Product Titers Research Articles

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.

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.

Plasmid-free production of the plant lignan pinoresinol in growing Escherichia coli cells

BackgroundThe high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use.ResultsIn this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells.ConclusionsFor efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.

Metabolic Engineering of Corynebacterium glutamicum for the Production of the Four-Carbon Platform Chemicals γ-Hydroxybutyrate and γ-Butyrolactone.

γ-Hydroxybutyrate (GHB) is an important C4 platform chemical, serving as a crucial precursor for the synthesis of various bulk chemicals, including γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BDO). In this study, we report the systematic metabolic engineering of Corynebacterium glutamicum for the biological production of GHB from glucose via the introduction of a glutamate-derived pathway. We showed that C. glutamicum is a promising host for producing GHB due to its higher tolerance to GHB as compared to other chassis. By screening key enzymes capable of converting glutamate into GHB and blocking byproduct synthesis pathways, an engineered C. glutamicum strain was developed that achieved a GHB production titer of 30.6 g/L. Comparative transcriptome analysis was subsequently employed to identify previously uncharacterized aldehyde dehydrogenases responsible for succinate accumulation, and knockout of the corresponding genes led to an increased GHB titer of 33.7 g/L. Ultimately, the integration of a phosphoketolase-mediated nonoxidative glycolysis (NOG) pathway further enhanced GHB production, resulting in an accumulation of 38.3 g/L of GHB with a yield of 0.615 mol/mol glucose during batch fermentation. The GHB in the fermentation broth can be efficiently converted into GBL by acid treatment with a yield of 0.970 mol/mol.

Monitoring small-scale bioreactor studies for media development using polarized total synchronous fluorescence spectroscopy (pTSFS) and synchronous light scattering (SyLS)

Biopharmaceutical process development often involves the use of small-scale bioreactors (SSBR) for optimizing media formulations and process conditions during scale up to commercial scale production. Two key process parameters (CPP) used in SSBR studies are protein titre and viable cell density (VCD). Here, we explore the efficacy of parallel polarized total synchronous fluorescence spectroscopy (TSFS||) and Synchronous Light Scattering (SyLS||) to qualitatively monitor these CPPs and quantitatively predict titre and VCD for a large-scale cell culture media optimization SSBR study. The study involved 71 different media formulations (50+ components each), and the bioprocess was run for 13 days or more. Samples were extracted at set times (Day 0, 3, 9, and 13) and clarified by centrifugation. TSFS|| spectra showed significant emission changes along with increased light scatter over the course of the bioprocess. SyLS|| measurements strongly correlated with particle size data obtained from Dynamic Light Scattering but did not correlate well with VCD probably because of the centrifugation-based sample preparation. Statistical and principal component analysis (PCA) of the pTSFS data showed that spectral variation was greater between media formulations than due to the evolving bioprocess. This prevented the development of accurate global prediction models for media performance (e.g., predicting product titre at day 9 from media spectra measured at day 0). However, classification methods were successfully used to select media subsets with better quantitative prediction accuracy based on spectral similarities. A practical binary (high/low performance) classification model based on Support Vector Machines was generated for media formulation screening. Combining emission and scatter measurements with multivariate data analysis provides a more holistic, multi-attribute bioprocess monitoring method that minimizes the need to use different offline analytical methods. This methodology can be used to monitor process trajectories and deviations, and ultimately be used to predict bioprocess CPPs when implemented on production scale processes where there is much less compositional variation in the media. We believe this SSBR-pTSFS/SyLS approach will provide a valuable resource to develop the design/parameter space for in-process monitoring at production scale from early-stage process/media development studies.

Microbial production of adipic acid from 6-hydroxyhexanoic acid for biocatalytic upcycling of polycaprolactone

To valorize waste polycaprolactone (PCL), one of the most widely used biodegradable plastics, into a value-added chemical, we upcycled 6-hydroxyhexanoic acid (6-HHA), the sole monomer of PCL, into adipic acid (AA) using a microbial method. Recombinant Escherichia coli strains expressing chnD (6-HHA dehydrogenase) and chnE (6-oxohexanoic acid dehydrogenase) genes from three bacteria were constructed, and all these strains successfully produced AA from 6-HHA. Among these, the E. coli strain harboring ChnDE genes from Acinetobacter strain SE19 (E. coli [pKK-AcChn]) showed the highest AA-producing ability. To increase the AA production titer, we optimized the culture temperature of this strain in flask culture and performed fed-batch fermentation in a 5 L bioreactor. After the fed-batch fermentation, the AA production titer increased to 15.6 g/L. As 6-HHA is a monomer of PCL, our results provide the groundwork for the development of a biocatalytic upcycling method of PCL.

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.

