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In this study, an exopolysaccharide (EPS) named EPS-RB was produced when the gene cluster ycjD-fabI-yciW-rnb were overexpressed in E. coli. Monosaccharide composition analysis revealed that EPS-RB is a novel EPS that consisted of L-fucose, L-arabinose, D-galactose/N-acetyl glucosamine, D-glucose, D-xylose, D-ribose, and D-glucuronic acid, and their molecular ratio was approximately 80:3:53:69:1:2:64. The content of carbohydrates, protein, and uronic acids in EPS-RB was 90.35 ± 1.35%, 2.62 ± 0.05% and 8.16 ± 1.00%, respectively. The interaction between EPS-RB and gut microbiota was investigated using an in vitro batch fermentation system. The results showed that ~96% of EPS-RB can be degraded by human fecal microbiota after 72 h fermentation, but few can be degraded by mouse cecal microbiota. Furthermore, high-throughput sequencing showed that EPS-RB regulates the human gut microbiota. The genera Collinsella, Butyricimonas, and Hafnia were enriched in group VIR (EPS-RB as a carbon source) when compared with group VI (no carbon source) and VIS (starch as a carbon source). Short-chain fatty acids (SCFAs) production analysis showed that their concentration was significantly higher in group VIR than groups VI and VIS after 72 h fermentation. In summary, an EPS-RB in E. coli was isolated and its regulatory function on gut microbiota was analyzed.

With the growing interest in enzyme applications, there is an urgent demand for economic, affordable, and flexible enzyme production processes. In the present paper, we developed a high cell density fed-batch process for the production of two cofactor-containing oxidase, 5-hydroxymethylfurfural oxidase (HMFO) and eugenol oxidase (EUGO). The approach involved the arabinose-inducible system to drive the expression while using mineral media. In order to overcome a major drawback of arabinose-inducible promoters, carbon catabolite repression, (CCR) by glucose, we developed a high cell density culture (HCDC), two-stage fed-batch protocol allowing us to reach cell densities exceeding 70 g/L of dry cell weight (DCW) using glucose as carbon source. Then, induction was achieved by adding arabinose, while changing the carbon source to glycerol. This strategy allowed us to obtain an eightfold increase in recombinant HMFO titer when compared with a reference batch fermentation in Erlenmeyer flasks using terrific broth (TB), typically used with arabinose-inducible strains. The optimized protocol was also tested for expression of a structurally unrelated oxidase, EUGO, where a similar yield was achieved. Clearly, this two-step protocol in which a relatively cheap medium (when compared to TB) can be used reduces costs and provides a way to obtain protein production levels similar to those of IPTG-based systems. • Arabinose promoters are not well suited for HCDC production due to CCR effect. • This drawback has been overcome by using a two-stage Fed-batch protocol. • Protein yield has been increased by an eightfold factor, improving process economics.

As petro-based production generates numerous environmental impacts and their associated technological concerns, bio-based production has been well recognized these days as a modern alternative to manufacture chemical products in a more renewable, environmentally friendly, and sustainable manner. Herein, we report the development of a microbial bioprocess for high-level and potentially economical production of 3-hydroxyvalerate (3-HV), a valuable special chemical with multiple applications in chemical, biopolymer, and pharmaceutical industries, from glycerol, which can be cheaply and renewably refined as a byproduct from biodiesel production. We used our recently derived 3-HV-producing Escherichia coli strains for bioreactor characterization under various culture conditions. In the parental strain, 3-HV biosynthesis was limited by the intracellular availability of propionyl-CoA, whose formation was favored by anaerobic conditions, which often compromised cell growth. With appropriate strain engineering, we demonstrated that 3-HV can be effectively produced under both microaerobic (close to anaerobic) and aerobic conditions, which determine the direction of dissimilated carbon flux toward the succinate node in the tricarboxylic acid (TCA) cycle. We first used the ∆sdhA single mutant strain, in which the dissimilated carbon flux was primarily directed to the Sleeping beauty mutase (Sbm) pathway (via the reductive TCA branch, with enhanced cell growth under microaerobic conditions, achieving 3.08 g L−1 3-HV in a fed-batch culture. In addition, we used the ∆sdhA-∆iclR double mutant strain, in which the dissimilated carbon flux was directed from the TCA cycle to the Sbm pathway via the deregulated glyoxylate shunt, for cultivation under rather aerobic conditions. In addition to demonstrating effective cell growth, this strain has shown impressive 3-HV biosynthesis (up to 10.6 g L−1), equivalent to an overall yield of 18.8% based on consumed glycerol, in aerobic fed-batch culture. This study not only represents one of the most effective bio-based production of 3-HV from structurally unrelated carbons to date, but also highlights the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production. Key points • TCA cycle engineering was applied to enhance 3-HV biosynthesis in E. coli. • Effects of oxygenic conditions on 3-HV in E. coli biosynthesis were investigated. • Bioreactor characterization of 3-HV biosynthesis in E. coli was performed.

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20-25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.

In the present study, the production of poly(3-hydroxybutyrate-co-3–hydroxyvalerate) (PHBV) by Azotobacter vinelandii was evaluated in shake flasks and bioreactors, utilizing different precursors and oxygen transfer rates (OTRs). In shake flask cultures, the highest PHBV yield from sucrose (0.16 g g–1) and 3-hydroxyvalerate (3HV) fraction in the PHA chain (27.4 mol%) were obtained with valerate (1.0 g L−1). In the bioreactor, the cultures were grown under oxygen-limited conditions, and the maximum OTR (OTRmax) was varied by adjusting the agitation rate. In the cultures grown at low OTRmax (4.3 mmol L−1 h−1), the intracellular content of PHBV (73% w w−1) was improved, whereas a maximum 3HV fraction (35 mol %) was obtained when a higher OTRmax (17.2 mmol L−1 h−1, to 600 rpm) was employed. The findings obtained suggest that the PHBV production and the content of 3HV incorporated into the polymer were affected by the OTR. Based on the evidence, it is possible to produce PHBV with a different composition by varying the OTR of the culture; thus, the approach in this study could be used to scale up PHBV production.

