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链霉菌光诱导基因表达系统。

Light inducible gene expression system for Streptomyces.

机构信息

Life Science Research Center, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, 252-0880, Japan.

出版信息

Sci Rep. 2024 Oct 28;14(1):25852. doi: 10.1038/s41598-024-76860-6.

DOI:10.1038/s41598-024-76860-6
PMID:39468183
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11519972/
Abstract

The LitR/CarH family comprises adenosyl B-based photosensory transcriptional regulators that control light-inducible carotenoid production in nonphototrophic bacteria. In this study, we established a blue-green light-inducible hyperexpression system using LitR and its partner ECF-type sigma factor LitS in streptomycin-producing Streptomyces griseus NBRC 13350. The constructed multiple-copy number plasmid, pLit19, carried five genetic elements: pIJ101rep, the thiostrepton resistance gene, litR, litS, and σ-recognized light-inducible crtE promoter. Streptomyces griseus transformants harboring pLit19 exhibited a light-dependent hyper-production of intracellular reporter enzymes including catechol-2,3-dioxygenase and β-glucuronidase, extracellular secreted enzymes including laccase and transglutaminase, and secondary metabolites including melanin, flaviolin, and indigoidine. Cephamycin-producing Streptomyces sp. NBRC 13304, carrying an entire actinorhodin gene cluster, exhibited light-dependent actinorhodin production after the introduction of the pLit19 shuttle-type plasmid with the pathway-specific activator actII-ORF4. Insertion of sti fragment derived from Streptomyces phaeochromogenes pJV1 plasmid into pLit19 increased its light sensitivity, allowing gene expression under weak light irradiation. The two constructed Escherichia coli-Streptomyces shuttle-type pLit19 plasmids were found to have abilities similar to those of pLit19. We successfully established an optogenetically controlled hyperproduction system for S. griseus NBRC 13350 and Streptomyces sp. NBRC 13304.

摘要

LitR/CarH 家族包括基于腺嘌呤核苷 B 的光感转录调控因子，它们控制非光养细菌中光诱导类胡萝卜素的产生。在本研究中，我们在链霉菌产生的灰色链霉菌 NBRC 13350 中建立了一个基于 LitR 和其伴侣 ECF 型σ因子 LitS 的蓝绿光诱导超表达系统。构建的多拷贝数质粒 pLit19 携带五个遗传元件：pIJ101rep、硫链丝菌素抗性基因、litR、litS 和 σ 识别的光诱导 crtE 启动子。携带 pLit19 的灰色链霉菌转化体表现出依赖光的细胞内报告酶（包括儿茶酚-2,3-双加氧酶和β-葡糖苷酸酶）、细胞外分泌酶（包括漆酶和转谷氨酰胺酶）和次级代谢产物（包括黑色素、黄酮醇和靛蓝）的超量产生。携带完整放线紫红素基因簇的头孢菌素产生链霉菌 NBRC 13304 在引入具有途径特异性激活子 actII-ORF4 的穿梭型 pLit19 质粒后，表现出依赖光的放线紫红素产生。将来源于灰色链霉菌 pJV1 质粒的 sti 片段插入 pLit19 中增加了其对光的敏感性，允许在弱光照射下进行基因表达。这两个构建的大肠杆菌-链霉菌穿梭型 pLit19 质粒被发现具有类似于 pLit19 的能力。我们成功地建立了灰色链霉菌 NBRC 13350 和链霉菌 NBRC 13304 的光控超量生产系统。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/adbab3a82f01/41598_2024_76860_Fig11_HTML.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/d2288a953634/41598_2024_76860_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/2af9d97fd7b3/41598_2024_76860_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/615246c8dc92/41598_2024_76860_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/f41f756ff22b/41598_2024_76860_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/7f840fddc9c6/41598_2024_76860_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/523306972fd1/41598_2024_76860_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/2fed446d71ca/41598_2024_76860_Fig9_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/ec521bdf89f8/41598_2024_76860_Fig10_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d485/11519972/adbab3a82f01/41598_2024_76860_Fig11_HTML.jpg

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