Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
Acies Bio, d.o.o, Tehnološki Park, Ljubljana, Slovenia.
Microb Cell Fact. 2021 Feb 17;20(1):47. doi: 10.1186/s12934-021-01522-5.
Natural products are a valuable source of biologically active compounds that have applications in medicine and agriculture. One disadvantage with natural products is the slow, time-consuming strain improvement regimes that are necessary to ensure sufficient quantities of target compounds for commercial production. Although great efforts have been invested in strain selection methods, many of these technologies have not been improved in decades, which might pose a serious threat to the economic and industrial viability of such important bioprocesses.
In recent years, introduction of extra copies of an entire biosynthetic pathway that encodes a target product in a single microbial host has become a technically feasible approach. However, this often results in minor to moderate increases in target titers. Strain stability and process reproducibility are the other critical factors in the industrial setting. Industrial Streptomyces rimosus strains for production of oxytetracycline are one of the most economically efficient strains ever developed, and thus these represent a very good industrial case. To evaluate the applicability of amplification of an entire gene cluster in a single host strain, we developed and evaluated various gene tools to introduce multiple copies of the entire oxytetracycline gene cluster into three different Streptomyces rimosus strains: wild-type, and medium and high oxytetracycline-producing strains. We evaluated the production levels of these engineered S. rimosus strains with extra copies of the oxytetracycline gene cluster and their stability, and the oxytetracycline gene cluster expression profiles; we also identified the chromosomal integration sites.
This study shows that stable and reproducible increases in target secondary metabolite titers can be achieved in wild-type and in high oxytetracycline-producing strains, which always reflects the metabolic background of each independent S. rimosus strain. Although this approach is technically very demanding and requires systematic effort, when combined with modern strain selection methods, it might constitute a very valuable approach in industrial process development.
天然产物是具有医学和农业应用价值的生物活性化合物的宝贵来源。天然产物的一个缺点是，为了确保目标化合物有足够的商业生产数量，需要进行缓慢且耗时的菌株改良方案。尽管人们在菌株选择方法上投入了大量的努力，但这些技术中的许多在几十年内都没有得到改进，这可能对这些重要生物过程的经济和工业可行性构成严重威胁。
近年来，在单个微生物宿主中引入编码目标产物的整个生物合成途径的额外拷贝已成为一种可行的技术方法。然而，这通常只会导致目标产量的轻微到中度增加。菌株稳定性和过程重现性是工业环境中的其他关键因素。用于生产土霉素的工业链霉菌是有史以来最具经济效率的菌株之一，因此它们是一个非常好的工业案例。为了评估在单个宿主菌株中扩增整个基因簇的适用性，我们开发并评估了各种基因工具，以将整个土霉素基因簇的多个拷贝引入三种不同的链霉菌：野生型、中产量和高产土霉素菌株。我们评估了这些具有额外土霉素基因簇拷贝的工程化链霉菌菌株的生产水平及其稳定性，以及土霉素基因簇的表达谱；我们还确定了染色体整合位点。
本研究表明，在野生型和高产土霉素菌株中，可以稳定且可重复地提高目标次生代谢物的产量，这始终反映了每个独立链霉菌菌株的代谢背景。尽管这种方法在技术上要求很高，需要系统的努力，但当与现代菌株选择方法结合使用时，它可能构成工业过程开发的一种非常有价值的方法。