Background Docosahexaenoic acid (DHA) is essential for human diet. However, high production cost of DHA using C. cohnii makes it currently less competitive commercially, which is mainly caused by low DHA productivity. In recent years, repeated fed-batch strategies have been evaluated for increasing the production of many fermentation products. The reduction in terms of stability of culture system was one of the major challenges for repeated fed-batch fermentation. However, the possible mechanisms responsible for the decreased stability of the culture system in the repeated fed-batch fermentation are so far less investigated, restricting the efforts to further improve the productivity. In this study, a repeated fed-batch strategy for DHA production using C. cohnii M-1-2 was evaluated to improve DHA productivity and reduce production cost, and then the underlying mechanisms related to the gradually decreased stability of the culture system in repeated fed-batch culture were explored through LC– and GC–MS metabolomic analyses. Results It was discovered that glucose concentration at 15–27 g/L and 80% medium replacement ratio were suitable for the growth of C. cohnii M-1-2 during the repeated fed-batch culture. A four-cycle repeated fed-batch culture was successfully developed and assessed at the optimum cultivation parameters, resulting in increasing the total DHA productivity by 26.28% compared with the highest DHA productivity of 57.08 mg/L/h reported using C. cohnii, including the time required for preparing seed culture and fermentor. In addition, LC– and GC–MS metabolomics analyses showed that the gradually decreased nitrogen utilization capacity, and down-regulated glycolysis and TCA cycle were correlated with the decreased stability of the culture system during the long-time repeated fed-batch culture. At last, some biomarkers, such as Pyr, Cit, OXA, FUM, l-tryptophan, l-threonine, l-leucine, serotonin, and 4-guanidinobutyric acid, correlated with the stability of culture system of C. cohnii M-1-2 were identified. Conclusions The study proved that repeated fed-batch cultivation was an efficient and energy-saving strategy for industrial production of DHA using C. cohnii, which could also be useful for cultivation of other microbes to improve productivity and reduce production cost. In addition, the mechanisms study at metabolite level can also be useful to further optimize production processes for C. cohnii and other microbes.

Recording the data necessary to assess the kinetics of a reaction can be labor-intensive. In this technology brief, we show a method to automate this task by utilizing parts of an ÄKTApurifier chromatography system to automatically take samples from a reaction vessel at predefined time intervals and place them in 96-well plates and also enable correlating the samples with in-line spectral data of the reaction solution. Automatic sampling can reduce experimental bottlenecks by enabling overnight reactions or a higher degree of parallelization. To demonstrate the feasibility of the method, we performed batch-PEGylation of lysozyme with varying conditions by changing the molar excess of the PEG reagent. We used analytical cation-exchange chromatography to analyze the samples taken during the batch reaction, determining the concentrations of the individual species present at each time step. Subsequently, we fitted a kinetic model on these data. Fitting the model to four different reaction conditions simultaneously yielded a regression coefficient of

To increase the productivity of rCHO cells, many cell engineering approaches have been demonstrated that over-express or knockout a specific gene to achieve increased titers. In this work, we present an alternate approach, based on the concept of evolutionary adaptation, to achieve cells with higher titers. rCHO cells, producing a monoclonal antibody, are adapted to ER-stress, by continuous culturing under increasing concentration of tunicamycin. A sustained higher productivity of at-least 2-fold was achieved in all the clones, in a concentration-dependent manner. Similarly, a 1.5-2 fold increase in final titers was also achieved in the batch culture. Based on metabolic analysis of the adapted cells, a fed-batch process was designed where significantly higher titersare achieved as compared to control. Metabolic flux analysis is employed in addition with gene expression analysis of key genes to understand the basis of increased performance of the adapted cells. Overall, this work illustrates how process modifications and cellular adaptation can be used in synergy to drive up product titers.

Background Exhausted sugar beet pulp pellets (ESBPP), a sugar industry by-product generated after sugar extraction in the sugar production process, have been used as a raw material for lactic acid (LA) production via hydrolysis and fermentation by Lactobacillus casei. To design a more cost-effective process, simultaneous saccharification and fermentation (SSF) of ESBPP is proposed in the present study. The effects of pH control, nutrient supplementation and solid addition in fed-batch SSF on lactic acid production were investigated. Results The highest LA concentration (26.88 g L-1 ) was reached in fed-batch SSF at a solid/liquid loading of 0.2 g mL-1 , with pH control (by adding 30 g L-1 CaCO3 to the medium) and nutrient supplementation (by adding 20 mL of MRS medium per 100 mL of buffer). Under these conditions, a maximum productivity of 0.63 g L-1 h-1 was achieved, which is 2.7 times higher than that attained in the control experiment (SSF inoculated at time 0 h). However, a slightly lower LA yield was obtained, revealing the need of an increasing dose of enzymes at high solid loading SSF. Conclusion An efficient fed-batch SSF strategy with pH control and MRS supplementation is described in the present study, attaining higher LA productivity compared to separate hydrolysis and fermentation and SSF. © 2020 Society of Chemical Industry.

AIM 3-Phenyllactic acid (3-PLA) has been widely used in food and material industries. Three Lactobacillus crustorum strains have shown greater 3-PLA production ability in our previous study. The objectives of this study were to further improve 3-PLA yields in batch and continuous fermentation systems using of free-whole-cells of the three L. crustorum strains. MATERIALS AND RESULTS The fermentation conditions of free-whole-cells of the three L. crustorum strains for 3-PLA production were optimized. Among these strains, L. crustorum NWAFU 1078 showed excellent reusability and significantly (P

In the present work, we used systematic engineering at transport and transcription levels to significantly enhance alkaline α-amylase production in Bacillus subtilis 168M. Signal peptide YwbN’ proved to be optimal. Alkaline α-amylase production was elevated by deleting a putative peptide segment of YwbN’. Insertion of arginine (R) between residues 5 and 6 of YwbN’∆p further increased the protein yield. Enhancing positive charges at sites 4 and 10 and decreasing the hydrophobicity of the H-region of YwbN’∆p were critical for improving alkaline α-amylase production in B. subtilis 168M. PHpaII was the optimal promoter, and deleting − 27T or − 31A from PHpaII enhanced the transcription of the target gene. Using a single-pulse feeding-based fed-batch system, alkaline α-amylase activity of B. subtilis 168M P∆−27T was increased by 250.6-fold, compared with B. subtilis 168M A1.

