF;Place M;Dietrich JJ;Xie D;Serate J;Gajbhiye S;Liu L;Vang-Smith M;Coon JJ;Zhang Y;Gasch AP;Amador-Noguez D;Hittinger CT;Sato TK;Pfleger BF
代谢工程策略已成功实施，以提高酿酒酵母中下一代生物燃料异丁醇的生产。在这里，我们探讨了这些策略中的两个，途径重新定位和氧化还原辅因子平衡，影响异丁醇生产菌株的性能和生理。我们为酵母配备了异丁醇盒，该异丁醇盒具有线粒体或胞质局部异丁醇途径，并使用氧化还原不平衡（NADPH依赖性）或氧化还原平衡（NADH依赖性）的酮醇酸还原异构酶。然后，我们进行了转录组学，蛋白质组学和代谢组学分析，以阐明工程菌株之间的分子差异。通路定位对异丁醇生产有很大影响，其中表达胞浆定位酶的菌株比表达胞质酶的菌株产生多3.8倍的异丁醇。辅因子平衡没有改善异丁醇滴度，相反，具有氧化还原不平衡途径的菌株产生的异丁醇比平衡版本多1.5倍，尽管总体途径通量低。功能基因组分析表明，细胞溶质途径菌株的不良性能部分是由于缺乏细胞溶质Fe-S簇，这是二羟酸脱氢酶所需的辅因子。然后，我们证明，这种辅因子的限制可能会部分恢复破坏铁稳态与fra 2突变，从而增加细胞铁水平。所得到的具有胞质溶胶定位的异丁醇途径的fra 2无效菌株的异丁醇滴度超过具有胞质溶胶定位的途径的菌株1.3倍，表明两种定位都可以支持向异丁醇的通量。
Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe–S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.
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DOI：
10.1093/bioinformatics/btu638
发表时间：
2015-01-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Anders S;Pyl PT;Huber W
通讯作者：
Huber W
影响因子：
--
作者：
Clasquin, Michelle F;Melamud, Eugene;Rabinowitz, Joshua D
通讯作者：
Rabinowitz, Joshua D
影响因子：
6.4
作者：
Ida K;Ishii J;Matsuda F;Kondo T;Kondo A
通讯作者：
Kondo A
影响因子：
6.3
作者：
Brat D;Weber C;Lorenzen W;Bode HB;Boles E
通讯作者：
Boles E
影响因子：
3
作者：
Feng, Ruiqi;Li, Jingzhi;Zhang, Aili
通讯作者：
Zhang, Aili