Department of Life Sciences, University of Turku, Turku, FIN, 20014, Finland.
Department of Microbiology, University of Helsinki, Helsinki, FIN, 00014, Finland.
Metab Eng. 2022 Sep;73:124-133. doi: 10.1016/j.ymben.2022.07.002. Epub 2022 Jul 7.
Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.
放线菌是药物和工业酶的重要生产者。然而，野生型菌株在工业应用之前需要经过艰苦的开发。在这里，我们提出了一种基于随机诱变、选择压力和单细胞分选的通用报告基因指导的代谢工程工具。我们开发了荧光激活细胞分选（FACS）方法，能够从液体培养物中直接从突变体群体中重复鉴定出表现良好的单个细胞。放线菌是分解代谢酶的重要来源，产物产量决定了工业可行性。我们展示了工业胆固醇氧化酶 ChoD 产生菌 Streptomyces lavendulae 的 5 倍产率提高，达到 20.4 U g，经过三轮。传统上，菌株开发后需要进行生产培养基优化，这是一个耗时的多参数问题，可能需要难以获得的成分。超高通量筛选使我们能够绕过培养基优化，并且可以直接从在预设培养条件下生长的突变文库中鉴定出高产 ChoD 的菌株。基于基因组挖掘的药物发现是生物活性化合物的有前途的来源，但观察到目标代谢途径在实验室条件下可能沉默，这使得情况变得复杂。我们通过在 Amycolatopsis 中激活沉默的 Mutaxanthene 代谢途径来证明我们的技术用于药物发现。我们将该方法应用于工业菌株的开发，并在第二轮突变体选择中将 Mutaxanthene 的产量提高了 9 倍，达到 99mg/L。总之，以单细胞形式筛选数千万个突变体的能力为放线菌的代谢工程提供了广泛的适用性，可用于激活沉默的代谢途径和提高蛋白质和天然产物的产量。
