Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.

Metabolic engineering of Escherichia coli for high-level production of free lipoic acid.

L-Lipoic acid (LA) is an important antioxidant with various industrial applications as a nutraceutical and therapeutic. Currently, LA is produced by chemical synthesis. Cell factory development is complex as LA and its direct precursors only occur naturally in protein-bound forms. Here we report a rationally engineered LA cell factory and demonstrate de novo free LA production from glucose for the first time in E. coli. The pathway represents a significant challenge as the three key enzymes, native Octanoyltransferase (LipB) and Lipoyl Synthase (LipA), and heterologous Lipoamidase (LpA), are all toxic to overexpress in E. coli. To overcome the toxicity of LipB, functional metagenomic selection was used to identify a highly active and non-toxic LipB and LipA from S. liquefaciens. Using high throughput screening, we balanced translation initiation rates and dual, orthogonal induction systems for the toxic genes, LipA and LpA. The optimized strain yielded 2.5 mg free LA per gram of glucose in minimal media, expressing carefully balanced LipB and LipA, Enterococcus faecalis LpA, and a truncated, native, Dihydrolipoyllysine-residue acetyltransferase (AceF) lipoylation domain. When the optimized cell factory strain was cultivated in a fed-batch fermentation, a titer of 87 mg/L free LA in the supernatant was reached after 48 h. This titer is ∼3000-fold higher than previously reported free LA titer and ∼8-fold higher than the previous best total, protein-bound LA titer. The strategies presented here could be helpful in designing, constructing and balancing biosynthetic pathways that harbor toxic enzymes with protein-bound intermediates or products.

Improved biotin, thiamine, and lipoic acid biosynthesis by engineering the global regulator IscR.

Biotin, thiamine, and lipoic acid are industrially important molecules naturally synthesized by microorganisms via biosynthetic pathways requiring iron-sulfur (FeS) clusters. Current production is exclusively by chemistry because pathway complexity hinders development of fermentation processes. For biotin, the main bottleneck is biotin synthase, BioB, a S-adenosyl methionine-dependent radical enzyme that converts dethiobiotin (DTB) to biotin. BioB overexpression is toxic, though the mechanism remains unclear. We identified single mutations in the global regulator IscR that substantially improve cellular tolerance to BioB overexpression, increasing Escherichia coli DTB-to-biotin biocatalysis by more than 2.2-fold. Based on proteomics and targeted overexpression of FeS-cluster biosynthesis genes, FeS-cluster depletion is the main reason for toxicity. We demonstrate that IscR mutations significantly affect cell viability and improve cell factories for de novo biosynthesis of thiamine by 1.3-fold and lipoic acid by 1.8-fold. We illuminate a novel engineering target for enhancing biosynthesis of complex FeS-cluster-dependent molecules, paving the way for industrial fermentation processes.

Reconstitution of Th17, Tc17 and Treg cells after paediatric haematopoietic stem cell transplantation: Impact of interleukin-7.

Successful reconstitution of T lymphocytes after allogeneic haematopoietic stem cell transplantation (HSCT) is needed to establish the graft-versus-leukaemia effect and an effective anti-microbial defense, but the ratio between functionally different T-cell subsets needs to be balanced to avoid graft-versus-host disease (GVHD). IL-7 is essential for T-cell generation in the thymus and peripheral T-cell homeostasis. High IL-7 levels have been associated with impaired T-cell reconstitution, increased risk of acute GVHD and treatment-related mortality, but the underlying cellular mechanisms behind these associations have not been investigated previously. We hypothesized that increased levels of IL-7 post-transplant alters the balance between immune-regulatory T cell subsets during the post-transplant lymphocyte recovery towards a more pro-inflammatory profile. We quantified Th17 cells, Tc17 cells and Tregs in 29 children following HSCT. Th17 cell and Treg counts rose significantly from day +90 to +180 post-HSCT, and prior acute GVHD was associated with significant changes in the concentration of Tregs (9.4×106/L vs. 1.3×106/L, P=0.0052) and the Th17/Treg ratio (1.5 vs. 4.2, P=0.025). The plasma level of IL-7 at day +90 correlated inversely with Th17 cell counts (rs=-0.65, P=0.0002) and the proportion of Tc17 cells (rs=0.64, P=0.0005) at day +90, but not with Tregs. Furthermore, high IL-7 levels at day +7 were predictive of a less naïve T-cell phenotype at day +90. These findings add further evidence that IL-7 is a key regulatory factor that may tune the balance between functionally different T-cell subsets following HSCT.