Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA.

Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTB mRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.

Yeast increases glycolytic flux to support higher growth rates accompanied by decreased metabolite regulation and lower protein phosphorylation.

Supply of Gibbs free energy and precursors are vital for cellular function and cell metabolism have evolved to be tightly regulated to balance their supply and consumption. Precursors and Gibbs free energy are generated in the central carbon metabolism (CCM), and fluxes through these pathways are precisely regulated. However, how fluxes through CCM pathways are affected by posttranslational modification and allosteric regulation remains poorly understood. Here, we integrated multi-omics data collected under nine different chemostat conditions to explore how fluxes in the CCM are regulated in the yeast Saccharomyces cerevisiae. We deduced a pathway- and metabolism-specific CCM flux regulation mechanism using hierarchical analysis combined with mathematical modeling. We found that increased glycolytic flux associated with an increased specific growth rate was accompanied by a decrease in flux regulation by metabolite concentrations, including the concentration of allosteric effectors, and a decrease in the phosphorylation level of glycolytic enzymes.

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.

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.

Investigation and development of transient production process for porcine circovirus Type-2 (PCV2) capsid protein in HEK293F cells.

Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model "difficult-to-express" virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90ß, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other "difficult-to-express" virus capsid proteins in the mammalian cell system.

Construction and testing of Yarrowia lipolytica recombinant protein expression chassis cells based on the high-throughput screening and secretome.

BACKGROUND: In the recombinant protein market with broad economic value, the rapid development of synthetic biology has made it necessary to construct an efficient exocrine expression system for the different heterologous proteins. Yarrowia lipolytica possesses unique advantages in nascent protein transport and glycosylation modification, so it can serve as a potential protein expression platform. Although the Po1 series derived from W29 is often used for the expression of the various heterologous proteins, the ability of W29 to secrete proteins has not been verified and the Po1 series has been found to be not convenient for further gene editing. RESULTS: A total of 246 Y. lipolytica strains were evaluated for their secretory capacity through performing high-throughput screening in 48-well plate. Thereafter, following two rounds of shake flask re-screening, a high-secreting protein starting strain DBVPG 5851 was obtained. Subsequently, combined with the extracellular protein types and relative abundance information provided by the secretome of the starting strain, available chassis cell for heterologous protein expression were preliminarily constructed, and it was observed that the most potential signal peptide was derived from YALI0D20680g. CONCLUSIONS: This study offers a novel perspective on the diversification of Y. lipolytica host cells for the heterologous protein expression and provides significant basis for expanding the selection space of signal peptide tools in the future research.

Micro-electrochemical DO sensor with ultra-micropore matrix fabricated with femtosecond laser processing successfully applied in on-line DO monitoring for yeast culture.

Accurate monitoring of dissolved oxygen (DO) is vital for aerobic fermentation process control. This work presents an autoclavable Micro-Dissolved oxygen Sensor (MDS) that can monitor real time DO. The proposed sensor is much cheaper to be manufactured (< $35) and can be adapted to varying measurement environments. An ultra-micropore matrix was created using femtosecond laser processing technology to reduce flow dependency of probe signals. The validity of the proposed DO sensor was verified by testing it under different DO levels. The result revealed consistency between the new designed sensor and a commercial DO sensor. The obtained sensitivity was- 7.93 µA·L·mg-1 (MDS with ultra-micropore matrix). Moreover, the MDS can function without an oxygen-permeable membrane and a solid electrolyte was used which reduced the response time (4.6 s). For real-time monitoring, the stability of the MDS was validated during a yeast batch fermentation carried out until 18 h.

Multiple feature fusion transformer for modeling penicillin fermentation process with unequal sampling intervals.

