Flavor is an important sensory characteristic that significantly influences the acceptance of various food products. Among the several flavor compounds, 2-acetyl-1-pyrroline (2AP) holds special attention from the scientific community and industry. The flavor of several foods such as cheese (Zehentbauer and Reineccius, 2002), popcorn (Schieberle, 1995), and wine (Herderich et al., 1995) are related to 2AP. However, 2AP is firmly linked with the fragrance of basmati rice aroma (Buttery et al., 1983). 2AP is the major volatile aroma compound responsible for the fragrance of basmati rice and it has been identified as contributing to the pleasant flavor in aromatic rice and other plant species such as aromatic mung bean, Pandanus amaryllifolius Roxb., Bassia latifolia Roxb., and Vallaris glabra Ktze, etc. (Buttery et al., 1983; Wakte et al., 2011; Wongpornchai et al., 2003). Interestingly besides plant species, fungi like Aspergillus awamori and Aspergillus nigricans (Rungsardthong and Noomhoom, 2005) and bacterial species like Bacillus cereus (Romanczyk et al., 1995), Lactobacillus brevis and, Lactobacillus hilgardii (Costello and Henschke, 2002) are also reported to synthesize 2AP. In a previous study, potential 2AP synthesizing rhizobacteria were isolated from the rhizosphere of aromatic rice varieties and assessed their plant growth-promoting potential and aroma (2AP) enhancement ability in two scented rice varieties namely, Ambemohar-157 and Dehradun basmati (Dhondge et al., 2021). These 2AP synthesizing rhizobacteria were found to promote plant growth and enhance aroma (2AP) content in aromatic rice on mono-inoculation as well as consortium inoculation (Dhondge et al., 2021).

Several studies on the understanding of the molecular mechanism of 2AP biosynthesis have revealed that 2AP biosynthesis is linked with the loss of function of the betaine aldehyde dehydrogenase 2 (BADH2) gene (Bradbury et al., 2005). However, BADH2-independent 2AP was also predicted via functional ∆1-pyrolline-5-carboxylic acid synthetase (P5CS) and Ornithine aminotransferase (OAT) genes, and higher ∆1-pyrolline-5-carboxylic acid synthetase (P5CS) content was found to play an important role in enhanced 2AP biosynthesis through proline synthesis (Huang et al., 2007). Bhatt et al. (2021) recently reported 2AP biosynthesis associated with the presence of a functional BADH2 gene in P. amaryllifolius.

The above-mentioned studies on the molecular mechanisms for 2AP biosynthesis were carried out in plant systems. However, the molecular mechanisms for 2AP biosynthesis in prokaryotic systems like bacteria are still unknown, and targeted efforts to understand 2AP biosynthesis routes in prokaryotic systems are lacking. Considering the facts that the presence of 2AP in many food products may increase their flavor quality and the potential importance of 2AP in the food industry, it would be helpful to gain more insights into the genetic basis of 2AP biosynthesis in prokaryotic systems to explore their future practical applications in the food industry. Therefore the present study primarily aimed to assess the functionality of BADH2 and the genetic basis for 2AP biosynthesis in a bacterial system through characterizing the BADH2 gene and enzyme in 2AP-synthesizing rice rhizospheric bacterial isolate. The secondary aim of the present study was to confirm and verify the identity of 2AP-synthesizing rice rhizospheric bacterial isolate with more reliable and accurate genomic identification. To achieve this, we carried out whole-genome sequencing of the 2AP synthesizing rhizobacterial isolate Bacillus cereus DB25 (isolated from the rhizosphere of the Dehradun basmati rice variety, Dhondge et al., 2021) and retrieved its full-length BADH2 gene sequence (hereafter denoted as BcBADH2) then characterized its functionality by in-silico protein docking studies and enzyme activity assay. Further, to gain more insights into the 2AP biosynthesis mechanism in the bacterial system the metabolite analysis of key 2AP precursor metabolites namely, proline and methylglyoxal was carried out along with enzyme assays.