背景:塑料是我们日常生活中不可或缺的一部分。然而,在其生命周期结束时管理不善会导致严重的环境后果。将这些聚合物微生物转化为新的增值产品提供了有希望的替代方案。在这项研究中,我们设计了土壤细菌Comamonas睾丸激素KF-1,一种对苯二甲酸的天然降解剂,用于将后者转化为高价值产物2-吡喃酮-4,6-二羧酸。
结果:为了将对苯二甲酸转化为2-吡喃酮-4,6-二羧酸,我们删除了天然PDC水解酶,仅观察到有限量的产物形成。为了测试这是否是碳源(即乙醇酸)抑制对苯二甲酸吸收生长的结果,在野生型菌株中监测两种碳源的消耗。两种碳源同时消耗,表明分解代谢物压抑不是这种情况。接下来,我们调查了野生型和突变株中途径酶的活性是否保持相同。又来了,没有观察到统计学差异.最后,我们假设降解操纵子中pmdK变体的存在可能是观察到的表型的原因,并创建了双缺失突变株。这种新产生的菌株在更大程度上积累了PDC,并再次消耗了两种碳源。然后将双缺失菌株用于生物反应器实验,导致在24小时内积累6.5g/L的产品,总生产率为0.27g/L/h。
结论:这项研究显示了通过工程化的C.testosteroniKF-1菌株从对苯二甲酸生产化学结构单元2-吡喃酮-4,6-二羧酸。观察到,需要天然PDC水解酶的缺失以及pmdK变体以实现高转化产率。在24小时内实现6.5g/L的产物滴度和0.27g/L/h的总生产率。
BACKGROUND: Plastics are an indispensable part of our daily life. However, mismanagement at their end-of-life results in severe environmental consequences. The microbial conversion of these polymers into new value-added products offers a promising alternative. In this study, we engineered the soil-bacterium Comamonas testosteroni KF-1, a natural degrader of terephthalic acid, for the conversion of the latter to the high-value product 2-pyrone-4,6-dicarboxylic acid.
RESULTS: In order to convert terephthalic acid to 2-pyrone-4,6-dicarboxylic acid, we deleted the native PDC hydrolase and observed only a limited amount of product formation. To test whether this was the result of an inhibition of terephthalic acid uptake by the carbon source for growth (i.e. glycolic acid), the consumption of both carbon sources was monitored in the wild-type strain. Both carbon sources were consumed at the same time, indicating that catabolite repression was not the case. Next, we investigated if the activity of pathway enzymes remained the same in the wild-type and mutant strain. Here again, no statistical differences could be observed. Finally, we hypothesized that the presence of a pmdK variant in the degradation operon could be responsible for the observed phenotype and created a double deletion mutant strain. This newly created strain accumulated PDC to a larger extent and again consumed both carbon sources. The double deletion strain was then used in a bioreactor experiment, leading to the accumulation of 6.5 g/L of product in 24 h with an overall productivity of 0.27 g/L/h.
CONCLUSIONS: This study shows the production of the chemical building block 2-pyrone-4,6-dicarboxylic acid from terephthalic acid through an engineered C. testosteroni KF-1 strain. It was observed that both a deletion of the native PDC hydrolase as well as a pmdK variant is needed to achieve high conversion yields. A product titer of 6.5 g/L in 24 h with an overall productivity of 0.27 g/L/h was achieved.