High Cell Density Cultivation Research Articles

Selective screening and characterization of plant growth promoting bacteria for growth enhancement of tomato, Lycopersicon esculentum.

Background and Objectives:Plant Growth-promoting Bacteria (PGPB) can replace the dangerous chemical fertilizers and pesticides. The aim of this study was to isolate the PGPBs for Lycopersicon esculentum plant and to determine the appropriate volume for inoculation.Materials and Methods:Plants samples were collected from tomato fields. Nitrogen fixing-PGPBs were isolated from rhizoplane and rhizosphere. Five isolates were screened based on their growth abilities and examined for PGPB traits including phosphate solubilization, and IAA, ammonia and HCN production. After high cell density cultivation, the cells were separated by centrifugation and freeze dried after resuspension in cryoprotectant. The powders were inoculated into sterile soil with a dose of 106, 107 and 108 CFUs/g. Tomato (Lycopersicon esculentum) seeds were sown in soil and after 42 days the shoot length was measured.Results:Most of the potent PGPBs with high growth capacity were isolated from rhizoplane. Maximum phosphate solubilization was 289.7 μg/ml by NFB12 which isolated from rhizoplane. This strain produced the maximum level of IAA. NFB12 produced ammonia without the ability of production of HCN. This strain enhanced shoot length in dosed dependent manner. Surprisingly, inoculation of soil with 108 CFUs/g dramatically decreased the shoot length by 21%. Based on molecular approach NFB12 was identified as Bacillus megaterium.Conclusion:Isolation of specific PGPBS is recommended for sustainable plant production. Our results showed that NBF12 improves tomato plant growth and its effect on tomato plant growth is does dependent. Maximum growth rate of tomato was observed with 107 CFUs/g soil inoculation of NFB12 while higher inoculation showed negative effect.

Application of robust ketoreductase from Hansenula polymorpha for the reduction of carbonyl compounds

Enzyme-catalysed asymmetric reduction of ketones is an attractive tool for the production of chiral building blocks or precursors for the synthesis of bioactive compounds. Expression of robust ketoreductase (KRED) from Hansenula polymorpha was upscaled and applied for the asymmetric reduction of 31 prochiral carbonyl compounds (aliphatic and aromatic ketones, diketones and β-keto esters) to the corresponding optically pure hydroxy compounds. Biotransformations were performed with the purified recombinant KRED together with NADP+ recycling glucose dehydrogenase (GDH, Bacillus megaterium), both overexpressed in Escherichia coli BL21(DE3). Maximum activity of KRED for biotransformation of ethyl-2-methylacetoacetate achieved by the high cell density cultivation was 2499.7 ± 234 U g–1DCW and 8.47 ± 0.40 U·mg–1E, respectively. The KRED from Hansenula polymorpha is a very versatile enzyme with broad substrate specificity and high activity towards carbonyl substrates with various structural features. Among the 36 carbonyl substrates screened in this study, the KRED showed activity with 31, with high enantioselectivity in most cases. With several ketones, the Hansenula polymorpha KRED catalysed preferentially the formation of the (R)-secondary alcohols, which is highly valued.

Enhanced human lysozyme production by Pichia pastoris via periodic glycerol and dissolved oxygen concentrations control.

In human lysozyme (hLYZ) production by Pichia pastoris, the glycerol fed-batch phase was generally implemented under the environment of "oxygen sufficient-glycerol limited" to achieve high cell-density cultivation during the cell growth phase. However, the structural and functional components in P. pastoris cells were irreversible damaged with more and more reactive oxygen species (ROS) accumulation when cells were exposed to the oxygen sufficient environments for long time, leading to a failure of hLYZ expression. In this study, a novel periodic glycerol and dissolved oxygen concentration (DO) control strategy was proposed to solve these problems. This strategy periodically switched the cultivation environments from "oxygen sufficient-glycerol limited" to "oxygen limited-glycerol sufficient" for 5 cycles. When using this strategy: (1) the highest dry cell weight (DCW) of 143.02 g-DCW/L and the lowest distribution of glycerol towards to cell maintenance (0.0400 1/h) were achieved during the glycerol feeding phase by maintaining ROS levels below 48.39 Fluorescence intensity/g-DCW; (2) the adaption time of P. pastoris cells to methanol induction environments was shortened for about 50%; (3) P. pastoris cell metabolic activities reflected by the activities of alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol consumption rate, etc., in the successive induction phase were largely enhanced; (4) hLYZ activity reached the highest level of 2.45 × 105 IU/mL, which was about 2-fold than that obtained with the strategy of "oxygen sufficient-glycerol limited," when the same methanol induction strategy was adopted. KEY POINTS: • A novel periodic glycerol feeding strategy proposed/used for P. pastoris cell growth. • Higher cell density was obtained by controlling ROS at low level via this strategy. • The highest hLYZ activity was achieved when initiating induction at higher cell density.

