A Self-Sustained System Spanning the Primary and Secondary Metabolism Stages to Boost the Productivity of Streptomyces.

Streptomyces species possess strong secondary metabolism, the switches of which from the primary metabolism are complex and thus a challenge to holistically optimize their productivities. To avoid the complex switches and to reduce the limitations of different metabolic stages on the synthesis of metabolites, we designed a Streptomyces self-sustained system (StSS) that contains two functional modules, the primary metabolism module (PM) and the secondary metabolism module (SM). The PM includes endogenous housekeeping sigma factor σhrdB and σhrdB-dependent promoters, which are used to express target genes in the primary metabolism phase. SM consists of the expression cassette of σhrdB under the control of a secondary metabolism promoter, which maintains continuous activity of the σhrdB-dependent promoters in the secondary metabolism phase. As a proof-of-principle, the StSS was used to boost the production of some non-toxic metabolites, including indigoidine, undecylprodigiosin (UDP), ergothioneine, and avermectin, in Streptomyces. All these metabolites can undergo a continuous production process spanning the primary and secondary metabolism stages instead of being limited to a specific stage. Scale-up of UDP fermentation in a 4 L fermentor indicated that the StSS is a stable and robust system, the titer of which was enhanced to 1.1 g/L, the highest at present. This study demonstrated that the StSS is a simple but powerful strategy to rationally engineer Streptomyces cell factories for the efficient production of non-toxic metabolites via reconstructing the relationships between primary and secondary metabolism.

In-situ reactive heat breaking cell wall by SO3 hydration: innovative cell-wall breaking technique to enhance extraction of cinnamaldehyde from cinnamon.

Cinnamaldehyde (CA) is one of the major active pharmaceutical ingredient of cinnamon bark. Hydrodistillation (HD) is usually used in CA extraction, however, the extraction yield is lower. The cell wall is a key factor limiting the extraction of essential oils. In-situ reactive heat breaking cell wall (RHB) could destroy the cell wall, which was conducive to the diffusion of CA. The aim of this work was to examine the effect of RHB pretreatment to HD extraction. Response surface methodology (RSM) was used to optimize RHB pretreatment parameters, and Box-Behnken Design (BBD) method was performed to evaluate the effects of different operating parameters. The maximum yield was increased to 3.31 ± 0.11% (w/w) from 2.08 ± 0.042% (w/w) after RSM optimization. Scanning electron microscopic (SEM) analysis showed that RHB destroyed and disrupted the cell wall of cinnamon bark. The GC analysis demonstrated that the purity of cinnamaldehyde was improved and no new components were presented in the extraction product from the cinnamon via RHB pretreatment. In conclusion, RHB is an effective pretreatment method for the CA extraction, and also may be used in the other herbal medicine extraction.

Engineering diverse eubacteria promoters for robust Gene expression in Streptomyces lividans.

Due to the lack of powerful gene regulation elements, the engineering development of Streptomyces is often limited. Here, we disclosed that the heterologous σ70 -dependent promoters, which have been reported as inefficient tools for gene expression in Streptomyces, could be efficiently recognized by Streptomyces housekeeping factor σhrdB. Therefore, an effective strategy was developed to engineer these promoters for robust gene expression in Streptomyces by fusing them with optimized 5'-untranslation regions (5'-UTRs). As a proof of concept, the widely used Ptac in E. coli was engineered by fusing its core promoter region with the 5'-UTRR15 from a relatively powerful Streptomyces promoter PkasO*R15 and resulted in Ptac*, the activity of which was 8.1-fold that of Ptac and 1.7-fold that of PkasO*R15 in S. lividans TK24. Next, the 5'-UTRR15 was optimized by randomizing the ribosome binding site (RBS). Based on the base biases of those RBSs with higher activity, eight artificial RBSs were rationally designed, and the optimal resulting promoter Ptac*RBS3 showed about 2.1, 3.6, and 17.6 times the activity of Ptac*, PkasO*R15, and Ptac, respectively, demonstrating that the heterologous Ptac was converted into a type of robust Streptomyces promoters. This study thus greatly expands promoter diversity for the engineering of Streptomyces.