{"title":"Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.","authors":"Takuma Kojima, Shinji Masuda","doi":"10.2323/jgam.2023.07.004","DOIUrl":null,"url":null,"abstract":"<p><p>5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (P<sub>rrnB</sub> or P<sub>rsp_7571</sub>) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where P<sub>rrnB</sub> was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where P<sub>rrnB</sub> was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where P<sub>rrnB</sub> was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":"270-277"},"PeriodicalIF":0.8000,"publicationDate":"2024-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of General and Applied Microbiology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2323/jgam.2023.07.004","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/7/24 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (PrrnB or Prsp_7571) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where PrrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where PrrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where PrrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.
5-Aminolevulinic acid(ALA)是血红素的前体,也是一种在大多数生物体细胞中合成的天然氨基酸。目前,ALA 被用作药品、保健品、化妆品、饲料、肥料和其他产品的成分。ALA 主要由光合细菌 Rhodobacter sphaeroides 通过工业发酵生产。在本研究中,我们尝试使用高表达 ALA 合成酶(ALAS)基因的遗传策略来提高水合根瘤菌的 ALA 生产率。我们在编码 ALAS(hemA 和/或 hemT)的基因上游插入了一个组成型启动子(PrrnB 或 Prsp_7571),以构建能组成型表达 ALAS 的菌株。在将 PrrnB 插入 hemA 启动子区域的菌株中观察到了最高的 hemA 转录水平,是野生型的 3.5 倍。在将 PrrnB 插入 hemT 启动子区域的菌株中,观察到 hemT 的转录水平最高,是野生型的 46 倍。在优化生长条件下,将 PrrnB 插入 hemT 启动子区域的菌株的粗细胞提取物中观察到的 ALAS 活性最高,是野生型的 2.7 倍。该菌株的 ALA 积累是野生型的 12 倍。因此,我们在不使用外源 DNA 序列的情况下提高了 ALA 的产量。未来,将这种方法应用于目前的工业 ALA 生产菌,有望进一步提高 ALA 的生产率。
期刊介绍:
JGAM is going to publish scientific reports containing novel and significant microbiological findings, which are mainly devoted to the following categories: Antibiotics and Secondary Metabolites; Biotechnology and Metabolic Engineering; Developmental Microbiology; Environmental Microbiology and Bioremediation; Enzymology; Eukaryotic Microbiology; Evolution and Phylogenetics; Genome Integrity and Plasticity; Microalgae and Photosynthesis; Microbiology for Food; Molecular Genetics; Physiology and Cell Surface; Synthetic and Systems Microbiology.