High cell density cultivation of Corynebacterium glutamicum by deep learning-assisted medium design and the subsequent feeding strategy.

To improve the cell productivity of Corynebacterium glutamicum, its initial specific growth rate was improved by medium improvement using deep neural network (DNN)-assisted design with Bayesian optimization (BO) and a genetic algorithm (GA). To obtain training data for the DNN, experimental design with an orthogonal array was set up using a chemically defined basal medium (GC XII). Based on the cultivation results for the training data, specific growth rates were observed between 0.04 and 0.3/h. The resulting DNN model estimated the test data with high accuracy (R2test ≥ 0.98). According to the validation cultivation, specific growth rates in the optimal media components estimated by DNN-BO and DNN-GA increased from 0.242 to 0.355/h. Using the optimal media (UCB_3), the specific growth rate, along with other parameters, was evaluated in batch culture. The specific growth rate reached 0.371/h from 3 to 12 h, and the dry cell weight was 28.0 g/L at 22.5 h. From the cultivation, the cell yields against glucose, ammonium ion, phosphate ion, sulfate ion, potassium ion, and magnesium ion were calculated. The cell yield calculation was used to estimate the required amounts of each component, and magnesium was found to limit the cell growth. However, in the follow-up fed-batch cultivation, glucose and magnesium addition was required to achieve the high initial specific growth rate, while appropriate feeding of glucose and magnesium during cultivation resulted in maintaining the high specific growth rate, and obtaining a cell yield of 80 g/Lini.

A novel sulfur supply strategy for maximizing lipid production in Tribonema minus (Xanthophyceae).

Tribonema minus, a promising filamentous oleaginous microalga, was cultured under different nutrient concentrations and different culture modes (fed-batch culture, two-step culture) to study the method of rapid regulation of its lipid metabolism. In contrast to many other oleaginous microalgae, T. minus did not show that nitrogen stress promoted lipid accumulation; however, sulfur deficiency promoted rapid lipid accumulation with a maximum lipid content of 54% of dry weight. Increasing the MgSO4 concentration significantly increased nitrogen uptake and biomass (10.09 g/L). Lipid productivity was significantly increased by the two-step culture using a medium with a high concentration of MgSO4 in the first step and a sulfur-free medium in the second step. In addition, it was found that the lipid content of T. minus was negatively correlated with the intracellular sulfur content when the intracellular sulfur content was below 0.6%. This study provides a new approach for industrial lipid production in T. minus.

Development of chemically defined media for Lactococcus lactis subsp. lactis YF11 to eliminate the influence of hyperosmotic stress.

Chemically defined media (CDM) can eliminate or lessen the interference that occurs in complex culture media (CCM) caused by the undefined substrate pools, and various CDM have been designed and employed for investigating microbial physiology and multiomics. Herein, using the measured amount of total amino acids in CCM and combined with the in vivo and in vitro amino acid content of Lactococcus lactis subsp. lactis YF11, new enriched CDM were designed and then optimized using a statistical design-of-experiment method coupling with fed-batch fermentation to eliminate or lessen the influence of hyperosmotic pressure. Cell volume was introduced as a target index to assess the performance of CDM, and average osmotic pressure (AOP) was employed to describe the osmotic pressure of CDM. The AOP was significantly decreased from 610 mOsm/kg·H2O in the initial CDM (I-CDM) to 360 mOsm/kg·H2O in fed-batch CDM (F-CDM), and the cell volume was increased from 0.142 ± 0.004 µm3 in I-CDM to 0.198 ± 0.008 µm3 in F-CDM, which was close to 0.206 ± 0.005 µm3 found in CCM, indicating that the strategy of designing and improving CDM followed by a statistical design-of-experiment coupling with fed-batch cultivation presented a promising pathway for extensive utilization of CDM. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03788-5.

Sugar Alcohol Sweetener Production by Yarrowia lipolytica Grown in Media Containing Glycerol.

