Špela Pikl1, Andrés Felipe Carrillo Rincón1, Lucija Slemc1, Dušan Goranovič2, Martina Avbelj1, Krešimir Gjuračić2, Hilda Sucipto3,4, Katja Stare5, Špela Baebler5, Martin Šala6, Meijin Guo7, Andriy Luzhetskyy3,4, Hrvoje Petković8, Vasilka Magdevska9. 1. Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia. 2. Acies Bio, d.o.o, Tehnološki Park, Ljubljana, Slovenia. 3. Pharmazeutische Biotechnologie, Universität des Saarlandes, Saarbrücken, Germany. 4. Helmholtz-Institut für Pharmazeutische Forschung Saarland, Saarbrücken, Germany. 5. National Institute of Biology, Večna pot 111, Ljubljana, Slovenia. 6. National Institute of Chemistry, Hajdrihova 19, SI-1000, Ljubljana, Slovenia. 7. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China. 8. Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia. hrvoje.petkovic@bf.uni-lj.si. 9. Acies Bio, d.o.o, Tehnološki Park, Ljubljana, Slovenia. vasilka.magdevska@aciesbio.com.
Abstract
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
Authors: Benjamin Kirm; Vasilka Magdevska; Miha Tome; Marinka Horvat; Katarina Karničar; Marko Petek; Robert Vidmar; Spela Baebler; Polona Jamnik; Štefan Fujs; Jaka Horvat; Marko Fonovič; Boris Turk; Kristina Gruden; Hrvoje Petković; Gregor Kosec Journal: Microb Cell Fact Date: 2013-12-17 Impact factor: 5.328