Product Titers Research Articles

Distillation for in situ recovery of volatile fermentation products.

Many fermentation products inhibit their own microbial production, which complicates industrial-scale fermentation development for these products. When a product is volatile, this inhibition can be circumvented by removing product during fermentation through evaporation in a loop around the bioreactor. Microbes can survive this loop if its temperature is reduced using vacuum. Then, regrowing of microbes is not required. From a separation efficiency viewpoint, the evaporation loop should not use a single equilibrium stage, but a multistage vacuum distillation column. Such in situ product removal (ISPR) by vacuum distillation has hardly been recognized as an option, however. Costs for this product removal with subsequent purification are modest, even when product titers are low. A prerequisite is the use of advanced energy integration and heat pumping methods.

A Fed-batch Process for the Production of Recombinant Adeno-Associated Virus (rAAV) Vectors Using the Sf9-Rhabdovirus-Negative Cell Line.

Gene therapy has been effectively applied in many biological studies and for the treatment of many genetic or cancer diseases. Currently, Recombinant Adeno- Associated Viruses (rAAVs) are one of the main types of delivery vectors used for gene therapy. rAAV vectors produced via the Sf9 cells have the advantages of high rAAV yields, easy scaleup, and low cost. Here, we used Sf9 rhabdovirus-negative (Sf9-RVN) cells to validate and optimize the rAAV production process, and the fed-batch process increased the rAAV production titre. In the fed-batch procedure, the cell density reached 12.9×106 cells/mL, which was approximately twice as high as in the batch culture process. The rAAV titre was also approximately 8-fold higher in the fed-batch process, reaching 1.5×1012 VG/mL. The optimized process was validated by generating rAAVs with various serotypes and genes of interest (GOI), all of which gave production titres greater than 1×1012 VG/mL. We used Sf9-RVN cells to develop a fed-batch rAAV production process that replaces Sf9 cells to meet regulatory standards. This process has good applicability, and the rAAV titre can reach at least 1×1012 VG/mL, which is higher than the level of 1011 VG/mL reported in the literature.

Metabolic engineering of Escherichia coli for enhanced production of p-coumaric acid via L-phenylalanine biosynthesis pathway.

p-Coumaric acid (p-CA), an invaluable phytochemical, has novel bioactivities, including antiproliferative, anxiolytic, and neuroprotective effects, and is the main precursor of various flavonoids, such as caffeic acid, naringenin, and resveratrol. Herein, we report the engineering of Escherichia coli for de novo production of p-CA via the PAL-C4H pathway. As the base strain, we used the E. coli H-02 strain, which was previously engineered for sufficient supplementation of L-phenylalanine, the main precursor of p-CA. For the bioconversion of L-Phe to p-CA, we constructed and optimized an expression system for phenylalanine ammonia lyase (SmPAL), codon-optimized cinnamate 4-hydroxylase (AtC4H), and its redox partner, cytochrome P450 reductase (AtCPR1). We confirmed that the engineered cell showed higher production of p-CA at 30°C and the addition of 0.5mM 5-aminolevulinic acid could increase the production titer further. Subsequently, the main pathways of acetic acid (poxB and pta-ackA) were eliminated to reduce its accumulation and restore cell growth. Next, to increase the available pool of cofactor (NADPH), the co-expression system of the zwf gene in the pentose phosphate pathway (PPP) was integrated into genome and the expression level was optimized with synthetic promoters. Finally, by optimizing fed-batch culture in a 5 L-scale bioreactor, the engineered strain achieved 1.5g/L p-CA with a productivity of 31.8mg/L/h.

Machine learning reveals novel compound for the improved production of chitooligosaccharides in Escherichia coli.

In order to improve predictability of outcome and reduce costly rounds of trial-and-error, machine learning models have been of increasing importance in the field of synthetic biology. Besides applications in predicting genome annotation, process parameters and transcription initiation frequency, such models have also been of help for pathway optimization. The latter is a common strategy in metabolic engineering and improves the production of a desirable compound by optimizing enzyme expression levels of the production pathway. However, engineering steps might not lead to sufficient improvement, and bottlenecks may remain hidden among the hundreds of metabolic reactions occurring in a living cell, especially if the production pathway is highly interconnected with other parts of the cell's metabolism. Here, we use the synthesis of chitooligosaccharides (COS) to show that the production from such complex pathways can be improved by using machine learning models and feature importance analysis to find new compounds with an impact on COS production. We screened Escherichia coli libraries of engineered transcription regulators with an expected broad range of metabolic diversity and trained several machine learning models to predict COS production titers. Subsequent feature analysis led to the finding of iron, whose addition we could show improved COS production in vivo up to 2-fold. Additionally, the analysis revealed important clues for future engineering steps.

