Araceli Flores-Sánchez, Juliana A Ramsay, Berenice Vergara-Porras, Fermin Pérez-Guevara, Bruce A Ramsay
Escuela de Ingeniería, Tecnológico de Monterrey, Campus Estado de México, Estado de México 52926, Mexico; Chemical Engineering, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Chemical Engineering, Queen's University, Kingston, Ontario K7L 3N6, Canada.
J Biotechnol. 2022 Apr 10;349:25-31. doi: 10.1016/j.jbiotec.2022.03.011. Epub 2022 Mar 24.
Recombinant Cupriavidus necator H16/pMPJAS03, expressing a P. putida KT2440 enoyl-CoA hydratase (phaJ), was able to synthesize short-chain-length/ medium-chain-length (scl-mcl) PHA copolymers with a high content of mcl subunits using its native poly(3-hydroxyalkanoate) synthase. The cells were cultivated on fructose with canola oil or canola oil/decanoic acid (DA) mixtures in fed-batch fermentations. The recombinant C. necator H16 (without any synthase modification) produced a polymer composed of 3-hydroxybutyrate (3HB) with mcl-subunits, including 3-hydroxyhexanoate (3HHx), and about 300-fold more 3-hydroxyoctanoate (3HO) than the yields reported in previous studies, as well as a significant amount of 3-hydroxydecanoate (3HD). Increasing the DA content in the feed from 0% to 15% v/v increased the molar content of the 3HD subunits from 1.2 to 2.1 mol%. The presence of larger monomers, such as 3HO and 3HD, decreased the crystallinity and melting temperature and modified the mechanical properties of the polymers. Thus, replacing either of the two gene products (phaJ or phaC1) required to produce PHA from CoA-3-hydroxy fatty acids with broader spectrum enzymes, is suitable for the production of commercially useful scl-mcl-PHA.
表达恶臭假单胞菌KT2440烯酰辅酶A水合酶（phaJ）的重组食酸戴尔福特菌H16/pMPJAS03，能够利用其天然的聚（3-羟基链烷酸）合酶合成具有高含量中链长度（mcl）亚基的短链长度/中链长度（scl-mcl）PHA共聚物。在补料分批发酵中，将细胞接种于添加了菜籽油或菜籽油/癸酸（DA）混合物的果糖培养基上进行培养。重组食酸戴尔福特菌H16（未对合酶进行任何修饰）产生了一种由3-羟基丁酸（3HB）与mcl亚基组成的聚合物，其中mcl亚基包括3-羟基己酸（3HHx），且3-羟基辛酸（3HO）的产量比之前研究报道的高出约300倍，同时还产生了大量的3-羟基癸酸（3HD）。将补料中DA的含量从0%（v/v）提高到15%（v/v），使3HD亚基的摩尔含量从1.2 mol%增加到2.1 mol%。较大单体如3HO和3HD的存在降低了聚合物的结晶度和熔点，并改变了聚合物的机械性能。因此，用具有更广泛底物谱的酶替代从辅酶A-3-羟基脂肪酸生产PHA所需的两种基因产物（phaJ或phaC1）中的任何一种，都适合于生产具有商业价值的scl-mcl-PHA。
