In this study, we developed a mechanism-assisted data-driven model to regulate substrate feedback to improve the production efficiency of sophorolipids (SLs). First, we used a variety of on-line biosensors to establish a multi-scale parameter detection system. We found that the production of SLs by fed-batch fermentation could be divided into three stages: a stage that was limited by cell production capacity, a stage that was inhibited by high product concentration, and a stage that was limited by oxygen supply. Subsequently, we used process parameters to develop a data-driven model, and this was then combined with the analysis of cell metabolic mechanisms. The optimal production of SLs was achieved in the first and second stages by the precise feedback regulation of substrate feeding, which increased the titer of SLs by 4.9%. The control error of the substrate was reduced from more than 15% to less than 5%. The mechanism-assisted data-driven model was then applied for semi-continuous fermentation during the production of SLs. This effectively alleviated the oxygen limitation during the third stage, and further increased the productivity of SLs to 2.30 g/L/h, 40.2% higher than the fed-batch fermentation method.

In this study, we formulate a rapid, scalable and reproducible in vitro model of 3D MTS for drug screening purposes, and the cytotoxicity results showed an enhanced 5-FU resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture.In order to reveal the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. We found that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs was significant, mainly in the heat shock protein family, with the upregulation of protein folding function in endoplasmic reticulum, which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration.

Cervical cancer is a serious health problem in women around the globe, with 600 thousand new cases each year. However, the use of clinical drug is seriously dampened by the development of drug resistance, which has been evidenced to be associated with metabolic reprogramming and heterogeneity in tumor cells. Efficient in vitro tumor model is essential to improve the efficiency of drug screening and the accuracy of clinical application. Multicellular tumor spheroids (MTSs) can in a way recapitulates tumor traits in vivo, thereby representing a powerful transitional model between 2D monolayer culture and xenograft. In this study, based on the liquid overlay method, a protocol for rapid generation of the MTSs with uniform size and high reproducibility in a high-throughput manner was established. As expected, the cytotoxicity results showed that there was enhanced 5-FU resistance of HeLa carcinoma cells in 3D MTSs than 2D monolayer culture with a resistance index of 5.72. In the presence of both glucose and glutamine, HeLa carcinoma cells preferentially used glutamine as a bioenergetic substrate to support cell proliferation and maintenance under all conditions, while the ubiquitous by-product ammonium might be only recycled in the 3D MTSs. Furthermore, in order to obtain a holistic view of the molecular mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a multi-omics study was applied to discover hidden biological regularities. We found that in the 3D MTSs mitochondrial function-related proteins and the metabolites of the tricarboxylic acid cycle (TCA cycle) were significantly decreased, and the cellular metabolism was shifted towards glycolysis. The differences in the protein synthesis, processing, and transportation between 2D monolayer cultures and 3D MTSs was significant, mainly in the heat shock protein family, with the upregulation of protein folding function in endoplasmic reticulum (ER) which promoted the maintenance of ER homeostasis in the 3D MTSs. In addition, at the transcript and protein level, the expression of extracellular matrix (ECM) proteins (e.g., laminin and collagen) were up-regulated in the 3D MTSs, which enhanced the physical barrier of drug penetration. Summarizing, this study formulates a rapid, scalable and reproducible in vitro model of MTS for drug screening purposes, and the findings establish a critical role of glycolytic metabolism, ER hemostasis and ECM proteins expression profiling in tumor chemoresistance of HeLa carcinoma cells towards 5-FU.

