Rational engineering strategies for achieving high-yield, high-quality and high-stability of natural product production in actinomycetes

Droplet-Microfluidic-Based Promoter Engineering and Expression Fine-Tuning for Improved Erythromycin Production in Saccharopolyspora erythraea NRRL 23338

“…Comparing transcriptional levels of targeted genes in different strains are usually employed to determine key under-expressed genes for constructing overproducing strains for improved yields ( Bu et al, 2021 ). To identify the potential limiting factors that restricted erythromycin biosynthesis in the wild-type strain, we performed comparative qRT-PCR analysis of the low-producing strain S. erythraea NRRL 23338 and a high-producing industrial strain S0 in our laboratory stock, which represented over 400-fold variation in erythromycin titers in 24-well-plate cultivation ( Figure 4A ).…”

“…Overexpressing the rate-limiting genes is usually a direct and effective way to improve the secondary biosynthesis and metabolite production ( Bu et al, 2021 ). In order to enhance erythromycin biosynthesis, we intended to combine promoter engineering and gene overexpression strategy to enhance the expression of the potential eight key limiting enzymes in S. erythraea NRRL 23338.…”

“…However, native actinomycete hosts usually show low productivities of target products that are far from ideal for large-scale production ( Li et al, 2019 ). The fast-developing synthetic biology provides many solutions to address this problem, such as optimizing the precursor availability, balancing intracellular cofactors, manipulating pathway-related genes, and enhancing metabolite efflux ( Bu et al, 2021 ). To achieve these purposes, promoter engineering and gene overexpression are the most direct strategies to coordinate target gene transcription, and strong promoters are often used to increase expression of rate-limiting enzymes ( Zhu et al, 2020 ; Bu et al, 2021 ).…”

“…The fast-developing synthetic biology provides many solutions to address this problem, such as optimizing the precursor availability, balancing intracellular cofactors, manipulating pathway-related genes, and enhancing metabolite efflux ( Bu et al, 2021 ). To achieve these purposes, promoter engineering and gene overexpression are the most direct strategies to coordinate target gene transcription, and strong promoters are often used to increase expression of rate-limiting enzymes ( Zhu et al, 2020 ; Bu et al, 2021 ). Generally speaking, high promoter activities can contribute to improved expression level of target genes, but it will also induce accompanying metabolic burdens or even genetic instability in host cells ( Sleight et al, 2010 ; Pasini et al, 2016 ).…”

Transcriptome-guided identification of a four-component system, SbrH1-R, that modulates milbemycin biosynthesis by influencing gene cluster expression, precursor supply, and antibiotic efflux

“…In recent decades, the regulatory mechanisms of CSRs or pleiotropic regulators in the control of antibiotic production by influencing the expression levels of biosynthetic gene clusters (BGCs) have been extensively studied [ 1 , 4 ]. Hence, regulator-based engineering methods such as activator overexpression, repressor deletion, promoter replacement and cluster refactoring and amplification are also widely used to promote overproduction of known antibiotics and activation of cryptic antibiotic BGCs [ [5] , [6] , [7] , [8] ]. As is well known, for commercially valuable antibiotics, generation of a high-titer strain is crucial for their large-scale industrial production and wide use.…”