A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum.

Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C.pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C.pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48gL-1, and this metabolite was not reported to be produced by C.pasteurianum before. The developed procedure expands the genetic toolkit for C.pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.

Engineered Passive Glucose Uptake in Pseudomonas taiwanensis VLB120 Increases Resource Efficiency for Bioproduction.

Glucose is the most abundant monosaccharide and a principal substrate in biotechnological production processes. In Pseudomonas, this sugar is either imported directly into the cytosol or first oxidised to gluconate in the periplasm. While gluconate is taken up via a proton-driven symporter, the import of glucose is mediated by an ABC-type transporter, and hence both require energy. In this study, we heterologously expressed the energy-independent glucose facilitator protein (Glf) from Zymomonas mobilis to replace the native energy-demanding glucose transport systems, thereby increasing the metabolic energy efficiency. The implementation of passive glucose uptake in engineered production strains significantly increased product titres and yields of the two different aromatic products, cinnamic acid (+10%-15%) and resveratrol (+26%; 18.1 mg/g) in batch cultures.

Pyrophosphate-free glycolysis in Clostridium thermocellum increases both thermodynamic driving force and ethanol titers

BackgroundClostridium thermocellum is a promising candidate for production of cellulosic biofuels, however, its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild-type organism, this reaction uses pyrophosphate (PPi) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor. Previously we showed that replacing the PPi-linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer.ResultsIn this study, we substituted PPi-pfk with ATP-pfk, deleted the other PPi-requiring glycolytic gene pyruvate:phosphate dikinase (ppdk), and expressed a soluble pyrophosphatase (PPase) and pyruvate kinase (pyk) genes to engineer PPi-free glycolysis in C. thermocellum. We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 to 21.0 g/L) by using PPi-free glycolysis.ConclusionsBy engineering PPi-free glycolysis in C. thermocellum, we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers.

Application of integrated full-membrane platform in antibody purification

AbstractAntibody products are promising therapeutic candidates for various diseases, such as cancers and autoimmune disorders. Owing to the improvement of cell culture technology in recent years, the production titers of antibodies can reach up to 10 g/L. With such high titers in upstream production, advancements in the development of downstream separation and purification techniques are highly desired. To ensure a balance between handling the increased product supply and maintaining the high purity required for biopharmaceutical applications, membrane chromatography is an efficient and cost-effective technique that can be used to separate and purify proteins and other biological components. In this study, we developed a full-membrane platform that uses affinity membrane chromatography to capture antibodies, which is followed by a two-step flow-through polishing process involving anion exchange membrane chromatography and hydrophobic interaction membrane chromatography. We tested five molecules using this platform, and the outcomes of membrane chromatography were comparable to those of column chromatography in terms of yield and product quality. Moreover, full-membrane chromatography might lead to a cost reduction of > 70% in chromatography media. Altogether, the full-membrane platform developed in this has potential applications in downstream processes of antibody production.

Selective Recruitment of a Synthetic Histone Acetyltransferase Can Boost CHO Cell Productivity.

Industrial production of biologics typically involves the integration of transgenes into host cell genomes, most often Chinese hamster ovary (CHO) cells. Epigenetic control of transgene expression is a major determinant of production titers. Although the cytomegalovirus (CMV) promoter has long been used to drive industrial transgene expression, we found that its associated histones are suboptimally acetylated in CHO cells, providing an opportunity to enhance productivity through epigenetic manipulation. Expression of monoclonal antibody mRNAs increased up to 12-fold when a CRISPR-dCas9 system delivered the catalytic domain of a histone acetyltransferase to the CMV promoter. This effect was far stronger than when promoter DNA was selectively demethylated using dCas9 fused to a 5-methylcytosine dioxygenase. Mechanistically, acetylation-mediated transcriptional activation involved heightened phosphorylation and activity of RNA polymerase II, enabling it to escape promoter-proximal pausing at the transgene. This approach almost doubled the titer and specific productivity of antibody-producing CHO cells, demonstrating the potential for biomanufacturing.

