Manipulation of two regulatory genes for efficient production of chromomycins in Streptomyces reseiscleroticus

Abstract

Background: Regulatory genes play critical roles in natural product biosynthetic pathways. Chromomycins are promising anticancer natural products from actinomycetes. This study is aimed to create an efficient strain for production of these molecules by manipulating the regulatory genes.

Results: A putative but silent chromomycin biosynthetic gene cluster was discovered in Streptomyces reseiscleroticus. Heterologous expression of the ketosynthase, chain length factor, and acyl carrier protein in Streptomyces lividans confirmed that they are responsible for the assembly of a decaketide. Two regulatory genes are present in this gene cluster, including SARP-type activator SrcmRI and PadR-like repressor SrcmRII. Either overexpression of SrcmRI or disruption of SrcmRII turned on the biosynthetic pathway of chromomycins. The production titers of chromomycin A₃/A₂ in R5 agar in these two strains reached 8.9 ± 1.2/13.2 ± 1.6 and 49.3 ± 4.3/53.3 ± 3.6 mg/L, respectively. An engineered strain was then constructed with both SrcmRII disruption and SrcmRI overexpression, which produced chromomycins A₃ and A₂ in R5 agar at 69.4 ± 7.6 and 81.7 ± 7.2 mg/L, respectively. Optimization of the culture conditions further increased the titers of chromomycins A₃ and A₂ respectively to 145.1 ± 15.3 and 158.3 ± 15.4 mg/L in liquid fermentation.

Conclusions: This work revealed the synergistic effect of manipulation of pathway repressor and activator genes in the engineering of a natural product biosynthetic pathway. The resulting engineered strain showed the highest production titers of chromomycins by a strain of Streptomyces, providing an efficient way to produce these pharmaceutically valuable molecules.

Keywords: Biosynthesis; Chromomycins; PadR-like transcription regulator; SARP regulator; Streptomyces reseiscleroticus.

Fig. 1

Structures of chromomycin A₃ (1) and A₂ (2)

Fig. 2

Biosynthetic gene cluster and proposed biosynthetic pathway of chromomycins. a Genetic organization of the srcm gene cluster. b Proposed biosynthetic pathway of chromomycins A₃ (1) and A₂ (2)

Fig. 3

Heterologous expression of SrcmPKS in S. lividans K4–114. a HPLC analysis of the extracts of S. lividans K4–114 harboring pSUN6 (i), pTZ3 (ii) and empty pRM5 vector (iii) respectively. Compounds were detected at 420 nm. b Formation of SEK15b (3) through spontaneous cyclization of the nascent decaketide chain

Fig. 4

Engineered production of chromomycins in S. roseiscleroticus. a HPLC analysis (420 nm) of chromomycins production by wild type S. roseiscleroticus (i), S. roseiscleroticus SR2 (ii, srcmRII disruption), S. roseiscleroticus SR1 (iii, srcmRI overexpression), S. roseiscleroticus SR3 (iv, srcmRI overexpression and srcmRII disruption) in R5 agar. b The disruption strategy of scrmRII in S. roseiscleroticus using a double crossover homologous recombination approach. c PCR conformation of the double crossover mutant (S. roseiscleroticus/ΔscrmRII). M: 1 kb plus DNA ladder; 1: PCR product from the mutant; 2: PCR product from the wild type

Fig. 5

Optimization of chromomycins production. a Production of 1 and 2 in two different solid media. b Time course analysis of chromomycins production of in liquid YEME medium. Error bars represent the standard deviation from three replicates