Yield improvement of enediyne yangpumicins in Micromonospora yangpuensis through ribosome engineering and fermentation optimization

Yangpumicins (YPMs), eg. YPM A, F, and G, are newly discovered enediynes from Micromonospora yangpuensis DSM 45577, which could be exploited as promising payloads of antibody-drug conjugates. However, the low yield of YPMs in the wild-type strain (~1 mg/L) significantly hampers their further drug development. In this study, a combined ribosome engineering and fermentation optimization strategy has been used for yield improvement of YPMs. One gentamycin-resistant M. yangpuensis DSM 45577 strain (MY-G-1) showed higher YPMs production (7.4 ± 1.0 mg/L), while it exhibits delayed sporulation and slender mycelium under scanning electron microscopy. Whole genome re-sequencing of MY-G-1 reveals several deletion and single nucleotide polymorphism mutations, which were confirmed by PCR and DNA sequencing. Further Box–Behnken experiment and regression analysis determined that the optimal medium concentrations of soluble starch, mannitol, and pharmamedia for YPMs production in shaking flasks (10.0 ± 0.8 mg/L). Finally, the total titer of YPM A/F/G in MY-G-1 reached to 15.0 ± 2.5 mg/L in 3-L fermenters, which was about 11-fold higher than the original titer of 1.3 ± 0.3 mg/L in wild-type strain. Our study may be instrumental to develop YPMs into a clinical anticancer drug, and inspire the use of these multifaceted strategies for yield improvement in Micromonospora species.

Orthogonal translation enables heterologous ribosome engineering in E. coli

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.

Ribosome-Engineered Lacticaseibacillus rhamnosus Strain GG Exhibits Cell Surface Glyceraldehyde-3-Phosphate Dehydrogenase Accumulation and Enhanced Adhesion to Human Colonic Mucin

ABSTRACT Differences in individual host responses have emerged as an issue regarding the health benefits of probiotics. Here, we applied ribosome engineering (RE) technology, developed in an actinomycete study, to Lacticaseibacillus rhamnosus GG (LGG). RE can effectively enhance microbial potential by using antibiotics to induce spontaneous mutations in the ribosome and/or RNA polymerase. In this study, we identified eight types of streptomycin resistance mutations in the LGG rpsL gene, which encodes ribosomal protein S12. Notably, LGG harboring the K56N mutant (LGG-MTK56N) expressed high levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the cell surface compared with the LGG wild type (LGG-WT). GAPDH plays a key role in colonic mucin adhesion. Indeed, LGG-MTK56N significantly increased type A human colonic mucin adhesion compared to LGG-WT in experiments using the Biacore system. The ability to adhere to the colon is an important property of probiotics; thus, these results suggest that RE is an effective breeding strategy for probiotic lactic acid bacteria. IMPORTANCE We sought to apply ribosome engineering (RE) to probiotic lactic acid bacteria and to verify RE’s impact. Here, we showed that one mutant of RE Lacticaseibacillus rhamnosus GG (LGG-MTK56N) bore a GAPDH on the cell surface; the GAPDH was exported via an ABC transporter. Compared to the wild-type parent, LGG-MTK56N adhered more strongly to human colonic mucin and exhibited a distinct cell size and shape. These findings demonstrate that RE in LGG-MTK56N yielded dramatic changes in protein synthesis, protein transport, and cell morphology and affected adherence to human colonic mucin.

Increasing production of the antibiotic-abyssomicin C in Micromonospora maris

Abyssomicin C is a polyketide antibiotic produced by Micromonospora maris AB-18-032. Previous work on abyssomicin C indicated that it inhibits growth of infectious pathogens such as Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). It does this by suppressing para-aminobenzoic acid (pABA) synthesis, which is required for folic acid biosynthesis in bacteria. This makes abyssomicin C an appealing antibiotic drug, as it is specific only to bacteria. However, its yield in chemical synthesis (4 %) and biosynthesis (60.0 mg/L) are low. Ribosome engineering, through the selection of streptomycin-resistant and rifampin-resistant mutants, of M. maris may result in strains with a higher titer of production. After screening by bioassay and sequencing, the mutant genes in six Ochi mutants were identified. Of these mutants, four of them are able to produce a higher titer of abyssomicin C. The other two strains were detected to produce the other secondary metabolite.