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Abstract BackgroundSilk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. ResultsIn this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in theBacillusgenus. Using an industrially relevantB. megateriumhost, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. ConclusionIt is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for anE. colihost producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in theBacillushost system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.
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Bacterial hemophilin homologs and their specific type eleven secretor proteins have conserved roles in heme capture and are diversifying as a family
ABSTRACT Cellular life relies on enzymes that require metals, which must be acquired from extracellular sources. Bacteria utilize surface and secreted proteins to acquire such valuable nutrients from their environment. These include the cargo proteins of the type eleven secretion system (T11SS), which have been connected to host specificity, metal homeostasis, and nutritional immunity evasion. This Sec-dependent, Gram-negative secretion system is encoded by organisms throughout the phylum Proteobacteria, including human pathogensNeisseria meningitidis, Proteus mirabilis, Acinetobacter baumannii,andHaemophilus influenzae. Experimentally verified T11SS-dependent cargo includetransferrin-bindingprotein B (TbpB), the hemophilin homologshemereceptorprotein C (HrpC),hemophilinA(HphA), the immune evasion proteinfactor-H bindingprotein (fHbp), and the host symbiosis factornematodeintestinallocalization protein C (NilC). Here, we examined the specificity of T11SS systems for their cognate cargo proteins using taxonomically distributed homolog pairs of T11SS and hemophilin cargo and explored the ligand binding ability of those hemophilin cargo homologs.In vivoexpression inEscherichia coliof hemophilin homologs revealed that each is secreted in a specific manner by its cognate T11SS protein. Sequence analysis and structural modeling suggest that all hemophilin homologs share an N-terminal ligand-binding domain with the same topology as the ligand-binding domains of theHaemophilus haemolyticusheme binding protein (Hpl) and HphA. We term this signature feature of this group of proteins the hemophilin ligand-binding domain. Network analysis of hemophilin homologs revealed five subclusters and representatives from four of these showed variable heme-binding activities, which, combined with sequence-structure variation, suggests that hemophilins are diversifying in function.IMPORTANCEThe secreted protein hemophilin and its homologs contribute to the survival of several bacterial symbionts within their respective host environments. Here, we compared taxonomically diverse hemophilin homologs and their paired Type 11 secretion systems (T11SS) to determine if heme binding and T11SS secretion are conserved characteristics of this family. We establish the existence of divergent hemophilin sub-families and describe structural features that contribute to distinct ligand-binding behaviors. Furthermore, we demonstrate that T11SS are specific for their cognate hemophilin family cargo proteins. Our work establishes that hemophilin homolog-T11SS pairs are diverging from each other, potentially evolving into novel ligand acquisition systems that provide competitive benefits in host niches.
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- PAR ID:
- 10574640
- Editor(s):
- Champion, Patricia A
- Publisher / Repository:
- Journal of Bacteriology
- Date Published:
- Journal Name:
- Journal of Bacteriology
- Volume:
- 206
- Issue:
- 6
- ISSN:
- 0021-9193
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/jb.00444-23
