Constitutive overexpression of asm2 and asm39 increases AP-3 production in the actinomycete Actinosynnema pretiosum
Constitutive overexpression of asm2 and asm39 increases AP-3 production in the actinomycete Actinosynnema pretiosum
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DOI：
10.1007/s10295-009-0619-7
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发表时间：
2009-07
影响因子：
3.4
通讯作者：
D. Ng;H. K. Chin;Victor Wong
中科院分区：
文献类型：
--
作者：
D. Ng;H. K. Chin;Victor Wong
研究了在放线菌的安丝菌素生物合成簇中组成性过表达调节剂作为增加安丝菌素-P3（AP-3）的产生的策略，安丝菌素-P3是一种临床上有前途的化疗剂。将推测的转录调控因子asm 2、asm 29和asm 34以及推测的调控蛋白asm 39分别克隆到单位点整合型载体pAP 40和多拷贝复制型载体pREP中，并转化到A. pretiosum中。在生物测定筛选中，在pREP中过表达asm 2和asm 39的转化体显示安丝菌素产量增加（超过亲本水平的1.3倍）。在摇瓶发酵中，theasm 2和asm 39过表达转化体分别达到33和52 mg/l的最大AP-3滴度，分别是空白载体对照的1.6倍和2.5倍。由于先前的报道表明Asm 2是一种转录抑制因子，因此，过表达Asm 2的AP-3产量的增加是出乎意料的。产量的增加似乎依赖于用复制型载体实现的高表达水平，这可能破坏了Asm 2的正常功能。定量逆转录聚合酶链反应（RT-PCR）证实，asm 2和asm 39在转化子中的转录水平显著高于对照，表明产量的提高是由于转化质粒的作用。本研究表明，调节基因的过度表达是一种可行的策略，以增加AP-3的生产在A. pretiosum。
Constitutive overexpression of regulators in the ansamitocin biosynthetic cluster ofActinosynnema pretiosumwas investigated as a strategy to increase the production of ansamitocin-P3 (AP-3), a clinically promising chemotherapeutic agent. Putative transcriptional regulatorsasm2,asm29, andasm34as well as the putative regulatory proteinasm39were cloned into a single-site integrative vector and a multicopy replicative vector, pAP40 and pREP, respectively, and then transformed intoA. pretiosum. Transformants overexpressingasm2andasm39in pREP showed an increase in ansamitocin production (1.3-fold over parental levels) in a bioassay screen. In shake-flask fermentations, theasm2andasm39overexpression transformants attained a maximum AP-3 titer of 33 and 52 mg/l, respectively, which were 1.6- and 2.5-fold higher than the blank vector control. The increase in AP-3 production for theasm2overexpression transformant was unexpected, since prior reports suggested that Asm2 was a transcriptional repressor. The increase in production appeared to be dependent on the high expression levels achieved with the replicative vector, which may have disrupted the normal function of Asm2. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) confirmed thatasm2andasm39transcription levels were significantly higher in the transformants relative to the control, suggesting that the yield improvement was due to the transformed plasmids. This study demonstrates that deregulated overexpression of regulatory genes is a feasible strategy to increase AP-3 production inA. pretiosum.