A chemoenzymatic cascade for sustainable production of chiral N-arylated aspartic acids from furfural and waste

Both biomass valorization and waste upcycling are important routes to sustain the circular bioeconomy. In this work, we present a chemoenzymatic cascade for selective synthesis of chiral N-arylated aspartic acids from biomass-derived furfural and waste nitrophenols (NPs) by merging robust photo- and electrocatalysis with stereoselective biocatalysis. Concurrent photoelectrocatalytic oxidation of furfural into maleic acid (MA) and fumaric acid (FA) was significantly enhanced by combining catalyst and reaction engineering strategies including identification of a powerful photocatalyst meso-tetra(4-carboxyphenyl)porphyrin, continuous flow technique, enhancing dissolved O2 and paired electrosynthesis. The overall space-time yield (STY) approached 2.8 g L−1 h−1 in a fed-batch process, with the product titer of 28.3 g L−1. Besides, photoelectrosynthesis of MA/FA was effectively fueled by sunlight, with the STY of up to 3.6 g L−1 h−1. Both MA selectivity and yield could be facilely improved to around 89% by reducing the buffer concentrations. Paired electrosynthesis strategy not only resulted in greatly improved MA production at the anode, but also enabled NPs upcycling into value-added aminophenols (APs) at the cathode. The products formed in the two electrode chambers were converted into N-arylated (S)-aspartic acids by a bienzymatic cascade. This work presents a multicatalytic approach for integrating selective biomass valorization and waste upcycling towards sustainable manufacture.

Multienzyme Cascade Synthesis of Rare Sugars From Glycerol in Bacillus subtilis.

Rare sugars are valuable and unique monosaccharides extensively utilized in the food, cosmetics, and pharmaceutical industries. Considering the high purification costs and the complex processes of enzymatic synthesis, whole-cell conversion has emerged as a significantly important alternative. The Escherichia coli strain was initially used in whole-cell synthesis of rare sugars. However, its pathogenic nature poses limitations to its widespread applications. Consequently, there is an urgent need to explore biologically safe strains for the efficient production of rare sugars. In this study, the generally regarded as safe (GRAS) strain Bacillus subtilis was employed as the chassis cells to produce rare sugars via whole-cell conversion. Three genes encoding alditol oxidase (AldO), L-rhamnulose-1-phosphate aldolase (RhaD), and fructose-1-phosphatase (YqaB) involved in rare sugars biosynthesis were heterogeneously expressed in B. subtilis to convert the only substrate glycerol into rare sugars. To enhance the expression levels of the relevant enzymes in B. subtilis, different promoters for aldO, rhaD, and yqaB were investigated and optimized in this system. Under the optimized reaction conditions, the maximum total production titer was 16.96g/L of D-allulose and D-sorbose with a conversion yield of 33.9% from glycerol. Furthermore, the engineered strain produced 26.68g/L of D-allulose and D-sorbose through fed-batch for the whole-cell conversion, representing the highest titer from glycerol reported to date. This study demonstrated an efficient and cost-effective method for thesynthesis of rare sugars, providing a food-grade platform with the potential to meet the growing demand for rare sugars in industries.

Dose sparing enabled by immunization with influenza vaccine using orally dissolving film

Influenza vaccine delivered by orally dissolving film vaccine (ODFV) is a promising approach. In this study, we generated three ODFVs each comprising pulluan and trehalose with different doses of inactivated A/Puerto Rico/8/34, H1N1 virus (ODFV I, II, III) to evaluate their dose-sparing effect in mice. The ODFVs were placed on the tongues of mice to elicit immunization and after 3 immunizations at 4-week intervals, mice were challenged with a lethal dose of A/PR/8/34 to assess vaccine-induced protection. The 3 ODFVs containing 50, 250, or 750 μg of inactivated viruses elicited virus-specific antibody responses and virus neutralization in a dose-dependent manner. Dose-dependent antibody responses were also observed from the mucosal tissue samples, and also from antibody-secreting cells of the lungs and spleens. ODFV-induced cellular immunity, particularly germinal center B cells and T cells were also dose-dependent. Importantly, all 3 ODFVs evaluated in this study provided complete protection by strongly suppressing the pro-inflammatory cytokine production and lung virus titers. None of the immunized mice underwent noticeable weight loss nor succumbed to death, a phenomenon that was only observed in the infection challenge controls. These results indicated that the protection conferred by a low dose influenza vaccine formulated in ODF is comparable to that of a high-dose vaccine, thereby enabling vaccine dose sparing effect.

Oxygen Consumption in Filamentous Pellets of Aspergillus niger: Microelectrode Measurements and Modeling.