Anti-CD40 antibody KPL-404 inhibits T cell-mediated activation of B cells from healthy donors and autoimmune patients

BackgroundCD40-CD40L is a key co-stimulatory pathway for B cell activation. As such, its blockade can inhibit pathogenic B cell responses in autoimmune diseases, such as Sjogren’s syndrome (SjS) and systemic lupus erythematosus (SLE). In this study, we examined the in vitro effects of KPL-404, a humanized anti-CD40 monoclonal antibody (Ab), on primary human B cells derived from either healthy donors (HD) or autoimmune patients and compared them to the effects of G28-5, a partially antagonistic anti-CD40 antibody.MethodsPBMCs from HD or SjS and SLE patients were cultured in high-density cell cultures in the presence of IgG4 isotype control or anti-CD40 Abs KPL-404 or G28-5. Cells were stimulated with anti-CD3/CD28 cross-linking reagent ImmunoCult (IC) to induce CD40L-CD40-mediated B cell responses. B cell proliferation and activation, measured by dilution of proliferation tracker dye and the upregulation of CD69 and CD86, respectively, were assessed by flow cytometry. Anti-CD40 Ab cell-internalization was examined by imaging flow cytometry. Cytokine release in the PBMC cultures was quantified by bead-based multiplex assay.ResultsKPL-404 binds to CD40 expressed on different subsets of B cells without inducing cell depletion, or B cell proliferation and activation in in vitro culture. Under the same conditions, G28-5 promoted proliferation of and increased CD69 expression on otherwise unstimulated B cells. KPL-404 efficiently blocked the CD40L-CD40-mediated activation of B cells from HD at concentrations between 1 and 10 μg/ml. Treatment with KPL-404 alone did not promote cytokine production and blocked the production of IFNβ in healthy PBMC cultures. KPL-404 efficiently blocked CD40L-CD40-mediated activation of B cells from patients with SjS and SLE, without affecting their anti-IgM responses or affecting their cytokine production. Consistent with the differences of their effects on B cell responses, KPL-404 was not internalized by cells, whereas G28-5 showed partial internalization upon CD40 binding.ConclusionsAnti-CD40 mAb KPL-404 showed purely antagonistic effects on B cells and total PBMCs. KPL-404 inhibited CD40L-CD40-mediated B cell activation in PBMC cultures from both healthy controls and autoimmune patients. These data support the therapeutic potential of CD40 targeting by KPL-404 Ab for inhibiting B cell responses in SjS and SLE.

High cell density cultivation enables efficient and sustainable recombinant polyamine production in the microalga Chlamydomonas reinhardtii

Modern chemical industry calls for new resource-efficient and sustainable value chains for production of key base chemicals such as polyamines. The green microalga Chlamydomonas reinhardtii offers great potential as an innovative green-cell factory by combining fast and inexpensive, phototrophic growth with mature genetic engineering. Here, overexpression of recombinant lysine decarboxylases in C. reinhardtii enabled the robust accumulation of the non-native polyamine cadaverine, which serves as building block for bio-polyamides. The issue of low cell densities, limiting most microalgal cultivation processes was resolved by systematically optimizing cultivation parameters. A new, easy-to-apply and fully phototrophic medium enables high cell density cultivations of C. reinhardtii with a 6-fold increase in biomass and cell count (20 g/L biomass dry weight, ~2·108 cells/mL). Application of high cell density cultivations in established photobioreactors resulted in a 10-fold increase of cadaverine yields, with up to 0.24 g/L after 9 days and maximal productivity of 0.1 g/L/d.