Most of the world's annual production of mannitol is by chemical means, but, due to increasing demand for natural sweeteners, alternative production methods are being sought. The aim of the study was to screen Yarrowia lipolytica yeast strains and select culture conditions for the efficient and selective biosynthesis of mannitol from glycerol. From 21 strains examined in the shake-flask culture for mannitol biosynthesis from glycerol (100 g/L), three strains were selected-S2, S3, and S4-and further evaluated in batch bioreactor cultures with technical and raw glycerol (150 g/L). The best production parameters were observed for strain S3, which additionally was found to be the most resistant to NaCl concentration. Next, strain S3 was examined in batch culture with regard to the initial glycerol concentration (from 50 to 250 g/L). It was found that the substrate concentrations of 50 and 75 g/L resulted in the highest mannitol selectivity, about 70%. The fed-batch culture system proposed in this paper (performed in two variants in which glycerol was dosed in four portions of about 50 or 75 g/L) resulted in increased mannitol production, up to 78.5 g/L.

Analysis of the effects of specific growth rate of Lactococcus lactis MG1363 on aerobic metabolism and its application to high-density culture.

Lactic acid bacteria (LAB) are known to produce a large amount of lactate when cultured under non-aerated conditions, which inhibits their growth at high concentrations. Our previous studies have shown that LAB can be cultured without lactate production under aerated conditions at a low specific growth rate. In this study, we investigated the effects of specific growth rate on cell yield and the specific production rates of metabolites in aerated fed-batch cultures of Lactococcus lactis MG1363. The results showed that lactate and acetoin production could be suppressed at specific growth rates below 0.2 h-1, whereas acetate production was the highest at a specific growth rate of 0.2 h-1. When LAB was cultured at a specific growth rate of 0.25 h-1 with the addition of 5 mg/L heme to assist ATP production by respiration, lactate and acetate production was suppressed, and cell concentration reached 19 g-dry-cell/L (5.6 × 10ˆ10 cfu/mL) with a high cell yield of 0.42 ± 0.02 g-dry-cell/g-glucose.

Experimental studies from shake flasks to 3 L stirred tank bioreactor of nutrients and oxygen supply conditions to improve the growth of the avian cell line DuckCelt®-T17.

BACKGROUND: To produce viral vaccines, avian cell lines are interesting alternatives to replace the egg-derived processes for viruses that do not grow well on mammalian cells. The avian suspension cell line DuckCelt®-T17 was previously studied and investigated to produce a live attenuated metapneumovirus (hMPV)/respiratory syncytial virus (RSV) and influenza virus vaccines. However, a better understanding of its culture process is necessary for an efficient production of viral particles in bioreactors. RESULTS: The growth and metabolic requirements of the avian cell line DuckCelt®-T17 were investigated to improve its cultivation parameters. Several nutrient supplementation strategies were studied in shake flasks highlighting the interest of (i) replacing L-glutamine by glutamax as main nutrient or (ii) adding these two nutrients in the serum-free growth medium in a fed-batch strategy. The scale-up in a 3 L bioreactor was successful for these types of strategies confirming their efficiencies in improving the cells' growth and viability. Moreover, a perfusion feasibility test allowed to achieve up to ~ 3 times the maximum number of viable cells obtained with the batch or fed-batch strategies. Finally, a strong oxygen supply - 50% dO2 - had a deleterious effect on DuckCelt®-T17 viability, certainly because of the greater hydrodynamic stress imposed. CONCLUSIONS: The culture process using glutamax supplementation with a batch or a fed-batch strategy was successfully scaled-up to 3 L bioreactor. In addition, perfusion appeared as a very promising culture process for subsequent continuous virus harvesting.

Developing a single-stage continuous process strategy for vitamin B12 production with Propionibacterium freudenreichii.

