Biosynthesis of polyhydroxybutyrate from methane and carbon dioxide using type II methanotrophs.

Methane (CH4) and carbon dioxide (CO2) are the dominant greenhouse gases (GHGs) that are increasing at an alarming rate. Methanotrophs have emerged as potential CH4 and CO2 biorefineries. This study demonstrated the synchronous incorporation of CH4 and CO2 into polyhydroxybutyrate (PHB) for the first time using 13C-labeling experiments in methanotrophs. By supplying substantial amounts of CO2, PHB content was enhanced in all investigated type II methanotrophic strains by 140 %, 146 %, and 162 %. The highest content of PHB from CH4 and CO2 in flask-scale cultivation reached 38 % dry cell weight in Methylocystis sp. MJC1, in which carbon percentage in PHB from CO2 was 45 %. Flux balance analysis predicted the critical roles of crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase in CO2 recycling. This study provided proof of the conversion of GHGs into a valuable and practical product using methanotrophic bacteria, contributing to addressing GHG emissions.

Methanotrophs as a reservoir for bioactive secondary metabolites: Pitfalls, insights and promises.

Methanotrophs are potent natural producers of several bioactive secondary metabolites (SMs) including isoprenoids, polymers, peptides, and vitamins. Cryptic biosynthetic gene clusters identified from these microbes via genome mining hinted at the vast and hidden SM biosynthetic potential of these microbes. Central carbon metabolism in methanotrophs offers rare pathway intermediate pools that could be further diversified using advanced synthetic biology tools to produce valuable SMs; for example, plant polyketides, rare carotenoids, and fatty acid-derived SMs. Recent advances in pathway reconstruction and production of isoprenoids, squalene, ectoine, polyhydroxyalkanoate copolymer, cadaverine, indigo, and shinorine serve as proof-of-concept. This review provides theoretical guidance for developing methanotrophs as microbial chassis for high-value SMs. We summarize the distinct secondary metabolic potentials of type I and type II methanotrophs, with specific attention to products relevant to biomedical applications. This review also includes native and non-native SMs from methanotrophs, their therapeutic potential, strategies to induce silent biosynthetic gene clusters, and challenges.

TSSNote-CyaPromBERT: Development of an integrated platform for highly accurate promoter prediction and visualization of Synechococcus sp. and Synechocystis sp. through a state-of-the-art natural language processing model BERT.

Since the introduction of the first transformer model with a unique self-attention mechanism, natural language processing (NLP) models have attained state-of-the-art (SOTA) performance on various tasks. As DNA is the blueprint of life, it can be viewed as an unusual language, with its characteristic lexicon and grammar. Therefore, NLP models may provide insights into the meaning of the sequential structure of DNA. In the current study, we employed and compared the performance of popular SOTA NLP models (i.e., XLNET, BERT, and a variant DNABERT trained on the human genome) to predict and analyze the promoters in freshwater cyanobacterium Synechocystis sp. PCC 6803 and the fastest growing cyanobacterium Synechococcus elongatus sp. UTEX 2973. These freshwater cyanobacteria are promising hosts for phototrophically producing value-added compounds from CO2. Through a custom pipeline, promoters and non-promoters from Synechococcus elongatus sp. UTEX 2973 were used to train the model. The trained model achieved an AUROC score of 0.97 and F1 score of 0.92. During cross-validation with promoters from Synechocystis sp. PCC 6803, the model achieved an AUROC score of 0.96 and F1 score of 0.91. To increase accessibility, we developed an integrated platform (TSSNote-CyaPromBERT) to facilitate large dataset extraction, model training, and promoter prediction from public dRNA-seq datasets. Furthermore, various visualization tools have been incorporated to address the "black box" issue of deep learning and feature analysis. The learning transfer ability of large language models may help identify and analyze promoter regions for newly isolated strains with similar lineages.

Reconstructing ethanol oxidation pathway in Pseudomonas putida KT2440 for bio-upgrading of ethanol to biodegradable polyhydroxybutanoates.

Ethanol has recently been demonstrated as a suitable carbon source for acetyl-CoA-derived products with high theoretical yield. Herein, the short-chain-length polyhydroxyalkanoates production pathway was constructed in an industrial platform P. putida KT2440, allowing the engineered strain to produce 674.97 ± 22.3 mg/L of Polyhydroxybutyrate (PHB) from ethanol as sole carbon source. Furthermore, the ethanol catabolic pathway was reconstructed to enhance the acetyl-coA pool by expressing the novel Aldehyde dehydrogenases from Klebsiella pneumonia and Dickeya zeae, resulting in a titer of 1385.34 ± 16.5 mg/L and 9300 ± 0.56 mg/L of PHB in shake flask and fermenter, respectively. Furthermore, transcriptome analysis was conducted to provide insights into the central metabolic pathways and different expression patterns in response to changes in substrate. Additionally, the production of co-polymer poly(3-hydroxybutyrate-co-3-hydroxypropionate) was shown using glycerol and ethanol as co-substrates from recombinant P. putida KT2440. This work demonstrates the potential of P. putida KT2440 as a promising industrial platform for short-chain-length PHAs production from structurally unrelated carbon sources.

Developments of Riboswitches and Toehold Switches for Molecular Detection-Biosensing and Molecular Diagnostics.

Riboswitches and toehold switches are considered to have potential for implementation in various fields, i.e., biosensing, metabolic engineering, and molecular diagnostics. The specific binding, programmability, and manipulability of these RNA-based molecules enable their intensive deployments in molecular detection as biosensors for regulating gene expressions, tracking metabolites, or detecting RNA sequences of pathogenic microorganisms. In this review, we will focus on the development of riboswitches and toehold switches in biosensing and molecular diagnostics. This review introduces the operating principles and the notable design features of riboswitches as well as toehold switches. Moreover, we will describe the advances and future directions of riboswitches and toehold switches in biosensing and molecular diagnostics.