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. 2025 Apr;301(4):108390.
doi: 10.1016/j.jbc.2025.108390. Epub 2025 Mar 10.

New routes for spermine biosynthesis

Bin Li  1 , Hamid R Baniasadi  1 , Jue Liang  1 , Margaret A Phillips  1 , Anthony J Michael  2
Affiliations

New routes for spermine biosynthesis

Bin Li et al. J Biol Chem. 2025 Apr.

Abstract

The polyamine spermine (Spm) is a flexible linear teraamine found in bacteria and eukaryotes and in all known cases is synthesized from triamine spermidine by addition of an aminopropyl group acquired from decarboxylated S-adenosylmethionine (dcAdoMet). We have now identified in bacteria a second biosynthetic route for Spm based on the formation of carboxyspermine from spermidine, dependent on aspartate β-semialdehyde (ASA). This route also produces thermospermine (Tspm) from spermidine via carboxythermospermine. Two enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase, are responsible for ASA-dependent production of spermidine, Spm, and Tspm from diamine putrescine. Production of Spm/Tspm from spermidine is controlled primarily by carboxyspermidine dehydrogenase, not carboxyspermidine decarboxylase. This new ASA-dependent Spm biosynthetic pathway is an example of convergent evolution, employing nonanalogous, nonhomologous enzymes to produce the same biosynthetic products as the dcAdoMet-dependent Spm pathway. We have also identified bacteria that encode hybrid Spm biosynthetic pathways dependent on both dcAdoMet and ASA. In the hybrid pathways, spermidine is produced from agmatine primarily by the ASA-dependent route, and Spm is synthesized from agmatine or spermidine by dcAdoMet-dependent modules. Both parts of the hybrid pathway initiate from agmatine and each produces N1-aminopropylagmatine, so that agmatine, N1-aminopropylagmatine, and spermidine are common, potentially shared metabolites. Bacteria such as Clostridium leptum that encode the hybrid pathway may explain the origin of Spm produced by the gut microbiota. This is the first example of convergent evolution of hybrid dcAdoMet- and ASA-dependent N1-aminopropylagmatine, spermidine, and Spm biosynthesis encoded in the same genomes and suggests additional polyamine biosynthetic diversification remains to be discovered.

Keywords: N(1)-aminopropylagmatine; aspartate β-semialdehyde; bacteria; biosynthesis; carboxyspermidine; carboxyspermine; convergent evolution; polyamine; spermidine; spermine; thermospermine.

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Conflict of interest statement

Conflict of interest The authors declare no conflict of interests with the contents of this article.

