Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, People's Republic of China.
SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
Microb Cell Fact. 2020 Feb 13;19(1):32. doi: 10.1186/s12934-020-01301-8.
Bleomycin is a broad-spectrum glycopeptide antitumor antibiotic produced by Streptomyces verticillus. Clinically, the mixture of bleomycin A2 and bleomycin B2 is widely used in combination with other drugs for the treatment of various cancers. As a secondary metabolite, the biosynthesis of bleomycin is precisely controlled by the complex extra-/intracellular regulation mechanisms, it is imperative to investigate the global metabolic and regulatory system involved in bleomycin biosynthesis for increasing bleomycin production.
N-acetylglucosamine (GlcNAc), the vital signaling molecule controlling the onset of development and antibiotic synthesis in Streptomyces, was found to increase the yields of bleomycins significantly in chemically defined medium. To mine the gene information relevant to GlcNAc metabolism, the DNA sequences of dasR-dasA-dasBCD-nagB and nagKA in S. verticillus were determined by chromosome walking. From the results of Real time fluorescence quantitative PCR (RT-qPCR) and electrophoretic mobility shift assays (EMSAs), the repression of the expression of nagB and nagKA by the global regulator DasR was released under induction with GlcNAc. The relief of blmT expression repression by BlmR was the main reason for increased bleomycin production. DasR, however, could not directly affect the expression of the pathway-specific repressor BlmR in the bleomycins gene cluster. With at the beginning of bleomycin synthesis, the supply of the specific precursor GDP-mannose played the key role in bleomycin production. Genetic engineering of the GDP-mannose synthesis pathway indicated that phosphomannose isomerase (ManA) and phosphomannomutase (ManB) were key enzymes for bleomycins synthesis. Here, the blmT, manA and manB co-expression strain OBlmT/ManAB was constructed. Based on GlcNAc regulation and assisted metabolic profiling analysis, the yields of bleomycin A2 and B2 were ultimately increased to 61.79 and 36.9 mg/L, respectively.
Under GlcNAc induction, the elevated production of bleomycins was mainly associated with the alleviation of the inhibition of BlmT, so blmT and specific precursor synthesis pathways were genetically engineered for bleomycins production improvement. Combination with subsequent metabolomics analysis not only effectively increased the bleomycin yield, but also extended the utilization of chitin-derived substrates in microbial-based antibiotic production.
博莱霉素是一种由产枝菌素产生的广谱糖肽抗肿瘤抗生素。临床上，博莱霉素 A2 和博莱霉素 B2 的混合物广泛与其他药物联合用于治疗各种癌症。作为一种次级代谢产物，博莱霉素的生物合成受到复杂的细胞外/细胞内调控机制的精确控制，因此，研究博莱霉素生物合成中涉及的全局代谢和调控系统对于提高博莱霉素的产量至关重要。
N-乙酰葡萄糖胺（GlcNAc）是控制链霉菌发育和抗生素合成起始的重要信号分子，在化学成分确定的培养基中发现它能显著提高博莱霉素的产量。为了挖掘与 GlcNAc 代谢相关的基因信息，通过染色体步行确定了产枝菌素 verticillus 中的 dasR-dasA-dasBCD-nagB 和 nagKA 的 DNA 序列。实时荧光定量 PCR（RT-qPCR）和电泳迁移率变动分析（EMSA）的结果表明，GlcNAc 诱导时，全局调控因子 DasR 对 nagB 和 nagKA 的表达抑制作用得到释放。BlmR 对 blmT 表达抑制的解除是博莱霉素产量增加的主要原因。然而，DasR 不能直接影响博莱霉素基因簇中途径特异性抑制剂 BlmR 的表达。在博莱霉素合成开始时，特异性前体 GDP-甘露糖的供应对博莱霉素的合成起着关键作用。GDP-甘露糖合成途径的遗传工程表明，磷酸甘露糖异构酶（ManA）和磷酸甘露糖变位酶（ManB）是博莱霉素合成的关键酶。在此，构建了 blmT、manA 和 manB 共表达菌株 OBlmT/ManAB。基于 GlcNAc 调控和辅助代谢谱分析，最终将博莱霉素 A2 和 B2 的产量分别提高到 61.79 和 36.9mg/L。
在 GlcNAc 诱导下，博莱霉素产量的提高主要与 BlmT 抑制的缓解有关，因此通过基因工程改造了 blmT 和特定前体合成途径，以提高博莱霉素的产量。结合后续的代谢组学分析，不仅有效地提高了博莱霉素的产量，而且扩展了微生物来源抗生素生产中壳聚糖衍生底物的利用。
