Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011

Abstract

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

Combination of traditional mutation and metabolic engineering to enhance ansamitocin P‐3 production in Actinosynnema pretiosum

“…RM7011 (Mo et al, 2013). Those reports and this work show that based on a high producing mutant from random mutagenesis, metabolic engineering could be further elaborated to feasibly improve the production of targeted secondary metabolites.…”

“…The production of FK506, a clinically used immuno suppressive agent, was improved by combining random mutagenesis and metabolically‐engineered precursor supply in Streptomyces sp. RM7011 (Mo et al, ). Those reports and this work show that based on a high producing mutant from random mutagenesis, metabolic engineering could be further elaborated to feasibly improve the production of targeted secondary metabolites.…”

“…Integration of classical mutagenesis with metabolic engineering has also been employed by a couple of groups to increase the biosynthesis of other secondary metabolites (Dmytruk et al, 2011;Jung et al, 2011;Mo et al, 2013;Shi et al, 2014). For example, Dmytruk et al (2011) combined classical mutagenesis and overexpression of regulator and pathway genes to enhance the production of riboflavin.…”

“…The facts show that traditional mutation is still a powerful and practical method for obtaining a high producing cell line. However, because of the laborious and tedious process in the traditional strain screening, it is necessary to introduce rational methods such as metabolic engineering (Dmytruk, Yatsyshyn, Sybirna, Fedorovych, & Sibirny, ; Jung et al, ; Lal et al, ; Mo, Lee, Jin, Oh, & Suh, ; Shi, Valle‐Rodríguez, Siewers, & Nielsen, ).…”

Development of FK506-hyperproducing strain and optimization of culture conditions in solid-state fermentation for the hyper-production of FK506

“…The spores were smeared on agar medium plates with 50 µg/mL FK506 for sequential adaptation and incubated at 28 o C for 5 days. Among the strains with resistant to FK506, an FK506 high-yielding strain was screened using the A. niger bioassay and high performance liquid chromatography (HPLC) analysis according to the methods of Mo et al (2013). The calibration curve of FK506 was created using an FK506 standard (Sigma-Aldrich, USA).…”