Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.
Metabolic engineering of a tyrosine-overproducing yeast platform using targeted metabolomics.
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DOI：
10.1186/s12934-015-0252-2
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发表时间：
2015-05-28
影响因子：
6.4
通讯作者：
Martin VJ
中科院分区：
文献类型：
--
作者：
Gold ND;Gowen CM;Lussier FX;Cautha SC;Mahadevan R;Martin VJ
L-酪氨酸是一系列有价值的次级代谢产物的共同前体，包括苄基异喹啉生物碱（BIA）和许多聚酮化合物。一个工业上易于处理的酵母菌株生产L-酪氨酸优化可以作为一个平台，为BIA和聚酮细胞工厂的发展。本研究应用靶向代谢组学方法评估代谢工程策略，以增加酿酒酵母CEN. PK细胞内L-酪氨酸的可用性。我们的工程策略结合了本地化的途径工程与全球工程的中央代谢，促进基因组规模的稳态建模。添加来自S.酿酒酵母与来自运动发酵单胞菌的酪氨酸反馈抗性酵母分支酸酯Aro 7、天然五功能芳香族蛋白Aro 1、天然预苯酸脱氢酶Tyr 1或环己二烯基脱氢酶Tyr C的过表达组合。通过消除苯丙酮酸脱羧酶Aro 10来限制芳香碳的损失。来自球形红细菌的TAL基因用于生产香豆酸，作为酪氨酸的异源副产物的简单测试情况。此外，使用代谢建模评估了用于工程化全局代谢以促进酪氨酸产生的多种策略。假设丙酮酸激酶Cdc 19的T21 E突变体减缓磷酸烯醇式丙酮酸向丙酮酸的转化，并将前者积累为莽草酸途径的前体。删除编码葡萄糖-6-磷酸脱氢酶的ZWF 1基因，以产生NADPH缺陷，该缺陷旨在迫使细胞通过过表达的NADP+依赖性预苯酸脱氢酶Tyr 1将其生长与酪氨酸产生偶联。我们的工程化Zwf 1 −菌株表达TYRC ARO 4FBR，并在甲硫氨酸存在下生长，在胞质溶胶中实现了高达520 μmol/g DCW或192 mM的细胞内L-酪氨酸积累，但发现通过该途径的持续通量取决于反馈抑制和降解途径的完全消除。我们的靶向代谢组学方法证实了DAHP合酶的可能调控位点，并确定了预苯酸脱氢酶的另一个可能的辅因子限制。此外，基因组规模的代谢模型确定的设计策略，有可能提高DAHP合酶的赤藓糖4-磷酸的可用性和预苯酸脱氢酶的辅因子的可用性。我们对这些策略进行了评价，并为进一步提高S.酿酒。本文的在线版本（doi：10.1186/s12934-015-0252-2）包含补充材料，可供授权用户使用。
L-tyrosine is a common precursor for a wide range of valuable secondary metabolites, including benzylisoquinoline alkaloids (BIAs) and many polyketides. An industrially tractable yeast strain optimized for production of L-tyrosine could serve as a platform for the development of BIA and polyketide cell factories. This study applied a targeted metabolomics approach to evaluate metabolic engineering strategies to increase the availability of intracellular L-tyrosine in the yeast Saccharomyces cerevisiae CEN.PK. Our engineering strategies combined localized pathway engineering with global engineering of central metabolism, facilitated by genome-scale steady-state modelling. Addition of a tyrosine feedback resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase Aro4 from S. cerevisiae was combined with overexpression of either a tyrosine feedback resistant yeast chorismate mutase Aro7, the native pentafunctional arom protein Aro1, native prephenate dehydrogenase Tyr1 or cyclohexadienyl dehydrogenase TyrC from Zymomonas mobilis. Loss of aromatic carbon was limited by eliminating phenylpyruvate decarboxylase Aro10. The TAL gene from Rhodobacter sphaeroides was used to produce coumarate as a simple test case of a heterologous by-product of tyrosine. Additionally, multiple strategies for engineering global metabolism to promote tyrosine production were evaluated using metabolic modelling. The T21E mutant of pyruvate kinase Cdc19 was hypothesized to slow the conversion of phosphoenolpyruvate to pyruvate and accumulate the former as precursor to the shikimate pathway. The ZWF1 gene coding for glucose-6-phosphate dehydrogenase was deleted to create an NADPH deficiency designed to force the cell to couple its growth to tyrosine production via overexpressed NADP+-dependent prephenate dehydrogenase Tyr1. Our engineered Zwf1− strain expressing TYRC ARO4FBR and grown in the presence of methionine achieved an intracellular L-tyrosine accumulation up to 520 μmol/g DCW or 192 mM in the cytosol, but sustained flux through this pathway was found to depend on the complete elimination of feedback inhibition and degradation pathways. Our targeted metabolomics approach confirmed a likely regulatory site at DAHP synthase and identified another possible cofactor limitation at prephenate dehydrogenase. Additionally, the genome-scale metabolic model identified design strategies that have the potential to improve availability of erythrose 4-phosphate for DAHP synthase and cofactor availability for prephenate dehydrogenase. We evaluated these strategies and provide recommendations for further improvement of aromatic amino acid biosynthesis in S. cerevisiae. The online version of this article (doi:10.1186/s12934-015-0252-2) contains supplementary material, which is available to authorized users.
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影响因子：
--
作者：
Mo ML;Palsson BO;Herrgård MJ
通讯作者：
Herrgård MJ
影响因子：
12.3
作者：
Blank LM;Kuepfer L;Sauer U
通讯作者：
Sauer U
影响因子：
3.5
作者：
MANNHAUPT, G;STUCKA, R;FELDMANN, H
通讯作者：
FELDMANN, H
影响因子：
9.9
作者：
Lun, Desmond S.;Rockwell, Graham;Guido, Nicholas J.;Baym, Michael;Kelner, Jonathan A.;Berger, Bonnie;Galagan, James E.;Church, George M.
通讯作者：
Church, George M.
影响因子：
3.2
作者：
HOHMANN, S
通讯作者：
HOHMANN, S