Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering

Abstract

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.

FIG. 1.

Effect of SsgA on morphology: representative phase-contrast micrographs of mycelia from various streptomycetes with control plasmid pSET152 (left) or with pGWS4-SD overproducing SsgA (right). For ATCC strain designations see Materials and Methods. Notice the strongly reduced pellet formation in S. coelicolor, S.lividans, and S. roseosporus due to the overexpression of ssgA. Bar, 10 μm.

FIG. 2.

Effect of enhanced SsgA production on growth of S. coelicolor in a pilot-scale fermentor. Results shown are from batch fermentation of SsgA-overexpressing S. coelicolor GSA2 (top) and its parent M145, harboring control plasmid pSET152 (bottom), performed in a 42-liter fermentor. Glutamate was consumed prior to glucose, and glucose consumption started after approximately 50 h. Glutamate concentrations were calculated from the amount of added acid required to neutralize the ammonia produced from the utilization of glutamate. Growth ceased when glucose was fully consumed; this was after 85 h for GSA2 and after 115 h for M145 (indicated by arrows). Morphology of the mycelia after preculturing (pre) and after fermentation (end) is shown in the inset photographs. Clearly visible is the reduced pellet size in the GSA2 transformant. Bar, 20 μm.

FIG. 3.

Effect of enhanced expression of ssgA on growth and antibiotic production of S. coelicolor. Batch fermentation of SsgA-overexpressing S. coelicolor GSA2 and its parent M145 (with control plasmid pSET152) was performed in TSBS medium, with control of pH (maintained at 6.7) and of dissolved oxygen concentration (maintained at 80%). Biomass accumulation of S. coelicolor M145 (▴) and GSA2 (•) was calculated from dry weight measurements, while undecylprodigiosin (Red) production (open symbols) was measured spectrophotometrically.

FIG. 4.

Effects of enhanced expression of ssgA on growth and tyrosinase production of S. lividans. (A) Tyrosinase production by S. lividans. S. lividans 1326 containing pIJ703 was transformed with pGWS4-SD (ssgA overproduction; strain GSAL1) or with pSET152 (control plasmid; strain 1326), and transformants were selected on R2YE agar plates. Representative plates with around 500 colonies are shown. Note the strongly enhanced tyrosinase production by the SsgA-overproducing transformant. As follows from the data in panel B, the SsgA-overproducing strain produced two to three times more tyrosinase than the control strain. Hence, enhanced expression of ssgA resulted in an important improvement of the tyrosinase yield. (B) Batch fermentation of SsgA-overproducing S. lividans GSAL1/pIJ703 and its parent, 1326/pIJ703 (harboring control plasmid pSET152), was performed in 4 liters of TSBS in a 5-liter fermentor. The pH was maintained at 6.7, and the dissolved oxygen concentration was maintained at 80%. Biomass accumulation of S. lividans 1326 (▴) and GSAL1 (•) was calculated from dry weight measurements. Tyrosinase production (for description of the assay, see Materials and Methods) is represented by the open symbols.