Depletion of Boric Acid and Cobalt from Cultivation Media: Impact on Recombinant Protein Production with Komagataella phaffii

The REACH regulation stands for “Registration, Evaluation, Authorization and Restriction of Chemicals” and defines certain substances as harmful to human health and the environment. This urges manufacturers to adapt production processes. Boric acid and cobalt dichloride represent such harmful ingredients, but are commonly used in yeast cultivation media. The yeast Komagataella phaffii (Pichia pastoris) is an important host for heterologous protein production and compliance with the REACH regulation is desirable. Boric acid and cobalt dichloride are used as boron and cobalt sources, respectively. Boron and cobalt support growth and productivity and a number of cobalt-containing enzymes exist. Therefore, depletion of boric acid and cobalt dichloride could have various negative effects, but knowledge is currently scarce. Herein, we provide an insight into the impact of boric acid and cobalt depletion on recombinant protein production with K. phaffii and additionally show how different vessel materials affect cultivation media compositions through leaking elements. We found that boric acid could be substituted through boron leakiness from borosilicate glassware. Furthermore, depletion of boric acid and cobalt dichloride neither affected high cell density cultivation nor cell morphology and viability on methanol. However, final protein quality of three different industrially relevant enzymes was affected in various ways.

Soluble Expression of hFGF19 without Fusion Protein through Synonymous Codon Substitutions and DsbC Co-Expression in E. coli.

Human fibroblast growth factor 19 (hFGF19) is a difficult-to-express protein that is frequently fused with another protein for soluble expression. However, residual amino acids after cleavage with protease represent one of the major problems in therapeutic protein development. Here, we introduced synonymous codon substitutions in the N-terminal region encoding sequence of hFGF19 and co-expressed disulfide bond isomerase (ΔssDsbC) to functionally express hFGF19 without any fusion protein. Synonymous codon substitution significantly increased hFGF19 expression. Subsequent co-expression of ΔssDsbC with a selected variant of hFGF19 (scvhFGF19) further increased the proportion of soluble hFGF19 expression in Escherichia coli XL1-Blue. Both total and soluble scvhFGF19 expression increased remarkably in the alternative host, E. coli Origami 2 with mutated thioredoxin reductase and glutathione reductase. scvhFGF19 purification by anion exchange and heparin affinity chromatography resulted in a yield of 6.5 mg/L under normal induction conditions in flask culture. As such, a high cell density culture is expected to achieve an even higher yield. The biological activities of purified scvhFGF19 were assessed based on its ability to activate ERK1/2 signaling pathway in HepG2 hepatocarcinoma cells. In conclusion, the strategy described here may represent an efficient alternative process for the production of hFGF19 and/or related proteins.

Enhancing microaerobic plasmid DNA production by chromosomal expression of Vitreoscilla hemoglobin in E. coli

• VHb reduced overflow metabolism. • VHb improved pDNA production under oxygen limitations. • VHb improved pDNA yields even at lower RNAII/RNAI ratios. • Higher lactate production may be caused by stronger NAD + demand. Oxygen availability and overflow metabolism are often limiting factors in high cell-density cultures. In this study, Vitreoscilla hemoglobin (VHb) was expressed in the chromosome of E. coli as a strategy to improve plasmid DNA (pDNA) production in biphasic fed-batch cultures. During the aerobic batch phase, the strain expressing VHb accumulated 28 % less acetate and 19 % more pDNA than the wild type strain. The culture was shifted from aerobic to microaerobic conditions during the fed-batch phase. pDNA yields increased consistently in the VHb-expressing strain during the whole culture, while decreased progressively for the wild type under microaerobic conditions. Unexpectedly, the ratio of positive/negative replication control molecules (RNAII/RNAI) were lower for the VHb-expressing than for the wild type strain. However, the final pDNA titer was ca. 74 % higher for the former. Flux balance analysis suggests that VHb presence increases the fluxes in anaplerotic pathways. The higher lactate production observed in VHb-expressing cells may be triggered by an increased demand of NAD + in glycolysis under microaerobic conditions.