Figures

Figure 1
Figure 1
Decarboxylated S-adenosylmethionine- and aspartate β-semialdehyde-dependent pathways for biosynthesis of spermidine and spermine/thermospermine in bacteria. CASDH, carboxyspermidine dehydrogenase; CASDC, carboxyspermidine decarboxylase.
Figure 2
Figure 2
Heterologous spermidine and spermine/thermospermine production by CASDH/CASDC homologs in E. coli BL21speD. Polyamines from cell extracts were benzoylated and analyzed by LC-MS. The extracted ion chromatograms (EICs) for tribenzoylated spermidine (mass tolerance window 457.94:458.94) and tetrabenzoylated spermine/thermospermine (619.02:620.02) are shown. Carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC) homologs from the indicated species were coexpressed from pETDuet-1 and pACYCDuet-1, respectively, in spermidine-void E.coli BL21speD (AdoMetDC gene deletion). Spermidine or spermine/thermospermine peaks are highlighted by red boxes. The y-axis represents arbitrary units of ion intensity, and all samples were grown, extracted, and analyzed together. AdoMetDC, S-adenosylmethionine decarboxylase.
Figure 3
Figure 3
A new pathway for spermine and thermospermine biosynthesis from putrescine via carboxyspermidine, carboxyspermine, and carboxythermospermine. CASDH, carboxyspermidine dehydrogenase; CASDC, carboxyspermidine decarboxylase.
Figure 4
Figure 4
Expression of the Deferribacter desulfuricans aminopropyltransferase in E. coli BL21speE and BL21speB. The D. desulfuricans aminopropyltransferase (APT) was expressed from pETDuet-1 in either spermidine-void BL21speE or BL21speB grown with 300 μM L-arginine. Polyamines from cell extracts were benzoylated and analyzed by LC-MS. Shown are the extracted ion chromatograms for tribenzoylated spermidine (457.94:458.94), tetrabenzoylated spermine/thermospermine (619.02:620.02), and tetrabenzoylated N1-aminopropylagmatine (605:606). The y-axis represents arbitrary units of ion intensity, and all samples were grown and analyzed in parallel.
Figure 5
Figure 5
Expression of diverse aminopropyltransferases in E. coli BL21speE. Aminopropyltransferases from genomes also encoding CASDH/CASDC homologs were expressed from pETDuet-1 in the spermidine-void E. coli BL21speE. Benzoylated polyamines from cell extracts were analyzed by LC-MS. The extracted ion chromatograms for tribenzoylated spermidine (457.94:458.94) and tetrabenzoylated spermine/thermospermine (619.02:620.02) are shown. The spermidine and spermine/thermospermine content of BL21speG is also shown for comparison. The y-axis represents arbitrary units of ion intensity, and all samples were grown and analyzed in parallel. CASDC, carboxyspermidine decarboxylase; CASDH, carboxyspermidine dehydrogenase.
Figure 6
Figure 6
Expression of diverse CASDH/CASDC pairs in E. coli BL21speB. CASDH-encoding open reading frames were expressed from pETDuet-1 and CASDC from pACYCDuet-1 in E. coli BL21speB grown with 300 μM L-arginine. Benzoylated polyamines from cell extracts were analyzed by LC-MS. Shown are the extracted ion chromatograms for tetrabenzoylated N1-aminopropylagmatine (605:606) and tetrabenzoylated spermine/thermospermine (619.02:620.02). The y-axis represents arbitrary units of ion intensity, and all samples were grown and analyzed in parallel. CASDC, carboxyspermidine decarboxylase; CASDH, carboxyspermidine dehydrogenase.
Figure 7
Figure 7
Expression of CASDH/CASDC pairs from Agrobacterium tumefaciens and Clostridium leptum in E. coli BL21speD and BL21speB . Carboxyspermidine dehydrogenase (CASDH)-encoding open reading frames were expressed from pETDuet-1 and carboxyspermidine decarboxylase (CASDC) from pACYCDuet-1 in spermidine-void BL21speD and in E. coli BL21speB grown with 300 μM L-arginine. Benzoylated polyamines from cell extracts were analyzed by LC-MS. Shown are the extracted ion chromatograms for tribenzoylated spermidine (457.94:458.94), tetrabenzoylated spermine/thermospermine (619.02:620.02), and tetrabenzoylated N1-aminopropylagmatine (605:606). The spermidine and spermine/thermospermine content of BL21speG is also shown for comparison. The y-axis represents arbitrary units of ion intensity, and all samples were grown and analyzed in parallel.
Figure 8
Figure 8
A hybrid pathway for spermine biosynthesis from agmatine via carboxyaminopropylagmatine, N1-aminopropylagmatine, and spermidine. Aspartate β-semialdehyde-dependent formation of N1-aminopropylagmatine via carboxyaminopropylagmatine converges on decarboxylated S-adenosylmethionine-dependent formation of N1-aminopropylagmatine for production of spermidine and spermine. CASDC, carboxyspermidine decarboxylase; CASDH, carboxyspermidine dehydrogenase.
Figure 9
Figure 9
Expression of diverse CASDH/CASDC and CASDH/CAPADC pairs in E. coli BL21speD. Carboxyspermidine dehydrogenase (CASDH)-encoding open reading frames were expressed from pETDuet-1 and carboxyspermidine decarboxylase (CASDC) or carboxyaminopropylagmatine decarboxylase (CAPADC) from pACYCDuet-1 in spermidine-void BL21speD. Benzoylated polyamines from cell extracts were analyzed by LC-MS. The extracted ion chromatograms for tribenzoylated spermidine (457.94:458.94) and tetrabenzoylated spermine/thermospermine (619.02:620.02) are shown. The y-axis represents arbitrary units of ion intensity, and all samples were grown and analyzed in parallel.
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