In various (industrial) conditions, cells are in a non-growing but metabolically active state in which de novo protein synthesis capacity is limited. The production of a metabolite by such non-growing cells is dependent on the cellular condition and enzyme activities, such as the amount, stability, and degradation of the enzyme(s). For industrial fermentations in which the metabolites of interest are mainly formed after cells enter the stationary phase, the investigation of prolonged metabolite production is of great importance. However, current batch model systems do not allow prolonged measurements due to metabolite accumulation driving product-inhibition. Here we developed a protocol that allows high-throughput metabolic measurements to be followed in real-time over extended periods (weeks). As a validation model, sugar utilization and arginine consumption by a low density of translationally blocked Lactococcus lactis was designed in a defined medium. In this system L. lactis MG1363 was compared with its derivative HB60, a strain described to achieve higher metabolic yield through a shift toward heterofermentative metabolism. The results showed that in a non-growing state HB60 is able to utilize more arginine than MG1363, and for both strains the decay of the measured activities were dependent on pre-culture conditions. During the first 5 days of monitoring a ∼25-fold decrease in acidification rate was found for strain HB60 as compared to a ∼20-fold decrease for strain MG1363. Such measurements are relevant for the understanding of microbial metabolism and for optimizing applications in which cells are frequently exposed to long-term suboptimal conditions, such as microbial cell factories, fermentation ripening, and storage survival.

中文翻译：

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一种长期分析非生长乳酸乳球菌中乳酸和氨产量的新方法揭示了预培养和菌株依赖性

在各种（工业）条件下，细胞处于非生长但代谢活跃的状态，在这种状态下，从头合成蛋白质的能力是有限的。这种非生长细胞产生的代谢物取决于细胞条件和酶活性，例如酶的量、稳定性和降解。对于感兴趣的代谢物主要在细胞进入稳定期后形成的工业发酵，延长代谢物产生的研究非常重要。然而，由于代谢物积累驱动产物抑制，当前的批量模型系统不允许长时间测量。在这里，我们开发了一个协议，允许在长时间（数周）内实时跟踪高通量代谢测量。作为验证模型，在确定的培养基中设计了低密度的翻译阻断乳酸乳球菌的糖利用和精氨酸消耗。在该系统中，乳酸乳球菌 MG1363 与其衍生物 HB60 进行了比较，HB60 菌株被描述为通过向异源发酵代谢转变来实现更高的代谢产量。结果表明，在非生长状态下，HB60 比 MG1363 能够利用更多的精氨酸，并且对于这两种菌株，所测量活性的衰减取决于预培养条件。在监测的前 5 天，发现菌株 HB60 的酸化率降低了约 25 倍，而菌株 MG1363 的酸化率降低了约 20 倍。此类测量与了解微生物代谢和优化细胞经常暴露于长期次优条件的应用有关，