维生素B12
生物
酿酒酵母
蛋氨酸合酶
甲钴胺
生物化学
酵母
钴胺素
代谢工程
蛋氨酸
大肠杆菌
微生物学
酶
基因
氨基酸
作者
Sandra Lehner,Eckhard Boles
出处
期刊:Fems Yeast Research
[Oxford University Press]
日期:2023-01-01
卷期号:23
标识
DOI:10.1093/femsyr/foad020
摘要
For decades, the industrial vitamin B12 (cobalamin) production has been based on bacterial producer strains. Due to limited methods for strain optimization and difficult strain handling, the desire for new vitamin B12-producing hosts has risen. As a vitamin B12-independent organism with a big toolbox for genomic engineering and easy-to-handle cultivation conditions, Saccharomyces cerevisiae has high potential for heterologous vitamin B12 production. However, the B12 synthesis pathway is long and complex. To be able to easily engineer and evolve B12-producing recombinant yeast cells, we have developed an S. cerevisiae strain whose growth is dependent on vitamin B12. For this, the B12-independent methionine synthase Met6 of yeast was replaced by a B12-dependent methionine synthase MetH from Escherichia coli. Adaptive laboratory evolution, RT-qPCR, and overexpression experiments show that additional high-level expression of a bacterial flavodoxin/ferredoxin-NADP+ reductase (Fpr-FldA) system is essential for in vivo reactivation of MetH activity and growth. Growth of MetH-containing yeast cells on methionine-free media is only possible with the addition of adenosylcobalamin or methylcobalamin. A heterologous vitamin B12 transport system turned out to be not necessary for the uptake of cobalamins. This strain should be a powerful chassis to engineer B12-producing yeast cells.
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