Limitations in mixing and mass transfer coupled with high hydrostatic pressures lead to significant spatial variations in dissolved oxygen (DO) concentrations in large-scale bioreactors. While traveling through different zones in the bioreactor, microbes are subjected to fluctuating DO conditions at the timescales of global circulation time. In this study, to mimic industrial-scale spatial DOT gradients, we present a scale-down model based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose-limited chemostat cultures of Penicillium chrysogenum. The results revealed that the exposure time to the low DO level (less than 10%) imposed a significant impact on the biomass growth and penicillin productivity was considerably reduced by a factor of two, while the averaged substrate consumption rates were comparable under the DO oscillation condition compared to that of 60% DO steady-state condition. Quantitative metabolomics data showed that the DO feast/famine induced a stable and repetitive pattern with a reproducible metabolic response in time. The dynamic response of intracellular metabolites under such DO oscillating conditions showed specific differences in comparison to repetitive substrate pulse experiments. Due to invariable the specific glucose uptake rate ( q s ) during a cycle, the variation in the intracellular pools size of amino acids, sugar phosphates and organic acids was less pronounced in terms of both coverage and magnitude under DO fluctuations than under repetitive substrate pulses featured with a marked variation in the q s . Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites to reserve carbon source and reducing equivalent, which likely provide short-term benefits in such changing environments. Furthermore, the calculated cytosolic NADH/NAD + ratio under the DO oscillating condition indicated a dynamic and higher redox state of the cytosol, which has been reported to negatively affect the maintenance of penicillin productivity. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased ATP supply. From an economical point of view, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho-hydroxyphenyl acetic acid (ο-OH-PAA) and degeneration of penicillin productivity induced by low extracellular glucose sensing. Furthermore, dynamic metabolic flux analysis based on constraining time-resolved metabolite data into genome-scale metabolic model showed that Penicillium chrysogenum metabolism shifted from penicillin production to maintaining biomass growth upon a reduction of oxygen supply. The relative decreasing fluxes of amino acid metabolic pathways and fatty acid biosynthetic pathways were assumed to relieve the energy demand for balanced cellular metabolism. Taken together, the metabolic responses of Penicillium chrysogenum to DOT gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale-up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for Hela carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (80% methanol, methanol: chloroform: water (1:1:1, v/v/v), 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent Hela carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids, adenosine nucleotides and coenzymes involved in central carbon metabolism. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile was found to be the most optimal method to acquire intracellular metabolites with minimal loss during sample preparation. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment.

Multi-parameters of physiological metabolism, environmental state, cell morphology, and manipulated variable, have mutual influences and constraints on coenzyme Q 10 (CoQ 10) biosynthesis of Rhodobater sphaeroides. In this work, a multi-parameter fusional correlation-analysis were applied to illuminate and optimize the crucial metabolism on CoQ 10 biosynthesis. The quantitative response model for accurate living cells concentration, morphological deformation, specific oxygen consumption rate (Q O2), and specific product biosynthesis rate were established, the results revealed that maintain the optimal electrical conductivity(Cond.) and Q O2 at 9.0±0.5 mS/cm and 0.06-0.08±0.01×10 -7mmol/cell/h, respectively, by adjusting the nutrient feeding and oxygen transfer rate, the cell’s morphology and oxygen uptake rate were strengthened. CoQ 10 production in 50L bioreactors was successfully improved by 35% to 3384mg/L than that of control, which would be more powerful and effective for scale up in the large-scale production of CoQ 10.

The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell-specific production rate (q p) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature-induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature-induced cell metabolism, we systematically assessed the differences in cell growth, antibody expression, and antibody quality between high-producing (HP) and low-producing (LP) CHO cell lines under both constant temperature (37°C) and temperature downshift (37°C→33°C) settings during fed-batch culture. Although the results showed that low-temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p<0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift leaded to a higher cellular viability, and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p<0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra- and intra-cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated TCA cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress-alleviating strategies. To experimentally address this, we developed two high-performance fluorescent biosensors, denoted SoNar and iNap1, for real-time monitoring of intracellular NAD +/NADH ratio and NADPH amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD +/NADH ratio, which might be ascribed to the re-consumption of lactate, and increased the intracellular NADPH amount (P<0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high-level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real-time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes.

