一个模块化多基酸合成酶thioesterase的定向进化,用于生成一个混合宏环环系统
Maria L Adrover-Castellano1, Brian J Curtis1, Jennifer J Schmidt1
1Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216 (USA).
概括
研究人员设计了铁酶 (TE) 酶,以改善新型杂交巨乳素的产生. 定向进化增强了非自然宏循环的产量,克服了聚基酸合成酶 (PKS) 路径工程中的一个关键挑战.
科学领域:
- 生物化学 生物化学
- 合成生物学 合成生物学
- 自然产品的合成自然产品的合成
背景情况:
- 模块化I型多基合成酶 (PKSs) 对于合成具有药用价值的多种天然产品至关重要.
- 铁酶 (TE) 域的选择性是使用PKS模块用于新型麦克罗拉克合成的一个主要限制.
- 之前的研究发现TE选择性是创造多样化的巨乳素的瓶.
研究的目的:
- 为了设计一个铁酶 (TE) 域,以提高对含有非天然胺的六基胺中间体的选择性.
- 通过PKS工程来产生新的混合12个成员的麦克罗拉克/乳糖环系统.
- 克服PKS路径工程中的局限性,用于复杂宏观循环的合成.
主要方法:
- 利用来自皮克罗米 (Pik) 生物合成途径的工程TE S148C突变.
- 在宏观循环合成中使用含有非天然胺的六甲基胺中间体.
- 开展了一项逐步指导的进化运动,以提高Pik TE变体的选择性和产量.
主要成果:
- 最初,与天然产品相比,非自然宏观循环的产量明显低于自然产品.
- 定向进化产生了Pik TE变体,它们在水解上对宏循环形成具有更好的选择性.
- 通过代突变组合,开发了一种复合变体,其混合宏循环的产量提高了六倍.
结论:
- 该研究确定了影响TE选择性和非自然基质产量的关键氨基酸残留物.
- 工程 TE 变种克服了关键的 PKS 路径守门员,使混合宏循环的高效合成成为可能.
- 这项工作为增强PKS工程用于新型天然产品模拟合成提供了洞察力.
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