Growth-coupled production of L-isoleucine in Escherichia coli via metabolic engineering

L-isoleucine, an essential amino acid, is widely used in the pharmaceutical and food industries. However, the current production efficiency is insufficient to meet the increasing demands. In this study, we aimed to develop an efficient L-isoleucine-producing strain of Escherichia coli. First, accumulation of L-isoleucine was achieved by employing feedback-resistant enzymes. Next, a growth-coupled L-isoleucine synthetic pathway was established by introducing the metA-metB-based α-ketobutyrate-generating bypass, which significantly increased L-isoleucine production to 7.4 g/L. Upon employing an activity-improved cystathionine γ-synthase mutant obtained from adaptive laboratory evolution, L-isoleucine production further increased to 8.5 g/L. Subsequently, the redox flux was improved by bypassing the NADPH-dependent aspartate aminotransferase pathway and employing the NADH-dependent pathway and transhydrogenase. Finally, L-isoleucine efflux was enhanced by modifying the transport system. After fed-batch fermentation for 48 h, the resultant strain, ISO-12, reached an L-isoleucine production titer of 51.5 g/L and yield of 0.29 g/g glucose. The strains developed in this study achieved a higher L-isoleucine production efficiency than those reported previously. These strategies will aid in the development of cell factories that produce L-isoleucine and related products.

CHO stable pool fed-batch process development of SARS-CoV-2 spike protein production: Impact of aeration conditions and feeding strategies.

Technology scale-up and transfer are a fundamental and critical part of process development in biomanufacturing. Important bioreactor hydrodynamic characteristics such as working volume, overhead gas flow rate, volumetric power input (P/V), impeller type, agitation regimen, sparging aeration strategy, sparger type, and kLa must be selected based on key performance indicators (KPI) to ensure a smooth and seamless process scale-up and transfer. Finding suitable operational setpoints and developing an efficient feeding regimen to ensure process efficacy and consistency are instrumental. In this investigation, process development of a cumate inducible Chinese hamster ovary (CHO) stable pool expressing trimeric SARS-CoV-2 spike protein in 1.8 L benchtop stirred-tank bioreactors is detailed. Various dissolved oxygen levels and aeration air caps were studied to determine their impact on cell growth and metabolism, culture longevity, and endpoint product titers. Once hydrodynamic conditions were tuned to an optimal zone, various feeding strategies were explored to increase culture performance. Dynamic feedings such as feeding based on current culture volume, viable cell density (VCD), oxygen uptake rate (OUR), and bio-capacitance signals were tested and compared to standard bolus addition. Increases in integral of viable cell concentration (IVCC) (1.25-fold) and protein yield (2.52-fold), as well as greater culture longevity (extension of 5 days) were observed in dynamic feeding strategies when compared to periodic bolus feeding. Our study emphasizes the benefits of designing feeding strategies around metabolically relevant signals such as OUR and bio-capacitance signals.

The fed-batch production of mannosylerythritol lipids by Ustilago maydis DSM 4500 from hydrophilic carbon sources

Glycolipids are a class of widely studied biosurfactants with excellent applicability in cosmetic and pharmaceutical formulations. This class of biosurfactants includes mannosylerythritol lipids (MELs), which have gained particular interest due to their moisturizing and healing activity for dry and damaged human skin, arising from conditions such as eczema. Traditionally, MELs have been produced by growing certain basidiomycetous yeasts on vegetable oils. However, oils are a comparatively expensive substrate, which negatively affects the economic performance of MEL production. In addition to this, vegetable oils significantly complicate the downstream processing required to produce a product with the required purity for most applications. To address these challenges, this study investigated MEL-A production exclusively from hydrophilic carbon sources by Ustilago maydis DSM 4500. By implementing a fed-batch production strategy, maximum MEL-A concentration of 0.87 g/L was achieved from glucose exclusively. Also, adding micronutrients (such as MnSO4) to MEL-A production showed a 24.1% increase in the product titer, implying other metabolites are formed, favoring MEL production.

Identification of genes associated with the high-temperature fermentation trait in the Saccharomyces cerevisiae natural isolate BCC39850.

The fermentative model yeast Saccharomyces cerevisiae has been extensively used to study the genetic basis of stress response and homeostasis. In this study, we performed quantitative trait loci (QTL) analysis of the high-temperature fermentation trait of the progeny from the mating of the S. cerevisiae natural isolate BCC39850 (haploid#17) and the laboratory strain CEN.PK2-1C. A single QTL on chromosome X was identified, encompassing six candidate genes (GEA1, PTK2, NTA1, NPA3, IRT1, and IML1). The functions of these candidates were tested by reverse genetic experiments. Deletion mutants of PTK2, NTA1, and IML1 showed growth defects at 42°C. The PTK2 knock-out mutant also showed significantly reduced ethanol production and plasma membrane H+ ATPase activity and increased sensitivity to acetic acid, ethanol, amphotericin B (AMB), and β-1,3-glucanase treatment. The CRISPR-Cas9 system was used to construct knock-in mutants by replacement of PTK2, NTA1, IML1, and NPA3 genes with BCC39850 alleles. The PTK2 and NTA1 knock-in mutants showed increased growth and ethanol production titers at 42°C. These findings suggest an important role for the PTK2 serine/threonine protein kinase in regulating plasma membrane H+ ATPase activity and the NTA1 N-terminal amidase in protein degradation via the ubiquitin-proteasome system machinery, which affects tolerance to heat stress in S. cerevisiae.

