PDF(Pubmed)
Abstract：
BACKGROUND: Polyketide synthases (PKSs) are classified into three types based on their enzyme structures. Among them, type III PKSs, catalyzing the iterative condensation of malonyl-coenzyme A (CoA) with a CoA-linked starter molecule, are important synthases of valuable natural products. However, low efficiency and byproducts formation often limit their applications in recombinant overproduction.
RESULTS: Herein, a rapid growth selection system is designed based on the accumulation and derepression of toxic acyl-CoA starter molecule intermediate products, which could be potentially applicable to most type III polyketides biosynthesis. This approach is validated by engineering both chalcone synthases (CHS) and host cell genome, to improve naringenin productions in Escherichia coli. From directed evolution of key enzyme CHS, beneficial mutant with ~ threefold improvement in capability of naringenin biosynthesis was selected and characterized. From directed genome evolution, effect of thioesterases on CHS catalysis is first discovered, expanding our understanding of byproduct formation mechanism in type III PKSs. Taken together, a whole-cell catalyst producing 1082 mg L-1 naringenin in flask with E value (evaluating product specificity) improved from 50.1% to 96.7% is obtained.
CONCLUSIONS: The growth selection system has greatly contributed to both enhanced activity and discovery of byproduct formation mechanism in CHS. This research provides new insights in the catalytic mechanisms of CHS and sheds light on engineering highly efficient heterologous bio-factories to produce naringenin, and potentially more high-value type III polyketides, with minimized byproducts formation.
RESULTS: Herein, a rapid growth selection system is designed based on the accumulation and derepression of toxic acyl-CoA starter molecule intermediate products, which could be potentially applicable to most type III polyketides biosynthesis. This approach is validated by engineering both chalcone synthases (CHS) and host cell genome, to improve naringenin productions in Escherichia coli. From directed evolution of key enzyme CHS, beneficial mutant with ~ threefold improvement in capability of naringenin biosynthesis was selected and characterized. From directed genome evolution, effect of thioesterases on CHS catalysis is first discovered, expanding our understanding of byproduct formation mechanism in type III PKSs. Taken together, a whole-cell catalyst producing 1082 mg L-1 naringenin in flask with E value (evaluating product specificity) improved from 50.1% to 96.7% is obtained.
CONCLUSIONS: The growth selection system has greatly contributed to both enhanced activity and discovery of byproduct formation mechanism in CHS. This research provides new insights in the catalytic mechanisms of CHS and sheds light on engineering highly efficient heterologous bio-factories to produce naringenin, and potentially more high-value type III polyketides, with minimized byproducts formation.
摘要：
背景：聚酮化合物合酶（PKS）根据其酶结构分为三种类型。其中,III型PKS，催化丙二酰辅酶A（CoA）与CoA连接的起始分子的迭代缩合，是有价值的天然产物的重要合成酶。然而,低效率和副产物形成通常限制了它们在重组过量生产中的应用。
结果：这里，基于有毒酰基CoA起始分子中间产物的积累和去抑制，设计了一种快速生长选择系统，这可能适用于大多数III型聚酮生物合成。这种方法是通过工程查尔酮合酶（CHS）和宿主细胞基因组验证，以提高柚皮素在大肠杆菌中的产量。从关键酶CHS的定向进化，选择并表征了柚皮素生物合成能力提高〜三倍的有益突变体。从定向基因组进化，首次发现了硫酯酶对CHS催化的作用，扩大我们对III型PKS副产物形成机制的理解。一起来看,获得了在烧瓶中产生1082mgL-1柚皮素的全细胞催化剂，其E值（评估产品特异性）从50.1％提高到96.7％。
结论：生长选择系统极大地促进了CHS中活性的增强和副产物形成机制的发现。这项研究为CHS的催化机理提供了新的见解，并为工程高效的异源生物工厂生产柚皮素提供了启示，和潜在的更高价值的III型聚酮化合物，最小化副产物的形成。
结果：这里，基于有毒酰基CoA起始分子中间产物的积累和去抑制，设计了一种快速生长选择系统，这可能适用于大多数III型聚酮生物合成。这种方法是通过工程查尔酮合酶（CHS）和宿主细胞基因组验证，以提高柚皮素在大肠杆菌中的产量。从关键酶CHS的定向进化，选择并表征了柚皮素生物合成能力提高〜三倍的有益突变体。从定向基因组进化，首次发现了硫酯酶对CHS催化的作用，扩大我们对III型PKS副产物形成机制的理解。一起来看,获得了在烧瓶中产生1082mgL-1柚皮素的全细胞催化剂，其E值（评估产品特异性）从50.1％提高到96.7％。
结论：生长选择系统极大地促进了CHS中活性的增强和副产物形成机制的发现。这项研究为CHS的催化机理提供了新的见解，并为工程高效的异源生物工厂生产柚皮素提供了启示，和潜在的更高价值的III型聚酮化合物，最小化副产物的形成。
