Biotechnology-enabled plastic valorization across enzymatic, microbial, and hybrid conversion systems: A review.

Esters are vital compounds with wide-ranging applications in food, fragrance, and pharmaceuticals, yet their sustainable supply increasingly relies on microbial biosynthesis. Acyltransferases (ATFs), particularly BAHD family members, catalyze the esterification of alcohols with acyl-CoA donors, a central step in generating structurally diverse esters in vivo. This review highlights recent progress in ATF-mediated microbial biosynthesis of three representative classes: short-chain fatty acid esters (SCFAEs), monoterpenyl esters (MTEs), and aromatic esters (AEs). Advances in enzyme discovery, protein engineering, and metabolic pathway reprogramming have expanded substrate scope, improved catalytic efficiency, and enabled titers from milligram to gram scale. Nonetheless, challenges remain in achieving high activity, selectivity, and stereochemical fidelity under industrial conditions. We summarize strategies including co-culture systems, non-natural precursor biosynthetic pathways, high-throughput screening, and rational enzyme engineering. Future perspectives emphasize developing a precision precursor supply for ATF-mediated esterification, elucidating ATF structure-function relationships, and optimizing downstream processing to achieve efficient, scalable, and sustainable microbial production of diverse natural esters.

The synthesis of secondary and tertiary amines through the reductive amination of carbonyl compounds is one of the most significant reactions in synthetic chemistry. Asymmetric reductive amination for the formation of chiral amines, which are required for the synthesis of pharmaceuticals and other bioactive molecules, is often achieved through transition metal catalysis, but biocatalytic methods of chiral amine production have also been a focus of interest owing to their selectivity and sustainability. The discovery of asymmetric reductive amination by imine reductase (IRED) and reductive aminase (RedAm) enzymes has served as the starting point for a new industrial approach to the production of chiral amines, leading from laboratory-scale milligram transformations to ton-scale reactions that are now described in the public domain. In this perspective we trace the development of the IRED-catalyzed reductive amination reaction from its discovery to its industrial application on kg to ton scale. In addition to surveying examples of the synthetic chemistry that has been achieved with the enzymes, the contribution of structure and protein engineering to the understanding of IRED-catalyzed reductive amination is described, and the consequent benefits for activity, selectivity and stability in the design of process suitable catalysts.

Strategic conversion of plastic containers with food waste into energy through thermochemical processes

Cytochrome P450 enzymes (CYPs) are versatile heme-containing monooxygenases involved in the metabolism of endogenous and exogenous compounds, as well as natural product biosynthesis. Their ability to catalyze regio- and stereoselective oxidation reactions makes them valuable in pharmaceuticals, fine chemicals, and biocatalysis. However, wild-type CYPs suffer from low catalytic efficiency, limited substrate specificity, and instability under industrial conditions. Recent advances in protein engineering—rational design, semi-rational design, and directed evolution—have enhanced their activity, stability, and substrate scope. These strategies have enabled CYPs to be engineered for applications like C–H functionalization, carbene transfer, and complex molecule biosynthesis. Despite progress, challenges remain in optimizing efficiency, expanding substrate ranges, and scaling production for industrial use. Future directions include integrating CYPs with other biocatalysts, improving high-throughput screening, and applying machine learning to enzyme design. This review highlights recent developments and the promising future of engineered CYPs in sustainable chemistry, drug development, and high-value chemical production.

Yeast surface display (YSD) is a powerful tool in biotechnology that links genotype to phenotype. In this review, the latest advancements in protein engineering and high-throughput screening based on YSD are covered. The focus is on innovative methods for overcoming challenges in YSD in the context of biotherapeutic drug discovery and diagnostics. Topics ranging from titrating avidity in YSD using transcriptional control to the development of serological diagnostic assays relying on serum biopanning and mitigation of unspecific binding are covered. Screening techniques against nontraditional cellular antigens, such as cell lysates, membrane proteins, and extracellular matrices are summarized and techniques are further delved into for expansion of the chemical repertoire, considering protein–small molecule hybrids and noncanonical amino acid incorporation. Additionally, in vivo gene diversification and continuous evolution in yeast is discussed. Collectively, these techniques enhance the diversity and functionality of engineered proteins isolated via YSD, broadening the scope of applications that can be addressed. The review concludes with future perspectives and potential impact of these advancements on protein engineering. The goal is to provide a focused summary of recent progress in the field.

The study of bacterial metabolism holds immense significance for improving human health and advancing agricultural practices. The prospective applications of genomically encoded bacterial metabolism present a compelling opportunity, particularly in the light of the rapid expansion of genomic sequencing data. Current metabolic inference tools face challenges in scaling with large datasets, leading to increased computational demands, and often exhibit limited inter-relatability and interoperability. Here, we introduce the Integrated Biosynthetic Inference Suite (IBIS), which employs deep learning models and a knowledge graph to facilitate rapid, scalable bacterial metabolic inference. This system leverages a series of Transformer based models to generate high quality, meaningful embeddings for individual enzymes, biosynthetic domains, and metabolic pathways. These embedded representations enable rapid, large-scale comparisons of metabolic proteins and pathways, surpassing the capabilities of conventional methodologies. The examination of evolutionary and functionally conserved metabolites across diverse bacterial species is facilitated by integrating the predictive capabilities of IBIS into a graph database enriched with comprehensive metadata. The consideration of both primary and specialized metabolism, combined with an embedding logic for enzyme discovery, uniquely positions IBIS to identify potential novel metabolic pathways. With the expansion of genomic data necessitating transformative approaches to advance molecular metabolism research, IBIS delivers an AI-driven holistic investigation of bacterial metabolism.