BACKGROUND: Vitamin B12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. Besides, the use of Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) microorganisms, like Propionibacterium freudenreichii, especially non-GMO wild-type producers, are becoming an interesting alternative in markets where many final consumers have high health and ecological awareness. In this study, the production of vitamin B12 using the Propionibacterium freudenreichii NBRC 12391 wild-type strain was characterized and optimized in shake flasks before assessing several scale-up strategies. RESULTS: Initial results established that: (i) agitation during the early stages of the culture had an inhibitory effect on the volumetric production, (ii) 5,6-dimethylbenzimidazole (DMBI) addition was necessary for vitamin B12 production, and (iii) kinetics of vitamin B12 accumulation were dependent on the induction time when DMBI was added. When scaling up in a bioreactor, both batch and fed-batch bioprocesses proved unsuitable for obtaining high volumetric productivities mainly due to carbon source limitation and propionic acid inhibition, respectively. To overcome these drawbacks, an anaerobic single-phase continuous bioprocess strategy was developed. This culture strategy was maintained stable during more than 5 residence times in two independent cultures, resulting in 5.7-fold increase in terms of volumetric productivity compared to other scale-up strategies. CONCLUSION: Overall, compared to previously reported strategies aimed to reduce propionic acid inhibition, a less complex anaerobic single-phase continuous and more scalable bioprocess was achieved.

Progress in fed-batch culture for recombinant protein production in CHO cells.

Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: • Fed-batch culture in CHO cells is reviewed. • Fed-batch has become a common technology for recombinant protein production. • Fed batch culture promotes recombinant protein production in CHO cells.

Adaptive control of the E. coli-specific growth rate in fed-batch cultivation based on oxygen uptake rate.

In this study, an automatic control system is developed for the setpoint control of the cell biomass specific growth rate (SGR) in fed-batch cultivation processes. The feedback signal in the control system is obtained from the oxygen uptake rate (OUR) measurement-based SGR estimator. The OUR online measurements adapt the system controller to time-varying operating conditions. The developed approach of the PI controller adaptation is presented and discussed. The feasibility of the control system for tracking a desired biomass growth time profile is demonstrated with numerical simulations and fed-batch culture E.coli control experiments in a laboratory-scale bioreactor. The procedure was cross-validated with the open-loop digital twin SGR estimator, as well as with the adaptive control of the SGR, by tracking a desired setpoint time profile. The digital twin behavior statistically showed less of a bias when compared to SGR estimator performance. However, the adaptation-when using first principles-was outperformed 30 times by the model predictive controller in a robustness check scenario.

Recent developments in miRNA based recombinant protein expression in CHO.

It is widely accepted that the growing demand for recombinant therapeutic proteins has led to the expansion of the biopharmaceutical industry and the development of strategies to increase recombinant protein production in mammalian cell lines such as SP2/0 HEK and particularly Chinese hamster ovary cells. For a long time now, most investigations have been focused on increasing host cell productivity using genetic manipulating of cellular processes like cell cycle, apoptosis, cell growth, protein secretory and other pathways. In recent decades MicroRNAs beside different genetic engineering tools (e.g., TALEN, ZFN, and Crisper/Cas) have attracted further attention as a tool in the genetic engineering of host cells to increase protein expression levels. Their ability to simultaneously target multiple mRNAs involved in one or more cellular processes made them a favorable tool in this field. Accordingly, this study aimed to review the methods of selecting target miRNA for cell line engineering, miRNA gain- or loss-of-function strategies, examples of laboratory and pilot studies in this field and discussed advantages and disadvantages of this technology.

Heterologous expression of novel SUMO proteases from Schizosaccharomyces pombe in E. coli: Catalytic domain identification and optimization of product yields.

Small ubiquitin-related modifier (SUMO) proteins are efficiently used to target the soluble expression of various difficult-to-express proteins in E. coli. However, its utilization in large scale protein production is restricted by the higher cost of Ulp, which is required to cleave SUMO fusion tag from protein-of-interest to generate an authentic N-terminus. This study identified and characterized two novel SUMO proteases i.e., Ulp1 and Ulp2 from Schizosaccharomyces pombe. Codon-optimized gene sequences were cloned and expressed in E. coli. The sequence and structure of SpUlp1 and SpUlp2 catalytic domains were deduced using bioinformatics tools. Protein-protein interaction studies predicted the higher affinity of SpUlp1 towards SUMO compared to its counterpart from Saccharomyces cerevisiae (ScUlp1). The catalytic domain of SpUlp1 was purified using Ni-NTA chromatography with 83.33% recovery yield. Moreover, In vitro activity data further confirmed the fast-acting nature of SpUlp1 catalytic domain, where a 90% cleavage of fusion proteins was obtained within 1 h of incubation, indicating novelty and commercial relevance of S. pombe Ulp1. Biophysical characterization showed 8.8% α-helices, 36.7% ß-sheets in SpUlp1SD. From thermal CD and fluorescence data, SpUlp1SD Tm was found to be 45 °C. Further, bioprocess optimization using fed-batch cultivation resulted in 3.5 g/L of SpUlp1SD production with YP/X of 77.26 mg/g DCW and volumetric productivity of 205.88 mg/L/h.

Optimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers Using Recombinant Escherichia coli.

Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction.

Escherichia coli coculture for de novo production of esters derived of methyl-branched alcohols and multi-methyl branched fatty acids.

BACKGROUND: A broad diversity of natural and non-natural esters have now been made in bacteria, and in other microorganisms, as a result of original metabolic engineering approaches. However, the fact that the properties of these molecules, and therefore their applications, are largely defined by the structural features of the fatty acid and alcohol moieties, has driven a persistent interest in generating novel structures of these chemicals. RESULTS: In this research, we engineered Escherichia coli to synthesize de novo esters composed of multi-methyl-branched-chain fatty acids and short branched-chain alcohols (BCA), from glucose and propionate. A coculture engineering strategy was developed to avoid metabolic burden generated by the reconstitution of long heterologous biosynthetic pathways. The cocultures were composed of two independently optimized E. coli strains, one dedicated to efficiently achieve the biosynthesis and release of the BCA, and the other to synthesize the multi methyl-branched fatty acid and the corresponding multi-methyl-branched esters (MBE) as the final products. Response surface methodology, a cost-efficient multivariate statistical technique, was used to empirical model the BCA-derived MBE production landscape of the coculture and to optimize its productivity. Compared with the monoculture strategy, the utilization of the designed coculture improved the BCA-derived MBE production in 45%. Finally, the coculture was scaled up in a high-cell density fed-batch fermentation in a 2 L bioreactor by fine-tuning the inoculation ratio between the two engineered E. coli strains. CONCLUSION: Previous work revealed that esters containing multiple methyl branches in their molecule present favorable physicochemical properties which are superior to those of linear esters. Here, we have successfully engineered an E. coli strain to broaden the diversity of these molecules by incorporating methyl branches also in the alcohol moiety. The limited production of these esters by a monoculture was considerable improved by a design of a coculture system and its optimization using response surface methodology. The possibility to scale-up this process was confirmed in high-cell density fed-batch fermentations.

A genome-scale nutrient minimization forecast algorithm for controlling essential amino acid levels in CHO cell cultures.

Mammalian cell culture processes rely heavily on empirical knowledge in which process control remains a challenge due to the limited characterization/understanding of cell metabolism and inability to predict the cell behaviors. This study facilitates control of Chinese hamster ovary (CHO) processes through a forecast-based feeding approach that predicts multiple essential amino acids levels in the culture from easily acquired viable cell density data. Multiple cell growth behavior forecast extrapolation approaches are considered with logistic curve fitting found to be the most effective. Next, the nutrient-minimized CHO genome-scale model is combined with the growth forecast model to generate essential amino acid forecast profiles of multiple CHO batch cultures. Comparison of the forecast with the measurements suggests that this algorithm can accurately predict the concentration of most essential amino acids from cell density measurement with error mitigated by incorporating off-line amino acids concentration measurements. Finally, the forecast algorithm is applied to CHO fed-batch cultures to support amino acid feeding control to control the concentration of essential amino acids below 1-2 mM for lysine, leucine, and valine as a model over a 9-day fed batch culture while maintaining comparable growth behavior to an empirical-based culture. In turn, glycine production was elevated, alanine reduced and lactate production slightly lower in control cultures due to metabolic shifts in branched-chain amino acid degradation. With the advantage of requiring minimal measurement inputs while providing valuable and in-advance information of the system based on growth measurements, this genome model-based amino acid forecast algorithm represent a powerful and cost-effective tool to facilitate enhanced control over CHO and other mammalian cell-based bioprocesses.

Development of a High-Titer Culture Medium for the Production of Cholesterol by Engineered Saccharomyces cerevisiae and Its Fed-Batch Cultivation Strategy.

