ABSTRACT

Acetic acid is one of the main inhibitors of lignocellulosic hydrolysates and acetic acid tolerance is crucial for the development of robust cell factories for conversion of biomass. As a precursor of acetyl-coenzyme A, it also plays an important role in central carbon metabolism. Thus, monitoring acetic acid levels is a crucial aspect when cultivating yeast. Transcription factor-based biosensors represent useful tools to follow metabolite concentrations. Here, we present the development of an acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1 that upon binding to acetic acid relocates to the nucleus. In the biosensor, a synthetic transcription factor consisting of Haa1 and BM3R1 from Bacillus megaterium was used to control expression of a reporter gene under a promoter containing BM3R1 binding sites. The biosensor did not drive expression under a promoter containing Haa1 binding sites and responded to acetic acid over a linear range spanning from 10 to 60 mM. To validate its applicability, the biosensor was integrated into acetic acid-producing strains. A direct correlation between biosensor output and acetic acid production was detected. The developed biosensor enables high-throughput screening of strains producing acetic acid and could also be used to investigate acetic acid-tolerant strain libraries.

中文翻译：

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基于 Haa1 的酿酒酵母乙酸传感生物传感器的开发

摘要

乙酸是木质纤维素水解产物的主要抑制剂之一，乙酸耐受性对于开发用于生物质转化的稳健细胞工厂至关重要。作为乙酰辅酶A的前体，它在中枢碳代谢中也起着重要作用。因此，在培养酵母时监测乙酸水平是一个关键方面。基于转录因子的生物传感器代表了跟踪代谢物浓度的有用工具。在这里，我们介绍了一种基于酿酒酵母转录因子 Haa1 的乙酸生物传感器的开发，该转录因子在与乙酸结合后重新定位到细胞核。在生物传感器中，由巨大芽孢杆菌的 Haa1 和 BM3R1 组成的合成转录因子用于控制报告基因在含有 BM3R1 结合位点的启动子下的表达。生物传感器在含有 Haa1 结合位点的启动子下不驱动表达，并且在 10 至 60 mM 的线性范围内对乙酸作出反应。为了验证其适用性，将生物传感器整合到产生乙酸的菌株中。检测到生物传感器输出与乙酸产量之间的直接相关性。开发的生物传感器能够对产生乙酸的菌株进行高通量筛选，也可用于研究耐乙酸菌株库。