The quality prediction of batch processes is an important task in the field of biological fermentation. However, dynamic nonlinearity, unequal sampling intervals, uneven duration, and multiple features of a batch process make this task challenging. Thus, the multiple-feature fusion transformer (MFFT) model is proposed for the time series quality prediction of a batch process. First, the application of sequence-to-sequence architecture enables MFFT to perform a wide range of sequence prediction tasks. Second, the transformer parallel operation model imposes no rigid requirement for the order of sequence input, allowing the model to deal with problems of unequal interval sampling and utilize the sequence information. Third, MFFT integrates a pretrained ResNet50 as a mycelium status classifier for fusing image information into the features. Moreover, a multiple-feature encoding structure is proposed to integrate sampling time and mycelium status. Finally, multiple tasks in penicillin fermentation have shown that MFFT significantly outperforms existing methods for time series prediction.

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.

Physiological metabolic topology analysis of Halomonas elongata DSM 2581T in response to sodium chloride stress.

The halophilic bacterium Halomonas elongata DSM 2581T generally adapts well to high level of salinity by biosynthesizing ectoine, which functions as an important compatible solute protecting the cell against external salinity environment. Halophilic bacteria have specific metabolic activities under high-salt conditions and are gradually applied in various industries. The present study focuses on investigating the physiological and metabolic mechanism of H. elongata DSM 2581T driven by the external salinity environment. The physiological metabolic dynamics under salt stress were investigated to evaluate the effect of NaCl stress on the metabolism of H. elongata. The obtained results demonstrated that ectoine biosynthesis transited from a nongrowth-related process to a growth-related process when the NaCl concentration varied from 1% to 13% (w/v). The maximum biomass (Xm = 41.37 g/L), and highest ectoine production (Pm = 12.91 g/L) were achieved under 8% NaCl. Moreover, the maximum biomass (Xm ) and the maximum specific growth rates (µm ) showed a first rising and then declining trend with the increased NaCl stress. Furthermore, the transcriptome analysis of H. elongata under different NaCl concentrations demonstrated that both 8% and 13% NaCl conditions resulted in increased expressions of genes involved in the pentose phosphate pathway, Entner-Doudoroff pathway, flagellar assembly pathway, and ectoine metabolism, but negatively affected the tricarboxylic acid cycle and fatty acid metabolism. At last, the proposed possible adaptation mechanism under the optimum NaCl concentration in H. elongata was described.

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger.

BACKGROUND: Our recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction. RESULTS: Based on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction. CONCLUSION: This study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

Urchin-like magnetic microspheres for cancer therapy through synergistic effect of mechanical force, photothermal and photodynamic effects.

BACKGROUND: Magnetic materials mediated by mechanical forces to combat cancer cells are currently attracting attention. Firstly, the magnetic force penetrates deeper into tissues than the NIR laser alone to destroy tumours. Secondly, the synergistic effect of nano-magnetic-material characteristics results in a viable option for the targeted killing of cancer cells. Therefore, mechanical force (MF) produced by magnetic nanomaterials under low frequency dynamic magnetic field combined with laser technology is the most effective, safe and efficient tool for killing cancer cells and tumour growth. RESULTS: In this study, we synthesized novel urchin-like hollow magnetic microspheres (UHMMs) composed of superparamagnetic Fe3O4. We demonstrated the excellent performance of UHMMs for killing laryngocarcinoma cancer cells through mechanical force and photothermal effects under a vibrating magnetic field and near-infrared laser, respectively. The killing efficiency was further improved after loading the synthesised UHMMs with Chlorin e6 relative to unloaded UHMMs. Additionally, in animal experiments, laryngocarcinoma solid tumour growth was effectively inhibited by UHMMs@Ce6 through magneto-mechanic force, photothermal and photodynamic therapy. CONCLUSIONS: The biocompatibility and high efficiency of multimodal integrated therapy with the UHMMs prepared in this work provide new insights for developing novel nano therapy and drug loading platforms for tumour treatment. In vivo experiments further demonstrated that UHMMs/Ce6 are excellent tools for strongly inhibiting tumour growth through the above-mentioned characteristic effects.

Effect of temperature on synthesis of clavulanic acid and impurity substance G during fermentation by Streptomyces clavuligerus.

