Ali Raza

Nowadays, enzyme engineering has moved from traditional structure-based mutagenesis and directed evolution to data-intensive, AI-assisted design paradigms that involve the rapid discovery and optimization of biocatalysts. Whereas classical approaches relied on rational design and experimental screening, advances in high-throughput sequencing, modeling, and machine learning have enabled predictive exploration of sequence–structure–function relationships in enzymes. Importantly, the latest protein language models and deep learning approaches enable accurate prediction of mutational outcomes, stability engineering, and functional annotation at an unprecedented scale. Generative AI models also enable the design of novel enzymes by predicting protein sequences with tailored catalytic functions and broadened substrate specificity. AI combined with design–build–test–learn (DBTL) automation and synthetic biology has enabled the creation of closed-loop engineering workflows for rapid, iterative optimization. This review examines enzyme engineering from classical methods to AI-assisted biocatalyst development, highlighting key advances, challenges, and emerging trends in autonomous laboratories, sustainable biocatalysis, and computational protein design.

Probiotics have long been recognized for their broad interests in regulating the immune system and promoting gut health. Recently, they have become a key component in the growing field of precision medicine. This review thoroughly examines the transition of probiotics from general health enhancers to advanced, targeted therapies designed to address the unique characteristics of different microbiomes, genetic profiles, and individual health statuses. It emphasizes various mechanisms by which probiotics affect host physiology, including the regulation of immune responses, the modulation of metabolism, and the promotion of intestinal interactions. This review further explores the integration of psychobiology, next-generation probiotics (NGP), modified strains, artificial intelligence, and synthetic biology technologies to develop personalized probiotic therapies. By integrating recent advances with cutting-edge technologies, this review redefines probiotics as a crucial tool in personalized medicine, highlighting the need for innovation and collaboration to maximize their therapeutic potential.

Viroids and viroid-like exogenous circular RNAs (ex-circRNAs) are small infectious RNA agents capable of autonomous replication within host cells, independent of any helper virus. In this study, we analyzed rRNA-depleted transcriptomic data sets from members of the Vitaceae , Solanaceae , and Rosaceae families to explore the diversity of these subviral elements. Using both homology-based and alignment-free ribozyme detection methods, we recovered 16 known viroids and identified 13 novel viroid-like RNAs from plant metatranscriptomic data sets. Among these, we report two new hammerhead ribozyme-containing viroid-like RNAs—CGHVd-RNA in Physalis peruviana and GPLMVd-RNA in Vitis vinifera —that display distinct secondary structures and limited sequence similarity to known viroids, supporting their classification as new species within the Avsunviroidae family. We also identified a symmetric twister ribozyme-containing ex-circRNA in peach, with validated in vitro self-cleavage activity. Additionally, three ambi-like circular RNAs were detected in grapevine and peach, encoding divergent RNA-dependent RNA polymerases, hairpin (HP), and delta virus ribozymes (DVRz). Finally, a novel Obelisk-like RNA with unique structural features was discovered in the Cape gooseberry. These results reveal the hidden complexity and broad host range of plant-associated viroid-like RNAs and suggest the existence of previously unrecognized subviral lineages with distinct evolutionary origins. IMPORTANCE: This study reveals remarkable diversity in plant-associated subviral RNA populations, identifying 13 novel viroid-like RNAs alongside 16 known viroids. Notable discoveries include new hammerhead ribozyme-containing species-level ex-circRNAs from Cape gooseberry and grapevine, a symmetric twister ribozyme ex-circRNA in peach, and, surprisingly, ambi-like circular RNAs with RNA-dependent RNA polymerases. These findings significantly expand our understanding of the complexity and evolutionary diversity of plant-associated subviral RNAs.

Rice viruses seriously threaten rice cultivation and cause significant economic losses, but they have not yet been systematically identified, with only 20 rice-infecting viruses reported. Here, we perform a large-scale analysis of 17,115 RNA-seq libraries spanning 24 Oryza species across 51 countries. Using de novo assembly and homology-based methods, we identify 810 complete or near-complete viruses, including 276 known viruses and 534 novel viruses. Given the high divergence and atypical genome organizations of novel viruses, more than a half of them are tentatively assigned to 1 new order, 61 new families, and at least 104 new genera. Utilizing homology-independent approaches, we additionally identify 49 divergent RNA-dependent RNA polymerases (RdRPs), which are confirmed by protein structural alignment. Furthermore, we analyze the metadata of related Sequence Read Archive (SRA) libraries and estimated viral abundance in each library, leading to the screening of 427 viruses closely associated with rice plants. Overall, our study vastly expands the viral diversity in rice plants, providing insights for the prevention and control of viral disease.

