Abstract

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.

中文翻译：

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大肠杆菌中单体比例调节的聚3-羟基丁酸酯--3-羟基戊酸酯的生物基生产

摘要

在这项研究中，我们应用代谢工程和生物处理策略，以提高异源生产的重要生物降解的共聚物，即，聚（3-羟基丁酸酯共-3-羟基戊酸酯）（PHBV），利用调制3-羟基戊酸酯（3-HV）在不同的氧气条件下，工程化大肠杆菌中甘油的结构无关碳的单体级分。我们使用先前衍生的产丙醇的（即产生1-丙醇的）大肠杆菌菌株和活化的基因组睡眠美容变异酶（Sbm）操纵子作为宿主来进行phaCAB的异源表达操纵子。通过调节从三羧酸（TCA）循环进入Sbm途径的异化碳通量通道来调节3-HV单体馏分，以生物合成丙酰辅酶A，这是（R的关键前体）-3-羟基戊酰基-CoA（3-HV-CoA）单体。通过操纵参与TCA循环的基因选择或改变细菌培养物的含氧条件来调节碳通量通道。通过实施这些整合策略，我们成功地实现了高水平的PHBV生物合成，其3-HV单体馏分的范围从〜4到50 mol％不等，从而有可能在生物合成阶段微调PHBV的机械性能。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。

关键点

• TCA循环工程应用于增强大肠杆菌中的3-HV单体含量。

•研究了氧气条件对大肠杆菌中3-HV掺入PHBV的影响。

•进行了细菌培养，以在工程化大肠杆菌中大量生产PHBV。