Filamentous fungi cultivated as biopellets are well established in biotechnology industries. A distinctive feature of filamentous fungi is that hyphal growth and fungal morphology affect product titers and require tailored process conditions. Within the pellet, mass transfer, substrate consumption, and biomass formation are intricately linked to the local hyphal fraction and pellet size. This study combined oxygen concentration measurements with microelectrode profiling and three-dimensional X-ray microtomography measurements of the same fungal pellets for the first time. This allowed for the precise correlation of micromorphological information with local oxygen concentrations of two Aspergillus niger strains (hyperbranching and regular branching). The generated results showed that the identified oxygen-penetrated outer pellet regions exhibited a depth of 90-290 µm, strain-specific, with the active part percentage in the pellet ranging from 18% to 69%, without any difference between strains. Using a 1D continuum diffusion consumption model, the oxygen concentration in the pellets was computed depending on the local hyphal fraction. The best simulation results were achieved by individually estimating the oxygen-related biomass yield coefficient of the consumption term within each examined pellet, with an average estimated value of 1.95 (± 0.72) kg biomass per kg oxygen. The study lays the foundation for understanding oxygen supply in fungal pellets and optimizing processes and pellet morphologies accordingly.

The Unveiled Novel regulator of Adeno-associated virus production in HEK293 cells

The field of gene therapy using Adeno-associated viral (AAV) vector delivery is rapidly advancing in the biotherapeutics industry. Despite its successes, AAV manufacturing remains a challenge due to limited production yields. The triple plasmid transfection of HEK293 cells represents the most extensively utilized system for AAV production. The regulatory factors and mechanisms underlying viral production in HEK293 cells are largely unknown. In this study, we isolated high-titer AAV production clones from a parental HEK293 population using a single limiting dilution step, and subsequently elucidating their underlying molecular mechanisms through whole transcriptome analysis. LncRNA TCONS_00160397 was upregulated in clones and shown to promoted HEK293 cells proliferation and improved the titer of AAV production. Mechanistically, results from proteomics and metabolomics indicated that TCONS_00160397 regulated the ABC transporters pathway. These findings furnish a rich repository of knowledge and actionable targets for the rational optimization of HEK293-based producer lines, thereby paving the way for tangible improvements in AAV vector output and expediting the broad implementation of gene therapies.

Efficient utilization of xylose requires CO2 fixation in Synechococcus elongatus PCC 7942

Cyanobacteria show great promise as autotrophic hosts for the renewable biosynthesis of useful chemicals from CO2 and light. While they can efficiently fix CO2, cyanobacteria are generally outperformed by heterotrophic production hosts in terms of productivity and titer. Photomixotrophy, or co-utilization of sugars and CO2 as carbon feedstocks, has been implemented in cyanobacteria to greatly improve productivity and titers of several chemical products. We introduced xylose photomixotrophy to a 2,3-butanediol producing strain of Synechococcus elongatus PCC 7942 and characterized the effect of gene knockouts, changing pathway expression levels, and changing growth conditions on chemical production. Interestingly, 2,3-butanediol production was almost completely inhibited in the absence of added CO2. Untargeted metabolomics implied that RuBisCO was a significant bottleneck, especially at ambient CO2 levels, restricting the supply of lower glycolysis metabolites needed for 2,3-butanediol production. The dependence of the strain on elevated CO2 levels suggests some practical limitations on how xylose photomixotrophy can be efficiently carried out in S. elongatus.

Characterization of the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

Improved reactor design enables productivity of microbial electrosynthesis on par with classical biotechnology

Microbial electrosynthesis (MES) converts (renewable) electrical energy into CO2-derived chemicals including fuels. To achieve commercial viability of this process, improvements in production rate, energy efficiency, and product titer are imperative. Employing a compact plate reactor with zero gap anode configuration and NiMo-plated reticulated vitreous carbon cathodes substantially improved electrosynthesis rates of methane and acetic acid. Electromethanogenesis rates exceeded 10 L L–1catholyte d–1 using an undefined mixed culture. Continuous thermophilic MES by Thermoanaerobacter kivui produced acetic acid at a rate of up to 3.5 g L−1catholyte h−1 at a titer of 14 g/L, surpassing continuous gas fermentation without biomass retention and on par with glucose fermentation by T. kivui in chemostats. Coulombic efficiencies reached 80 %–90 % and energy efficiencies up to 30 % for acetate and methane production. The performance of this plate reactor demonstrates that MES can deliver production rates that are competitive with those of established biotechnologies.

