Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis

Abstract

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are widely used in the field of animal health, agriculture, and human health. Here we have adopted a practical approach to successfully improve avermectin production in an industrial overproducer. Transcriptional levels of the wild-type strain and industrial overproducer in production cultures were monitored using microarray analysis. The avermectin biosynthetic genes, especially the pathway-specific regulatory gene, aveR, were up-regulated in the high-producing strain. The upstream promoter region of aveR was predicted and proved to be directly recognized by sigma(hrdB) in vitro. A mutant library of hrdB gene was constructed by error-prone PCR and selected by high-throughput screening. As a result of evolved hrdB expressed in the modified avermectin high-producing strain, 6.38 g/L of avermectin B1a was produced with over 50% yield improvement, in which the transcription level of aveR was significantly increased. The relevant residues were identified to center in the conserved regions. Engineering of the hrdB gene can not only elicit the overexpression of aveR but also allows for simultaneous transcription of many other genes. The results indicate that manipulating the key genes revealed by reverse engineering can effectively improve the yield of the target metabolites, providing a route to optimize production in these complex regulatory systems.

Fig. 1.

Expression levels of aveAI, aveAIII, and aveR in S. avermitilis 3-115 relative to those in ATCC 31267. Samples were collected from 3-115 and ATCC 31267 after 36 h (vegetative growth) and 96 h (stationary phase and avermectin producing). We used 16 s rDNA as an RNA integrity control. Standard deviations are marked with error bars (n = 3). All the genes were examined by relative quantification real-time RT-PCR with gene-specific primers. ^∗∗P < 0.01 vs. control strain.

Fig. 2.

Recognition of the aveRp promoter by σ^hrdB-containing RNA polymerase. (A) Comparison of the putative aveRp promoter of S. avermitilis with rrnD-p2 and dagA-p4 of S. coelicolor. The conserved nucleotides within the -10 and -35 regions as well as transcription initiation sites are marked in bold. (B) In vitro transcription assay of the aveRp promoter by RNA polymerase containing σ^hrdB (Eσ^hrdB). The promoter of the rrnD-homologous gene in S. avermitilis was used as a positive control. In all lanes, the Eσ^hrdB holoenzyme was reconstituted by 0.25 pmol E. coli RNA polymerase core enzyme (EPICENTRE) with the indicated amounts of σ^hrdB. The ratio of sigma factor to the core RNA polymerase is indicated.

Fig. 3.

Screening of avermectin B1a overproduction mutants transformed with optimal mutant hrdB gene. S. avermitilis 3-115 was used as the control (dashed line -100%), and the error bars represent standard deviations. All samples were cultured in Erlenmeyer flasks and measured in triplicate. C1: strain containing a blank pSET152; C2: strain containing an additional copy of wild-type hrdB gene.

Fig. 4.

Comparative analysis of in vitro transcriptional assay of reconstituted holoenzyme with HrdB¹¹⁵ protein or its mutant HrdB^A56 protein on aveRp promoter. In all lanes, the holoenzyme was reconstituted by 0.25 pmol E. coli RNA polymerase core enzyme (EPICENTRE) with the indicated amounts of σ^hrdB. The ratio of sigma factor to the core RNA polymerase is indicated.

Fig. 5.

Mutations for the best clone A56 and A393 isolated from the hrdB mutant library are shown mapped onto a schematic of critical functional components of σ^hrdB. Conserved regions and their proposed functions are shown. The hrdB from parent strain 3-115 is also shown.

Fig. 6.

Time course of avermectin B1a production of parent strain 3-115 and mutant A56 in a 180-m³ fermentor. (One of the representative datasets is shown.)