S-adenosyl-l-methionine (SAM) has been attracting increasing attention because of its significance in the pharmaceutical industry; however, the high cost of this compound limits its application. Tofu yellow serofluid exhibits high nutritional value and is not costly; therefore, it can be utilized as a substrate in the fermentation industry. In the current study, Saccharomyces cerevisiae was cultured in the tofu yellow serofluid fermentation medium for the SAM biosynthesis. The optimum tofu yellow serofluid fermentation medium contained 70 g/L of glucose, 30 % of yellow serofluid, 20 g/L of l-methionine, and 2.5 g/L of ammonium citrate. Under these conditions, the optimum feeding strategy was established. The results revealed that the dry cell weight (DCW) reached 123.1 g/L, the maximum production of SAM was 16.14 g/L, the highest SAM productivity reached 1.048 g/L/h, and SAM content was determined at 131.1 mg/g DCW. Furthermore, addition of tofu yellow serofluid reduced the average cost of SAM by 31.9 % to compare with the culture process without addition of tofu yellow serofluid. Thus, the tofu yellow serofluid fermentation medium improved the production of SAM and significantly reduced the production costs.

Background Biomass growth of Pencillium chrysogenum is characterised by a distinct pellet morphology consisting of compact hyphal agglomerates. Fungal pellets are advantageous in industrial process control due to rheological advantages but lead to biomass degradation due to diffusional limitations of oxygen and substrate in the pellet’s core. Several fermentation parameters are known to affect key pellet characteristics regarding morphology, viability and productivity. Pellet morphology and size are affected by agitation. Biomass viability and productivity are tightly interlinked with substrate uptake and dissolved oxygen concentration. Results The goal of this study was to study the impact of the fermentation parameters power input, dissolved oxygen content and specific substrate uptake rate on morphology, biomass viability and productivity. A design of experiments (DoE) approach was conducted and corresponding responses were analysed using novel morphological descriptors analysed by a previously established flow cytometry method. Results clearly display inverse correlations between power input and pellet size, specific morphological parameters related to pellet density can be increased in direct proportion to power input. Biomass viability and productivity are negatively affected by high specific substrate uptake rates. Conclusions Based upon multiple linear regression, it was possible to obtain an optimal design space for enhanced viability and productivity at beneficial morphological conditions. We could maintain a high number of pellets with favourable morphology at a power input of 1500 W/m3. A sound compromise between viability and high productivity is possible at a specific glucose uptake rate of 0.043 g/g/h at dissolved oxygen levels of 40% minimum.

5-Hydroxytryptophan (5-HTP) is the precursor of the neurotransmitter serotonin and has been used for the treatment of various diseases such as depression, insomnia, chronic headaches, and binge eating associated obesity. The production of 5-HTP had been achieved in our previous report, by the development of a recombinant strain containing two plasmids for biosynthesis of L-tryptophan (L-trp) and subsequent hydroxylation. In this study, the L-trp biosynthetic pathway was further integrated into the E. coli genome, and the promoter strength of 3-deoxy-7-phosphoheptulonate synthase, which catalyzes the first step of L-trp biosynthesis, was engineered to increase the production of L-trp. Hence, the 5-HTP production could be manipulated by the regulation of copy number of L-trp hydroxylation plasmid. Finally, the 5-HTP production was increased to 1.61 g/L in the shaking flasks, which was 24% improvement comparing to the original producing strain, while the content of residual L-trp was successfully reduced from 1.66 to 0.2 g/L, which is beneficial for the downstream separation and purification. Our work shall promote feasible progresses for the industrial production of 5-HTP.

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.

β-Alanine is a naturally occurring β-amino acid that has been widely applied in the life and health field. Although microbial fermentation is a promising method for industrial production of β-alanine, an efficient microbial cell factory is still lacking. In this study, a new metabolically engineered Escherichia coli strain for β-alanine production was developed through a series of introduction, deletion, and overexpression of genes involved in its biosynthesis pathway. First, the L-aspartate a-decarboxylase gene, BtADC, from Bacillus tequilensis, with higher catalytic activity to produce β-alanine from aspartate, was constitutively expressed in E. coli, leading to an increased production of β-alanine up to 2.76 g/L. Second, three native aspartate kinase genes, akI, akII, and akIII, were knocked out to promote the production of β-alanine to a higher concentration of 4.43 g/L by preventing from bypass loss of aspartate. To increase the amount of aspartate, the native AspC gene was replaced with PaeAspDH, a L-aspartate dehydrogenase gene from Pseudomonas aeruginosa, accompanied with the overexpression of the native AspA gene, to further improve the production level of β-alanine to 9.27 g/L. Last, increased biosynthesis of oxaloacetic acid (OAA) was achieved by a combination of overexpression of the native PPC, introduction of CgPC, a pyruvate decarboxylase from Corynebacterium glutamicum, and deletion of ldhA, pflB, pta, and adhE in E. coli, to further enhance the production of β-alanine. Finally, the engineered E. coli strain produced 43.12 g/L β-alanine in fed-batch fermentation. Our study will lay a solid foundation for the promising application of β-alanine in the life and health field. KEY POINTS: • Overexpression of BtADC resulted in substantial accumulation of β-alanine. • The native AspC was replaced with PaeAspDH to catalyze the transamination of OAA. • Deletion of gluDH prevented from losing carbon flux in TCA recycle. • A 43.12-g/L β-alanine production in fed-batch fermentation was achieved. Graphical abstract.

