Uncovering the Role of Hydroxycinnamoyl Transferase in Boosting Chlorogenic Acid Accumulation in Carthamus tinctorius Cells under Methyl Jasmonate Elicitation.

Chlorogenic acids (CGAs) are bioactive compounds widely used in the food, pharmaceutical, and cosmetic industries. Carthamus tinctorius is an important economic crop, and its suspension cells are rich in CGAs. However, little is known about the biosynthesis and regulation of CGAs in Carthamus tinctorius cells. This study first elucidated the regulatory mechanism of CGA biosynthesis in methyl jasmonate (MeJA)-treated Carthamus tinctorius cells and the role of the MeJA-responsive hydroxycinnamoyl transferase (HCT) gene in enhancing their CGA accumulation. Firstly, temporal changes in intracellular metabolites showed that MeJA increased the intracellular CGA content up to 1.61-fold to 100.23 mg·g-1. Meanwhile, 31 primary metabolites showed significant differences, with 6 precursors related to increasing CGA biosynthesis. Secondly, the transcriptome data revealed 3637 new genes previously unannotated in the Carthamus tinctorius genome and 3653 differentially expressed genes. The genes involved in the plant signaling pathway and the biosynthesis of CGAs and their precursors showed a general up-regulation, especially the HCT gene family, which ultimately promoted CGA biosynthesis. Thirdly, the expression of a newly annotated and MeJA-responsive HCT gene (CtHCT, CtNewGene_3476) was demonstrated to be positively correlated with CGA accumulation in the cells, and transient overexpression of CtHCT enhanced CGA accumulation in tobacco. Finally, in vitro catalysis kinetics and molecular docking simulations revealed the ability and mechanism of the CtHCT protein to bind to various substrates and catalyze the formation of four hydroxycinnamic esters, including CGAs. These findings strengthened our understanding of the regulatory mechanism of CGA biosynthesis, thereby providing theoretical support for the efficient production of CGAs.

Temporal dynamics of stress response in Halomonas elongata to NaCl shock: physiological, metabolomic, and transcriptomic insights.

BACKGROUND: The halophilic bacterium Halomonas elongata is an industrially important strain for ectoine production, with high value and intense research focus. While existing studies primarily delve into the adaptive mechanisms of this bacterium under fixed salt concentrations, there is a notable dearth of attention regarding its response to fluctuating saline environments. Consequently, the stress response of H. elongata to salt shock remains inadequately understood. RESULTS: This study investigated the stress response mechanism of H. elongata when exposed to NaCl shock at short- and long-time scales. Results showed that NaCl shock induced two major stresses, namely osmotic stress and oxidative stress. In response to the former, within the cell's tolerable range (1-8% NaCl shock), H. elongata urgently balanced the surging osmotic pressure by uptaking sodium and potassium ions and augmenting intracellular amino acid pools, particularly glutamate and glutamine. However, ectoine content started to increase until 20 min post-shock, rapidly becoming the dominant osmoprotectant, and reaching the maximum productivity (1450 ± 99 mg/L/h). Transcriptomic data also confirmed the delayed response in ectoine biosynthesis, and we speculate that this might be attributed to an intracellular energy crisis caused by NaCl shock. In response to oxidative stress, transcription factor cysB was significantly upregulated, positively regulating the sulfur metabolism and cysteine biosynthesis. Furthermore, the upregulation of the crucial peroxidase gene (HELO_RS18165) and the simultaneous enhancement of peroxidase (POD) and catalase (CAT) activities collectively constitute the antioxidant defense in H. elongata following shock. When exceeding the tolerance threshold of H. elongata (1-13% NaCl shock), the sustained compromised energy status, resulting from the pronounced inhibition of the respiratory chain and ATP synthase, may be a crucial factor leading to the stagnation of both cell growth and ectoine biosynthesis. CONCLUSIONS: This study conducted a comprehensive analysis of H. elongata's stress response to NaCl shock at multiple scales. It extends the understanding of stress response of halophilic bacteria to NaCl shock and provides promising theoretical insights to guide future improvements in optimizing industrial ectoine production.

Mechanistic insight for improving butenyl-spinosyn production through combined ARTP/UV mutagenesis and ribosome engineering in Saccharopolyspora pogona.

Butenyl-spinosyn is a highly effective, wide-spectrum and environmentally-friendly biological insecticide produced by Saccharopolyspora pogona. However, its scale-up is impeded due to its lower titer in wild-type strains. In this work, ARTP/UV mutagenesis and ribosome engineering were employed to enhance the butenyl-spinosyn production, and a stable mutant Saccharopolyspora pogona aG6 with high butenyl-spinosyn yield was successfully obtained. For the first time, the fermentation results in the 5 L bioreactor demonstrated that the butenyl-spinosyn produced by mutant Saccharopolyspora pogona aG6 reached the maximum value of 130 mg/L, almost 4-fold increase over the wild-type strain WT. Furthermore, comparative genomic, transcriptome and target metabolomic analysis revealed that the accumulation of butenyl-spinosyn was promoted by alterations in ribosomal proteins, branched-chain amino acid degradation and oxidative phosphorylation. Conclusively, the proposed model of ribosome engineering combined with ARTP/UV showed the improved biosynthesis regulation of butenyl-spinosyn in S. pogona.