Consolidated bioprocessing of lignocellulosic wastes in Northwest China for D-glucaric acid production by an artificial microbial consortium.

D-glucaric acid is a platform chemical of great importance and the consolidated bioprocessing (CBP) of lignocellulose by the microbial consortium of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 features prospects in biomanufacturing it. Here we compared some representative lignocelluloses in Northwest China including corn stover, wheat straw and switchgrass, and the leading pretreatments including steam explosion, subcritical water pretreatment, sodium hydroxide pretreatment, aqueous ammonia pretreatment, lime pretreatment, and diluted sulfuric acid pretreatment. It was found that sodium hydroxide pretreated switchgrass (SHPSG) was the best substrate for D-glucaric acid production, resulting in the highest D-glucaric acid titers, 11.69 ± 0.73g/L in shake flask and 15.71 ± 0.80g/L in 10L airlift fermenter, respectively. To the best of our knowledge, this is the highest D-glucaric acid production titer from lignocellulosic biomass. This work offers a paradigm of producing low-cost D-glucaric acid for low-carbon polyethylene 2,5-furandicarboxylate (PEF) and a reference on developing biorefinery in Northwest China.

Pathway reconstruction and metabolic engineering for the de novo and enhancing production of monacolin J in Pichia pastoris.

The statin is the primary cholesterol-lowering drug. Monacolin J (MJ) is a key intermediate in the biosynthetic pathway of statin. It was obtained in industry by the alkaline hydrolysis of lovastatin. The hydrolysis process resulted in multiple by-products and expensive cost of wastewater treatment. In this work, we used Pichia pastoris as the host to produce the MJ. The biosynthesis pathway of MJ was built in P. pastoris. The stable recombinant strain MJ2 was obtained by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 genome-editing tool, and produced the MJ titer of 153.6 ± 2.4mg/L. The metabolic engineering was utilized to enhance the production of MJ, and the fermentation condition was optimized. The MJ titer of 357.5 ± 5.0mg/L was obtained from the recombinant strain MJ5-AZ with ATP-dependent citrate lyase (ACL), glucose-6-phosphate dehydrogenase (ZWF1) and four lovB genes, 132.7% higher than that from the original strain MJ2. The recombinant strain MJ5-AZ was cultured in a 7-L fermenter, and the MJ titer of 1493.0 ± 9.2mg/L was achieved. The results suggested that increasing the gene dosage of rate-limiting step in the biosynthesis pathway of chemicals could improve the titer of production. It might be applicable to the production optimization of other polyketide metabolites.

Toxicants improve glycerol production in the fermentation of undetoxified hydrolysate by Candida glycerinogenes.

Toxicants inhibit microbial fermentation and reduce product titres. This work investigated the glycerol production characteristics of Candida glycerinogenes in highly toxic unwashed undetoxified hydrolysate and provided new ideas for high glycerol production from hydrolysates. The unwashed hydrolysate contains higher concentrations of toxicants, such as furfural, acetic acid, phenols and NaCl than the washed alkali-treated bagasse hydrolysate. C. glycerinogenes fermented unwashed undetoxified hydrolysate yielded 36.1g/L glycerol, 15.8% higher than the washed hydrolysate, suggesting that the toxicants stimulated glycerol synthesis. qRT-PCR analysis showed that toxicants of unwashed undetoxified hydrolysates greatly up-regulated the transcript levels of the genes GPD1, HXT4 and MSN4 et al. Overexpressing the above genes increased glycerol production by 27.9% to 46.1g/L. And it was further increased by 8.8% to 50.1g/L in a 5L bioreactor. This result proves that toxicants in lignocellulosic hydrolysates can increase the titre of microbial glycerol production.

Microbial Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoate (mcl-PHA) from Waste Cooking Oil.