Acetyl-CoA is a precursor for phloroglucinol (PG), and pyruvate is one of the sources of intracellular acetyl-CoA. Therefore, enhancing intracellular pyruvate levels may help to improve the anabolic pathway of PG. In this study, the effects of phosphoenolpyruvate carboxykinase (PckA, encoded by pckA) or triosephosphate isomerase (TpiA, encoded by tpiA) overexpression on the production of PG were studied. Overexpression of pckA or tpiA could enhance the pyruvate anabolic pathway in shake-flask culture compared to the control strain, and the concentration of PG also increased by 44% and 92%, respectively. In addition, the acetate levels were all down regulated by the overexpression of the two genes to some extent and lower acetate level resulted in lower ATP pool and higher survival rate. These results indicate that overexpression of pckA or tpiA can enhance the pyruvate “pool” and PG production in Escherichia coli, which provides a new reference for further increasing the production of PG.

To isolate a novel cis-epoxysuccinate hydrolase (CESH)-producing fungus for production of l( +)-tartaric acid, before this, all strains were selected from bacteria. A CESH-producing fungus was first isolated from soil and identified as Aspergillus niger WH-2 based on its morphological properties and ITS sequence. The maximum activity of hyphaball and fermentation supernatants was 1278 ± 64 U/g and 5.6 ± 0.3 U/mL, respectively, in a 5 L fermenter based on the conditions optimized on the flask. Almost 70% of CESH was present in hyphaball, which maintained 40% residual activity at pH 4.0 and showed a good acid stability (pH 3.0–10.0), high conversion rate (> 98%), and enantioselectivity (EE > 99.6%). However, the reported CESHs from bacteria can’t be catalyzed under acidic conditions. The Aspergillus niger WH-2 was the first reported CESH-producing fungus, which could biosynthesize l ( +)-tartaric acid under acidic conditions and provide an alternative catalyst and process.

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with PHXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.

Hyaluronidases that break down hyaluronan are widely used for preparation of low molecular weight hyaluronan. Leech hyaluronidase (LHyal) is a newly discovered hyaluronidase with outstanding enzymatic properties. The Pichia pastoris expression system of LHyal that depends on AOX1 promoter (PAOX1) has been constructed. However, the addition of the toxic inducer methanol is a big safety concern. Here, a combinational strategy was adopted for constitutive expression of LHyal to high level in P. pastoris. By optimizing the combination of promoters PGAP, PGAP(m), and PTEF1 and signal peptides α-factor, nsB, and sp23, the enzyme activity of extracellular LHyal reached 1.38 × 105 U/mL in shake flasks. N-terminal engineering with neutral polar amino acids further increased LHyal activity to 2.06 × 105 U/mL. In addition, the impact of overexpressing transcription factors Aft1, Gal4-like, and Yap1 on LHyal production was also investigated. We found the co-expression of Aft1 significantly enhanced the expression of LHyal to 3.03 × 105 U/mL. Finally, LHyal activity of 2.12 × 106 U/mL was achieved in a 3-L fermenter, with a high productivity of 1.96 × 104 U/mL/h. The engineered LHyal-producing Pichia pastoris strains will be more attractive for production of hyaluronidase on industrial scale.

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O2 (DO) and dissolved CO2 (dCO2) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small‐scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real‐time monitoring of DO and dCO2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO2 at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results., Surface aeration intensification was implemented in shake flasks enabled with in house developed monitoring sensors for pH, dissolved oxygen and dissolved CO2. Efficient removal of CO2 from the shake flasks and enhanced O2 supply resulted in increased biomass growth and protein yield. Improved surface aeration in shake flasks can translate into better scale down models and smoother scale up activities, and thus will bridge the gap that exists in process development.

The Crabtree effect involves energy management in which yeasts utilize glycolysis as the terminal electron acceptor instead of oxygen, despite the presence of sufficient dissolved oxygen, when oxygen concentrations exceed a certain limit. The Crabtree effect is detrimental to bakery yeast production, because it results in lower cellular glucose yields. Batch culture of Saccharomyces cerevisiae, a Crabtree positive yeast, decreased the cell yield of glucose and produced large amounts of ethanol despite a high specific glucose consumption rate compared to Candida utilis, a Crabtree negative yeast. This study investigated the effect of these characteristics on metabolite levels. We performed metabolome analysis of both yeasts during each growth phase of batch culture using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Principle component analysis of metabolome data indicated that the Crabtree effect affected metabolites related to NADH synthesis in central metabolism. The amount of these metabolites in S. cerevisiae was lower than that in C. utilis. However, to maintain the specific glucose consumption rate at high levels, yeasts must avoid depletion of NAD+, which is essential for glucose utilization. Our results indicated that NADH was oxidized by converting acetaldehyde to ethanol in S. cerevisiae, which is in accordance with previous reports. Therefore, the specific NADH production rates of S. cerevisiae and C. utilis did not show a difference. This study suggested that NAD+/NADH ratio is disrupted by the Crabtree effect, which in turn influenced central metabolism and that S. cerevisiae maintained the NAD+/NADH ratio by producing ethanol.

Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2-44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.

The 2 biotypes of Vibrio cholerae O1 serogroup strains—classical and El Tor—use glucose in distinct ways. Classical biotype strains perform organic acid-producing fermentation and eventually lose viability due to the self-induced creation of an acidic environment, whereas El Tor biotype strains use an alternative neutral fermentation pathway, which confers them with survival advantages. However, we report that the neutral fermentation pathway has only been recruited in prototype Wave 1 El Tor biotype strains, which have not been isolated since the mid-1990s. Current Wave 2 and Wave 3 atypical El Tor strains contain a single-base deletion in a gene that directs bacteria toward neutral fermentation, resulting in the loss of neutral fermentation and an appearance that is similar to classical biotype strains. Moreover, when sufficient glucose was supplied, Wave 1 El Tor strains maintained their use of acid-producing fermentation, in parallel with neutral fermentation, and thus lost viability in the late stationary phase. The global replacement of Wave 1 El Tor strains by Wave 2 and 3 atypical El Tor strains implies that the acidic fermentation pathway may not be disadvantageous to V. cholerae. The characteristics that we have reported might improve oral rehydration in the treatment of cholera.

