Rerouting phytosterol degradation pathway for directed androst-1,4-diene-3,17-dione microbial bioconversion.

Steroid-based drugs are now mainly produced by the microbial transformation of phytosterol, and a two-step bioprocess is adopted to reach high space-time yields, but byproducts are frequently observed during the bioprocessing. In this study, the catabolic switch between the C19- and C22-steroidal subpathways was investigated in resting cells of Mycobacterium neoaurum NRRL B-3805, and a dose-dependent transcriptional response toward the induction of phytosterol with increased concentrations was found in the putative node enzymes including ChoM2, KstD1, OpccR, Sal, and Hsd4A. Aldolase Sal presented a dominant role in the C22 steroidal side-chain cleavage, and the byproduct was eliminated after sequential deletion of opccR and sal. Meanwhile, the molar yield of androst-1,4-diene-3,17-dione (ADD) was increased from 59.4 to 71.3%. With the regard of insufficient activity of rate-limiting enzymes may also cause byproduct accumulation, a chromosomal integration platform for target gene overexpression was established supported by a strong promoter L2 combined with site-specific recombination in the engineered cell. Rate-limiting steps of ADD bioconversion were further characterized and overcome. Overexpression of the kstD1 gene further strengthened the bioconversion from AD to ADD. After subsequential optimization of the bioconversion system, the directed biotransformation route was developed and allowed up to 82.0% molar yield with a space-time yield of 4.22 g·L-1·day-1. The catabolic diversion elements and the genetic overexpression tools as confirmed and developed in present study offer new ideas of M. neoaurum cell factory development for directed biotransformation for C19- and C22-steroidal drug intermediates from phytosterol. KEY POINTS: • Resting cells exhibited a catabolic switch between the C19- and C22-steroidal subpathways. • The C22-steroidal byproduct was eliminated after sequential deletion of opccR and sal. • Rate-limiting steps were overcome by promoter engineering and chromosomal integration.

[Role of transcription factor ETS-1 and B cell in the pathogenesis of systemic lupus erythematosus].

OBJECTIVE: To explore the effects of transcription factor ETS-1 mRNA and B lymphocyte-associated cytokines on the differentiation of B cells in systemic lupus erythematosus (SLE) patients and explore its pathogenic and clinical significance. METHODS: Thirty-one SLE patients (20 active and 11 inactive) and 15 healthy controls were enrolled. CD19+ B cells were isolated with magnetic beads. The levels of ETS-1 mRNA in B cells were determined by real-time polymerase chain reaction (PCR). Flow cytometry was used to detect the altered ratio of CD19-CD138 + plasma cells and CD19 + B cells. And enzyme-linked immunosorbent assay (ELISA) was performed to detect the serum levels of B cell differentiation-related cytokines interleukin-10 (IL-10) and APRIL. RESULTS: Compared to the healthy controls, SLE patients showed decreased mRNA expression level of ETS-1 in B cells (Z = -4.218, P < 0.01) . Moreover, the expression level of ETS-1 mRNA was significantly negatively correlated with the proportion of CD19-CD138+ plasma cells (r = -0.359, P < 0.05) and negatively correlated with the CD19-CD138 +B cells/CD19+ plasma cells ratio (r = -0.493, P < 0.01) . However, there was no correlation in normal controls. Significant negative correlation existed between the expression level of ETS-1 mRNA in B cells and the serum levels of IL-10 and APRIL in active SLE patients. But no correlation existed in inactive group. CONCLUSION: ETS-1 may participate in the pathogenesis of SLE through its effects on the differentiation of B cells and cooperation with IL-10 and APRIL.