Accelerated production of α2,8- and α2,9-linked polysialic acid in recombinant Escherichia coli using high cell density cultivation

Polysialic acid (polySia) are α2,8- and/or α2,9-linked homopolymers with interesting properties for meningococcal vaccine development or the cure of human neurodegenerative disorders.With the goal to avoid large scale production of pathogenic bacteria, we compare in the current study the efficacy of conventional polySia production to recombinant approaches using the engineered laboratory safety strain E. coli BL21. High cell density cultivation (HCDC) experiments were performed in two different bioreactor systems. Increased cell densities of up to 11.3 (±0.4) g/L and polySia concentrations of up to 774 (±18) mg/L were reached in E. coli K1. However, cultivation of engineered E. coli BL21 strains delivered comparable cell densities but a maximum of only 133 mg/L polySia. Using established downstream procedures, host cell DNA and proteins were removed. All recombinant polySia products showed an identical degree of polymerization >90. Polymers with different glycosidic linkages could be successfully differentiated by nuclear magnetic resonance spectroscopy.

Aqueous Two‐Phase Extraction of Monoclonal Antibodies from High Cell Density Cell Culture

AbstractImprovements of the upstream process for the production of monoclonal antibodies (mAb) has resulted in mammalian cell cultures with significantly increased cell densities. These high cell density (HCD) broths pose severe challenges for established downstream unit operations due to the increased biomass load. To meet these challenges, aqueous two‐phase extraction was examined as a potential method for the clarification, as well as first stage purification of a Chinese hamster ovary expressed mAb with cell densities up to 70 · 106 cells mL−1. A clarification with 99 % yield of mAb and 68 % DNA removal was accomplished, which contributes to enable a foundation to the industrial application of aqueous two‐phase extraction for manufacturing of mAb from HCD cultivations.

High Cell Density Cultivation of Saccharomyces cerevisiae with Intensive Multiple Sequential Batches Together with a Novel Technique of Fed-Batch at Cell Level (FBC)

High cell density cultivation (HCDC) is developed for the production of microbial biomasses and their products. They must be produced from high concentrations of substrate, e.g., glucose or sucrose. In batch culture, a high concentration of those sugars >40–50% (w/v) cannot efficiently be utilized because of a dissolved O2 limitation causing the Crabtree effect that produces toxic by-products, i.e., ethanol and/or acetate, that inhibit cell growth. To prevent this effect, the HCDC is conducted with the fed-batch strategies. However, it has many disadvantages, i.e., complicated operations. To overcome those problems, this study was designed to use a new, efficient C-source (carbon source) substrate, namely dextrin, an oligomer of glucose. It can be utilized by yeast at a very high concentration of ~100 g/L although using just batch cultivation. As it is gradually hydrolyzed to release glucose molecules and gradually assimilated into the cells as “fed-batch at the cell level” (FBC), it prevents the yeast cell system from undergoing the Crabtree effect. In this research, the types of medium, the types of sugar compared with dextrin, and the concentrations of yeast extract (YE) were studied. The batch production medium (BPM) with dextrin and YE performed very good results. The concentrations of dextrin for yeast cultivation were studied in the aerobic batch 5-L bioreactors. Its optimum concentration was at 90 g/L with 9 g/L of YE in 3× BPM. It was operated at 3 W/kg energy dissipation rate per unit mass (ε¯T) and 3 vvm airflow rate. Further, the intensive multiple sequential batch (IMSB) technique of high intensities of agitation speed and airflow was developed to achieve higher yield and productivity. The maximum values of cell biomass, specific growth rate, yield coefficient, productivity, and efficiency were at 55.17 g/L, 0.21 h−1, 0.54 g/g, 2.30 g/L/h, and 98.18%, respectively. The studies of cell growth kinetics, biochemical engineering mass balances, and fluid dynamics for the design of impeller speeds of the 5-L bioreactors during the cultivations of yeast using dextrin at the high concentrations were successful. The results can be used for the scale-up of bioreactor for the industrial production of yeast cell biomass at high concentrations.

Evaluating Cost-Effective Culture Media for Nutraceutics Production from Microalgae Using Computer-Aided Large Scale Predictions

The purpose of this research consist of microalgae biomass productivity prediction in a large scale plant from Chlorella vulgaris using SuperPro Designer software. Two culture media were evaluated to identify their potential use in large-scale processes: (a) the Bold Basal medium enriched with sodium acetate and (b) a growth medium for high cell density cultures proposed from references. Results found in this research suggest that simulated plant should reach a capacity to generate 1.4 kg/h of biomass at a cost of 1.44 USD/g using Bold Basal medium. In contrast, a productivity of 28 kg/h at a cost of 0.14 USD/g was found using a medium proliferation of highly dense microalgae cultures. These findings suggest that Bold Basal medium could negatively impact operating costs, limiting its use in large-scale processes regarding nutraceutics production. Results found here demonstrate the potential usage of simulators for estimating costs and production which allows predicting the bioprocess feasibility.

A General Process-Based Model for Describing the Metabolic Shift in Microbial Cell Cultures.

The metabolic shift between respiration and fermentation at high glucose concentration is a widespread phenomenon in microbial world, and it is relevant for the biotechnological exploitation of microbial cell factories, affecting the achievement of high-cell-densities in bioreactors. Starting from a model already developed for the yeast Saccharomyces cerevisiae, based on the System Dynamics approach, a general process-based model for two prokaryotic species of biotechnological interest, such as Escherichia coli and Bacillus subtilis, is proposed. The model is based on the main assumption that glycolytic intermediates act as central catabolic hub regulating the shift between respiratory and fermentative pathways. Furthermore, the description of a mixed fermentation with secondary by-products, characteristic of bacterial metabolism, is explicitly considered. The model also represents the inhibitory effect on growth and metabolism of self-produced toxic compounds relevant in assessing the late phases of high-cell density culture. Model simulations reproduced data from experiments reported in the literature with different strains of non-recombinant and recombinant E. coli and B. subtilis cultured in both batch and fed-batch reactors. The proposed model, based on simple biological assumptions, is able to describe the main dynamics of two microbial species of relevant biotechnological interest. It demonstrates that a reductionist System Dynamics approach to formulate simplified macro-kinetic models can provide a robust representation of cell growth and accumulation in the medium of fermentation by-products.

Kinetic study and optimization of recombinant human tumor necrosis factor-alpha (rhTNF-α) production in Escherichia coli

Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that plays a major role in immune regulation, homeostatic function, and cellular organization. The present study was undertaken to overproduce recombinant human TNF-α (rhTNF-α) in Escherichia coli (E.coli) in high cell density culture. The use of a codon-optimized gene and strong promoter-based (T7) expression system, choice of Terrific Broth (TB) as medium, and subsequent optimization of culture conditions in shake flasks resulted in production of 0.95 g/L insoluble rhTNF-α comprising upto 50% of total cellular protein (TCP) The protein yield further increased upto 1.26 g/L in 1 L TB medium batch culture in bioreactor with the controlled temperature, pH, and dissolved oxygen. In a series of chemostats operated at dilution rates of 0.2 h−1, 0.3 h−1, 0.4 h−1 and 0.5 h−1 the specific growth rate (μ) positively correlated with specific yield (Yp / x ) and a maximum yield of 164 mg/g DCW was obtained at μ = 0.4 h−1 within 4 h post-induction. A fed-batch cultivation in TB with an exponential feeding profile (μ = ∼0.4 h−1) of concentrated feed resulted in an accumulation of 5.5 g/L of rhTNF-α within 14 h of cultivation which accounted for ∼29% of TCP.

Expression of a novel α-glucosidase from Aspergillus neoniger in Pichia pastoris and its efficient recovery for synthesis of isomaltooligosaccharides

A gene conferring α-glucosidase (AG) with high transglycosylation activity from Aspergillus neoniger (a non-niger strain belonging to section Nigri) was cloned and expressed in Pichia pastoris. As the cDNA construction retained intronic portions due to alternative splicing, the exonic portions of the gene were stitched using restriction digestion and overlap extension PCR. Pre-determined open-loop exponential feeding strategies were evaluated for methanol dosage to improve the recombinant enzyme synthesis during high-cell density cultivation in 5 L bioreactor. Specific growth rate of 0.1 h−1 resulted in the highest enzyme activity of 182.3 mU/mL in the supernatant, whereas the activity of 3.8 U/g dry cell weight was obtained in the cell pellet. There was negligible enzyme activity in the cell lysate, indicating that approximately 80 % accumulation of total enzyme is in the periplasm. Later, this unreleased fraction was extracted to 90 % yield using 25 mM cysteine. The enzyme was purified and validated using western blot analysis and MS/MS profile. The SDS PAGE analysis revealed three bands corresponding to 80, 38, and 33 kDa indicating the multimeric nature of the enzyme. Thus, obtained enzyme was utilized in synthesis of a potential prebiotic molecule, isomaltooligosaccharides (IMOs), which can be used as a sweetener and bulk filler in the food industry. This is the first report to demonstrate challenges in cloning and expression of transglycosylating α-glucosidase from Aspergillus neoniger.

Expression of VHb Improved Lipid Production in Rhodosporidium toruloides

The oleaginous yeast Rhodosporidium toruloides has emerged as a robust host for production of microbial lipids as alternative biofuel feedstocks. Oxygen supply is a limiting factor for microbial lipid production, as lipid biosynthesis is highly oxygen-demanding. Vitreoscilla hemoglobin (VHb) is a protein capable of promoting oxygen delivery for anabolism. In this study, we developed R. toruloides with VHb expression for improved lipid production. The VHb expression cassette was integrated into the R. toruloides chromosome via the Agrobacterium-mediated transformation. In shake flask cultures, the engineered strain 4#-13 produced 34% more lipids than the parental strain did. Results obtained under reduced aeration conditions in 3 L bioreactor showed that lipid titer and lipid yield of the engineered strain 4#-13 were 116% and 71%, respectively, higher than those of the parental strain. Under high cell density culture conditions, the engineered strain 4#-13 grew faster and produced 72% more lipids. Our results demonstrated that the VHb gene is functional in R. toruloides for promoting lipid production. The strains described here may be further engineered by integrating extra genetic parts to attain robust producers for more valuable products. This should improve the economics of microbial lipids to facilitate a sustainable production of biodiesel and other lipid-based biofuels.

Activation of Energy Metabolism through Growth Media Reformulation Enables a 24-Hour Workflow for Cell-Free Expression.

Cell-free protein synthesis (CFPS) platforms have undergone numerous workflow improvements to enable diverse applications in research, biomanufacturing, and education. The Escherichia coli cell extract-based platform has been broadly adopted due to its affordability and versatility. The upstream processing of cells to generate crude cell lysate remains time-intensive and technically nuanced, representing one of the largest sources of cost associated with the biotechnology. To overcome these limitations, we have improved the processes by developing a long-lasting autoinduction media formulation for CFPS that obviates human intervention between inoculation and harvest. The cell-free autoinduction (CFAI) media supports the production of robust cell extracts from high cell density cultures nearing the stationary phase of growth. As a result, the total mass of cells and the resulting extract volume obtained increases by 400% while maintaining robust reaction yields of reporter protein, sfGFP (>1 mg/mL). Notably, the CFAI workflow allows users to go from cells on a streak plate to completing CFPS reactions within 24 h. The CFAI workflow uniquely enabled us to elucidate the metabolic limits in CFPS associated with cells grown to stationary phase in the traditional 2× YTPG media. Metabolomics analysis demonstrates that CFAI-based extracts overcome these limits due to improved energy metabolism and redox balance. The advances reported here shed new light on the metabolism associated with highly active CFPS reactions and inform future efforts to tune the metabolism in CFPS systems. Additionally, we anticipate that the improvements in the time and cost-efficiency of CFPS will increase the simplicity and reproducibility, reducing the barriers for new researchers interested in implementing CFPS.

Fusion and secretory expression of an exo-inulinase and a d-allulose 3-epimerase to produce d-allulose syrup from inulin.

This study developed a feasible catalytic method for d-allulose syrup production using a fusion enzyme, either in free or immobilized form, through hydrolysis of inulin extracted from Jerusalem artichoke tubers. d-Allulose 3-epimerase (DAE) was actively expressed in secretory form by fusing with the extracellular exo-inulinase CSCA in Escherichia coli BL21 (DE3). The best linker ligating the two enzymes was a flexible peptide containing 12 residues (GSAGSAAGSGEF). At 55 °C and pH 8.0, and as with the addition of 1 mmol L-1 Mn2+ , the CSCA-linkerE-DAE fusion enzyme obtained through high cell-density cultivation displayed a maximal exo-inulinase activity of 21.8 U mg-1 and resulted in a yield of 6.3 g L-1 d-allulose and 39.2 g L-1 d-fructose using 60 g L-1 inulin as the raw material. Catechol-modified alginate with titanium ions (Alg(Ti)PDA) was found to be a promising immobilization material for the fusion enzyme. After conversion for 8 days, the Alg(Ti)PDA-immobilized CSCA-linkerE-DAE (8 U g-1 ) completed 24 reaction cycles and retained over 80% of its original activity. Each reaction obtained an average of 19.8 g L-1 d-allulose and 32.7 g L-1 D-fructose from 60 g L-1 inulin. This study shed light on a feasible and cost-effective approach for the production of syrup containing d-allulose and D-fructose with inulin as the raw material via the use of a CSCA and DAE fusion enzyme. This syrup is of added value as a functional sweetener. © 2020 Society of Chemical Industry.

Investigation into the impact of tyrosine on the product formation and quality attributes of mAbs in rCHO cell cultures.

Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality attributes in fed-batch cultures of recombinant Chinese hamster ovary (rCHO) cells. However, the relation between tyrosine and these aspects is not yet fully defined. In order to further elucidate such a relation, two groups of fed-batch experiments with high tyrosine (H-T) or low tyrosine (L-T) additions producing an IgG1 monoclonal antibody against CD20 were implemented to investigate the intracellular and extracellular effects of tyrosine on the culture performance. It was found that the scarcity of tyrosine led to the distinctive reduction in both viable cell density and antibody specific production rate, hence the sharply reduced titer, possibly related to the impaired translation efficiency caused by the substrate limitation of tyrosine. In addition, alterations to the critical quality attributes were detected in the L-T group, compared to those in the H-T condition. Notable decrease in the contents of intact antibody was found under the L-T condition because of the elevated reductive level in the supernatant. Moreover, the aggregate content in the L-T condition was also reduced, probably resulting from the accumulation of extracellular cystine. In particular, the lysine variant content noticeably increased with tyrosine limitation owing to the downregulation of two carboxypeptidases, i.e., CpB and CpH. Overall, understanding the role of tyrosine in these aspects is fundamental to the increase of product titers and control of critical quality attributes in the monoclonal antibody production of rCHO cell fed-batch cultures. KEY POINTS: • Tyrosine is essential in the maintenance of product titers and the control of product qualities in high cell density cultivations in rCHO cell. • This study revealed the bottleneck of decreased qmAbupon the deficiency of tyrosine. • The impact of tyrosine on the critical product qualities and the underlying mechanisms were also thoroughly assessed.

Increasing PHB production with an industrially scalable hardwood hydrolysate as a carbon source

In this study, we report an industrially feasible conversion of wood hydrolysate from a pilot-scale forest biorefinery. The wood hydrolysate was compared to a synthetic hydrolysate with a similar content of carbon sources (glucose, xylose, and acetate). To maximize the PHB concentration, high-cell density cultivations were conducted in bioreactors for 52 h using the bacterium Paraburkholderia sacchari IPT 101 LMG 19450. The conversion of wood hydrolysate yielded a maximum PHB concentration of 34.5 g/L, which is among the highest reported for lignocellulose hydrolysates. In comparison, the use of a synthetic hydrolysate resulted in substantially lower PHB concentration (22.0 g/L). This could be attributed to a higher maximum specific growth rate of 0.36 vs. 0.33 per h for cells grown in wood hydrolysate vs. synthetic hydrolysate. For the wood hydrolysate, the final PHB content per cell mass reached 58 % g/g and a maximum PHB productivity of 0.72 g/(Lh), while the synthetic hydrolysate reached 55 % g/g and 0.46 g/(Lh) respectively. Given the increase in bacterial growth and PHB productivity when using the wood hydrolysate, the chosen lignocellulose conversion process is beneficial for subsequent biotechnological conversion to key bioproducts.