Niacin (vitamin B3), an essential nutrient and precursor in the biosynthesis of NAD(P), exists primarily in two forms: nicotinic acid and nicotinamide [1]. It plays a vital role in numerous metabolic processes, including DNA repair and cellular stress responses [2]. A niacin deficiency can lead to pellagra, a condition characterized by dermatitis, diarrhea, and dementia. Beyond preventing deficiency-related diseases, niacin has also been associated with the prevention and management of several chronic conditions, such as cardiovascular disease, diabetes, Alzheimer’s disease, and Parkinson’s disease [3], [4], [5]. Due to its broad range of benefits, niacin is widely utilized in food products, dietary supplements, pharmaceuticals, and cosmetic formulations[2]. It also plays a key role in animal nutrition, supporting the health and development of livestock [6]. The global market for nicotinic acid, one of niacin’s primary forms, was valued at USD 614 million in 2019. By 2021, it had nearly doubled to USD 1.2 billion, with projections estimating it will reach USD 2.5 billion by 2032 [6].

Conventional nicotinic acid production relies on chemical synthesis that uses 3-methylpyridine as the starting material (Scheme 1A). This process often requires harsh oxidation conditions and produces undesirable byproducts such as pyridine oxide [6]. In the last two decades, developing environmentally friendly and sustainable processes has become a priority, leading to increased interest in biocatalytic approaches that utilize 3-cyanopyridine as the starting material (Scheme 1B) [7]. There are two applicable pathways. In the first biocatalytic pathway, 3-cyanopyridine is initially converted to nicotinamide through hydration of the cyano group catalyzed by nitrile hydratase (NHase) [8], then nicotinamide is hydrolyzed to produce nicotinic acid by amidase (AMase) [9]. The second pathway employs a single enzyme, nitrilase [10], to directly convert 3-cyanopyridine to nicotinic acid. BASF and Lonza have achieved significant industrial developments of this second pathway since Rhodococcus rhodochrous nitrilase was identified in 1988. Following that, this enzyme was discovered in various microorganisms, including Rhodococcus sp. [11], Bacillus pallidus Dac521 [12], and Nocardia rhodochrous [13]. Generally, this transformation was conducted by utilizing resting cells in the whole-cell biocatalytic setup. Recombinant E. coli strain overexpressing the nitrilase from Gibberella intermedia [14] or Alcaligenes faecalis MTCC 126 [15] has also been reported. However, 3-cyanopyridine is primarily synthesized from 3-methylpyridine (3-MP) with ammonia through ammoxidation under harsh conditions [16]. Therefore, exploring a new biocatalytic pathway that starts with 3-MP could be valuable.

Intrigued by the chemical synthesis using 3-MP, we proposed a pathway that begins with the oxidation of the C-H bond in the methyl group of 3-MP to generate 3-pyridinemethanol (3-POH). Subsequently, 3-POH undergoes oxidation to yield 3-pyridinecarboxaldehyde (3-PCHO), which is then converted to nicotinic acid (Scheme 1C). Nature uses P450 enzymes and non-heme di-iron monooxygenase to catalyze C-H oxidation of various natural products [17]. P. putida was known to utilize hydrocarbons as the carbon source by converting them to carboxylic acids [18]. Its catabolic pathway is encoded in the plasmid of pWWO [19]. Further characterization revealed that the initial step catalyzed by XMO oxidizes the methyl group of toluene and xylenes, producing the corresponding alcohols in the presence of oxygen [20], [21], [22], [23]. XMO consists of two enzymes, namely XylM and XylA. XylM, identified as xylene monooxygenase, has been characterized to catalyze the C-H oxidation aerobically [20], [21], [22]. The active XylM requires XylA, annotated as NADH-dependent reductase, to transfer electrons from NADH to reduce the di-iron cofactor of XylM [23]. The second step is catalyzed by XylB, annotated as benzyl alcohol dehydrogenase (BADH), facilitating the oxidation of alcohols to aldehydes [24]. Finally, XylC, annotated as benzaldehyde dehydrogenase (BZDH), catalyzes the oxidation of aldehydes to carboxylic acids [25]. Both XylB and XylC proteins were characterized to use NAD+ as the cofactor.

In 1992, a P. putida strain was reported to oxidize methyl groups on aromatic heterocycles, including 3-MP [26]. The cells were cultured in a mineral salt medium containing toxic p-xylene as the gene inducer and the carbon source. However, this biotransformation was not thoroughly characterized or optimized. Recently, the recombinant E. coli overexpressing P. putida XMO has been applied to oxidize the methyl groups of 2,6-lutidine to 2,6-bis(hydroxymethyl)pyridine [27]. This result intrigued us to revisit the biotransformation of 3-MP to nicotinic acid in E. coli, which is noted for its advantages in biotechnology, such as rapid growth, ease of genetic engineering, and low cultivation costs [28], [29]. To simplify the study, the biosynthetic pathway was divided into two modules to identify necessary enzymes and suitable E. coli hosts (Scheme 1C). The results showed that in the first module, using resting cells of E. coli MG1655(DE3) overexpressing XMO (E. coli XMO), catalyzes the oxidation of 9 mM 3-MP to generate 8.38 ± 0.57 mM 3-POH under the optimized condition for 12 h. The resultant supernatant was transferred to the second module using the resting cells of E. coli MG1655 RARE overexpressing XylB (RARE B) to convert 3-POH to nicotinic acid completely after 24 h. However, this cascade reaction is not practical. Therefore, we integrated the results from two modules to create a single recombinant strain of E. coli MG1655 RARE co-overexpressing XMO and XylB. After harvesting, the resting cells biotransformed 9 mM 3-MP into 8.28 ± 0.35 mM nicotinic acid under the optimized condition after 12 h. This study presents a straightforward pathway to convert 3-MP to nicotinic acid and provides guidance for researchers to engineer this system in the future.