Steroids are a class of compounds with cyclopentane polyhydrophenanthrene as the parent nucleus, and they usually have unique biological and pharmacological activities. Most of the biosynthesis of steroids is completed by a series of enzymatic reactions starting from cholesterol. Synthetic biology can be used to synthesize cholesterol in engineered microorganisms, but the production of cholesterol is too low to further produce other high-value steroids from cholesterol as the raw material and precursor. In this work, combinational strategies were established to increase the production of cholesterol in engineered Saccharomyces cerevisiae RH6829. The basic medium for high cholesterol production was selected by screening 8 kinds of culture media. Single-factor optimization of the carbon and nitrogen sources of the culture medium, and the addition of calcium ions, zinc ions and citric acid, further increased the cholesterol production to 192.53 mg/l. In the 5-L bioreactor, through the establishment of strategies for glucose and citric acid feeding and dissolved oxygen regulation, the cholesterol production was further increased to 339.87 mg/l, which was 734% higher than that in the original medium. This is the highest titer of cholesterol produced by microorganisms currently reported. The fermentation program has also been conducted in a 50-L bioreactor to prove its stability and feasibility.

Catechol 1,2-Dioxygenase From Paracoccus sp. MKU1-A Greener and Cleaner Bio-Machinery for cis, cis-Muconic Acid Production by Recombinant E. coli.

Cis, cis-muconic acid (ccMA) is known for its industrial importance as a precursor for the synthesis of several biopolymers. Catechol 1,2-dioxygenase (C12O) is involved in aromatic compounds catabolism and ccMA synthesis in a greener and cleaner way. This is the first study on C12O gene from a metabolically versatile Paracoccus sp. MKU1, which was cloned and expressed in E. coli to produce ccMA from catechol. From the E. coli transformant, recombinant C12O enzyme was purified and found to be a homotrimer with a subunit size of 38.6 kDa. The apparent K m and V max for C12O was 12.89 µM and 310.1 U.mg-1, respectively, evidencing high affinity to catechol than previously reported C12Os. The predicted 3D-structure of C12O from MKU1 consisted of five α-helices in N-terminus, one α-helix in C-terminus, and nine ß-sheets in C-terminus. Moreover, a unique α-helix signature 'EESIHAN' was identified in C-terminus between 271 and 277 amino acids, however the molecular insight of conservative α-helix remains obscure. Further, fed-batch culture was employed using recombinant E. coli expressing C12O gene from Paracoccus sp. MKU1 to produce ccMA by whole-cells catalyzed bioconversion of catechol. With the successive supply of 120 mM catechol, the transformant produced 91.4 mM (12.99 g/L) of ccMA in 6 h with the purity of 95.7%. This single step conversion of catechol to ccMA using whole-cells reactions of recombinants did not generate any by-products in the reaction mixtures. Thus, the recombinant E. coli expressing high activity C12O from Paracoccus sp. MKU1 holds promise as a potential candidate for yielding high concentrations of ccMA at faster rates in low cost settings.

The effect of growth rate on the production and vitality of non-Saccharomyces wine yeast in aerobic fed-batch culture.

Non-Saccharomyces wine yeasts are of increasing importance due to their influence on the organoleptic properties of wine and thus the factors influencing the biomass production of these yeasts, as starter cultures, are of commercial value. Therefore, the effects of growth rates on the biomass yield (Yx/s) and fermentation performance of non-Saccharomyces yeasts at bench and pilot scale were examined. The fermentative performance and (Yx/s) were optimised, in aerobic fed-batch cultivations, to produce commercial wine seed cultures of Lachancea thermotolerans Y1240, Issatchenkia orientalis Y1161 and Metschnikowia pulcherrima Y1337. Saccharomyces cerevisiae (Lalvin EC1118) was used as a benchmark. A Crabtree positive response was shown by L. thermotolerans in a molasses-based industrial medium, at growth rates exceeding 0.21 h-1 (µcrit), resulting in a Yx/s of 0.76 g/g at 0.21 h-1 (46% of µmax) in the aerobic bioreactor-grown fed-batch culture at bench scale. At pilot scale and 0.133 h-1 (36% of µmax), this yeast exhibited ethanol concentrations reaching 10.61 g/l, as a possible result of substrate gradients. Crabtree negative responses were observed for I. orientalis and M. pulcherrima resulting in Yx/s of 0.83 g/g and 0.68 g/g, respectively, below 32% of µmax. The Yx/s of M. pulcherrima, I. orientalis and L. thermotolerans was maximised at growth rates between 0.10 and 0.12 h-1 and the fermentative capacity of these yeasts was maximised at these lower growth rates.