Kluyveromyces marxianus is a rapidly growing thermotolerant yeast that secretes a variety of lytic enzymes, utilizes different sugars, and produces ethanol. The probiotic potential of this yeast has not been well explored. To evaluate its probiotic potential, the yeast strain Kluyveromyces marxianus DMKU3-1042 was analyzed using next-generation sequencing technology. Analysis of the genomes showed that the yeast isolates had a GC content of 40.10–40.59%. The isolates had many genes related to glycerol and mannose metabolism, as well as genes for acetoin and butanediol metabolism, acetolactate synthase subunits, and lactic acid fermentation. The strain isolates were also found to possess genes for the synthesis of different vitamins and Coenzyme A. Genes related to heat and hyperosmotic shock tolerance, as well as protection against reactive oxygen species were also found. Additionally, the isolates contained genes for the synthesis of lysine, threonine, methionine, and cysteine, as well as genes with anticoagulation and anti-inflammatory properties. Based on our analysis, we concluded that the strain DMKU3-1042 possesses probiotic properties that make it suitable for use in food and feed supplementation.

Viruses are ubiquitous in nature and exist in a variety of habitats. The advancement in sequencing technologies has revolutionized the understanding of viral biodiversity associated with plant diseases. Deep sequencing combined with metagenomics is a powerful approach that has proven to be revolutionary in the last decade and involves the direct analysis of viral genomes present in a diseased tissue sample. This protocol describes the details of RNA extraction and purification from wild rice plant and their yield, RNA purity, and integrity assessment. As a final step, bioinformatics data analysis including demultiplexing, quality control, de novo transcriptome assembly, taxonomic allocation and read mapping following Illumina HiSeq small and total RNA sequencing are described. Furthermore, the total RNAs extraction protocol and an additional ribosomal rRNAs depletion step which are significantly important for viral genomes construction are provided.

Viroids are single-stranded circular RNA molecules that cause diseases in plants and do not encode any protein. Classical approaches for the identification of new viroids are challenging for many plant pathology laboratories as viroid cDNA synthesis and sequencing require purification and enrichment of the naked viroid RNA by two-dimensional gel electrophoresis. Conventional metagenomic approaches are not effective for viroid discovery because the total number of known viroids is small, and distinct viroids share limited nucleotide sequence similarity. In this chapter, we describe a homology-independent approach for the identification of both known and new viroids in disease samples. It is known that viroid infection of plants triggers production of overlapping viroid-derived small interfering RNAs (siRNAs) targeting the entire genome with high densities and that replication of viroids occurs via a rolling-circle mechanism to yield head-to-tail multiple-repeat replicative intermediates. Our approach involves deep sequencing of either long or small RNAs in a disease sample followed by viroid identification with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Among the sequenced total small RNAs, PFOR retains viroid-derived siRNAs for viroid genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, a unique feature of viroid RNA replication. In contrast, long RNAs sequenced after depletion of ribosomal RNAs are cut into 21-nucleotide virtual overlapping small RNAs with the algorithm SLS (splitting longer read into shorter fragments) before PFOR. We show that new viroids or viroids from the two known families are readily identified and their full-length sequences recovered by PFOR from long or small RNAs sequenced directly from infected plants. We propose that our approach can be used for viroid discovery in both plants and potentially animals since PFOR identifies viroids by searching for circular RNAs or a unique replication intermediate of the viroid genome in a sequence homology-independent manner.

The consumption of dietary supplements to treat health complications or to improve overall health conditions has become a globally increasing trend that leads to the development of a large number of health-related novel products and expands the associated manufacturing industries around the world. In the current study, we applied selective culturing combined with next-generation sequencing to examine the microbial viability in terms of its culturability on culture medium, composition, and possible contamination in the selected 17 commercial probiotic products sold in the mainland China market. Additionally, the relative abundance of each individual bacterial content was also evaluated by using the generated sequencing reads. The tested probiotic product samples were subjected to Illumina HiSeq-2000 sequencing platform and thoroughly analyzed by the in-house developed bioinformatics pipeline. The comprehensive culturing and sequencing analysis revealed both viability and composition inaccuracy among the several tested probiotic products, however, no contaminant was identified during the analysis. Among the total, five probiotic products (29.41%) were found with an inaccurate or lower colony-forming unit (CFU) counts on culture media while four probiotic products (23.52%) have inaccurately labeled classification. This study provides an ideal qualitative and quantitative assessment approach, which can be used as a diagnostic tool for the accurate assessment of commercial probiotic supplements.