Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, stands as one of the most versatile enzymatic cofactors in nature, indispensable for a vast array of transformations including transaminations, decarboxylations, and racemizations [1], [2]. Its significance extends beyond fundamental metabolism to the modulation of pathways implicated in cardiovascular, neurological, and oncological disorders [3], [4]. In industrial biotechnology, PLP-dependent enzymes are pivotal for the stereoselective synthesis of high-value chiral amines and non-canonical amino acids, underpinning sustainable manufacturing routes for pharmaceuticals and agrochemicals [5], [6], [7], [8], [9]. Despite its broad utility, the commercial production of PLP remains dominated by multi-step chemical synthesis from pyridoxine or pyridoxamine precursors, processes that are typically resource-intensive and generate substantial environmental waste (Supplementary materials, Section S1), highlighting a critical need for more sustainable and efficient alternatives [10], [11].

Natural biosynthetic routes to PLP, comprising de novo and salvage pathways, present inherent advantages of enzymatic catalysis but face practical limitations for industrial implementation. The de novo pathways can be complex, often involving multiple enzymatic steps, suffering from inherent metabolic flux constraints, and risking the accumulation of phosphorylated intermediates [12], [13], [14]. The salvage pathway, which reactivates B6 vitamers, is frequently hampered by the catalytic inefficiency of pyridoxine phosphate oxidase (PNPO) and a dependency on specific substrate availability [6], [15]. These intrinsic bottlenecks have motivated the development of more streamlined, engineered biocatalytic systems for direct PLP production [9], [16].

A particularly attractive target is pyridoxal kinase (PLK), which catalyzes the single-step phosphorylation of inexpensive pyridoxal (PL) directly to PLP [17]. This reaction represents the most concise and direct route from a stable precursor to the active coenzyme. However, the industrial deployment of PLK has been constrained by two major hurdles: First, the identification and engineering of robust PLK variants with properties suitable for process-scale catalysis remains a challenge [9], [16], [18], [19], [20]; Second, and more critically, the reaction demands a stoichiometric supply of the expensive and labile cofactor adenosine triphosphate (ATP), rendering the process economically prohibitive for large-scale applications.

The high cost of ATP has made in situ ATP regeneration a cornerstone of practical cofactor-dependent biocatalysis. Conventional strategies, such as those employing phosphoenolpyruvate, are often prohibitively expensive and generate by-products that complicate downstream processing [21], [22], [23], [24]. In this context, polyphosphate kinase (PPK) has emerged as a superior solution [25]. PPK utilizes inorganic polyphosphate (polyP) —an inexpensive (∼$18/kg), stable, and abundant polymer—as a phosphate donor to regenerate ATP from ADP. This translates to a dramatic cost reduction, providing phosphate equivalents at a small fraction of the cost of ATP (>$4000/kg) [26]. This cost-effectiveness is further enhanced by the ability of many PPKs to regenerate ATP directly from AMP, improving the system's atom economy and versatility. This system offers exceptional operational simplicity and a compelling economic advantage; the cost of phosphate equivalents from polyP is orders of magnitude lower than that from ATP itself [27]. The ability of certain PPKs to regenerate ATP from AMP further enhances the system's versatility and atom economy.

The phosphorylation of pyridoxal (PL) to pyridoxal 5′-phosphate (PLP) and the regeneration of ATP from polyphosphate are established enzymatic processes. However, their efficient integration into a co-localized, ATP-autonomous cascade remains a challenge. To address this, we constructed an Escherichia coli-based system co-expressing pyridoxal kinase (PLK) and polyphosphate kinase (PPK). This design creates a self-sufficient catalytic platform where PL phosphorylation is directly coupled with continuous ATP regeneration from ADP using inexpensive polyphosphate (See Fig. 1.). By eliminating the need for exogenous ATP, this integrated approach offers a practical and cost-effective route for PLP synthesis, overcoming key limitations of previous methods. This work demonstrates high-yield production through engineered cascade catalysis, establishing a scalable enzymatic process for this essential cofactor.