Adaptive laboratory evolution and metabolic engineering of Cupriavidus necator for improved catabolism of volatile fatty acids

Bioconversion of high-volume waste streams into value-added products will be an integral component of the growing bioeconomy. Volatile fatty acids (VFAs) (e.g., butyrate, valerate, and hexanoate) are an emerging and promising waste-derived feedstock for microbial carbon upcycling. Cupriavidus necator H16 is a favorable host for conversion of VFAs into various bioproducts due to its diverse carbon metabolism, ease of metabolic engineering, and use at industrial scales. Here, we report that a common strategy to improve product titers in C. necator, deletion of the polyhydroxybutyrate (PHB) biosynthetic operon, results in a significant growth defect on VFA substrates. Using adaptive laboratory evolution, we identify mutations to the regulator gene phaR, the two-component response regulator-histidine kinase pair encoded by H16_A1372/H16_A1373, and the tripartite transporter assembly encoded by H16_A2296-A2298 as causative for improved growth on VFA substrates. Deletion of phaR and H16_A1373 led to significantly reduced NADH abundance accompanied by large changes to expression of genes involved in carbon metabolism, balance of electron carriers, and oxidative stress tolerance that may be responsible for improved growth of these engineered strains. These results provide insight into the role of PHB biosynthesis in carbon and energy metabolism and highlight a key role for the regulator PhaR in global regulatory networks. By combining mutations, we generated platform strains with significant growth improvements on VFAs, which can enable improved conversion of waste-derived VFA substrates to target bioproducts.

Lignin valorization to bioplastics with an aromatic hub metabolite-based autoregulation system

Exploring microorganisms with downstream synthetic advantages in lignin valorization is an effective strategy to increase target product diversity and yield. This study ingeniously engineers the non-lignin-degrading bacterium Ralstonia eutropha H16 (also known as Cupriavidus necator H16) to convert lignin, a typically underutilized by-product of biorefinery, into valuable bioplastic polyhydroxybutyrate (PHB). The aromatic metabolism capacities of R. eutropha H16 for different lignin-derived aromatics (LDAs) are systematically characterized and complemented by integrating robust functional modules including O-demethylation, aromatic aldehyde metabolism and the mitigation of by-product inhibition. A pivotal discovery is the regulatory element PcaQ, which is highly responsive to the aromatic hub metabolite protocatechuic acid during lignin degradation. Based on the computer-aided design of PcaQ, we develop a hub metabolite-based autoregulation (HMA) system. This system can control the functional genes expression in response to heterologous LDAs and enhance metabolism efficiency. Multi-module genome integration and directed evolution further fortify the strain’s stability and lignin conversion capacities, leading to PHB production titer of 2.38 g/L using heterologous LDAs as sole carbon source. This work not only marks a leap in bioplastic production from lignin components but also provides a strategy to redesign the non-LDAs-degrading microbes for efficient lignin valorization.

High-throughput optimization of organic carbon provision strategies enables enhanced arachidonic acid production in novel microalgae

BackgroundMicroalgae are potential sustainable resources for the production of value-added chemicals that can be used as biofuels, pharmaceuticals, and nutritional supplements. Arachidonic acid (ARA), a omega-6 fatty acid, plays a crucial role in infant development and immune response, and can be used in cosmetics and pharmaceuticals. Demand for industrial-scale ARA production is continuously increasing because of its broad applicability. To address this demand, there has been a significant shift towards microorganism-based ARA production. To accelerate large-scale ARA production, it is crucial to select suitable strains and establish optimal culture conditions.ResultsHere, we isolated a novel microalga Lobosphaera incisa CFRC-1, a valuable strain that holds promise as a feedstock for ARA production. Optimal cultivation conditions were investigated using a high-throughput screening method to enhance ARA production in this novel strain. Out of 71 candidates, four organic carbon substrates were identified that could be utilized by L. incisa CFRC-1. Through flask-scale verification, fructose was confirmed as the optimal organic carbon substrate for promoting microalgal growth, total lipid accumulation, and ARA production. Subsequently, we investigated appropriate substrate concentration and cultivation temperature, confirming that the optimal conditions were 30 g L− 1 of fructose and 27 ℃ of temperature. Under these optimized conditions, biomass and ARA production reached 13.05 ± 0.40 g L− 1 and 97.98 ± 7.33 mg L− 1, respectively, representing 9.6-fold and 5.3-fold increases compared to the conditions before optimization conditions. These results achieved the highest biomass and ARA production in flask-scale cultivation, indicating that our approach effectively improved both production titer and productivity.ConclusionsThis study presents a novel microalgae and optimized conditions for enhancing biomass and ARA production, suggesting that this approach is a practical way to accelerate the production of valuable microalgae-based chemicals. These findings provide a basis for large-scale production of ARA-utilizing microalgae for industrial applications.