Lactate produced by lactic acid bacteria inhibits their growth. To suppress lactate production, it is necessary to regenerate NAD+ consumed by glycolysis with alternative pathways other than lactate dehydrogenase. In a heterofermentative lactic acid bacterium, Lactobacillus reuteri JCM1112, suppression of lactate production by regenerating NAD+ when producing 1,3-propanediol from glycerol was investigated. The bacterium produced lactate with a yield of 4.7 ± 0.8 g·g-cell−1 in a batch culture using glucose as the sole carbon source. When glycerol was added to glucose at a molar ratio (rGly/Glc) of three in the batch culture, the bacterium produced 1,3-propanediol at 1.6 ± 0.7 g·g-cell−1·h−1 and the lactate yield decreased to 3.6 ± 0.5 g·g-cell−1. When glycerol was co-fed with glucose exponentially to give a target specific growth rate of 0.1 h−1 (rGly/Glc = 3), the lactate yield decreased to 1.5 ± 0.2 g·g-cell−1. The lactate production when glycerol was added together with glucose was reduced to one-third of that observed in the batch culture using glucose as a carbon source.

The production of microbial protein in the form of yeast grown on lignocellulosic sugars and nitrogen-rich industrial residues is an attractive approach for reducing dependency on animal and plant protein. Growth media composed of enzymatically saccharified sulfite-pulped spruce wood, enzymatic hydrolysates of poultry by-products and urea were used for the production of single-cell protein. Strains of three different yeast species,Cyberlindnera jadinii,Wickerhamomyces anomalusandBlastobotrys adeninivorans,were cultivated aerobically using repeated fed-batch fermentation up to 25 L scale.Wickerhamomyces anomaluswas the most efficient yeast with yields of 0.6 g of cell dry weight and 0.3 g of protein per gram of glucose, with cell and protein productivities of 3.92 g/L/h and 1.87 g/L/h, respectively. Using the conditions developed here for producingW. anomalus, it would take 25 industrial (200 m3) continuously operated fermenters to replace 10% of the fish feed protein used in Norway.

The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, β-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 μM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of β-glucuronidase from zinc-supplemented bioreactors.

The mechanism and effect of C/N ratios on the aerobic granules simultaneous nitrification, denitrification and phosphorus removal (SNDPR) system are still unclear. The reactor performance and microbial community dynamics of the system were investigated under variable C/N ratios (20, 10 and 5). The COD, TP and NH4+-N removal remained unaffected with variable C/N ratios. The decreased C/N ratio of five strongly influenced the nitrogen removal. Further investigations revealed that Candidatus_Accumulibacter, Acinetobacter, Candidatus_Competibacter were the predominant genera. Classification of key groups involved in nitrogen and phosphorus removal indicated the lowest C/N ratio resulting in a large microbial community shift. This study might contribute to the application of SNDPR system for the treatment of wastewater. Different C/N ratios led to shift on the microbial community and the dominant was phosphorus-accumulating bacteria. The nitrogen removal efficiency decreased while the removal of COD, TP and NH4+-N remained remarkable with the decreased C/N ratios.

The CRISPR-Cas9 system is a new tool that has been extensively used for genome editing. The system is composed of a Cas9 endonuclease, which has the function of cleaving DNA at a specific site, and a guide RNA (gRNA), which contains the sequence of the cleavage site that is the target of editing. Despite the great interest that has been generated because of the utility of Cas 9 as a molecular tool and a potential therapeutic protein, the production of the 158 kDa recombinant Cas9 protein derived from Streptococcus pyogenes remains a challenge. Here, we systematically evaluated the expression of recombinant Cas9 protein in two different E. coli strains in complex and defined media. The recombinant protein showed improved expression in E. coli BL21(DE3), while only traces of Cas9 protein could be detected in the Rosetta (DE3) strain as a result of much lower mRNA levels. The greatest Cas9 protein expression in defined media containing glucose was observed at an induction temperature of 30 °C and with 8 h of post induction time using IPTG in shake flasks. The protein concentration obtained during a batch bioreactor culture was approximately 420.1 mg/L with 6 h of post induction time. The results demonstrated the possibility of efficient Cas9 protein expression in batch mode using E. coli BL21(DE3) and a simple defined medium and also showed the potential for further improvements that could facilitate large-scale production.

Psilocybin, the prodrug of the psychoactive molecule psilocin, has demonstrated promising results in clinical trials for the treatment of addiction, depression, and post-traumatic stress disorder. The development of a psilocybin production platform in a highly engineerable microbe could lead to rapid advances towards the bioproduction of psilocybin for use in ongoing clinical trials. Here, we present the development of a modular biosynthetic production platform in the model microbe, Escherichia coli. Efforts to optimize and improve pathway performance using multiple genetic optimization techniques were evaluated, resulting in a 32-fold improvement in psilocybin titer. Further enhancements to this genetically superior strain were achieved through fermentation optimization, ultimately resulting in a fed-batch fermentation study, with a production titer of 1.16 g/L of psilocybin. This is the highest psilocybin titer achieved to date from a recombinant organism and a significant step towards demonstrating the feasibility of industrial production of biologically-derived psilocybin.