Advances and opportunities for the design of self-sufficient and spatially organized cell-free biocatalytic systems

With the advent of green chemistry, the use of enzymes in industrial processes serves as an alternative to the conventional chemical catalysts. A high demand for sustainable processes for catalysis has brought a significant attention to hunt for novel enzymes. Among various hydrolases, the α-amylase has a gamut of biotechnological applications owing to its pivotal role in starch-hydrolysis. Industrial demand requires enzymes with thermostability and to ameliorate this crucial property, various methods such as protein engineering, directed evolution and enzyme immobilisation strategies are devised. Besides the traditional culture-dependent approach, metagenome from uncultured bacteria serves as a bountiful resource for novel genes/biocatalysts. Exploring the extreme-niches metagenome, advancements in protein engineering and biotechnology tools encourage the mining of novel α-amylase and its stable variants to tap its robust biotechnological and industrial potential. This review outlines α-amylase and its genetics, its catalytic domain architecture and mechanism of action, and various molecular methods to ameliorate its production. It aims to impart understanding on mechanisms involved in thermostability of α-amylase, cover strategies to screen novel genes from futile habitats and some molecular methods to ameliorate its properties.

The advent of biologic therapies, particularly antibody therapeutics, has revolutionized the pharmacological treatment of many rheumatic diseases. Antibody discovery began with the immunization of mice for the production of rodent immunoglobulins, but advances in protein and genetic engineering have now made it possible to generate fully human antibodies, which are better tolerated by patients. For most clinical applications in rheumatology, antibodies have been used as blocking agents capable of neutralizing the function of pro-inflammatory proteins, such as TNF. The latest strategies involve antibody products armed with effector moieties, such as anti-inflammatory drugs or cytokines, or antibody products that are specific for multiple targets for the selective inhibition of inflammation at sites of disease. Antibodies are some of the best-selling drugs in the world, and with further advances in antibody development, engineering of armed antibodies and bispecific products will have an important role in the treatment of rheumatic diseases.

The characterization of enzyme structure and function was essential for understanding biochemical pathways and developing effective biotechnological applications, particularly in environmental bioremediation. Traditional experimental methods for protein analysis were often labor-intensive and limited by the inability to culture certain microorganisms. In this study, an in silico approach was employed to predict the structure and function of a putative degradative enzyme identified from metagenomic analysis of landfill leachate. Using a combination of bioinformatics tools, including sequence alignment, domain annotation, secondary structure prediction and three-dimensional (3D) structural modeling, the target enzyme was analyzed for its catalytic potential and stability. Conserved motifs and active sites were identified, suggesting its involvement in the degradation of xenobiotic compounds such as polychlorinated biphenyls (PCBs). The 3D structure model revealed a typical fold associated with oxygenase or dehydrogenase activities, with predicted metal-binding sites critical for catalyses. These findings demonstrate the power of computational methods to accelerate the discovery and characterization of novel enzymes, especially from unculturable microbial communities. This approach provides a valuable foundation for future functional validation, protein engineering and the development of environmentally sustainable biocatalysts.

The discovery and engineering of new enzymes is important across the bioeconomy, with diverse applications from foods to pharmaceuticals, sensors to agriculture. However, enzyme engineering, in particular machine learning-guided engineering, is hampered by a lack of data. Currently there exists no database designed to capture and interpret datasets created in this domain, nor are there easy analysis and visualisation tools. We developed the Enzyme Engineering Database to provide a centralized resource and an online analysis tool to consolidate sequence-function data from enzyme engineering campaigns, thereby making three contributions: (i) a database into which researchers can deposit public data, (ii) visualisation and analysis tools for protein engineers to analyse their own data or compare enzyme variants to other engineering campaigns, and (iii) a gold-standard dataset for benchmarking automated extraction along with the first large language model extraction pipeline specific for enzyme engineering campaigns. The Enzyme Engineering Database is accessible at http://enzengdb.org/.

Chapter 4 - Robust enzymes designing for efficient biocatalysis

Microbiome and metabolism: Advancements in microbiome engineering

Mammalian synthetic biology in the age of genome editing and personalized medicine

Advances in protein engineering have made the industrial use of enzymes feasible, though, a more wide-spread application is hampered by the limited stability of enzymes under often required harsh conditions. We developed a protein stabilization technology that involves the in situ cyclization of proteins (INCYPRO) and facilitates the design and semi-synthesis of multicyclic enzymes. These synthetically enhanced durable enzymes add to the current efforts towards a sustainable and green economy.