An Efficient High-Throughput Screening of High Gentamicin-Producing Mutants Based on Titer Determination Using an Integrated Computer-Aided Vision Technology and Machine Learning.

The "design-build-test-learn" (DBTL) cycle has been adopted in rational high-throughput screening to obtain high-yield industrial strains. However, the mismatch between build and test slows the DBTL cycle due to the lack of high-throughput analytical technologies. In this study, a highly efficient, accurate, and noninvasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. First, a self-made tool was established to obtain data sets in 24-well plates based on the color of the cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with an R2 value of 0.98430 for the same batch. Finally, a stable genetically high-yield strain (998 U/mL) was successfully screened out from 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new data sets were updated on the model database in order to improve the learning ability of the DBTL cycle.

Advances in sustainable approaches utilizing orange peel waste to produce highly value-added bioproducts.

Orange peel waste (OPW), a discarded part of orange fruit, is a rich source of essential constituents that can be transformed into highly value-added bioproducts. OPW is being generated in million tonnes globally and returns to the environment without complete benefit. Thus, a high volume of annually produced OPW in the industry requires effective valorization. In this regard, limited data is available that summarizes the broader spectrum for the sustainable fate of OPW to produce value-added bioproducts. The main objective of this treatise is to explore the sustainable production of bioproducts from OPW. Therefore, this review covers all the aspects of OPW, from its production to complete valorization. The review encompasses the extraction technologies employed for extracting different valuable bioactive compounds, such as: essential oil (EO), pectin, and carotenoids, from OPW. Furthermore, the suitability of bioconversion technologies (digestion/fermentation) in transforming OPW to other useful bioproducts, such as: biochemicals (lactic acid and succinic acid), biopolysaccharides (xanthan and curdlan gum), and bioenergy (biomethane and bioethanol) is discussed. Also, it includes the concept of OPW-based biorefineries and their development that shall play a definite role in future to cover demands for: food, chemicals, materials, fuels, power, and heat. Lastly, this review focuses on OPW-supplemented functional food products such as: beverages, yogurts, and extruded products. In conclusion, insights provided in this review maximize the potential of OPW for commercial purposes, leading to a safe, and waste-free environment.

Development of a novel noninvasive quantitative method to monitor Siraitia grosvenorii cell growth and browning degree using an integrated computer-aided vision technology and machine learning.

The rapid, accurate and noninvasive detection of biomass and plant cell browning can provide timely feedback on cell growth in plant cell culture. In this study, Siraitia grosvenorii suspension cells were taken as an example, a phenotype analysis platform was successfully developed to predict the biomass and the degree of cell browning based on the color changes of cells in computer-aided vision technology. First, a self-made laboratory system was established to obtain images. Then, matrices were prepared from digital images by a self-developed high-throughput image processing tool. Finally, classification models were used to judge different cell types, and then a semi-supervised classification to predict different degrees of cell browning. Meanwhile, regression models were developed to predict the plant cell mass. All models were verified with a good agreement by biological experiments. Therefore, this method can be applied for low-cost biomass estimation and browning degree quantification in plant cell culture.

Enhanced Biosynthesis of Chlorogenic Acid and Its Derivatives in Methyl-Jasmonate-Treated Gardenia jasminoides Cells: A Study on Metabolic and Transcriptional Responses of Cells.

Chlorogenic acid and its derivatives (CQAs) are considered as important bioactive secondary metabolites in Gardenia jasminoides Ellis (G. jasminoides). However, few studies have investigated the biosynthesis and regulation of CQAs in G. jasminoides. In this study, methyl jasmonate (MeJA) was used to enhance CQAs accumulation in cultured G. jasminoides cells. Moreover, the possible molecular mechanism of MeJA-mediated accumulation of CQAs is also explored. To this end, time-course transcriptional profiles of G. jasminoides cells responding to MeJA were used to investigate the mechanism from different aspects, including jasmonate (JAs) biosynthesis, signal transduction, biosynthesis of precursor, CQAs biosynthesis, transporters, and transcription factors (TFs). A total of 57,069 unigenes were assembled from the clean reads, in which 80.7% unigenes were successfully annotated. Furthermore, comparative transcriptomic results indicated that differentially expressed genes (DEGs) were mainly involved in JAs biosynthesis and signal transduction (25 DEGs), biosynthesis of precursor for CQAs (18 DEGs), CQAs biosynthesis (19 DEGs), and transporters (29 DEGs). Most of these DEGs showed continuously upregulated expressions over time, which might activate the jasmonic acid (JA) signal transduction network, boost precursor supply, and ultimately stimulate CQAs biosynthesis. Additionally, various TFs from different TF families also responded to MeJA elicitation. Interestingly, 38 DEGs from different subgroups of the MYB family might display positive or negative regulations on phenylpropanoids, especially on CQAs biosynthesis. Conclusively, our results provide insight into the possible molecular mechanism of regulation on CQAs biosynthesis, which led to a high CQAs yield in the G. jasminoides cells under MeJA treatment.