Desalination of cheese whey by electrodialysis yields saline wastewater (SWW). The goal was to test this as the basis of a culture medium and to prove experimentally the concept that it was a suitable resource for heterotrophic cultivation of the freshwater green microalga Chlorella vulgaris. Optimization of glucose concentration, nitrogen source and medium salinity for microalgal growth was first carried out in defined medium (DM) and shake flasks. These results were then adopted in shake flask cultivation experiments using pre-treated SWW medium (PSWW). Subsequently, microalgal growth under optimized conditions was tested in bioreactors. Various media such as DM, PSWW and diluted PSWW (DPSWW) were compared. Volumetric biomass productivities decreased in the order DM (0.371 g L−1 h−1, urea) > DPSWW (0.315 g L−1 h−1, soy peptone) > PSWW (0.152 g L−1 h−1, soy peptone). Although biomass productivities in DPSWW and PSWW media were significantly lower than in DM, these media required the addition of only 66 and 33% of DM N sources, respectively. No other added DM component was necessary in (D)PSWW to achieve microalgal growth. Although the optimized cultivation of freshwater microalgae on alternative medium based on SWW resulted in biomass productivities lower than those on DM, the required addition of N sources was also lower. Potentially lower production costs of Chlorella biomass and the meaningful use of SWW are the main outcomes of this work.

Seasonal and pandemic influenza respiratory infections are still a major public health issue. Vaccination is the most efficient way to prevent influenza infection. One option to produce influenza vaccines is cell-culture based virus propagation. Different host cell lines, such as MDCK, Vero, AGE1.CR or PER.C6 cells have been shown to be a good substrate for influenza virus production. With respect to the ease of scale-up, suspension cells should be preferred over adherent cells. Ideally, they should replicate different influenza virus strains with high cell-specific yields. Evaluation of new cell lines and further development of processes is of considerable interest, as this increases the number of options regarding the design of manufacturing processes, flexibility of vaccine production and efficiency. Here, PBG.PK2.1, a new mammalian cell line that was developed by ProBioGen AG (Germany) for virus production is presented. The cells derived from immortal porcine kidney cells were previously adapted to growth in suspension in a chemically-defined medium. Influenza virus production was improved after virus adaptation to PBG.PK2.1 cells and optimization of infection conditions, namely multiplicity of infection and trypsin concentration. Hemagglutinin titers up to 3.24 log10(HA units/100 µL) were obtained in fed-batch mode in bioreactors (700 mL working volume). Evaluation of virus propagation in high cell density culture using a hollow-fiber based system (ATF2) demonstrated promising performance: Cell concentrations of up to 50 × 106 cells/mL with viabilities exceeding 95%, and a maximum HA titer of 3.93 log10(HA units/100 µL). Analysis of glycosylation of the viral HA antigen expressed showed clear differences compared to HA produced in MDCK or Vero cell lines. With an average cell-specific productivity of 5000 virions/cell, we believe that PBG.PK2.1 cells are a very promising candidate to be considered for next-generation influenza virus vaccine production.

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL.

Driven by the concept of plug-and-play cell culture-based viral vaccine production using disposable bioreactors, we evaluated an orbital shaken bioreactor (OSB) for human influenza A virus production at high cell concentration. Therefore, the OSB model SB10-X was coupled to two hollow fiber-based perfusion systems, namely, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). The AGE1.CR.pIX avian suspension cells grew to 50 × 106 cells/mL in chemically defined medium, maintaining high cell viabilities with an average specific growth rate of 0.020 h−1 (doubling time = 32 h). Maximum virus titers in the range of 3.28–3.73 log10(HA units/100 µL) were achieved, corresponding to cell-specific virus yields of 1000–3500 virions/cell and productivities of 0.5–2.2 × 1012 virions/L/d. This clearly demonstrates the potential of OSB operation in perfusion mode, as results achieved in a reference OSB batch cultivation were 2.64 log10(HA units/100 µL), 1286 virions/cell and 1.4 × 1012 virions/L/d, respectively. In summary, the SB10-X bioreactor can be operated with ATF and TFF systems, which is to our knowledge the first report regarding OSB operation in perfusion mode. Moreover, the results showed that the system is a promising cultivation system for influenza A virus vaccine production. The OSB disposable bioreactor has the potential for simplifying the scale-up from shake flasks to the large-scale bioreactor, facilitating rapid responses in the event of epidemics or pandemics.

The cost of polyhydroxyalkanoates (PHAs) can be reduced by improving their productivity and recovery. In this study, we attempted to obtain a high cell density culture from a 13 L bioreactor and subsequently improved the recently developed biological recovery process using mealworms to obtain the PHA granules. A cell dry weight of 161 g/L containing 68-70 wt% P(3HB) was obtained. The freeze-dried cells contained a significant amount of mineral salts from the culture medium which reduced the cells' palatability for the mealworms. A simple washing procedure with water was sufficient to remove the residual mineral salts and this improved the cells' consumption by up to 12.5% of the mealworms' body weight. As a result, one kilogram of mealworms consumed 125 g of the washed cells daily and 87.2 g of feacal pellets were recovered, which was almost twice the weight of the unwashed cells. In addition, it also improved the purity of the PHA in the faecal pellets to a value90% upon washing with water to remove the water-soluble compounds. This study has demonstrated a significant improvement in the production and recovery of PHA. In addition, the resulting mealworms showed a significant increase in protein content up to 79% and a decrease in fat content down to 8.3% of its dry weight.

Secreted recombinant proteins can aggregate during cell culture. We studied a poorly-behaved bispecific scaffold that increasingly aggregated (up to 62% high molecular weight species, HMW) as a function of culture time in a fed-batch and intensified cell culture processes. We identified that protein aggregates increased with accumulated protein concentration inside the bioreactor. Furthermore, results indicated that a maximum product concentration was reached beyond which no additional soluble protein accumulated in culture even when doubling the integrated viable cell density with the intensified process, suggesting additional secreted protein was precipitating. To overcome this limitation and maintain the cell-specific productivity (qp) in culture, we explored a perfusion process where recombinant protein was continuously removed from the bioreactor to maintain low product concentration and consequently, minimize protein aggregation. We studied different viable cell densities (VCDs) inside the bioreactor (one to five-fold) and found a corresponding two-fold modulation of monomer levels. In all VCD conditions, qp was maintained. On the contrary, the previous intensified process showed an "apparent" 2.5-fold decrease in qp at the end of culture because of the presumed limited protein solubility at higher concentrations. The combination of lower aggregate levels and constant qp resulted in up to four to five-fold increase in recoverable product (i.e., monomer) with the improved perfusion process.

