Utilization of in vitro porphyrin biosynthesis

Shelby Olivia Pernell

doi:10.17918/00011131

The human gastrointestinal tract is home to a diverse population of microorganisms that interact with each other and their surrounding environment to make up what is known as the gut microbiome. This intestinal ecosystem plays a role in assisting with vital functions such as the digestion of macromolecules, training the immune system and stimulating the production of hormones. Gut bacteria rely on complex cross-talking networks and the exchange of essential molecules to mediate community dynamics and regulate vital biochemical processes that influence the stabilization of important human physiological processes. Amongst these molecules are cobamides, which are essential organometallic cofactors involved in various prokaryotic metabolic functions including methionine biosynthesis, amino acid synthesis, and carbon metabolism. Vitamin B12, also known as cobalamin, is the most known cobamide involved in methionine metabolism. Gut bacteria can synthesize cobamides through the expression of cobamide-dependent enzymes involved in cross-feeding interactions and lower ligand remodeling. Because only select bacteria can produce specific cobamides, gut microbiome communities rely on each other for cobamide production and have designated roles in the biosynthetic pathway. Gut bacteria can be categorized as de novo producers, salvagers, remodelers, and utilizers. Early salvagers will uptake incomplete cobamide precursors that share a porphyrin ring to form a complete cobamide. To analyze and further characterize "early salvaging" in gut microbial communities, we initiated a project in which we supplement bacteria in vitro with intermediates and observe cobamide(s) production via liquid chromatography mass spectrometry. However, none of the porphyrin intermediates are commercially available. Porphyrin intermediates are shared metabolites between heme, chlorophyll and cobamide biosynthesis. The early stages of the porphyrin biosynthetic pathway involve various enzymes that catalyzes the biosynthesis of 5-aminolevulinic acid (ALA), porphobilinogen (PBG), hydroxymethyl bilane (HMB), uroporphyrinogen III (uroIII), precorrin-2, and sirohydrochlorin. We heterologously expressed HemB, HemC, HemD, CysGa, and characterized their activities in vitro. We successfully produced early intermediates in one-pot reactions and for the first time, showed detailed mass spectrometry on products and various side-products of the porphyrin biosynthetic enzymes. Next, we used these intermediates to showcase select "early salvaging" gut bacteria to further elucidate how the exchange of cobamide metabolites plays a role in the stabilization of gut microbe community dynamics.

Utilization of in vitro porphyrin biosynthesis

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Utilization of in vitro porphyrin biosynthesis

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