Effect of temperature on synthesis of Clavulanic acid (CA) and impurity substance G during fermentation by Streptomyces clavuligerus were investigated. Results show that fermentation at 24 °C is the most favorable for CA synthesis though the fermentation duration was 20-30 hours longer than fermentation at 26 and 28 °C. Meanwhile, the impurity substance G was only 110 mg/L in the end broth of fermentation at 24 °C, which was significantly lower than 148 and 180 mg/L of fermentation at 26 and 28 °C, respectively. Correlation of specific growth rate and CA synthesis was statistically analyzed based on data of 10 batches of industrial fermentation. Two temperature-shift strategies were investigated in 50 L fermenter. Fermentation with 26-24 °C temperature strategy achieved 5097 mg/L CA titer, meanwhile the fermentation duration was shortened 24 hours comparing with fermentation at constant 24 °C. Fermentation with 26-24 °C control strategy was validated in a 60 m3 industrial fermenter, in which 4960 mg/L of CA was achieved while impurity G substance was decreased to titer 65 mg/L from 200 to 300 mg/L of normal production.

Dynamic response of Aspergillus niger to periodical glucose pulse stimuli in chemostat cultures.

In industrial large-scale bioreactors, microorganisms encounter heterogeneous substrate concentration conditions, which can impact growth or product formation. Here we carried out an extended (12 h) experiment of repeated glucose pulsing with a 10-min period to simulate fluctuating glucose concentrations with Aspergillus niger producing glucoamylase, and investigated its dynamic response by rapid sampling and quantitative metabolomics. The 10-min period represents worst-case conditions, as in industrial bioreactors the average cycling duration is usually in the order of 1 min. We found that cell growth and the glucoamylase productivity were not significantly affected, despite striking metabolomic dynamics. Periodical dynamic responses were found across all central carbon metabolism pathways, with different time scales, and the frequently reported ATP paradox was confirmed for this A. niger strain under the dynamic conditions. A thermodynamics analysis revealed that several reactions of the central carbon metabolism remained in equilibrium even under periodical dynamic conditions. The dynamic response profiles of the intracellular metabolites did not change during the pulse exposure, showing no significant adaptation of the strain to the more than 60 perturbation cycles applied. The apparent high tolerance of the glucoamylase producing A. niger strain for extreme variations in the glucose availability presents valuable information for the design of robust industrial microbial hosts.

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.

Integration of enzyme constraints in a genome-scale metabolic model of Aspergillus niger improves phenotype predictions.

BACKGROUND: Genome-scale metabolic model (GSMM) is a powerful tool for the study of cellular metabolic characteristics. With the development of multi-omics measurement techniques in recent years, new methods that integrating multi-omics data into the GSMM show promising effects on the predicted results. It does not only improve the accuracy of phenotype prediction but also enhances the reliability of the model for simulating complex biochemical phenomena, which can promote theoretical breakthroughs for specific gene target identification or better understanding the cell metabolism on the system level. RESULTS: Based on the basic GSMM model iHL1210 of Aspergillus niger, we integrated large-scale enzyme kinetics and proteomics data to establish a GSMM based on enzyme constraints, termed a GEM with Enzymatic Constraints using Kinetic and Omics data (GECKO). The results show that enzyme constraints effectively improve the model's phenotype prediction ability, and extended the model's potential to guide target gene identification through predicting metabolic phenotype changes of A. niger by simulating gene knockout. In addition, enzyme constraints significantly reduced the solution space of the model, i.e., flux variability over 40.10% metabolic reactions were significantly reduced. The new model showed also versatility in other aspects, like estimating large-scale [Formula: see text] values, predicting the differential expression of enzymes under different growth conditions. CONCLUSIONS: This study shows that incorporating enzymes' abundance information into GSMM is very effective for improving model performance with A. niger. Enzyme-constrained model can be used as a powerful tool for predicting the metabolic phenotype of A. niger by incorporating proteome data. In the foreseeable future, with the fast development of measurement techniques, and more precise and rich proteomics quantitative data being obtained for A. niger, the enzyme-constrained GSMM model will show greater application space on the system level.

