Miscevic Dragan, Mao Ju-Yi, Mozell Bradley, Srirangan Kajan, Abedi Daryoush, Moo-Young Murray, Chou C Perry
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada.
Department of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan.
Appl Microbiol Biotechnol. 2021 Feb;105(4):1435-1446. doi: 10.1007/s00253-021-11108-1. Epub 2021 Jan 23.
In this study, we applied metabolic engineering and bioprocessing strategies to enhance heterologous production of an important biodegradable copolymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a modulated 3-hydroxyvalerate (3-HV) monomeric fraction from structurally unrelated carbon of glycerol in engineered Escherichia coli under different oxygenic conditions. We used our previously derived propanologenic (i.e., 1-propanol-producing) E. coli strain with an activated genomic Sleeping beauty mutase (Sbm) operon as a host for heterologous expression of the phaCAB operon. The 3-HV monomeric fraction was modulated by regulating dissimilated carbon flux channeling from the tricarboxylic acid (TCA) cycle into the Sbm pathway for biosynthesis of propionyl-CoA, which is a key precursor to (R)-3-hydroxyvaleryl-CoA (3-HV-CoA) monomer. The carbon flux channeling was regulated either by manipulating a selection of genes involved in the TCA cycle or varying oxygenic condition of the bacterial culture. With these consolidated strategies being implemented, we successfully achieved high-level PHBV biosynthesis with a wide range of 3-HV monomeric fraction from ~ 4 to 50 mol%, potentially enabling the fine-tuning of PHBV mechanical properties at the biosynthesis stage. We envision that similar strategies can be applied to enhance bio-based production of chemicals derived from succinyl-CoA. KEY POINTS: • TCA cycle engineering was applied to enhance 3-HV monomeric fraction in E. coli. • Effects of oxygenic conditions on 3-HV incorporation into PHBV in E. coli were investigated. • Bacterial cultivation for high-level PHBV production in engineered E. coli was performed.
在本研究中，我们应用代谢工程和生物加工策略，在不同的有氧条件下，增强重要的可生物降解共聚物聚（3-羟基丁酸酯-co-3-羟基戊酸酯）（PHBV）在工程化大肠杆菌中的异源生产，该共聚物中3-羟基戊酸酯（3-HV）单体部分由甘油中结构不相关的碳进行调控。我们使用先前构建的具有激活的基因组睡美人变位酶（Sbm）操纵子的产丙醇（即产1-丙醇）大肠杆菌菌株作为phaCAB操纵子异源表达的宿主。通过调节从三羧酸（TCA）循环到Sbm途径的异化碳通量，用于丙酰辅酶A的生物合成，从而调节3-HV单体部分，丙酰辅酶A是（R）-3-羟基戊酰辅酶A（3-HV-CoA）单体的关键前体。通过操纵参与TCA循环的一系列基因或改变细菌培养的有氧条件来调节碳通量。通过实施这些综合策略，我们成功实现了高水平的PHBV生物合成，其3-HV单体部分范围广泛，从约4至50摩尔％，这可能在生物合成阶段实现对PHBV机械性能的微调。我们设想可以应用类似的策略来增强源自琥珀酰辅酶A的化学品的生物基生产。要点：•应用TCA循环工程增强大肠杆菌中3-HV单体部分。•研究了有氧条件对大肠杆菌中3-HV掺入PHBV的影响。•进行了工程化大肠杆菌中高水平PHBV生产的细菌培养。