Waste cooking oil is a common byproduct in the culinary industry, often posing disposal challenges. This study explores its conversion into the valuable bioplastic material, medium-chain-length polyhydroxyalkanoate (mcl-PHA), through microbial biosynthesis in controlled bioreactor conditions. Twenty-four bacterial isolates were obtained from oil-contaminated soil and waste materials in Mahd Ad-Dahab, Saudi Arabia. The best PHA-producing isolates were identified via 16S rDNA analysis as Neobacillus niacini and Metabacillus niabensis, with the sequences deposited in GenBank (accession numbers: PP346270 and PP346271). This study evaluated the effects of various carbon and nitrogen sources, as well as environmental factors, such as pH, temperature, and shaking speed, on the PHA production titer. Neobacillus niacini favored waste cooking oil and yeast extract, achieving a PHA production titer of 1.13 g/L, while Metabacillus niabensis preferred waste olive oil and urea, with a PHA production titer of 0.85 g/L. Both strains exhibited optimal growth at a neutral pH of 7, under optimal shaking -flask conditions. The bioreactor performance showed improved PHA production under controlled pH conditions, with a final titer of 9.75 g/L for Neobacillus niacini and 4.78 g/L for Metabacillus niabensis. Fourier transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) confirmed the biosynthesized polymer as mcl-PHA. This research not only offers a sustainable method for transforming waste into valuable materials, but also provides insights into the optimal conditions for microbial PHA production, advancing environmental science and materials engineering.

Metabolic engineering of fast growing cyanobacteria for phototrophic production of 2,3-butanediol

Metabolic engineering of cyanobacteria holds great potential for sustainable photosynthetic production of platform chemicals from CO2. However, product titers have been significantly low due to the slow growth rates of available model strains and lack of adequate synthetic biology tools. Here, we engineered three newly isolated fast growing Synechococcus elongatus strains PCC 11801, PCC 11802, and IITB6 for production of the platform chemical 2,3-butanediol. Importantly, we used native cyanobacterial promoters to enable inducer-free gene expression and 2,3-butanediol production. Different combinations of these native promoters were employed to optimize expression of the three-gene 2,3-butanediol synthesis pathway. Among the strains tested in this study, the highest 2,3-butanediol titer of 1.62 g L−1 (130 mg L−1 Day−1) was obtained in IITB6 (PcpcB300:alsS::PpsbAIII:alsD::PrbcL:adh), with the highest reported photosynthetic productivity in cyanobacteria cultivated on minimal media. The findings from our study highlight the potential of using fast growing S. elongatus isolates with native cyanobacterial promoters for metabolic engineering applications.

Recombinant Antibody-Producing Stable CHOK1 Pool Stability Study.

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.

High-density perfusion cultures of the marine bacterium Rhodovulum sulfidophilum for the biomanufacturing of oligonucleotides

Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs. However, low viable cell densities and product titer were reported so far. In this work, perfusion cell cultures were established for the intensification of ON biomanufacturing. First, the perfusion conditions were simulated in 50 mL spin tubes, selected as a scale-down model of the process, with the aim of optimizing the medium composition and process parameters. This optimization stage led to an increase in the cell density by 44 % compared to the reference medium formulation. In addition, tests at increasing perfusion rates were conducted until achieving the maximum viable cell density (VCDmax), allowing the determination of the minimum cell-specific perfusion rate (CSPRmin) required to sustain the cell culture. Intriguingly, we discovered in this system also a maximum CSPR, above which growth inhibition starts. By leveraging this process optimization, we show for the first time the conduction of perfusion cultures of R. sulfidophilum in bench-scale bioreactors. This process development pipeline allowed stable cultures for more than 20 days and the continuous biomanufacturing of ONs, testifying the great potential of perfusion processes.

Balancing pH and yield: exploring itaconic acid production in Ustilago cynodontis from an economic perspective

BackgroundItaconic acid is a promising bio-based building block for the synthesis of polymers, plastics, fibers and other materials. In recent years, Ustilago cynodontis has emerged as an additional itaconate producing non-conventional yeast, mainly due to its high acid tolerance, which significantly reduces saline waste coproduction during fermentation and downstream processing. As a result, this could likely improve the economic viability of the itaconic acid production process with Ustilaginaceae.ResultsIn this study, we characterized a previously engineered itaconate hyper-producing Ustilago cynodontis strain in controlled fed-batch fermentations to determine the minimal and optimal pH for itaconate production. Under optimal fermentation conditions, the hyper-producing strain can achieve the theoretical maximal itaconate yield during the production phase in a fermentation at pH 3.6, but at the expense of considerable base addition. Base consumption is strongly reduced at the pH of 2.8, but at cost of production yield, titer, and rate. A techno-economic analysis based on the entire process demonstrated that savings due to an additional decrease in pH control reagents and saline waste costs cannot compensate the yield loss observed at the highly acidic pH value 2.8.ConclusionsOverall, this work provides novel data regarding the balancing of yield, titer, and rate in the context of pH, thereby contributing to a better understanding of the itaconic acid production process with Ustilago cynodontis, especially from an economic perspective.