Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi-shi, Ishikawa, Japan.
TechnoPro, Inc., Roppongi Hills Mori Tower 35th floor, 6-10-1 Roppongi, Minatoku, Tokyo, Japan.
Appl Microbiol Biotechnol. 2021 Jul;105(13):5433-5447. doi: 10.1007/s00253-021-11401-z. Epub 2021 Jun 28.
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 BH, 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.
我们构建了一个基于大肠杆菌的平台，使用多达 14 个基因来生产（S）-黎藤碱，这是苯并异喹啉生物碱（BIAs）的重要中间体。（S）-黎藤碱可以从葡萄糖和甘油等简单碳源通过 L-DOPA 合成，L-DOPA 是通过反应的限速步骤之一 L-酪氨酸的羟化合成的。有三种酶催化酪氨酸羟化：酪氨酸酶（TYR）、酪氨酸羟化酶（TH）和 4-羟基苯乙酸 3-单加氧酶（HpaBC）。在这里，为了进一步提高（S）-黎藤碱的产量，我们从这三种酪氨酸羟化酶（两种 TYR、四种 TH 和两种 HpaBC）中选择了八种来自不同生物体的酶，并研究了哪种酶最适合大肠杆菌中的（S）-黎藤碱生产。黑腹果蝇的 TH 是在测试的实验条件下最适合（S）-黎藤碱生产的酶。我们通过基因组整合过量表达 L-酪氨酸的基因簇、引入 TH 的辅因子 BH 的再生途径以及添加蛋氨酸来增强 S-腺苷甲硫氨酸供应，提高了生产力。结果，（S）-黎藤碱的产量从葡萄糖达到了 384μM。此外，我们发现了三个不一致的现象：由于额外基因表达引起的抑制作用、实验条件之间的冲突以及来自额外下游酶的上游酶的干扰。基于这些结果，我们讨论了使用微生物整合多个酶基因进行物质生产的未来展望和挑战。
