Identification of Bromelain Subfamily Proteases Encoded in the Pineapple Genome

Abstract

Papain (aka C1A) family proteases, including bromelain enzymes, are widespread across the plant kingdom and play critical regulatory functions in protein turnover during development. The proteolytic activity exhibited by papain family proteases has led to their increased usage for a wide range of cosmetic, therapeutic, and medicinal purposes. Bromelain enzymes, or bromelains in short, are members of the papain family that are specific to the bromeliad plant family. The only major commercial extraction source of bromelain is pineapple. The importance of C1A family and bromelain subfamily proteases in pineapple development and their increasing economic importance led several researchers to utilize available genomic resources to identify protease-encoding genes in the pineapple genome. To date, studies are lacking in screening bromelain genes for targeted use in applied science studies. In addition, the bromelain genes coding for the enzymes present in commercially available bromelain products have not been identified and their evolutionary origin has remained unclear. Here, using the newly developed MD2 v2 pineapple genome, we aimed to identify bromelain-encoding genes and elucidate their evolutionary origin. Orthologous and phylogenetic analyses of all papain-family proteases encoded in the pineapple genome revealed a single orthogroup (189) and phylogenetic clade (XIII) containing the bromelain subfamily. Duplication mode and synteny analyses provided insight into the origin and expansion of the bromelain subfamily in pineapple. Proteomic analysis identified four bromelain enzymes present in two commercially available bromelain products derived from pineapple stem, corresponding to products of four putative bromelain genes. Gene expression analysis using publicly available transcriptome data showed that 31 papain-family genes identified in this study were up-regulated in specific tissues, including stem, fruit, and floral tissues. Some of these genes had higher expression in earlier developmental stages of different tissues. Similar expression patterns were identified by RT-qPCR analysis with leaf, stem, and fruit. Our results provide a strong foundation for future applicable studies on bromelain, such as transgenic approaches to increase bromelain content in pineapple, development of bromelain-producing bioreactors, and studies that aim to determine the medicinal and/or therapeutic viability of individual bromelain enzymes.

Genome wide identification of bromelain-like cysteine proteases in Puya raimondii

“…Species have different numbers of C1A protease genes. The best-studied genome in the Bromeliaceae family is A. comosus, which has 71 protease genes, four of which have been related to recognized activities and named as true bromelains (Yow et al 2023). In our study, we found six PrBLCP genes in P. raimondii.…”

“…This analysis included the three UniProt database bromelain sequences, ve Agave tequilana sequences with the highest PrBLCPs identity, and genomic annotations F153, MD2v1, and MD2v2, previously grouped in the same orthogroup by Yow et al (2023). The PrBLCP1, PrBLCP2, and PrBLCP3 sequences cluster closely with each other and with ACMD2v2_05.02955 (AcC1A31), ACMD2v2_05.02951 (AcC1A28), and ACMD2v2_05.00101 (AcC1A22), all de ned as real bromelains by Yow et al (2023). PrBLCP4, PrBLCP5, and PrBLCP6 were more closely linked to A. tequilana proteins than PrBLCP1, PrBLCP2, and PrBLCP3.…”

“…Two pineapple varieties have genome annotations: genome F153 (organized up to the chromosomal level) in the Phytozome database(Ming et al 2015) and MD2 (organized at the scaffold level) in the NCBI database(Redwan et al 2016). The new MD2 assembly (MD2v2) by Yow et al (2021) increased gene count Yow et al (2023). found bromelain-family proteases in A. comosus using this information.…”