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

Abstract

Erythromycin is a clinically important drug produced by the rare actinomycete Saccharopolyspora erythraea. In the wide-type erythromycin producer S. erythraea NRRL 23338, there is a lack of systematical method for promoter engineering as well as a well-characterized promoter panel for comprehensive metabolic engineering. Here we demonstrated a systematical promoter acquiring process including promoter characterization, engineering and high-throughput screening by the droplet-microfluidic based platform in S. erythraea NRRL 23338, and rapidly obtained a panel of promoters with 21.5-fold strength variation for expression fine-tuning in the native host. By comparative qRT-PCR of S. erythraea NRRL 23338 and a high-producing strain S0, potential limiting enzymes were identified and overexpressed individually using two screened synthetic promoters. As a result, erythromycin production in the native host was improved by as high as 137.24 folds by combinational gene overexpression. This work enriches the accessible regulatory elements in the important erythromycin-producing strain S. erythraea NRRL 23338, and also provides a rapid and systematic research paradigm of promoter engineering and expression fine-tuning in the similar filamentous actinomycete hosts.

CRISPR/Cas9-Mediated Multi-Locus Promoter Engineering in ery Cluster to Improve Erythromycin Production in Saccharopolyspora erythraea

“…To enhance transcription of these six genes, three heterologous promoters of different strengths were selected as candidates for further in situ promoter substitution in four loci, among which p kasO is a well-known strong promoter used in many Streptomyces strains [ 17 , 18 , 19 ], and the other two promoters, p ermE*_s23 and p 2101_s32 , derived from p ermE* and p SACE_2101 , respectively, were synthetic promoters characterized in our previous study [ 4 ] ( Table S3 ). The strengths of these three heterologous promoters were also evaluated using the eGFP reporter system, and around 1.3-, 4.3-, and 13.3-fold higher GFP fluorescence was observed from the p 2101_s32 -, p ermE*_s23 -, and p kasO -harboring strains, respectively, than the average level of the strains carrying native ones ( Figure 1 B), demonstrating the varied and stronger activities of the heterologous promoters.…”

“…In our previous work, we have identified that six eryB and eryC genes were low-expressing in the wild-type strain compared to the high producer [ 4 ], and their key limiting roles were verified by the results of gene overexpression and promoter engineering. It is reported that, in erythromycin biosynthesis, the formation rates of the intermediates MEB and ErD are lower than that of EB, which suggests that the whole pathway is limited by the synthesis and addition of two monosaccharides catalyzed by EryB and EryC enzymes [ 1 ].…”

“…Secondary biosynthesis may be restricted in many aspects such as poor precursor supply, low expression of biosynthetic genes, and complex inherent regulation [ 3 ]. In the previous work, by the comparative transcriptional analysis of the high- and low-producing strains, we have demonstrated that the insufficient expression of ery genes is the main cause leading to the low production of the native producer and six genes representing extremely low transcription levels, SACE_0716 , SACE_0717 , SACE_0718 , SACE_0719 , SACE_0720 , and SACE_0731 , encoding EryCIV, EryBVI, EryCVI, EryBV, EryBIV, and EryBIII, respectively, were characterized to play important roles in limiting erythromycin biosynthesis in S. erythraea NRRL 23338 [ 4 ].…”

Modulating Sensitivity of an Erythromycin Biosensor for Precise High-Throughput Screening of Strains with Different Characteristics

“…S. erythraea strain fermentation was performed in 24-well plates at 32 °C and 250 rpm, and the components of seed and fermentation media were as described in the previous study. 19 Colonies were inoculated in 3 mL of seed medium for 3 days of cultivation, and then 300 μL of seed culture was transferred into 3 mL of fermentation medium for 7 days of cultivation. The fermentation broth was mixed with an equal volume of ethyl acetate for erythromycin extraction.…”

“…To demonstrate the application potential of the biosensors with varied sensitivities, we set to apply them in the droplet microfluidic-based high-throughput screening of erythromycin high-producing mutants from libraries with different initial productivities. Two random libraries starting from the wild-type strain S. erythraea NRRL 23338 and the industrial strain S0 (abbreviated as SE_lib and S0_lib, respectively), whose erythromycin production significantly differed in our previous study, 19 were generated by atmospheric and room temperature plasma (ARTP) mutagenesis, and ∼50 000 variants of each library were obtained. Two biosensors, Top10/pSense_BBaJ61132 and Top10/ pSense_apFAB916, which represented over a 10-fold difference in their sensitivities, were co-cultivated with SE_lib and S0_lib, respectively, in droplets for 3 days before the high-throughput screening by FADS (Figure 5A).…”