Escherichia coli was engineered to produce d-pantothenic acid via systematic metabolic engineering. Firstly, genes of acetohydroxy acid synthase II, pantothenate synthetase, 3-methyl-2-oxobutanoate hydroxymethyltransferase, 2-dehydropantoate 2-reductase and ketol-acid reductoisomerase were edited in E. coli W3110 with a resulting d-pantothenic acid yield of 0.49 g/L. Expressions of valine-pyruvate aminotransferase and branched-chain-amino-acid aminotransferase were then attenuated to decrease the carbon flux in l-valine biosynthetic pathway which is a competing pathway to the d-pantothenic acid biosynthetic pathway, and the yield increased to 1.48 g/L. Mutagenesis of pantothenate kinase and deletion of threonine deaminase further increased the production to 1.78 g/L. Overexpressions of panC and panB from Corynebacterium glutamicum enhanced the production by 29%. In fed-batch fermentations, strain DPA-9/pTrc99a-panBC(C.G) exhibited a highest d-pantothenic acid yield of 28.45 g/L. The findings in this study demonstrate the systematic metabolic engineering in Escherichia coli W3110 would be a promising strategy for industrial production of d-pantothenic acid.

Ebola virus disease is an urgent international priority. Promising results for several vaccine candidates have been reported in non-human primate studies and clinical trials with the most promising being the rVSV-ZEBOV vaccine. In this study, we sought to produce rVSV-ZEBOV in HEK 293SF cells in suspension and serum-free media. The purpose of this study was to establish a process using the HEK 293SF production platform, optimise the production titre, demonstrate scalability and the efficiency of the generated material to elicit an immune reaction in an animal model. Critical process parameters were evaluated to maximize production yield and process robustness and the following operating conditions: 1–2 × 106 cells/mL grown in HyClone HyCell TransFx-H media infected at an MOI of 0.001 with a temperature shift to 34 °C during the production phase and a harvest of the product after 48 h. Using these conditions, scalability in a 3.5 L controlled bioreactor was shown reaching a titre of 1.19 × 108 TCID50/mL at the peak of production, the equivalent of 4165 doses of vaccine per litre. The produced virus was shown to be thermostable in the culture media and, when concentrated, purified and administered to mice, demonstrated the ability to induce a ZEBOV-specific immune response.

Penicillium glabrum GQ1-3 and Aspergillus oryzae HGPA20 isolated from home-made soybean pastes were separately inoculated into soybean paste subjected to brine fermentation for 90 days. The amino acid nitrogen contents of the two fermentation systems were detected every 10 days, and both reached the maximum level at 40 days. The samples fermented for 40 days were analyzed via gas chromatography-time of flight mass spectrometry. Using univariate, multivariate and KEGG analyses, 72 differential metabolites were obtained, and 7 metabolic pathways closely related to fermentation were screened. The relative contents of 2-oxoglutarate, ornithine, glutamine, and citrulline were higher in GQ1-3, whereas those of l-homoserine, aspartic acid, and asparagine were higher in HGPA20. These findings indicate that α-ketoglutaric acid-derived amino acid synthesis is preponderant in GQ1-3, whereas oxaloacetate-derived type is predominant in HGPA20. The different pathways of amino acid synthesis lead to the distinct nutrients and umami substances in the fermented soybean pastes.

The effect of sodium selenite on batch culture of Candida utilis CCTCC M 209298 was investigated. Cell growth was inhibited while glutathione biosynthesis and secretion were improved during selenium enrichment. To reveal the mechanism underlying the decrease in biomass and the increase in glutathione, both metabolic flux analysis of key intermediates involved in glutathione metabolic pathway and transcriptome analysis of C. utilis by RNA-seq were carried out for selenized cells and the control without selenium enrichment. Results indicated that sodium selenite decreased carbon fluxes towards biomass but increased fluxes towards amino acids for the biosynthesis of glutathione and related amino acids. Selenium enrichment down-regulated a large number of genes involved in cell components and the cell cycle, resulting in decreased biomass as well as increased cell permeability. Moreover, several genes associated with transportation, binding, and mitochondrial and ribosomal functions for energy metabolism and protein synthesis were up-regulated in the presence of sodium selenite. All of these results disclosed the physiological response of C. utilis to sodium selenite.

High volumetric productivities can be achieved when perfusion processes are operated at high cell densities. Yet it is fairly challenging to keep high cell density cultures in a steady state over an extended period. Aiming for robust processes, cultures were operated at a constant biomass specific perfusion rate (BSPR) in this study. The cell density was monitored with a capacitance probe and a continuous bleed maintained the targeted viable cell volume. Despite our tightly controlled BSPR, a gradual accumulation of ammonium and changes in cell diameter were observed during the production phase for three different monoclonal antibodies. Although a lot of efforts in media optimization have been made to reduce ammonium in fed-batch process, less examples are known about how media components impact the cellular metabolism and thus the quality of monoclonal antibodies in continuous processes. In this study, we show that a continuous Na-pyruvate feed (2 g/L/day) strongly reduced ammonium production and stabilized fucosylation, sialylation and high mannose content for three different mAbs.

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L−1 h−1.

