Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

中文翻译：

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Cupriavidus necator 的适应性实验室进化和代谢工程改善挥发性脂肪酸的分解代谢


将大体积废物流生物转化为增值产品将成为不断增长的生物经济不可或缺的组成部分。挥发性脂肪酸 （VFA）（例如丁酸盐、戊酸盐和己酸盐）是一种新兴且有前途的微生物碳升级回收废物衍生原料。Cupriavidus necator H16 是 VFA 转化为各种生物制品的有利宿主，因为它具有多样化的碳代谢、易于代谢工程和工业规模的使用。在这里，我们报道了提高 C. necator 中产物滴度的常见策略，即删除多羟基丁酸酯 （PHB） 生物合成操纵子，导致 VFA 底物上出现显着的生长缺陷。使用适应性实验室进化，我们确定了调节基因 phaR、H16_A1372/H16_A1373 编码的双组分反应调节因子-组氨酸激酶对以及 H16_A2296-A2298 编码的三联转运蛋白组装的突变，这些突变是改善 VFA 底物生长的原因。phaR 和 H16_A1373 的缺失导致 NADH 丰度显著降低，伴随着参与碳代谢、电子载体平衡和氧化应激耐受性的基因表达的巨大变化，这可能是改善这些工程菌株生长的原因。这些结果提供了对 PHB 生物合成在碳和能量代谢中的作用的见解，并突出了调节因子 PhaR 在全球监管网络中的关键作用。通过结合突变，我们生成了在 VFA 上具有显着生长改善的平台菌株，这可以提高废物衍生的 VFA 底物向目标生物制品的转化。