Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one in Mycolicibacterium smegmatis.

The 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) is a C22 steroid synthon of pharmaceutical interest that can be produced as a lateral end-product of the catabolism of natural sterols (e.g., cholesterol or phytosterols). This work studies the role of an aldehyde dehydrogenase coded by the MSMEG_6563 gene of Mycolicibacterium smegmatis, named msRed, in 4-HBC production. This gene is located contiguously to the MSMEG_6561 encoding the aldolase msSal which catalyses the retroaldol elimination of acetyl-CoA of the metabolite intermediate 22-hydroxy-3-oxo-cholest-4-ene-24-carboxyl-CoA to deliver 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA). We have demonstrated that msRed reduces 3-OPA to 4-HBC. Moreover, the role of msOpccR reductase encoded by MSMEG_1623 was also explored confirming that it also performs the reduction of 3-OPA into 4-HBC, but less efficiently than msRed. To obtain a M. smegmatis 4-HBC producer strain we deleted MSMEG_5903 (hsd4A) gene in strain MS6039-5941 (ΔkshB1, ΔkstD1) that produces 4-androstene-3,17-dione (AD) from natural sterols (cholesterol or phytosterols). The triple MS6039-5941-5903 mutant was able to produce 9 g/L of 4-HBC from 14 g/L of phytosterols in 2 L bioreactor, showing a productivity of 0.140 g/L h-1. To improve the metabolic flux of sterols towards the production of 4-HBC we have cloned and overexpressed the msSal and msRed enzymes in the MS6039-5941-5903 mutant rendering a production titter of 12.7 g/L with a productivity of 0.185 g/L h-1, and demonstrating that the new recombinant strain has a great potential for its industrial application.

Surgery for Crohn's Disease Is Associated With a Dysbiotic Microbiome and Metabolome: Results From Two Prospective Cohorts.

BACKGROUND & AIMS: Crohn's disease is associated with alterations in the gut microbiome and metabolome described as dysbiosis. We characterized the microbial and metabolic consequences of ileal resection, the most common Crohn's disease surgery. METHODS: Patients with and without intestinal resection were identified from the Diet to Induce Remission in Crohn's Disease and Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease studies. Stool samples were analyzed with shotgun metagenomics sequencing. Fecal butyrate was measured with 1H nuclear magnetic resonance spectroscopy. Fecal bile acids and plasma 7α-hydroxy-4-cholesten-3-one (C4) was measured with mass spectrometry. RESULTS: Intestinal resection was associated with reduced alpha diversity and altered beta diversity with increased Proteobacteria and reduced Bacteroidetes and Firmicutes. Surgery was associated with higher representation of genes in the KEGG pathway for ABC transporters and reduction in genes related to bacterial metabolism. Surgery was associated with reduced concentration of the But gene but this did not translate to reduced fecal butyrate concentration. Surgery was associated with decreased abundance of bai operon genes, with increased plasma C4 concentration, increased primary bile acids and reduced secondary bile acids, including isoLCA. Additionally, Egerthella lenta, Adlercreutzia equalofaciens, and Gordonibacter pamelaeae were lower in abundance among patients with prior surgery in both cohorts. CONCLUSIONS: In 2 different populations, prior surgery in Crohn's disease is associated with altered fecal microbiome. Patients who had undergone ileal resection had reduction in the potentially beneficial bacteria E lenta and related actinobacteria and secondary bile acids, including isoLCA, suggesting that these could be biomarkers of patients at higher risk for disease progression.