N-(3-Aminopropyl)-1,4-butanediamine, also known as spermidine, is a naturally occurring polyamine compound ubiquitously present in living organisms and possesses diverse biologically active functions. Within biological systems, spermidine can trigger autophagy by upregulating the expression of various autophagy-related genes, thereby attenuating the rates of oxidative stress and necrosis, contributing to lifespan extension [1], [2]. Additionally, spermidine helps maintain cardiac health by enhancing autophagy in cardiomyocytes and improving mitochondrial function [3]. Recently, spermidine was also implicated in crucial physiological roles within the human body, including participation in cell signaling, regulation of cell proliferation and differentiation, maintenance of cellular morphology, anti-inflammatory effects, anticancer activity, and stabilization of blood pressure [4], [5], [6].

As illustrated in Fig. 1, in most eukaryotes, the spermidine biosynthesis pathway primarily relies on arginine as the key precursor. In this process, arginine is hydrolyzed by arginase 1, converting it to ornithine. Subsequently, ornithine is further decarboxylated by ornithine decarboxylase to yield putrescine [7] (Fig. 1A). Concurrently, S-adenosylmethionine is decarboxylated by S-adenosylmethionine decarboxylase (SAMDC) to generate decarboxylated S-adenosylmethionine, which serves as the aminopropyl donor. Finally, spermidine synthase (SPDS) catalyzes the condensation of putrescine with decarboxylated S-adenosylmethionine to form spermidine [8] (Fig. 1A). In prokaryotic cells, the aminopropyl donor is also decarboxylated S-adenosylmethionine. However, the pathway for putrescine synthesis differs: Arginine undergoes decarboxylation catalyzed by arginine decarboxylase, converting it to agmatine. Subsequently, agmatine is hydrolyzed by agmatinase to yield putrescine. Ultimately, SPDS catalyzes the conversion of putrescine to spermidine [9], [10]. (Fig. 1A). Notably, in many bacterial phyla, the aminopropyl is not derived from S-adenosylmethionine, but rather from L-aspartate β-semialdehyde (L-Asa). This pathway generates carboxyspermidine, which is subsequently decarboxylated to yield spermidine [11] (Fig. 1B).

Based on established biosynthetic pathways, diverse enzymatic catalysis and metabolic engineering strategies have been employed for spermidine production. For instance, Xinxin Liang from our research group developed a self-sufficient cofactor regeneration system for spermidine synthesis through an HSD/CASDH/CASDC multi-enzyme cascade using L-homoserine and putrescine as substrates [11]. Alternatively, a dual-enzyme cascade system combining S-adenosylmethionine decarboxylase with SPDS in vitro achieved spermidine production at 3.7 g/L from 2.5 g/L putrescine and 15 g/L S-adenosylmethionine [12]. In Saccharomyces cerevisiae-based systems, spermidine titers reached 224 mg/L through fermentation optimization [13]. Similarly, a spermidine titer of 105.2 mg/L was reported in liquid fermentation via metabolic engineering of Bacillus amyloliquefaciens [14]. Nevertheless, these approaches exhibit limitations: Multi-enzyme cascades require numerous catalytic steps resulting in suboptimal efficiency, while S. cerevisiae-based systems contend with complex metabolic pathways and significant byproduct formation.

As depicted in Fig. 2, a novel spermidine biosynthetic pathway was recently identified in Synechocystis sp. PCC 6803. This pathway diverges from the conventional mechanism involving aminopropylation of putrescine, and instead utilizing carboxyaminopropylagmatine (CAPA) as the key metabolic intermediate [15]. The CAPA pathway initiates with agmatine and L-Asa. These substrates undergo reductive condensation catalyzed by carboxyaminopropylagmatine dehydrogenase (CAPADH) to form CAPA, a NADPH-dependent reaction. Subsequently, CAPA decarboxylase (CAPADC) converts CAPA to aminopropylagmatine (APA). Finally, APA ureohydrolase (APAUH) cleaves APA to yield spermidine and urea (Fig. 2). This putrescine-independent route represents an alternative metabolic strategy for spermidine biosynthesis. We heterologously expressed three key enzymes (CAPADH, CAPADC, APAUH, encoded by Slr0049, Sll0873, Sll0228 respectively) of the CAPA pathway in E coli BL21 (DE3). Combinatorial optimization of these enzymes was performed alongside precursor enhancement (L-Asa and agmatine). Competitive pathways were disrupted and repressor genes deleted to optimize flux. Heterologous overexpression of a spermidine transporter further increased production, achieving titers of 163.11 mg/L in shake flask cultures and 1164.22 mg/L in a 5 L bioreactor.