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To improve the screening efficiency of high-yield neomycin sulfate (NM) Streptomyces fradiae strains after mutagenesis, a high-throughput screening method using streptomycin resistance prescreening (8 μg/mL) and a 24-deep well plates/microplate reader (trypan blue spectrophotometry) rescreening strategy was developed. Using this approach, we identified a high-producing NM mutant strain, Sf6-2, via six rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and screening. The mutant displayed a NM potency of 7780 ± 110 U/mL and remarkably stable genetic properties over six generations. Furthermore, the key components (soluble starch, peptone, and (NH4)2SO4) affecting NM potency in fermentation medium were selected using Plackett-Burman and optimized by Box-Behnken designs. Finally, the NM potency of Sf6-2 was increased to 10,849 ± 141 U/mL at the optimal concentration of each factor (73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively), and it exhibited about a 40% and 100% enhancement when compared with before optimization conditions and the wild-type strain, respectively. In this study, we provide a new S. fradiae NM production strategy and generate valuable insights for the breeding and screening of other microorganisms.

Methylobacterium extorquens AM1, which can be used as a methylotrophic cell factory (MeCF) for the production of fine chemicals from methanol, is the most extensively studied model methylotrophic strain. However, its low tolerance for methanol limits the development of bioprocesses and there have been no reports of improved methanol tolerance of M. extorquens AM1. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis, in combination with adaptive laboratory evolution (ALE), is used to generate a mutant with high methanol tolerance (referred to as CLY-2533). The final cell density of CLY-2533 is 7.10 times higher than that of the wild-type strain in medium containing 5% (v/v) methanol. Through comparative genomics analysis and overexpression of the exploited putative genes, seven mutated genes are identified as being closely related to the higher methanol tolerance of CLY-2533. Additionally, the mvt operon, which contains genes related to the biosynthesis of mevalonate acid (MEV), is introduced into CLY-2533. This recombinant strain shows significant improvements in both MEV production and cell growth in 5% methanol medium. These findings will be helpful in rational design of methanol-utilizing strain for an improved host platform for methanol based biomanufacturing.

As a natural biological macromolecule, γ-polyglutamic acid (γ-PGA) plays a significant role in medicine, food, and cosmetic industries owing to its unique properties of biocompatibility, biodegradability, water solubility, and viscosity. Although many strategies have been adopted to increase the yield of γ-PGA in Bacillus subtilis, the effectiveness of these common approaches is not high because the strong viscosity affects cell growth. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. A modular PhrQ-RapQ-DegU QS system is developed based on promoter PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system in B. subtilis 168. We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU QS system to dynamically control the synthesis of γ-PGA in BS168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of γ-PGA to 6.53-fold of that by static regulation in a 3 L bioreactor, which verified the effectiveness of this strategy. In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which can be stretched to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

The mangrove ecosystem is a rich resource for the discovery of actinomycetes with potential applications in pharmaceutical science. Besides the genus Streptomyces, Micromonospora is also a source of new bioactive agents. We screened Micromonospora from the rhizosphere soil of mangrove plants in Fujian province, China, and 51 strains were obtained. Among them, the extracts of 12 isolates inhibited the growth of human lung carcinoma A549 cells. Strain 110B exhibited better cytotoxic activity, and its bioactive constituents were investigated. Consequently, three new isoflavonoid glycosides, daidzein-4′-(2-deoxy-α-l-fucopyranoside) (1), daidzein-7-(2-deoxy-α-l-fucopyranoside) (2), and daidzein-4′,7-di-(2-deoxy-α-l-fucopyranoside) (3) were isolated from the fermentation broth of strain 110B. The structures of the new compounds were determined by spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESIMS). The result of medium-changing experiments implicated that these new compounds were microbial biotransformation products of strain M. aurantiaca 110B. The three compounds displayed moderate cytotoxic activity to the human lung carcinoma cell line A549, hepatocellular liver carcinoma cell line HepG2, and the human colon tumor cell line HCT116, whereas none of them showed antifungal or antibacterial activities.