Background Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (PPDF, PUPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Results Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. Conclusions As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

Microbial lipids-derived biodiesel is garnering much attention owing to its potential to substitute diesel fuel. In this study, lipid accumulation by Yarrowia lipolytica from volatile fatty acids (VFAs) was studied in a lab-scale stirred tank bioreactor. In batch cultures, Y. lipolytica NCYC 2904 was able to grow in 18 g·L

The mechanisms controlling entry into and exit from the death phase in the bacterial life cycle remain unclear. Although bacterial growth studies in batch cultures traditionally focus on the first three phases during incubation, two additional phases, the death phase and the long-term stationary phase, are less understood. Although there are a number of stressors that arise during long-term batch culture, including nutrient depletion and the accumulation of metabolic toxins such as reactive oxidative species, their roles in cell death are not well-defined. By manipulating the environmental conditions of Escherichia coli incubated in long-term batch culture through chemical and mechanical means, we investigated the role of volatile metabolic toxins in modulating the onset of the death phase. Here, we demonstrate that with the introduction of substrates with high binding affinities for volatile compounds, toxic by-products of normal cell metabolism, into the headspace of batch cultures, cells display a prolonged stationary phase and delayed entry into the death phase. The addition of these substrates allows cultures to maintain a high cell density for hours to days longer than cultures incubated under standard growth conditions. A similar effect is observed when the gaseous headspace in culture flasks is continuously replaced with sterile air, mechanically preventing the accumulation of metabolic by-products in batch cultures. We establish that toxic compound(s) are produced during the exponential phase, demonstrate that buildup of toxic by-products influence entry into the death phase, and present a novel tool for improving high-density growth in batch culture that may be used in future research or industrial or biotechnology applications. IMPORTANCE Bacteria, such as Escherichia coli, are routinely used in the production of biomaterials because of their efficient and sustainable capacity for synthesis of bioproducts. Industrial applications of microbial synthesis typically utilize cells in the stationary phase, when cultures have the greatest density of viable cells. By manipulating culture conditions to delay the transition from the stationary phase to the death phase, we can prolong the stationary phase on a scale of hours to days, thereby maintaining the maximum density of cells that would otherwise quickly decline. Characterization of the mechanisms that control entry into the death phase for the model organism E. coli not only deepens our understanding of the bacterial life cycle but also presents an opportunity to enhance current protocols for batch culture growth and explore similar effects in a variety of widely used bacterial strains.

In fed-batch culture, numerous factors, such as initial culture conditions and feeding strategies, affect the culture efficiency. Among the factors, the effect of initial culture medium is rarely investigated. In this work, Rhodiola sachalinensis cells were cultured in the fed-batch bioreactor system and the effects of volume, medium strength, and sucrose concentration of initial culture medium on biomass and accumulation of salidroside, polysaccharides, flavonoids, and phenolics were investigated. The results showed that an initial medium volume of 3 L significantly (p 0.05) increased biomass and the four bioactive compound contents. The maximum biomass and the highest contents of different bioactive compounds were determined at various MS medium strengths. Therefore, analytic hierarchy process (AHP) - technique for order preference by similarity to ideal solution (TOPSIS) was implemented and half-strength MS medium was selected. Furthermore, the effect of sucrose concentration was examined and 30 g/L sucrose in the initial medium was optimal, at which concentration, 39.45 mg/g DW of salidroside, 531.25 mg/g DW of total polysaccharides, 3.89 mg/g DW of total flavonoids, and 10.84 mg/g DW of total phenolics were produced. The findings of the present study provided a reference for further establishing the fed-batch culture system of R. sachalinensis cells.

Fed-batch processes are commonly used in industry to obtain sufficient biomass and associated recombinant protein or plasmids. In research laboratories, it is more common to use batch cultures, as the setup of fed-batch processes can be challenging. This method outlines a robust and reliable means to generate Escherichia coli biomass in a minimum amount of fermentation time using a standardized fed-batch process. Final cell densities can reach over 50g dry cell weight per liter (gdcw/L) depending on the strain. This method uses a predefined exponential feeding strategy and conservative induction protocol to achieve these targets without multiple trial and error studies. If desired, productivity can be optimized by balancing the induction time and feed rates. This method utilizes cost-efficient defined media, minimizes process control complexity, and potentially aids downstream purification.

Environmental growth inhibition conditions (GICs) have used extensively for increasing cell-specific productivity (qP ) of Chinese hamster ovary (CHO) cells, with the most common being temperature downshift and sodium butyrate (NaBu) treatment. BLIMP1 overexpression in CHO cells can also inhibit cell growth and increase product titres and qP . Given the similar responses, this study evaluated the individual and combined effects of BLIMP1 expression, low temperature and NaBu treatment on culture performance, cell metabolism and recombinant protein production of CHO cells. As expected, all three interventions decreased cell growth, arrested cells in G1/G0 cell cycle phase and increased qP . However, CHO cells presented different responses when considering cell viability, recombinant gene expression and cell metabolism that indicated differences in the molecular loci by which BLIMP1 and GICs generated higher productivities. Combinations of BLIMP1 expression and GICs acted synergistically to inhibit cell growth and maximise r-protein production, with the BLIMP1/NaBu condition leading to the most significant improvements in product titres and qP . This latter condition also proved to substantially increase product yields (up to 9.8 g IgG1 /L and 2.2 g EPO-Fc/L) and qP (up to 179 pcd for IgG1 and 30 pcd for EPO-Fc) in high-density perfusion cultures. These findings offered mechanistic insights about the productivity-enhancing effects of BLIMP1 and GICs, as well as their complementarity for generating highly productive processes. This article is protected by copyright. All rights reserved.

Fed-batch cultivation is a well-known type of submerged fermentation that is frequently used in manufacture of recombinant proteins and various kinds of enzymes, owing to its ability to produce products with high concentrations and high efficiency. In fed-batch culture, several issues must be considered; most of them are also presented in batch culture. However, feed flow rate calculation only corresponds to fed-batch fermentation and its value has a significant impact on productivity, efficiency, final concentration of product, formation of by-products, and viscosity of the culture. From this background, the present review article is an effort to gather the information on feeding strategies for fed-batch cultivation of