Repurposing carrimycin as an antiviral agent against human coronaviruses, including the currently pandemic SARS-CoV-2

COVID-19 pandemic caused by SARS-CoV-2 infection severely threatens global health and economic development. No effective antiviral drug is currently available to treat COVID-19 and any other human coronavirus infections. We report herein that a macrolide antibiotic, carrimycin, potently inhibited the cytopathic effects (CPE) and reduced the levels of viral protein and RNA in multiple cell types infected by human coronavirus 229E, OC43, and SARS-CoV-2. Time-of-addition and pseudotype virus infection studies indicated that carrimycin inhibited one or multiple post-entry replication events of human coronavirus infection. In support of this notion, metabolic labelling studies showed that carrimycin significantly inhibited the synthesis of viral RNA. Our studies thus strongly suggest that carrimycin is an antiviral agent against a broad-spectrum of human coronaviruses and its therapeutic efficacy to COVID-19 is currently under clinical investigation.

Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques / 生物工程学报

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.

Advances in metabolic engineering of macrolide antibiotics / 生物工程学报

14- to 16-membered macrolide antibiotics (MA) are clinically important anti-infective drugs. With the rapid emergence of bacterial resistance, there is an urgent need to develop novel MA to counter drug-resistant bacteria. The targeted optimization of MA can be guided by analyzing the interaction between the MA and its ribosomal targets, and the desired MA derivatives can be obtained efficiently when combining with the rapidly developed metabolic engineering approaches. In the past 30 years, metabolic engineering approaches have shown great advantages in engineering the biosynthesis of MA to create new derivatives and to improve their production. These metabolic engineering approaches include modification of the structural domains of the polyketide synthase (PKS) and post-PKS modification enzymes as well as combinatorial biosynthesis. In addition, the R&D (including the evaluation of its antimicrobial activities and the optimization through metabolic engineering) of carrimycin, a new 16-membered macrolide drug, are described in details in this review.

Construction of a new isovalerylspiramycin I producing strain by CRISPR-Cas9 system / 生物工程学报

Isovalerylspiramycin (ISP)Ⅰ, as a major component of bitespiramycin (BT), exhibits similar antimicrobial activities with BT and has advantages in quality control and dosage forms. It has been under preclinical studies. The existing ISPⅠ producing strain, undergoing three genetic modifications, carries two resistant gene markers. Thus, it is hard for further genetic manipulation. It is a time-consuming and unsuccessful work to construct a new ISPⅠ strain without resistant gene marker by means of the classical homologous recombination in our preliminary experiments. Fortunately, construction of the markerless ISPⅠ strain, in which the bsm4 (responsible for acylation at 3 of spiramycin) gene was replaced by the Isovaleryltansferase gene (ist) under control of the constitutive promoter ermEp*, was efficiently achieved by using the CRISPR-Cas9 gene editing system. The mutant of bsm4 deletion can only produce SPⅠ. Isovaleryltransferase coded by ist catalyzes the isovalerylation of the SPⅠat C-4" hydroxyl group to produce ISPⅠ. As anticipated, ISPⅠ was the sole ISP component of the resultant strain (ΔEI) when detected by HPLC and mass spectrometry. The ΔEI mutant is suitable for further genetic engineering to obtain improved strains by reusing CRISPR-Cas9 system.

Expression of 4"-O-isovaleryltransferase gene from Streptomyces thermotolerans in Streptomyces lividans TK24 / 生物工程学报

4"-O-isovaleryltransferase gene (ist) was regulated by positive regulatory genes of midecamycin 4"-O-propionyltransferase gene (mpt) in Streptomyces lividans TK24. A BamH I ~8.0 kb fragment from Streptomyces mycarofaciens 1748 was proved that it contained mpt gene and linked with two positive regulatory genes, orf27 and orf28. Orf of mpt was replaced by orf of ist and linked with two regulatory genes or orf27 single, and individually cloned into the vectors pKC1139 or pWHM3 (high copy number), and then transformed into S. lividans TK24. The levels of mpt and ist expression were evaluated by the bio-tramsformation efficacy of spiramycin into 4"-O-acylspiramycins in these transformants. The results showed that 4"-O-isovalerylspiramycins could be detected only in the transformants containing the plasmids constructed with pWHM3. The efficacy of bio-transformation of the transformants containing two regulatory genes was higher than that of orf27 single. So, the positive regulatory genes system of mpt gene could enhance ist gene expression.

Analysis of geldanamycin analogues in trace amounts by LC-MS/MS / 生物工程学报

Ansamycins, such as rifamycin and ansamitocin, usually consist of a group of structural similar components. Geldanamycin, a benzenic ansamycin, has been found to consist of four structural similar components. We analyzed the geldanamycin (GDM) preparation from Streptomyces hygroscopicus 17997 by LC-ESI(+)-MS/MS, and discovered five novel and one known GDM analogues in trace amounts. Based on the ESI(+)-MS/MS spectra of these GDM analogues, and the present understanding of GDM biosynthesis, we proposed the possible chemical structures of these GDM analogues. Three novel GDM analogues, all having the same molecular formula of C29H42N2O10, were GDM biosynthetic derivatives with one of the three C-C double bonds between C2-C3, C4-C5 and C8-C9 in GDM changed to mono-hydroxylated C-C single bond. The other two novel GDM analogues, having the same molecular formula of C28H38N2O8, were 17(or 12, or 4)-desmethoxylgeldanamycin and 4,5-dihydro-10,11-dehydrate-17-desmethyl-17-hydroxylgeldanamycin, respectively. The known GDM analogue, having the molecular formula of C29H42N2O9, was 4, 5-dihydrogeldanamycin, an intermediate in GDM biosynthesis. The discovery of novel GDM analogues provided us new insights in understanding the biosynthetic details of GDM, and clues of obtaining GDM derivatives by gene-disruption and combinatorial biosynthesis.

Regulatory genes of geldanamycin biosynthesis / 生物工程学报

Two LAL family regulatory genes, gdmRI and gdmRII, were identified in the geldanamycin biosynthetic gene cluster of Streptomyces hygroscopicus 17997. Disruption of the two regulatory genes resulted in absolute elimination of geldanamycin biosynthesis. The complementation experiments using a single wild-type gene could restore geldanamycin production. These results indicated that both gdmRI and gdmRII were positive regulatory genes of the geldanamycin biosynthesis.

Roles of geldanamycin biosynthetic genes in Streptomyces hygroscopicus 17997 / 生物工程学报

Geldanamycin (Gdm), an inhibitor of heat shock protein 90 (Hsp90), shows antitumor and antivirus bioactivity. Most Geldanamycin biosynthetic genes have been cloned from the genome library of Streptomyces hygroscopicus 17997. In this report, polyketide synthase (pks) gene, mono-oxygenase (gdmM) gene and carbamoyltransferase gene (gdmN) were subjected to inactivation. Three gene disrupted mutants (deltapks, deltagdmM and deltagdmN) were obtained by double crossover. No Geldanamycin production was detected in three mutant strains cultured in fermentation broth. Gene complementation experiments excluded the possible polar effect of gene disruption on other genes. These results confirmed that pks, gdmM and gdmN genes were essential for Geldanamycin biosynthesis.