Pyeon Hye-Rim, Nah Hee-Ju, Kang Seung-Hoon, Choi Si-Sun, Kim Eung-Soo
Department of Biological Engineering, Inha University, Incheon, 402-751, South Korea.
Microb Cell Fact. 2017 May 31;16(1):96. doi: 10.1186/s12934-017-0708-7.
Heterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes.
To validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem clusters of pikromycin biosynthetic gene clusters.
The 60 kb pikromycin biosynthetic gene cluster was isolated in a single integration pSBAC vector. Introduction of the pikromycin biosynthetic gene cluster into the pikromycin non-producing strains resulted in higher pikromycin production. The utility of the pSBAC system as a precise cloning tool for large-sized biosynthetic gene clusters was verified through heterologous expression of the pikromycin biosynthetic gene cluster. Moreover, this pSBAC-driven heterologous expression strategy was confirmed to be an ideal approach for production of low and inconsistent natural products such as pikromycin in S. venezuelae, implying that this strategy could be employed for development of a custom overexpression scheme of natural product biosynthetic gene clusters in actinomycetes.
天然微生物产物生物合成基因簇的异源表达已成为提高各种潜在有价值天然产物产量和进行途径工程的重要策略。一种链霉菌人工染色体接合载体pSBAC，先前已成功应用于大型聚酮化合物互隔交链孢酚（TMC）生物合成基因簇的精确克隆和串联整合（Nah等人，《微生物细胞工厂》，2015年，第14卷第1期，第1页），这意味着该策略可用于开发放线菌中天然产物途径簇的定制过表达方案。
为验证pSBAC系统作为链霉菌中大型聚酮生物合成基因簇通用的异源过表达系统，另一种模型聚酮化合物——苦霉素生物合成基因簇，被精确克隆并使用pSBAC系统进行异源表达。在委内瑞拉链霉菌染色体的苦霉素生物合成基因簇的一个边界区域精确插入一个独特的HindIII限制性酶切位点，随后pSBAC位点特异性重组到苦霉素基因簇的侧翼区域。与之前的克隆过程不同，通过pSBAC修饰跳过了一个HindIII位点整合步骤。含有苦霉素生物合成基因簇的pSBAC载体pPik001被直接导入两种异源宿主——变铅青链霉菌和天蓝色链霉菌，从而产生了主要的苦霉素衍生物10-脱氧甲炔诺酮。当将两个完整的苦霉素生物合成基因簇串联导入变铅青链霉菌染色体时，证实了10-脱氧甲炔诺酮的过量产生以及之前未检测到的苦霉素的存在。此外，比较定量逆转录-聚合酶链反应（qRT-PCR）结果证实，在含有苦霉素生物合成基因簇串联簇的变铅青链霉菌中，苦霉素生物合成基因的转录显著上调。
60 kb的苦霉素生物合成基因簇被分离到单个整合的pSBAC载体中。将苦霉素生物合成基因簇导入不产生苦霉素的菌株中可提高苦霉素产量。通过苦霉素生物合成基因簇的异源表达，验证了pSBAC系统作为大型生物合成基因簇精确克隆工具的实用性。此外，这种由pSBAC驱动的异源表达策略被证实是在委内瑞拉链霉菌中生产低产量且产量不稳定的天然产物（如苦霉素）的理想方法，这意味着该策略可用于开发放线菌中天然产物生物合成基因簇的定制过表达方案。