Application of a New Engineered Strain of Yarrowia lipolytica for Effective Production of Calcium Ketoglutarate Dietary Supplements.

The present study aimed to develop a technology for the production of dietary supplements based on yeast biomass and α-ketoglutaric acid (KGA), produced by a new transformant of Yarrowia lipolytica with improved KGA biosynthesis ability, as well to verify the usefulness of the obtained products for food and feed purposes. Transformants of Y. lipolytica were constructed to overexpress genes encoding glycerol kinase, methylcitrate synthase and mitochondrial organic acid transporter. The strains were compared in terms of growth ability in glycerol- and oil-based media as well as their suitability for KGA biosynthesis in mixed glycerol-oil medium. The impact of different C:N:P ratios on KGA production by selected strain was also evaluated. Application of the strain that overexpressed all three genes in the culture with a C:N:P ratio of 87:5:1 allowed us to obtain 53.1 g/L of KGA with productivity of 0.35 g/Lh and yield of 0.53 g/g. Finally, the possibility of obtaining three different products with desired nutritional and health-beneficial characteristics was demonstrated: (1) calcium α-ketoglutarate (CaKGA) with purity of 89.9% obtained by precipitation of KGA with CaCO3, (2) yeast biomass with very good nutritional properties, (3) fixed biomass-CaKGA preparation containing 87.2 µg/g of kynurenic acid, which increases the health-promoting value of the product.

A fed-batch feeding with succinic acid strategy for astaxanthin and lipid hyper-production in Haematococcus pluviualis.

Effects of succinic acid (SA) in fed-batch feeding mode on astaxanthin and lipids biopoduction of Haematococcus pluvialis against abiotic stresses were explored. By comparison with the control, the initial addition of SA on day 0 increased the production of astaxanthin by 71.61%. More importantly, the maximum values of astaxanthin (35.88 mg g-1) and lipid (54.79%) contents were obtained after supplementation of SA on day 7. Meanwhile, under SA treatment, the chlorophyll, carbohydrate, and protein levels were reduced, but the intracellular levels of SA and reactive oxygen species (ROS), the transcription levels of astaxanthin and fatty acids biosynthesis-, and antioxidant system-related genes were increased. Furthermore, scaling-up cultivation in bioreactor further enhanced the astaxanthin productivity from H. pluvialis. Generally, this study proved the intermittent SA feeding method in fed-batch culture as a potent strategy that facilitated massive astaxanthin and lipids production in algae.

Systematic metabolic tools reveal underlying mechanism of product biosynthesis in Chromochloris zofingiensis.

This study comprehensively explored underlying mechanism of fed-batch culture on product biosynthesis in Chromochloris zofingiensis by dynamic model, targeted metabolite determination, enzyme activity analysis, and 13C tracer-based metabolic flux analysis. Based on dynamic models of cell growth and product formation, exponential fed-batch culture and fed-batch culture based on pH changes were established to increase biomass concentration by 20.05-fold and 18.28-fold, respectively. Exponential fed-batch culture exhibited great potentials in biodiesel and protein productions from microalgae. Systematic metabolic tools revealed fed-batch culture limited photosynthetic efficiency by inhibiting photosystem and Rubisco activity, while strengthened respiratory action to provide more substances and energy for product biosynthesis. Fed-batch culture elevated biosynthetic capability for carotenoid and lipid by promoting related metabolic flux and contents of pyruvate and ace-CoA. Finally, economic analysis revealed biomass cost was decreased to 1.99 $/kg from 2.39 $/kg, suggesting fed-batch culture was a cost-effective strategy to improve economic viability of microalgal production.