State Key Laboratory of Animal Biotech Breeding and College of Biological Sciences, China Agricultural University, Beijing, China.
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Microb Biotechnol. 2024 Nov;17(11):e70038. doi: 10.1111/1751-7915.70038.
Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics.
达托霉素（DAP）是一种新型的环状脂肽抗生素，由玫瑰孢链霉菌产生，对治疗多药耐药革兰阳性病原体引起的感染具有重要的临床意义，但产量低阻碍了其大规模的工业生产。在这里，我们描述了一种组合代谢工程策略，用于构建 DAP 高产菌株。最初，我们通过使用 CRISPRi 分别抑制天冬氨酸（Asp）降解和竞争途径基因，以及使用强启动子 kasOp过表达 Asp 合成途径基因，来增强玫瑰孢链霉菌野生型（WT）菌株中的 Asp 前体供应。结果表明，所有这些菌株的 DAP 产量都有所提高。同时抑制 acsA4、pta、pyrB 和 pyrC 可使 DAP 产量提高到 167.4μg/mL（比 WT 值高 73.5%）。协同过表达 aspC、gdhA、ppc 和 ecaA 可使 DAP 产量达到 168μg/mL（比 WT 值高 75.7%）。同时，我们构建了一个有利于 DAP 生产的底盘菌株，通过消除副产物的产生（即删除红色素生物合成基因簇（BGC）的 21.1kb 区域）和工程化 DAP BGC（即用 kasOp替换其天然的 dptEp）。底盘菌株的产量达到了 185.8μg/mL。将我们的 Asp 前体供应策略应用于底盘菌株进一步将 DAP 产量提高到 302μg/mL（比 WT 值高 2.1 倍）。随后，我们克隆了工程化的 DAP BGC，并将其在底盘菌株中进行了复制，使 DAP 产量达到 274.6μg/mL。上述策略的组合使 DAP 产量达到了 350.7μg/mL（比 WT 值高 2.6 倍），是摇瓶发酵中报道的最高 DAP 产量。这些发现为提高 DAP 产量提供了一种有效的组合策略，也可以很容易地应用于其他 Asp 相关抗生素的过量生产。
