Shake Flask Cultures Research Articles

Biosynthesis of Sialyllacto-N-tetraose c in Engineered Escherichia coli.

Human milk oligosaccharides (HMOs) have attracted considerable interest for their vital role in supporting infant health. Among these, sialyllacto-N-tetraose c (LST c), a pentasaccharide with the structure Neu5Ac(α2,6)Gal(β1,4)GlcNAc(β1,3)Gal(β1,4)Glc, stands out due to its critical importance in the development and application of complex HMOs. In this study, we employed multivariate modular metabolic engineering (MMME) to screen for efficient sialyltransferases and balance metabolic fluxes, successfully constructing strains capable of LST c biosynthesis. Additionally, by blocking competing pathway genes, enhancing the supply of UDP-GlcNAc and UDP-Gal precursors, and establishing a CTP cofactor regeneration system, we developed a high-yielding Escherichia coli strain, W15. This strain achieved an LST c titer of 220.9 mg/L in shake flask cultures. In a 3-L fed-batch fermentation, the LST c concentration reached 922.2 mg/L, with a productivity of 10.25 mg/L/h and a specific yield of 38.70 mg/g DCW. This research provides an effective strategy for producing LST c in microbial cell factories.

A New Enzyme for Biodiesel Production and Food Applications: Lipase of Bacillus megateriumF25 Isolated From an Aquatic Insect Rhantus suturalis

ABSTRACTThis study aimed to isolate, purify, and characterize a lipase from the gut symbiont Bacillus megaterium F25 (GenBank accession: MF597792) of the aquatic insect Rhantus suturalis, with a focus on its potential applications in biodiesel and food industries. Under optimized culture conditions, B. megaterium F25 could produce 583 U/L of lipase in shaking flask culture. The purified lipase (PL) exhibited a specific activity with 113.89 U/mg, and its molecular weight was determined as 34 kDa. The activity of PL was enhanced by methanol, ethanol, Tween‐80, Triton X‐100, Ca2+, and Mg2+, while β‐mercaptoethanol, EDTA, SDS, Fe2+, Mn2+, and Cu2+ were inhibitory. PL showed optimal activity and stability at neutral and slightly acidic pHs, as well as in a temperature range of 20°C–30°C. PL displayed strong hydrolytic activity toward plant oils and animal fats, indicating its potency for both the food industry and the remediation of oil‐contaminated environments. When tested as a catalyst, PL provided biodiesel production with a transesterification yield of 86.8% under optimized conditions (36 h reaction time, 4 mL enzyme solution, 30°C, pH 7.0, and waste cooking oil:methanol ratio of 10 mL/40 mL). This is the first report on the lipase‐producing potential of gut microbial symbionts of aquatic insects. Furthermore, B. megaterium lipase was tested for the first time as a biocatalyst for biodiesel production.

Production of phenolic compounds, flavonoids, and lupeol by Lycium schweinfurthii suspension cultures in shake flasks and a rocking-motion bioreactor

AbstractLycium schweinfurthii, a wild shrub of the Solanaceae family, has received increasing attention in the last decade for its therapeutic potential in traditional medicine due to its diverse array of secondary metabolites, including phenolic substances and terpenoids. The aim of this study was to investigate the accumulation of phenolics, flavonoids, and the terpenoid lupeol in L. schweinfurthii cell suspension shake flask cultures and a single-use 2-dimensional rocking motion bioreactor. Three different media formulations were compared for in vitro cell cultures. Various parameters, such as biomass accumulation, settled cell volume, cell viability (assessed via a 2,3,5-triphenyl tetrazolium chloride assay), and sucrose consumption were determined as indicators of cell activity and growth. Total phenolic and flavonoid contents were estimated spectrophotometrically, lupeol was quantified via High-Performance Thin Layer Chromatography (HPTLC). Although a higher fresh biomass concentration of 464 g L− 1 was obtained in MS medium supplemented with a combination of each, 1 mg L− 1 of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-Naphthaleneacetic acid (NAA), the rocking-motion bioreactor cultivation was performed with 2 mg L− 1 NAA due to its superior reproducibility in viability, productivity, and content of bioactive compounds (e.g., phenolics, flavonoids, lupeol). A final fresh biomass concentration of 185 g L− 1 was achieved in a 16 L cultivation scale with a notable increase in the concentration of phenolics (1.4-fold) and flavonoids (1.7-fold). Most importantly, the concentration of lupeol, a pentacyclic triterpenoid known for its anti-inflammatory, antibacterial, and anti-atherogenic properties, exhibited a remarkable 5.5-fold increase in the bioreactor cultivation (585 µg g− 1) compared to shake flask cultivations (106 µg g− 1). The current study demonstrated the profound impact of media composition and non-limited fed-batch conditions in a rocking-motion bioreactor on the accumulation of bioactive compounds. The findings are also relevant to other plant cell cultures.

Discovering a novel glycosyltransferase gene CmUGT1 enhances main metabolites production of Cordyceps militaris.

Cordyceps militaris is a filamentous fungus used for both medicinal and culinary purposes. It exhibits a wide range of pharmacological activities due to its valuable contents of cordycepin, polysaccharides, carotenoids, terpenoids and other metabolites. However, C. militaris strains are highly susceptible to irreversible degradation in agricultural production, which is often manifested as a prolonged color change period and a significant decrease in the production of secondary metabolites. UDP-glycosyltransferases are an important enzyme family that participates in the synthesis of terpenoids by performing the glycosylation of key residues of enzymes or molecules. However, few studies have focused on its effect on the regulation of metabolite production in C. militaris. Therefore, in this study, we performed transcriptome analysis across four different developmental stages of C. militaris to target the putative glycosyltransferase gene CmUGT1, which plays important roles in metabolite production. We further constructed and screened a CmUGT1-overexpressing strain by Agrobacterium tumefaciens-mediated infestation of C. militaris spores. The major metabolite production of the wild-type and CmUGT1-overexpressing C. militaris strains was determined after short-term shake-flask cultivation of mycelia. The results showed that the yields of carotenoids and polysaccharides in the mycelia of the CmUGT1-overexpressing strains were 3.8 and 3.4 times greater than those in the mycelia of the wild type, respectively (p < 0.01). The levels of intracellular and extracellular cordycepin produced by the overexpression strain were 4.4 and 8.0 times greater than those produced by the wild-type strain (p < 0.01). This suggests that the overexpression of CmUGT1 in C. militaris enhances the synthesis activities of the main enzymes related to metabolite production, which provides a guide for obtaining excellent recombinant strains of C. militaris.

Impact of hydromechanical stress on CHO cells’ metabolism and productivity: Insights from shake flask cultivations with online monitoring of the respiration activity

The hydromechanical stress is a relevant parameter for mammalian cell cultivations, especially regarding scale-up processes. It describes the mechanical forces exerted on cells in a bioreactor. The maximum local energy dissipation rate is a suitable parameter to characterize hydromechanical stress. In literature, different studies deal with the effects of hydromechanical stress on CHO cells in stirred tank reactors. However, they often focus on lethal effects. Furthermore, systematic examinations in smaller scales like shake flasks are missing. Thus, this study systematically considers the influence of hydromechanical stress on CHO DP12 cells in shake flask cultivations. By utilizing online monitoring of the oxygen transfer rate, the study simplifies and enhances the resolution of examinations. Results indicate that while lethal effects are absent, numerous sub-lethal effects emerge with increasing hydromechanical stress: The process time is prolonged. The time of glucose and glutamine depletion, and the lactate switch correlate positively linear with the logarithmic average energy dissipation rate while the maximum specific growth rate correlates negatively. Strikingly, the final antibody concentration only declines at the highest tested average energy dissipation rate of 3.84Wkg-1 (only tested condition with a turbulent flow regime and therefore a higher maximal local energy dissipation rate) from about 250mgL-1 to about 180mgL-1. This study presents a straightforward method to examine the impact of hydromechanical stress in shake flasks, easily applicable to any other suspension cell line. Additionally, it offers valuable insights for scale-up processes, for example into stirred tank reactors.

Improving trans‐cinnamic acid production in a model cyanobacterium

Abstracttrans‐Cinnamic acid (tCA) is a precursor in the synthesis of many high‐value compounds with bio‐active qualities useful in applications like medicine, polymers, and cosmetics. Currently tCA is produced by industrial chemical synthesis from fossil fuels or cost‐prohibitive isolation from terrestrial plants. Cyanobacteria, a type of photosynthetic bacteria, can be readily engineered to convert sunlight and carbon dioxide into metabolites of interest at relatively high amounts compared to terrestrial plants. The purpose of this study is to advance the industrial and commercial value of cyanobacteria as a biological factory for renewable production of tCA. Production of tCA has previously been demonstrated in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) via expression of non‐native phenylalanine ammonia lyase (PAL) from various organisms. This project focuses on developing and characterizing a new high‐titer strain of S. 6803 expressing a plant PAL gene controlled by an inducible promoter. We assessed production in shake flasks under constant light, a 12 h:12 h light:dark cycle, and environmental photobioreactors (ePBRs) with a sinusoidal, rapidly fluctuating light environment. Our strain demonstrates a four‐fold increase in tCA production to ~500 mg L−1 by 14 days compared to previously reported titers in S. 6803 under shake flask cultivation and a 30–50% improved average tCA production per culture density (60 mg·L−1·OD730−1) in ePBRs over comparable previously reported culture methods. Our study progresses S. 6803 tCA bioproduction into higher culture volumes, up to 500 mL, while further validating the strength of an inducible system for tCA production in S. 6803.