The ‘design-build-test-learn’ (DBTL) cycle has been adopted in rational high-throughput screening for obtaining 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 non-invasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. Firstly, a self-made tool was established to obtain datasets in 24-well ​based on the coloring of cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with 98.5% for the training and 91.3% for verification. Finally, a stable genetic high-yield strain (998U/mL) was successfully screened out in 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new datasets were updated to the model database in order to improve learning ability of DBTL cycle.

Sophorolipids (SLs) are regarded as one of the most promising biosurfactants. However, high production costs are the main obstacle to extended SLs application. Semi-continuous fermentation, which is based on in-situ separation, is a promising technology for achieving high SLs productivity. In this study, the sedimentation mechanism of SLs was analyzed. The formation of a hydrophobic mixture of SLs and rapeseed oil was a key factor in sedimentation. And the hydrophobicity and density of the mixture determined SLs sedimentation rate. On this basis, ultrasonic enhanced sedimentation technology (UEST) was introduced, by which the sedimentation rates were increased by 46.9% to 485.4% with different ratio of rapeseed oil to SLs. UEST-assisted real-time in-situ separation and semi-continuous fermentation were performed. SLs productivity and yield were 2.15 g/L/h and 0.58 g/g, respectively, simultaneously the loss ratio of cells, glucose, and rapeseed oil were significantly reduced. This study provides the new horizon for optimization of the SLs fermentation process.

Sophorolipids (SLs) are regarded as one of the most promising biosurfactants. They have a low toxicity and are easily degradable without polluting the environment. However, high production costs are the main obstacle to extended SLs application. Semi-continuous fermentation is a promising technology for achieving high SLs productivity, which is based on in-situ separation. In this study, the sedimentation mechanism of SLs was analyzed. The formation of a hydrophobic mixture of SLs and oil was a key factor in sedimentation. The hydrophobicity and density of the mixture determined SLs sedimentation rate. According to the mechanism, ultrasonic enhanced sedimentation technology (UEST) was introduced, by which the sedimentation rates were increased by 46.9% to 485.4% with different Oil/SLs ratios. UEST-assisted real-time rational in-situ separation and semi-continuous fermentation were performed. We observed that SLs productivity and yield were 2.15 g/L and 0.58 g/g, whereas the loss ratio of cells, glucose, and oil was reduced by 68.2%, 16.2%, and 65.5%, respectively. In-situ SLs separation efficiency and rate were increased by 34.5% and 26.4%, respectively. This study provides the foundation and new horizon for the optimization of the SLs fermentation process.

A well known \emph{in silico} analysis in metabolic flux analysis is the structural identifiability analysis. It comes from the fact that some enrichment measurement sets cannot uniquely elucidate all intracellular fluxes. To the best of our knowledge, the structural identifiability analysis of dynamic isotope experiments is not available in the literature. In this work, it is shown that if one measurement plan makes the dynamic isotopic fractions balance equations structurally identifiable then for any arbitrary small time interval the plan also makes the equations structurally identifiable. Based on the fact, in order to resolve the local structural identifiablity problem of the dynamic isotopic fractions balance equations approximated with piecewise affine intracellular fluxes, one should check the local structural identifiablity for the corresponding cascaded linear time invariant system at each sampling point with the approach proposed in our earlier work (Lin \emph{et al.}, Math Biosci. 2018; 300:122-12).

A well known issue in metabolic flux analysis with labelling experiments is the structural identifiability analysis. It comes from the fact that some enrichment measurement sets cannot uniquely elucidate all intracellular fluxes. To the best of our knowledge, the structural identifiability analysis of dynamic isotope experiments is not available in the literature. In this work, it is shown that if one measurement plan makes the dynamic isotopic fractions balance equations structurally identifiable then for any arbitrary small time interval the plan also makes the equations structurally identifiable. Based on the fact, in order to resolve the local structural identifiablity problem of the dynamic isotopic fractions balance equations approximated with piecewise affine intracellular fluxes, one should check the local structural identifiablity for the corresponding cascaded linear time invariant system at each sampling point with the approach proposed in our earlier work (Lin \emph{et al.}, Math Biosci. 2018; 300:122-129).