A new strategy for dynamic metabolic flux estimation by integrating transient metabolome data into genome-scale metabolic models.

Metabolic flux analysis (MFA) is a powerful tool for studying microbial cell physiology. Isotope-based MFA is the accepted standard for studying metabolic fluxes under steady-state conditions. However, its application under dynamic extracellular conditions is limited due to lack of proper techniques, such as rapid sampling and quenching, high cost and laborious execution. Here, we propose a new strategy to tackle this through incorporating dynamic metabolite abundance data into genome-scale metabolic models (GEM). First, a dummy extracellular pool concept is proposed for each dynamically changing metabolite, which represents a "sink" or "source", with corresponding dummy reactions coded into the GEM model. The dynamic model (expressed as differential equations) is then transformed into a quasi-steady-state model (expressed as linear equations), which can be easily solved by constraining the GEM model with the dynamic metabolite quantification data. For this, common linear-programming optimization algorithms were utilized to estimate the dynamic fluxes. Dynamic high-accuracy metabolite abundance data were obtained through the Isotope Dilution Mass Spectrometry (IDMS) method and high-speed sampling-quenching, and it was demonstrated that the newly proposed strategy could be successfully applied to obtain intracellular dynamic fluxes of Aspergillus niger under regimes of single and periodic extracellular glucose pulses. The applicability of the new method was also tested on dynamic fluxes estimation in a glucose pulse-response study of Saccharomyces cerevisiae. The proposed method provides a powerful tool to investigate cell physiology under dynamic conditions, especially relevant for bioprocess scale-up to industrial-scale bioreactors.

Coupled metabolic-hydrodynamic modeling enabling rational scale-up of industrial bioprocesses.

Metabolomics aims to address what and how regulatory mechanisms are coordinated to achieve flux optimality, different metabolic objectives as well as appropriate adaptations to dynamic nutrient availability. Recent decades have witnessed that the integration of metabolomics and fluxomics within the goal of synthetic biology has arrived at generating the desired bioproducts with improved bioconversion efficiency. Absolute metabolite quantification by isotope dilution mass spectrometry represents a functional readout of cellular biochemistry and contributes to the establishment of metabolic (structured) models required in systems metabolic engineering. In industrial practices, population heterogeneity arising from fluctuating nutrient availability frequently leads to performance losses, that is reduced commercial metrics (titer, rate, and yield). Hence, the development of more stable producers and more predictable bioprocesses can benefit from a quantitative understanding of spatial and temporal cell-to-cell heterogeneity within industrial bioprocesses. Quantitative metabolomics analysis and metabolic modeling applied in computational fluid dynamics (CFD)-assisted scale-down simulators that mimic industrial heterogeneity such as fluctuations in nutrients, dissolved gases, and other stresses can procure informative clues for coping with issues during bioprocessing scale-up. In previous studies, only limited insights into the hydrodynamic conditions inside the industrial-scale bioreactor have been obtained, which makes case-by-case scale-up far from straightforward. Tracking the flow paths of cells circulating in large-scale bioreactors is a highly valuable tool for evaluating cellular performance in production tanks. The "lifelines" or "trajectories" of cells in industrial-scale bioreactors can be captured using Euler-Lagrange CFD simulation. This novel methodology can be further coupled with metabolic (structured) models to provide not only a statistical analysis of cell lifelines triggered by the environmental fluctuations but also a global assessment of the metabolic response to heterogeneity inside an industrial bioreactor. For the future, the industrial design should be dependent on the computational framework, and this integration work will allow bioprocess scale-up to the industrial scale with an end in mind.

Improving cytosolic aspartate biosynthesis increases glucoamylase production in Aspergillus niger under oxygen limitation.

BACKGROUND: Glucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce. RESULTS: In the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids (alanine, glutamate, glycine and aspartate) as the limited precursors for glucoamylase production in A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids (An11g02620, An04g00990, An05g00410, An04g06380 and An16g05570). Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5 L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased. CONCLUSION: Our study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains.