Culture Flasks Research Articles

Production, optimization and characterization of partially purified anti-mycotic compound from marine soil derived streptomycetes originating at unexplored region of Bay of Bengal, India

Sixty-eight morphologically distinct isolates of marine actinomycetes were derived from seashore, mangrove, and saltpan ecosystems located between the Palk Strait and Gulf of Mannar region, Bay of Bengal, Tamilnadu. Twenty-five (36.8%) isolates exhibited anti-mycotic activity against Candida albicans and Cryptococcus neoformans in preliminary screening, and 4 isolates with prominent activity were identified and designated at the genus level as Streptomyces sp. VPTS3-I, Streptomyces sp. VPTS3-2, Streptomyces sp. VPTSA1-4 and Streptomyces sp. VPTSA1-8. All the potential antagonistic isolates were further characterized with phenotypic and genotypic properties including 16S rRNA gene sequencing and identified species level as Streptomyces afghaniensis VPTS3-1, S. matensis VPTS3-2, S. tuirus VPTSA1-4 and S. griseus VPTSA1-8. In addition, the active fractions from the potential antagonistic streptomycetes were extracted with organic solvents by shake flask culture method and the anti-mycotic efficacies were evaluated. The optimization parameters for the production of the anti-mycotic compound were found to be pH between 7 and 8, the temperature at 30ᵒC, the salinity of 2%, incubation of 9 days, and starch and KNO3 as the suitable carbon and nitrogen sources respectively in starch casein medium.

Do milt storage techniques affect the quality of carp spermatozoa during short-term storage?

Short-term storage and management of milt are crucial parts of hatchery practice. There is speculation that sperm stored in a small Eppendorf could not retain their optimal performance compared to those stored in a 50 ml cell culture flask (CCF), thus raising the question of discrepancy between laboratory and hatchery conditions. Therefore, this study aimed to compare sperm quality parameters, such as osmolality and the ionic composition of seminal plasma, spermatozoa motility and its ability to maintain the volume under various osmotic conditions (310, 230, 150, 90, and 20 mOsm/kg) in fresh sperm and after its storage for 24 h in 1.5 ml Eppendorf or 50 ml CCF at 4 °C without any extender. Freshly collected sperm had higher motility percentages at each osmotic condition than 24 h stored sperm in Eppendorf and CCF. There was no significant difference in spermatozoa motility at various osmotic conditions except 90 mOsm/kg osmolality. Besides, VCL, VSL, VAP, and LIN of spermatozoa, osmolality, and ionic composition of seminal plasma did not significantly differ between these two containers. This study suggested no difference between sperm stored in an Eppendorf or CCF regarding sperm functionality.

Convergent Synthesis of Two Heterogeneous Fluxes from Glucose and Acetate for High-Yield Citramalate Production.

Biological production of citramalate has garnered attention due to its wide application for food additives and pharmaceuticals, although improvement of yield is known to be challenging. When glucose is used as the sole carbon source, carbon loss through decarboxylation steps for providing acetyl-CoA from pyruvate is inevitable. To avoid this, we engineered a strain to co-utilize glucose and cost-effective acetate while preventing carbon loss for enhancing citramalate production. The production pathway diverged to independently supply the precursors required for the synthesis of citramalate from glucose and acetate, respectively. Moreover, the phosphotransferase system was inactivated and the acetate assimilation pathway and the substrate ratio were optimized to enable the simultaneous and efficient utilization of both carbon sources. This yielded results (5.0 g/L, 0.87 mol/mol) surpassing the yield and titer of the control strain utilizing glucose as the sole carbon source in flask cultures, demonstrating an economically efficient strain redesign strategy for synthesizing various products.

The serine/threonine protein kinase MpSTE1 directly governs hyphal branching in Monascus spp.

Monascus spp. are commercially important fungi due to their ability to produce beneficial secondary metabolites such as the cholesterol-lowering agent lovastatin and natural food colorants azaphilone pigments. Although hyphal branching intensively influenced the production of these secondary metabolites, the pivotal regulators of hyphal development in Monascus spp. remain unclear. To identify these important regulators, we developed an artificial intelligence (AI)–assisted image analysis tool for quantification of hyphae-branching and constructed a random T-DNA insertion library. High-throughput screening revealed that a STE kinase, MpSTE1, was considered as a key regulator of hyphal branching based on the hyphal phenotype. To further validate the role of MpSTE1, we generated an mpSTE1 gene knockout mutant, a complemented mutant, and an overexpression mutant (OE::mpSTE1). Microscopic observations revealed that overexpression of mpSTE1 led to a 63% increase in branch number while deletion of mpSTE1 reduced the hyphal branching by 68% compared to the wild-type strain. In flask cultures, the strain OE::mpSTE1 showed accelerated growth and glucose consumption. More importantly, the strain OE::mpSTE1 produced 9.2 mg/L lovastatin and 17.0 mg/L azaphilone pigments, respectively, 47.0% and 30.1% higher than those of the wild-type strain. Phosphoproteomic analysis revealed that MpSTE1 directly phosphorylated 7 downstream signal proteins involved in cell division, cytoskeletal organization, and signal transduction. To our best knowledge, MpSTE1 is reported as the first characterized regulator for tightly regulating the hyphal branching in Monascus spp. These findings significantly expanded current understanding of the signaling pathway governing the hyphal branching and development in Monascus spp. Furthermore, MpSTE1 and its analogs were demonstrated as promising targets for improving production of valuable secondary metabolites.Key points• MpSTE1 is the first characterized regulator for tightly regulating hyphal branching• Overexpression of mpSTE1 significantly improves secondary metabolite production• A high-throughput image analysis tool was developed for counting hyphal branching

Culture of Hoffa fat pad mesenchymal stem/stromal cells on microcarrier suspension in vertical wheel bioreactor for extracellular vesicle production

BackgroundMesenchymal stromal cells (MSCs) are increasingly employed in regenerative medicine approaches for their immunomodulatory and anti-inflammatory properties, which are encoded in their secretome including extracellular vesicles (EVs). The Hoffa fat pad (HFP) located infrapatellarly harbours MSCs that could assist in tissue homeostasis in osteoarthritic joints. Intraarticular injection therapies based on blood products could modulate the populations of released HFP-MSC-EVs in a quantitative manner.MethodsTo obtain amounts of HFP-MSC-derived EVs that allow pre-clinical evaluation, suitable EV production systems need to be developed. This work investigates the release of EVs from primary HFP-MSCs cultivated in a 3D environment using microcarrier suspension culture in a vertical wheel bioreactor in comparison to conventional 2D culture. To simulate an intraarticular blood product therapy, cultures were treated with citrate-anticoagulated platelet-rich plasma (CPRP) or hyperacute serum (hypACT) before EV collection. HFP-MSC-EVs are enriched via ultrafiltration and characterised via Western Blot, nanoparticle tracking analysis in scatter as well as fluorescence mode. EV potency was determined via RT-qPCR analysing the expression of type II and X collagen (COL2 and COL10), as well as inducible nitric oxide synthase (iNOS) in primary OA chondrocytes.ResultsBlood product supplementation elevated HFP-MSC metabolic activity as determined via XTT assay over the course of 14 days. 3D culture resulted in a roughly 100-fold EV yield compared to 2D culture and elevated number of EVs released per cell. Total protein content correlated with the EV concentration. While typical EV marker proteins such as CD9, CD63 or Alix were detected in total protein extracts, CD9 and CD73 colocalised on individual EVs highlighting their cell origin. The type of blood product treatment did not affect the size or concentration of EVs obtained from HFP-MSCs. Assessing potency of 3D culture EVs in comparison to 2D EVs revealed superior biological activity with regard to inhibition of inflammation, inhibition of chondrocyte hypertrophy and induction of cartilage-specific ECM production.ConclusionsHFP-MSCs proliferate in presence of human blood products indicating that animal serum in culture media can be avoided in the future. The culture of HFP-MSCs in the employed bioreactor was successfully used to generate quantities of EVs that could allow evaluation of HFP-MSC-EV-mediated effects in pre-clinical settings. In addition, EV potency of 3D EVs is superior to EVs obtained in conventional 2D culture flasks.

In vitro Wound Healing Effect of a Siddha Formulation: Gandhaga Thailam

In vitro Wound Healing Effect of a Siddha Formulation: Gandhaga Thailam

Isolation and Characterization of Bacterial Contaminants from Bone Marrow-Derived Mesenchymal Stem Cell Cultures

This study aimed to assess and manage bacterial contamination in multiple batches of mesenchymal stem cell (MSC) cultures derived from rabbit bone marrow. Routine visual inspection and microscopic examination were employed for the detection of the contaminated cultures. The contaminated cultures were inoculated on Nutrient agar and multiple isolated colonies were subjected to Gram staining and biochemical characterization. Further, molecular identification of the bacterial isolates was performed using polymerase chain reaction. The determination of antibiotic susceptibility patterns was conducted using the Kirby-Bauer disc diffusion method. Among the 351 mesenchymal stem cell culture (SCC) flasks monitored, only 1.42% were found to be contaminated. Based on the phenotypic and biochemical characterization, the major bacterial contaminants were identified as Staphylococcus aureus, Bacillus spp., and Escherichia coli infiltrating during various stages of cell processing. Antibiotic susceptibility patterns revealed varying responses among isolates, crucial for effective antimicrobial strategies and maintaining aseptic conditions in SCCs. The study emphasizes the importance of regular monitoring to maintain sterile environments, validate culture quality, and uphold safety standards. The findings indicate the need to establish stringent quality control measures, crucial for the successful translation of MSC research into clinical applications. The research advocates for continuous monitoring, adherence to SOPs, and further investigations into preventive strategies for ensuring the safety and efficacy of MSC-based therapies.

Isolation and Culture of Primary Human Dental Pulp Cells-A Description of Technical and Methodological Steps to Maximise Predictability and Yield.

The dental pulp has critical functions in tooth development as well as an ongoing role in promoting and maintaining the vitality of teeth. In particular, its regenerative ability allows dental tissues to be restored following damage caused by traumatic injury or caries. Regenerative endodontic procedures aim to utilise these processes to stimulate dental pulp repair in a minimally invasive manner and reduce the need for more invasive procedures such as root canal treatment. Dental pulp is a source of dental pulp cells (DPCs), which has a subpopulation of dental pulp stem cells (DPSCs), which are attractive for use in regenerative medicine due to their high proliferation rate, ability to differentiate into multiple cell types, and their preserved vitality following cryopreservation. The development of next-generation clinical therapeutics that maximise the potential of dental pulp relies on strong empirical evidence arising from in vitro experimentation. Here, we describe a modified method for the efficient isolation of primary human DPCs from sound third molar teeth for culture using an explant outgrowth method on basement membrane-coated flasks, as well as using high-resolution macro-photography to illustrate the methods. Critically, steps are taken to minimise potential physical and mechanical trauma to the cells and maximise yield. Human DPCs cultured using this method can be further expanded in cell culture flasks to facilitate their use in various in vitro experimental procedures.

Carbohydrates Are Associated with the Flowering Ability of Oncidesa Gower Ramsey ‘Honey Angel’

Oncidesa Gower Ramsey ‘Honey Angel’ is a cut flower crop of high economic value worldwide. The regulation of flowering is important for cut flower production scheduling. However, its flowering transition mechanism is still unclear. Oncidesa usually flowers at the end of the growth cycle for each pseudobulb; this timing is probably related to carbohydrate accumulation. During this study, we investigated the carbohydrates in the pseudobulbs from juvenile plants to adult plants and compared the carbohydrates in flowering and nonflowering adult plants. The current pseudobulb and back pseudobulbs of the plants at 0, 0.5, 1.0, 1.5, and 2.0 years after having been moved out of the tissue culture flask were collected. The first pseudobulb formed at 0.5 years, and plants had fulfilled four growth cycles and flowered at 2.0 years. Each successive current shoot grew larger and the back shoot number progressively increased after each new growth cycle. The concentration of total soluble sugars in the current shoot increased from 5.5% of dry weight at 0.5 years to 20.2% of dry weight at 1.5 years. Conversely, the starch concentration decreased in the current pseudobulb as the plants matured. The starch concentration in the back pseudobulbs did not change when the plant grew a new shoot. The starch concentrations in the back pseudobulbs ranged from 33.2% to 57.5% of dry weight, but the combined content of starch in all of the back pseudobulbs increased significantly from 168 mg at 0.5 years to 4608 mg at 2.0 years because of the increasing number of back shoots. The starch in the first back pseudobulb of the nonflowering adult plants accounted for 18.0% of dry weight, which was lower than that of the flowering plants (48.3%). There was no significant difference in total soluble sugars in the current pseudobulb of the nonflowering and flowering plants. Overall, we revealed that the increase in the back shoot number increased the total amount of reserve carbohydrates as the plant reached reproductive maturity. A low starch level was observed in nonflowering adult plants. In both cases, flowering plants had higher starch storage in the back pseudobulbs, suggesting that carbohydrates might regulate the flowering of Oncidesa Gower Ramsey ‘Honey Angel’.

Coordinated optimization of the polymerization and transportation processes to enhance the yield of exopolysaccharide heparosan

Heparosan as the precursor for heparin biosynthesis has attracted intensive attention while Escherichia coli Nissle 1917 (EcN) has been applied as a chassis for heparosan biosynthesis. Here, after uncovering the pivotal role of KfiB in heparosan biosynthesis, we further demonstrate KfiB is involved in facilitating KpsT to translocate the nascent heparosan polysaccharide chain. As a result, an artificial expression cassette KfiACB was constructed with optimized RBS elements, resulting in 0.77 g/L heparosan in shake flask culture. Moreover, in view of the intracellular accumulation of heparosan, we further investigated the effects of overexpression of the ABC transport system proteins on heparosan biosynthesis. By co-overexpressing KfiACB with KpsTME, the heparosan production in flask cultures was increased to 1.03 g/L with an extracellular concentration of 0.96 g/L. Eventually, the engineered strain EcN/pET-kfiACB3-galU-kfiD-glmM/pCDF-kpsTME produced 12.2 g/L heparosan in 5-L fed-batch cultures while the extracellular heparosan was about 11.2 g/L. The results demonstrate the high-efficiency of the strategy for co-optimizing the polymerization and transportation for heparosan biosynthesis. Moreover, this strategy should be also available for enhancing the production of other polysaccharides.

Unlocking Nature’s Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter

BackgroundKomagataella phaffii (a.k.a. Pichia pastoris) harbors a glutamate utilization pathway in which synthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase (PEPCK) is induced by glutamate. Glutamate-inducible synthesis of these enzymes is regulated by Rtg1p, a cytosolic, basic helix-loop-helix protein. Here, we report food-grade monosodium glutamate (MSG)-inducible recombinant protein production from K. phaffii PEPCK promoter (PPEPCK) using green fluorescent protein (GFP) and receptor binding domain of SARS-CoV-2 virus (RBD) as model proteins.ResultsPPEPCK-RBD/GFP expression cassette was integrated at two different sites in the genome to improve recombinant protein yield from PPEPCK. The traditional, methanol-inducible alcohol oxidase 1 promoter (PAOX1) was used as the benchmark. Initial studies carried out with MSG as the inducer resulted in low recombinant protein yield. A new strategy employing MSG/ethanol mixed feeding improved biomass generation as well as recombinant protein yield. Cell density of 100–120 A600 units/ml was achieved after 72 h of induction in shake flask cultivations, resulting in recombinant protein yield from PPEPCK that is comparable or even higher than that from PAOX1.ConclusionsWe have designed an induction medium for recombinant protein production from K. phaffii PPEPCK in shake flask cultivations. It consists of 1.0% yeast extract, 2.0% peptone, 0.17% yeast nitrogen base with ammonium sulfate, 100 mM potassium phosphate (pH 6.0), 0.4 mg/L biotin, 2.0% MSG, and 2% ethanol. Substitution of ammonium sulphate with 0.5% urea is optional. Carbon source was replenished every 24 h during 72 h induction period. Under these conditions, GFP and RBD yields from PPEPCK equaled and even surpassed those from PAOX1. Compared to the traditional methanol-inducible expression system, the inducers of glutamate-inducible expression system are non-toxic and their metabolism does not generate toxic metabolites such as formaldehyde and hydrogen peroxide. This study sets the stage for MSG-inducible, industrial scale recombinant protein production from K. phaffii PPEPCK in bioreactors.

Dual Enzyme Sequential Digestion Protocol for Isolation of Human Primary Chondrocytes From the Articular Cartilage Derived From Knee Osteoarthritis Patients

Introduction: Majority of available protocols for isolation of chondrocytes from articular cartilage tissue rely on the enzymatic digestion of the tissue by collagenase type 2. The yield of chondrocytes in such protocols is low. Herein, we designed a novel indigenous sequential digestion by dual enzyme Pronase and Collagenase Type 1 for isolating human Chondrocytes from articular cartilage. Methods: Articular cartilage of Osteoarthritis (OA) patients undergoing total knee replacement were collected for the isolation of chondrocyte cells and subjected to sequential digestion by Pronase for three hours followed by Collagenase 1 overnight. Pellet of cells collected after digestion was plated on culture flask in 5% CO2 incubator. Results: From day three onwards, round to elongated cells adhered to the flask were visible which developed into elongated cell population of homogenous morphology, expressed Aggrecan (Agg), Collagen 2a (Col2a) and SRY-box transcription factor (Sox9) and had chondrogenic differentiation similar to a commercially available healthy chondrocyte. These cells were negative for Alizarin red stain, thus confirming the purity of chondrocytes. Conclusion: We have successfully established a sequential dual enzyme digestion-based culture technique for isolating the human chondrocytes from the articular cartilage biopsy derived from OA knee joints.

Reconstructing curcumin biosynthesis in yeast reveals the implication of caffeoyl-shikimate esterase in phenylpropanoid metabolic flux

Curcumin is a polyphenolic natural product from the roots of turmeric (Curcuma longa). It has been a popular coloring and flavoring agent in food industries with known health benefits. The conventional phenylpropanoid pathway is known to proceed from phenylalanine via p-coumaroyl-CoA intermediate. Although hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) plays a key catalysis in the biosynthesis of phenylpropanoid products at the downstream of p-coumaric acid, a recent discovery of caffeoyl-shikimate esterase (CSE) showed that an alternative pathway exists. Here, the biosynthetic efficiency of the conventional and the alternative pathway in producing feruloyl-CoA was examined using curcumin production in yeast. A novel modular multiplex genome-edit (MMG)-CRISPR platform was developed to facilitate rapid integrations of up to eight genes into the yeast genome in two steps. Using this MMG-CRISPR platform and metabolic engineering strategies, the alternative CSE phenylpropanoid pathway consistently showed higher titers (2–19 folds) of curcumin production than the conventional pathway in engineered yeast strains. In shake flask cultures using a synthetic minimal medium without phenylalanine, the curcumin production titer reached up to 1.5 mg/L, which is three orders of magnitude (∼4800-fold) improvement over non-engineered base strain. This is the first demonstration of de novo curcumin biosynthesis in yeast. Our work shows the critical role of CSE in improving the metabolic flux in yeast towards the phenylpropanoid biosynthetic pathway. In addition, we showcased the convenience and reliability of modular multiplex CRISPR/Cas9 genome editing in constructing complex synthetic pathways in yeast.

A deep-well plate enabled automated high-throughput cell line development platform.

Cell line development (CLD) plays a crucial role in the manufacturing process development of therapeutic biologics. Most biologics are produced in Chinese hamster ovary (CHO) cell. Because of the nature of random transgene integration in CHO genome and CHO's inherent plasticity, stable CHO transfectants usually have a vast diversity in productivity, growth, and product quality. Thus, we often must resort to screening a large number of cell pools and clones to increase the probability of identifying the ideal production cell line, which is a very laborious and resource-demanding process. Here we have developed a deep-well plate (DWP) enabled high throughput (DEHT) CLD platform using 24-well DWP (24DWP), liquid handler, and other automation components. This platform has capabilities covering the key steps of CLD including cell passaging, clone imaging and expansion, and fed-batch production. We are the first to demonstrate the suitability of 24DWP for CLD by confirming minimal well-to-well and plate-to-plate variability and the absence of well-to-well cross contamination. We also demonstrated that growth, production, and product quality of 24DWP cultures were comparable to those of conventional shake flask cultures. The DEHT platform enables scientists to screen five times more cultures than the conventional CLD platform, thus significantly decreases the resources needed to identify an ideal production cell line for biologics manufacturing.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

Efficient bioproduction of poly(3-hydroxypropionate) homopolymer using engineered Escherichia coli strains

This study focuses on the development of a scalable method for producing poly(3-hydroxypropionate), a homopolymer with significant physico-mechanical properties, through the use of metabolically-engineered Escherichia coli K12 (MG1655) and externally supplied 3-hydroxypropionate. The polymer synthesis pathway was established and optimized through synthetic biology techniques, including the effects of overexpressing phasin and cell division proteins. The optimized strain achieved unprecedented production titers of 9.5 g/L in flask cultures and 80 g/L in fed-batch bioreactors within 45 h. The analysis of poly(3-hydroxypropionate) polymer properties revealed it possesses excellent elasticity (Young’s modulus < 6 MPa) and tensile strength (∼80 MPa), positioning it within the category of elastomers or flexible plastics. These findings suggest a viable path for the sustainable, large-scale production of the poly(3-hydroxypropionate) biopolymer.

Advanced biomanufacturing and evaluation of adeno-associated virus

Recombinant adeno-associated virus (rAAV) has been developed as a safe and effective gene delivery vehicle to treat rare genetic diseases. This study aimed to establish a novel biomanufacturing process to achieve high production and purification of various AAV serotypes (AAV2, 5, DJ, DJ8). First, a robust suspensive production process was developed and optimized using Gibco Viral Production Cell 2.0 in 30–60 mL shaker flask cultures by evaluating host cells, cell density at the time of transfection and plasmid amount, adapted to 60–100 mL spinner flask production, and scaled up to 1.2–2.0-L stirred-tank bioreactor production at 37 °C, pH 7.0, 210 rpm and DO 40%. The optimal process generated AAV titer of 7.52–8.14 × 1010 vg/mL. Second, a new AAV purification using liquid chromatography was developed and optimized to reach recovery rate of 85–95% of all four serotypes. Post-purification desalting and concentration procedures were also investigated. Then the generated AAVs were evaluated in vitro using Western blotting, transmission electron microscope, confocal microscope and bioluminescence detection. Finally, the in vivo infection and functional gene expression of AAV were confirmed in tumor xenografted mouse model. In conclusion, this study reported a robust, scalable, and universal biomanufacturing platform of AAV production, clarification and purification.

Squalene production under oxygen limitation by Schizochytrium sp. S31 in different cultivation systems

The triterpene squalene is widely used in the food, cosmetics and pharmaceutical industries due to its antioxidant, antistatic and anti-carcinogenic properties. It is usually obtained from the liver of deep sea sharks, which are facing extinction. Alternative production organisms are marine protists from the family Thraustochytriaceae, which produce and store large quantities of various lipids. Squalene accumulation in thraustochytrids is complex, as it is an intermediate in sterol biosynthesis. Its conversion to squalene 2,3-epoxide is the first step in sterol synthesis and is heavily oxygen dependent. Hence, the oxygen supply during cultivation was investigated in our study. In shake flask cultivations, a reduced oxygen supply led to increased squalene and decreased sterol contents and yields. Oxygen-limited conditions were applied to bioreactor scale, where squalene accumulation and growth of Schizochytrium sp. S31 was determined in batch, fed-batch and continuous cultivation. The highest dry matter (32.03 g/L) was obtained during fed-batch cultivation, whereas batch cultivation yielded the highest biomass productivity (0.2 g/L*h−1). Squalene accumulation benefited from keeping the microorganisms in the growth phase. Therefore, the highest squalene content of 39.67 ± 1.34 mg/g was achieved by continuous cultivation (D = 0.025 h−1) and the highest squalene yield of 1131 mg/L during fed-batch cultivation. Volumetric and specific squalene productivity both reached maxima in the continuous cultivation at D = 0.025 h−1 (6.94 ± 0.27 mg/L*h−1 and 1.00 ± 0.03 mg/g*h−1, respectively). Thus, the choice of a suitable cultivation method under oxygen-limiting conditions depends heavily on the process requirements.Key points• Measurements of respiratory activity and backscatter light of thraustochytrids• Oxygen limitation increased squalene accumulation in Schizochytrium sp. S31• Comparison of different cultivation methods under oxygen-limiting conditions

