背景:苯丙素类是一类具有多种生物学功能的植物次生代谢产物,源自芳香氨基酸。蓝细菌是可持续生产植物类苯丙素的有前途的宿主生物。我们以前设计了集胞藻。PCC6803生产反式肉桂酸(tCA)和对香豆酸(pCou),苯丙素途径的第一个中间体,通过过表达苯丙氨酸和酪氨酸氨裂解酶。在这项研究中,我们的目的是提高集胞藻中目标化合物tCA和pCou的产量。
结果:我们消除了4-羟基苯基丙酮酸双加氧酶(HPPD)活性,这是一个消耗酪氨酸的竞争途径,可能,用于生育酚合成的苯丙氨酸。此外,莽草酸途径末端步骤的几个基因单独或在操纵子中过表达,如芳香转氨酶,来自运动发酵单胞菌的反馈不敏感的环己二烯基脱氢酶(TyrC)和来自大肠杆菌的融合的分支酸变位酶/预苯酸脱水酶的分支酸变位酶(CM)结构域。与亲本生产菌株相比,敲除HPPD后,获得的工程菌株的tCA和pCou产量提高了近1.5倍,光合自养生长7天后,积累了138±3.5mgL-1的tCA和72.3±10.3mgL-1的pCou。然而,当任何通路基因过表达时,没有进一步的改善.最后,我们使用先前获得的AtPRM8和TsPRM8集胞藻菌株,这些菌株具有shikimate途径的失调,作为过表达具有ppd敲除的合成构建体的背景。
结论:消除HPPD可在相似程度上提高tCA和pCou生产力。使用基于PRM8的菌株作为过表达合成构建体的背景,然而,没有提高tCA和pCou滴度,这表明对苯丙氨酸和酪氨酸合成的末端步骤进行了严格的调节。这项工作有助于建立蓝细菌作为苯丙烷生产的宿主。
BACKGROUND: Phenylpropanoids are a large group of plant secondary metabolites with various biological functions, derived from aromatic amino acids. Cyanobacteria are promising host organisms for sustainable production of plant
phenylpropanoids. We have previously engineered Synechocystis sp. PCC 6803 to produce trans-cinnamic acid (tCA) and p-coumaric acid (pCou), the first intermediates of phenylpropanoid pathway, by overexpression of phenylalanine- and tyrosine ammonia lyases. In this study, we aimed to enhance the production of the target compounds tCA and pCou in Synechocystis.
RESULTS: We eliminated the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, which is a competing pathway consuming tyrosine and, possibly, phenylalanine for tocopherol synthesis. Moreover, several genes of the terminal steps of the shikimate pathway were overexpressed alone or in operons, such as aromatic transaminases, feedback insensitive cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase (CM) domain of the fused chorismate mutase/prephenate dehydratase enzyme from Escherichia coli. The obtained engineered strains demonstrated nearly 1.5 times enhanced tCA and pCou production when HPPD was knocked out compared to the parental production strains, accumulating 138 ± 3.5 mg L-1 of tCA and 72.3 ± 10.3 mg L-1 of pCou after seven days of photoautotrophic growth. However, there was no further improvement when any of the pathway genes were overexpressed. Finally, we used previously obtained AtPRM8 and TsPRM8 Synechocystis strains with deregulated shikimate pathway as a background for the overexpression of synthetic constructs with ppd knockout.
CONCLUSIONS: HPPD elimination enhances the tCA and pCou productivity to a similar extent. The use of PRM8 based strains as a background for overexpression of synthetic constructs, however, did not promote tCA and pCou titers, which indicates a tight regulation of the terminal steps of phenylalanine and tyrosine synthesis. This work contributes to establishing cyanobacteria as hosts for phenylpropanoid production.