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.

Bio-upgrading of ethanol to fatty acid ethyl esters by metabolic engineering of Pseudomonas putida KT2440.

Fatty acid ethyl esters (FAEEs) have gained increasing attention as a replacement for traditional fossil fuels in the recent years. Here, we report the efficient upgrading of ethanol to FAEEs from Pseudomonas putida KT2440, using ethanol as the sole carbon source. First, the wax synthase (WS) encoded by the atfA gene from Acinetobacter baylyi ADP1 was expressed in P. putida KT2440. Second, the flux from ethanol towards acetyl-CoA was increased by expression of the acetaldehyde dehydrogenase (ada) from Dickeya zeae. By using dodecane overlay to capture FAEEs, 1.2 g/L of FAEEs with a yield of 152.09 mg FAEEs/g ethanol were produced. Culture optimization enhanced the FAEEs contents up to 1.6 g/L in shake flask and 4.3 g/L in a fed-batch fermenter. In summary, our study provides a basis for combining the bioethanol production process with the efficient upgrading of ethanol to biodiesel.

Co-upgrading of ethanol-assisted depolymerized lignin: A new biological lignin valorization approach for the production of protocatechuic acid and polyhydroxyalkanoic acid.

This study presents a promising biological co-upgrading of ethanol-assisted depolymerized lignin (EDL) into protocatechuic acid (PCA) and polyhydroxyalkanoic acid (PHA) without any separation process. A depolymerized alkali lignin containing various G-lignin-type monomers at a concentration of 77 mg/mL was used for co-upgrading. An engineered Pseudomonas putida KT2440 strain was constructed by knocking out the protocatechuate 3, 4-dioxygenase, expression of the formaldehyde utilization pathway, and the expression of aldehyde dehydrogenase to enhance the efficiency of the ethanol utilization pathway. The growth and production of value-added bioproducts have been promoted by the utilization of formaldehyde, resulted in 6.73 ± 0.26 mg/L of PCA with a 17.5% (w/w) yield of total lignin monomers, and 303.66 ± 26.75 mg/L of PHA with 21.26% (w/w) of dry cell weight from 0.5 mL EDL. Moreover, the ethanol solvent used for lignin depolymerization was also utilized along with depolymerized lignin for co-upgrading to value-added products.

Biological conversion of methane to putrescine using genome-scale model-guided metabolic engineering of a methanotrophic bacterium Methylomicrobium alcaliphilum 20Z.

BACKGROUND: Methane is the primary component of natural gas and biogas. The huge abundance of methane makes it a promising alternative carbon source for industrial biotechnology. Herein, we report diamine compound, putrescine, production from methane by an industrially promising methanotroph Methylomicrobium alcaliphilum 20Z. RESULTS: We conducted adaptive evolution to improve putrescine tolerance of M. alcaliphilum 20Z because putrescine highly inhibits the cell growth. The evolved strain 20ZE was able to grow in the presence of 400 mM of putrescine dihydrochloride. The expression of linear pathway ornithine decarboxylase genes from Escherichia coli and Methylosinus trichosporium OB3b allowed the engineered strain to produce putrescine. A higher putrescine titer of 12.44 mg/L was obtained in the strain 20ZE-pACO with ornithine decarboxylase from M. trichosporium OB3b. For elimination of the putrescine utilization pathway, spermidine synthase (MEALZ_3408) was knocked out, resulting in no spermidine formation in the strain 20ZES1-pACO with a putrescine titer of 18.43 mg/L. Next, a genome-scale metabolic model was applied to identify gene knockout strategies. Acetate kinase (MEALZ_2853) and subsequently lactate dehydrogenase (MEALZ_0534) were selected as knockout targets, and the deletion of these genes resulted in an improvement of the putrescine titer to 26.69 mg/L. Furthermore, the putrescine titer was improved to 39.04 mg/L by overexpression of key genes in the ornithine biosynthesis pathway under control of the pTac promoter. Finally, suitable nitrogen sources for growth of M. alcaliphilum 20Z and putrescine production were optimized with the supplement of 2 mM ammonium chloride to nitrate mineral salt medium, and this led to the production of 98.08 mg/L putrescine, almost eightfold higher than that from the initial strain. Transcriptome analysis of the engineered strains showed upregulation of most genes involved in methane assimilation, citric acid cycle, and ammonia assimilation in ammonia nitrate mineral salt medium, compared to nitrate mineral salt medium. CONCLUSIONS: The engineered M. alcaliphilum 20ZE4-pACO strain was able to produce putrescine up to 98.08 mg/L, almost eightfold higher than the initial strain. This study represents the bioconversion of methane to putrescine-a high value-added diamine compound.

Biological conversion of methane to chemicals and fuels: technical challenges and issues.

Methane is a promising next-generation carbon feedstock for industrial biotechnology due to its low price and huge availability. Biological conversion of methane to valuable products can mitigate methane-induced global warming as greenhouse gas. There have been challenges for the conversion of methane into various chemicals and fuels using engineered non-native hosts with synthetic methanotrophy or methanotrophs with the reconstruction of synthetic pathways for target products. Herein, we analyze the technical challenges and issues of potent methane bioconversion technology. Pros and cons of metabolic engineering of methanotrophs for methane bioconversion, and perspectives on the bioconversion of methane to chemicals and liquid fuels are discussed.