Oxidative stress resistance prompts pyrroloquinoline quinone biosynthesis in Hyphomicrobium denitrificans H4-45

Pyrroloquinoline quinone (PQQ) is a natural antioxidant with diverse applications in food and pharmaceutical industries. A lot of effort has been devoted toward the discovery of PQQ high-producing microbial species and characterization of biosynthesis, but it is still challenging to achieve a high PQQ yield. In this study, a combined strategy of random mutagenesis and adaptive laboratory evolution (ALE) with fermentation optimization was applied to improve PQQ production in Hyphomicrobium denitrificans H4-45. A mutant strain AE-9 was obtained after nearly 400 generations of UV-LiCl mutagenesis, followed by an ALE process, which was conducted with a consecutive increase of oxidative stress generated by kanamycin, sodium sulfide, and potassium tellurite. In the flask culture condition, the PQQ production in mutant strain AE-9 had an 80.4% increase, and the cell density increased by 14.9% when compared with that of the initial strain H4-45. Moreover, batch and fed-batch fermentation processes were optimized to further improve PQQ production by pH control strategy, methanol and H2O2 feed flow, and segmented fermentation process. Finally, the highest PQQ production and productivity of the mutant strain AE-9 reached 307 mg/L and 4.26 mg/L/h in a 3.7-L bioreactor, respectively. Whole genome sequencing analysis showed that genetic mutations in the ftfL gene and thiC gene might contribute to improving PQQ production by enhancing methanol consumption and cell growth in the AE-9 strain. Our study provided a systematic strategy to obtain a PQQ high-producing mutant strain and achieve high production of PQQ in fermentation. These practical methods could be applicable to improve the production of other antioxidant compounds with uncleared regulation mechanisms.Key points• Improvement of PQQ production by UV-LiCl mutagenesis combined with adaptive laboratory evolution (ALE) and fermentation optimization.• A consecutive increase of oxidative stress could be used as the antagonistic factor for ALE to enhance PQQ production.• Mutations in the ftfL gene and thiC gene indicated that PQQ production might be increased by enhancing methanol consumption and cell growth.

Biosynthetic ability of diverse basidiomycetous yeast strains to produce the natural antioxidant ergothioneine

Sixteen strains of basidiomycetous yeasts were evaluated for their capability to produce ergothioneine (EGT), an amino acid derivative with strong antioxidant activity. The cells were cultured in either two synthetic media or yeast mold (YM) medium for 72 h, after which cytosolic constituents were extracted from the cells with hot water. After analyzing the extracts via liquid chromatography-mass spectrometry (LC-MS), we found that all strains produced varying amounts of EGT. The EGT-producing strains, including Ustilago siamensis, Anthracocystis floculossa, Tridiomyces crassus, Ustilago shanxiensis, and Moesziomyces antarcticus, were subjected to flask cultivation in YM medium. U. siamensis CBS9960 produced the highest amount of EGT at 49.5 ± 7.0 mg/L after 120 h, followed by T. crassus at 30.9 ± 1.8 mg/L. U. siamensis was also cultured in a jar fermenter and produced slightly higher amounts of EGT than under flask cultivation. The effects of culture conditions, particularly the addition of precursor amino acids, on EGT production by the selected strains were also evaluated. U. siamensis showed a 1.5-fold increase in EGT production with the addition of histidine, while U. shanxiensis experienced a 1.8-fold increase in EGT production with the addition of methionine. These results suggest that basidiomycetous yeasts could serve an abundant source for natural EGT producers.