羟基化
酪氨酸羟化酶
酪氨酸
酪氨酸酶
生物化学
酶
大肠杆菌
化学
酪氨酸3-单加氧酶
生物
基因
作者
Akira Nakagawa,Shinya Nakamura,Eitaro Matsumura,Yurino Yashima,Mizuki Takao,Sachiyo Aburatani,Katsuro Yaoi,Takane Katayama,Hiromichi Minami
标识
DOI:10.1007/s00253-021-11401-z
摘要
We have constructed an Escherichia coli-based platform producing (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids (BIAs), using up to 14 genes. (S)-reticuline was produced from a simple carbon source such as glucose and glycerol via L-DOPA, which is synthesized by hydroxylation of L-tyrosine, one of the rate-limiting steps of the reaction. There are three kinds of enzymes catalyzing tyrosine hydroxylation: tyrosinase (TYR), tyrosine hydroxylase (TH), and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, to further improve (S)-reticuline production, we chose eight from these three kinds of tyrosine hydroxylation enzymes (two TYRs, four THs, and two HpaBCs) derived from various organisms, and examined which enzyme was optimal for (S)-reticuline production in E. coli. TH from Drosophila melanogaster was the most suitable for (S)-reticuline production under the experimental conditions tested. We improved the productivity by genome integration of a gene set for L-tyrosine overproduction, introducing the regeneration pathway of BH4, a cofactor of TH, and methionine addition to enhance the S-adenosylmethionine supply. As a result, the yield of (S)-reticuline reached up to 384 μM from glucose in laboratory-scale shake flask. Furthermore, we found three inconsistent phenomena: an inhibitory effect due to additional gene expression, conflicts among the experimental conditions, and interference of an upstream enzyme from an additional downstream enzyme. Based on these results, we discuss future perspectives and challenges of integrating multiple enzyme genes for material production using microbes. Graphical abstract The optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli KEY POINTS: • There are three types of enzymes catalyzing tyrosine hydroxylation reaction: tyrosinase, tyrosine hydroxylase, and 4-hydroxyphenylacetate 3-monooxygenase. • Tyrosine hydroxylase from Drosophila melanogaster exhibited the highest activity and was suitable for (S)-reticuline production in E. coli. • New insights were provided on constructing an alkaloid production system with multi-step reactions in E. coli.
科研通智能强力驱动
Strongly Powered by AbleSci AI