Underground metabolism facilitates the evolution of novel pathways for vitamin B6 biosynthesis

Abstract

The term vitamin B6 is a designation for the vitamers pyridoxal, pyridoxamine, pyridoxine and the respective phosphate esters pyridoxal-5'-phosphate (PLP), pyridoxamine-5'-phosphate and pyridoxine-5'-phosphate. Animals and humans are unable to synthesise vitamin B6. These organisms have to take up vitamin B6 with their diet. Therefore, vitamin B6 is of commercial interest as a food additive and for applications in the pharmaceutical industry. As yet, two naturally occurring routes for de novo synthesis of PLP are known. Both routes have been genetically engineered to obtain bacteria overproducing vitamin B6. Still, major genetic engineering efforts using the existing pathways are required for developing fermentation processes that could outcompete the chemical synthesis of vitamin B6. Recent suppressor screens using mutants of the Gram-negative and Gram-positive model bacteria Escherichia coli and Bacillus subtilis, respectively, carrying mutations in the native pathways or heterologous genes uncovered novel routes for PLP biosynthesis. These pathways consist of promiscuous enzymes and enzymes that are already involved in vitamin B6 biosynthesis. Thus, E. coli and B. subtilis contain multiple promiscuous enzymes causing a so-called underground metabolism allowing the bacteria to bypass disrupted vitamin B6 biosynthetic pathways. The suppressor screens also show the genomic plasticity of the bacteria to suppress a genetic lesion. We discuss the potential of the serendipitous pathways to serve as a starting point for the development of bacteria overproducing vitamin B6. KEY POINTS: • Known vitamin B6 routes have been genetically engineered. • Underground metabolism facilitates the emergence of novel vitamin B6 biosynthetic pathways. • These pathways may be suitable to engineer bacteria overproducing vitamin B6.

Keywords: Enzyme promiscuity; Enzyme specificity; Genetic suppression; Promiscuous enzyme; Pyridoxal 5′-phosphate.

Fig. 1

a The B6 vitamers pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and the respective phosphate esters pyridoxal-5′-phosphate (PLP), pyridoxine-5′-phosphate (PNP) and pyridoxamine-5′-phosphate (PMP). b The deoxyxylulose 5-phosphate (DXP)-dependent and DXP-independent vitamin B6 biosynthetic routes and the salvage pathway for the interconversion of the B6 vitamers. Epd, erythrose 4-phosphate dehydrogenase; PdxB, 4-phosphoerythronate dehydrogenase; SerC, 3-phosphoserine aminotransferase; PdxA, 4-phosphohydroxy-l-threonine dehydrogenase; PdxJ, PNP synthase; Dxs, 1-deoxyxylulose 5-phosphate synthase; PdxH, PNP oxidase; PdxS (PLP synthase subunit) and PdxT (glutaminase subunit) form the PdxST PLP synthase complex; PdxK, PL kinase present in B. subtilis and E. coli; PdxY, PL kinase present in E. coli. PdxK from B. subtilis has PN, PL and PM kinase activity; PdxP, PNP and PLP phosphatase from S. meliloti; YbhA, PLP phosphatase from E. coli; PdxI, PL reductase from E. coli

Fig. 2

Serendipitous pathways for vitamin B6 synthesis and enzymes that feed into the DXP-dependent pathway in E. coli. Epd, erythrose 4-phosphate dehydrogenase; PdxB, 4-phosphoerythronatedehydrogenase; SerC, 3-phosphoserine aminotransferase; PdxA, 4-phosphohydroxy-l-threonine dehydrogenase; PdxJ, pyridoxine 5′-phosphate synthase; Dxs, 1-deoxyxylulose 5-phosphate synthase; PdxH, pyridoxine 5′-phosphate oxidase; SerA, phosphoglycerate dehydrogenase; NudL, putative NUDIX hydrolase; LtaE, l-allo-threonine aldolase; ThrB (and DUF1537), homoserine kinase; DUF2257, l-threonine dioxygenase; AroB, 3-dehydroquinate synthase; HisB, imidazoleglycerolphosphate dehydratase and histidinol phosphatase; Php, unknown function; YjbQ, unknown function; ThiG, thiazole synthase. RsgA is a GTPase involved in ribosome maturation in E. coli (Campbell and Brown 2008). RsgA shares 38% overall sequence identify with the B. subtilis CpgA protein, which was shown to dephosphorylate 4-phosphoerythronate (Sachla and Helmann 2019). It is tempting to speculate that RsgA is also capable of dephosphorylating 4-phosphoerythronate

Fig. 3

Putative serindipitous pathways for vitamin B6 synthesis in B. subtilis. GapA, glyceraldehyde 3-phosphate dehydrogenase; CpgA, phosphatase; SerA, phosphoglycerate dehydrogenase; SerC, 3-phosphoserine aminotransferase; ThrB, homoserine kinase; PdxJ, pyridoxine 5′-phosphate synthase; Dxs, 1-deoxyxylulose 5-phosphate synthase; PdxH, pyridoxine 5′-phosphate oxidase; PdxS (PLP synthase subunit) and PdxT (glutaminase subunit) form the PdxST PLP synthase complex; YtoQ, a protein of unknown function