Cha Seungwoo, Cho Yong-Joon, Lee Jong Kwan, Hahn Ji-Sook
School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
Department of Molecular Bioscience, College of Biomedical Science, Kangwon National University, 1 Gangwondaehakgil, Chuncheon, Gangwon-do, 24341, Republic of Korea.
Biotechnol Biofuels Bioprod. 2023 Jul 18;16(1):114. doi: 10.1186/s13068-023-02364-6.
Methanotrophs have emerged as promising hosts for the biological conversion of methane into value-added chemicals, including various organic acids. Understanding the mechanisms of acid tolerance is essential for improving organic acid production. WatR, a LysR-type transcriptional regulator, was initially identified as involved in lactate tolerance in a methanotrophic bacterium Methylomonas sp. DH-1. In this study, we investigated the role of WatR as a regulator of cellular defense against weak organic acids and identified novel target genes of WatR.
By conducting an investigation into the genome-wide binding targets of WatR and its role in transcriptional regulation, we identified genes encoding an RND-type efflux pump (WatABO pump) and previously unannotated small open reading frames (smORFs), watS1 to watS5, as WatR target genes activated in response to acetate. The watS1 to watS5 genes encode polypeptides of approximately 50 amino acids, and WatS1 to WatS4 are highly homologous with one predicted transmembrane domain. Deletion of the WatABO pump genes resulted in decreased tolerance against formate, acetate, lactate, and propionate, suggesting its role as an efflux pump for a wide range of weak organic acids. WatR repressed the basal expression of watS genes but activated watS and WatABO pump genes in response to acetate stress. Overexpression of watS1 increased tolerance to acetate but not to other acids, only in the presence of the WatABO pump. Therefore, WatS1 may increase WatABO pump specificity toward acetate, switching the general weak acid efflux pump to an acetate-specific efflux pump for efficient cellular defense against acetate stress.
Our study has elucidated the role of WatR as a key transcription factor in the cellular defense against weak organic acids, particularly acetate, in Methylomonas sp. DH-1. We identified the genes encoding WatABO efflux pump and small polypeptides (WatS1 to WatS5), as the target genes regulated by WatR for this specific function. These findings offer valuable insights into the mechanisms underlying weak acid tolerance in methanotrophic bacteria, thereby contributing to the development of bioprocesses aimed at converting methane into value-added chemicals.
甲烷营养菌已成为将甲烷生物转化为包括各种有机酸在内的高附加值化学品的有前景的宿主。了解耐酸机制对于提高有机酸产量至关重要。WatR是一种LysR型转录调节因子，最初被鉴定为参与甲烷营养菌甲基单胞菌属DH-1中的乳酸耐受性。在本研究中，我们研究了WatR作为细胞对弱有机酸防御调节因子的作用，并鉴定了WatR的新靶基因。
通过对WatR的全基因组结合靶点及其在转录调控中的作用进行研究，我们鉴定出编码RND型外排泵（WatABO泵）和先前未注释的小开放阅读框（smORF）watS1至watS5的基因，作为响应乙酸盐而被激活的WatR靶基因。watS1至watS5基因编码约50个氨基酸的多肽，并且WatS1至WatS4与一个预测的跨膜结构域高度同源。WatABO泵基因的缺失导致对甲酸、乙酸、乳酸和丙酸的耐受性降低，表明其作为多种弱有机酸外排泵的作用。WatR抑制watS基因的基础表达，但在乙酸盐胁迫下激活watS和WatABO泵基因。仅在存在WatABO泵的情况下，watS1的过表达增加了对乙酸盐的耐受性，但对其他酸没有增加耐受性。因此，WatS1可能增加WatABO泵对乙酸盐的特异性，将一般的弱酸外排泵转变为乙酸盐特异性外排泵，以有效地对细胞进行乙酸盐胁迫防御。
我们的研究阐明了WatR作为关键转录因子在甲基单胞菌属DH-1中对弱有机酸特别是乙酸盐的细胞防御中的作用。我们鉴定出编码WatABO外排泵和小多肽（WatS1至WatS5）的基因，作为受WatR调控以实现此特定功能的靶基因。这些发现为甲烷营养菌中弱酸耐受性的潜在机制提供了有价值的见解，从而有助于开发旨在将甲烷转化为高附加值化学品的生物过程。