Metabolic engineering of Halomonas bluephagenesis for the production of ethylene glycol and glycolate from xylose

Halophilic Halomonas bluephagenesis, a natural producer of poly-3-hydroxybutyrate (PHB), was metabolically engineered to synthesize ethylene glycol and glycolate from xylose. Xylose utilization was achieved by overexpressing either the xylonate pathway or the ribulose-1-phosphate pathway. The key genes encoding for xylonate dehydratase and 2-keto-3-deoxy-xylonate aldolase in the xylonate pathway were screened. With further overexpressing aldehyde reductase gene yjgB, ethylene glycol accumulation was improved to 0.91g/L, accompanied with 1.48g/L of PHB accumulation. The disruption of native glycolate oxidase was found to be essential for glycolate production, and the defective recombinant strain produced 0.80g/L glycolate with 1.14g/L PHB in shake flask cultures. These results indicated that H. bluephagenesis has the potential to produce diverse metabolic chemicals from xylose.

ISOLATION AND IDENTIFICATION OF LIGNIN-DEGRADING BACTERIAL STRAINS

Screening bacterial strains with lignin-degrading ability is very necessary for processing agricultural and forestry waste. In this experiment, we collected soil samples from the Qinling Mountains in China and isolated 99 bacterial strains. Further, 18 strains of bacteria were screened out using alkaline lignin solid medium and cultured at 30°C for 48 hours, and they were molecularly identified. After that, lignin liquid culture medium was used for shake flask culture, and a microplate reader was used to measure the absorbance changes of lignin at visible light at 280 nm before and after culture. A total of 12 strains of bacteria with lignin degradation ability were screened out, respectively. For QL-D3, QL-D5, QL-D6, QL -D7, QL-D8, QL-D9, QL-D11, QL-D13, QL-D14, QL-D16, QL-D17, QL -D18. Their lignin degradation rates after culturing in lignin 1,5 g/L liquid medium for 7 days were 13,01%, 16,76%, 10,48%, 23,3%, 6,88%, 12,48%, 22,07%, 11,84%, 18,62%, 17,88%, 13,95%, 16,28% respectively. Scanning electron microscopy was used to observe the morphology of the two bacterial strains QL-D7 and QL-D11 with the highest lignin degradation rate.

Improvement of cellobiose 2-epimerase expression in Bacillus subtilis for efficient bioconversion of lactose to epilactose

Epilactose, a lactose derivative known for its prebiotic properties and potential health benefits, has garnered significant interest. Cellulose 2-epimerase (CEase) is responsible for catalyzing the conversion of lactose to epilactose. In this study, the enhancement of food-grade CEase expression in Bacillus subtilis WB600 was systematically investigated. Among seven selected epilactose-producing CEases, Rhodothermus marinus CEase (RmCE) exhibited the highest epimerization activity when expressed in B. subtilis. Translational and transcriptional regulations were employed to enhance CEase expression by screening effective N-terminal coding sequences (NCSs) and promoters. The final strain demonstrated efficient production of CEase, with epimerization activity reaching 273.6 ± 6.5 U/mL and 1255 ± 26.4 U/mL in shake-flask and fed-batch cultivation, respectively. Utilizing only 0.25 % (V/V) of the fed-batch cultivation broth for lactose biotransformation, epilactose was efficiently produced from 300 g/L of lactose within 4 h, achieving a yield of 29.5 %. These findings provide significant support for the potential industrialization of enzymatic epilactose production.

Unraveling the impact of pH, sodium concentration, and medium osmolality on Vibrio natriegens in batch processes

BackgroundVibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens’ growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking.ResultsThis study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium.ConclusionThis study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens’ physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens.

Microbial production of 5-epi-jinkoheremol, a plant-derived antifungal sesquiterpene.

Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. In the present study, we constructed a microbial platform for the high-level production of a sesquiterpene from Catharanthus roseus, 5-epi-jinkoheremol, which exhibits strong fungicidal activity. First, the mevalonate and sterol biosynthesis pathways were optimized in engineered yeast to increase the metabolic flux toward the biosynthesis of the precursor farnesyl pyrophosphate. Then, the transcription factor Hac1- and m6A writer Ime4-based metabolic engineering strategies were implemented in yeast to increase 5-epi-jinkoheremol production further. Next, protein engineering was performed to improve the catalytic activity and enhance the stability of the 5-epi-jinkoheremol synthase TPS18, resulting in the variant TPS18I21P/T414S, with the most improved properties. Finally, the titer of 5-epi-jinkoheremol was elevated to 875.25 mg/L in a carbon source-optimized medium in shake flask cultivation. To the best of our knowledge, this is the first study to construct an efficient microbial cell factory for the sustainable production of this antifungal sesquiterpene.IMPORTANCEBiofungicides represent a new and sustainable tool for the control of crop fungal diseases. However, hindered by the high cost of biofungicide production, their use is not as popular as expected. Synthetic biology using microbial chassis is emerging as a powerful tool for the production of natural chemicals. We previously identified a promising sesquiterpenoid biofungicide, 5-epi-jinkoheremol. Here, we constructed a microbial platform for the high-level production of this chemical. The metabolic engineering of the terpene biosynthetic pathway was firstly employed to increase the metabolic flux toward 5-epi-jinkoheremol production. However, the limited catalytic activity of the key enzyme, TPS18, restricted the further yield of 5-epi-jinkoheremol. By using protein engineering, we improved its catalytic efficiency, and combined with the optimization of regulation factors, the highest production of 5-epi-jinkoheremol was achieved. Our work was useful for the larger-scale efficient production of this antifungal sesquiterpene.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of l-Pipecolic Acid from Glucose.

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain Saccharomyces cerevisiae BY4743. Initially, an optimized biosynthetic pathway was constructed within S. cerevisiae, converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask in vivo. Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.

Analysis of secondary metabolites and morphology in Streptomyces rimosus microparticle-enhanced cultivation (MPEC) at various initial organic nitrogen concentrations

The influence of talc microparticles on metabolism and morphology of S. rimosus at various initial organic nitrogen concentrations was investigated. The shake flask cultivations were conducted in the media with yeast extract (nitrogen source) concentration equal to 1 g YE L− 1 and 20 g YE L− 1. Two talc microparticle concentrations of 5 g TALC L− 1 and 10 g TALC L− 1 were tested in microparticle-enhanced cultivation (MPEC) runs. A high nitrogen concentration of 20 g YE L− 1 promoted the development of small agglomerates (pellets) of projected area lower than 105 µm2 and dispersed pseudohyphae. A low nitrogen concentration of 1 g YE L− 1 led to the limitation of S. rimosus growth and, in consequence, the development of the smaller number of large pseudohyphal agglomerates (pellets) of projected area higher than 105 µm2 compared to the culture containing a high amount of nitrogen source. In both cases talc microparticles were embedded into pellets and caused the decrease in their sizes. The lower amount of talc (5 g TALC L− 1) usually caused the weaker effect on S. rimosus morphology and metabolite production than the higher one. This correlation between the microparticles effect on morphology and metabolism of S. rimosus was especially noticeable in the biosynthesis of oxytetracycline, 2-acetyl-2-dicarboxamide oxytetracycline (ADOTC) and spinoxazine A. Compared to the control run, in MPEC their levels increased 4-fold, 5-fold and 1.6-fold respectively. The addition of talc also improved the production of 2-methylthio-cis-zeatin, lorneic acid J and milbemycin A3.

The future of single-use plastics in life science: Sterile printing of PLA reduces greenhouse gas emissions by 80% and enables carbon neutrality

The European Union’s Green Deal emphasizes life science and biotechnology as key drivers for achieving a circular economy. However, the prevalent use of non-sustainable single-use plastics derived from petrochemical sources in life science research and development contradicts this ecological goal. This study presents a viable alternative through on-site sterile 3D printing of single-use plastics using poly(lactic acid) (PLA). The sterile printing of PLA demonstrates a substantial reduction in CO2eq emissions per shake flask, decreasing from 260 g CO2eq to 145 g CO2eq. Suitability for cell culture was demonstrated for sterile printed PLA, autoclaved high-temperature PLA and for suspension cell cultures in shake flasks using CHO and insect cells as well as adherent Vero cell cultures in cell culture wells. Further optimization of sustainable shake flasks is achieved by utilizing recycled PLA, resulting in a remarkable reduction to 73 g CO2eq, constituting a 72% decrease in CO2eq emissions compared to conventional single-use plastics. Moreover, by employing geometric optimization to minimize material usage, emissions can be further reduced to 44 g CO2eq, representing an 83% reduction in CO2eq emissions. Anticipated advancements in PLA production suggest future carbon net negative PLA production, potentially achieving zero carbon emissions for single-use plastics using PLA. The exceptionally cost-effective nature of 3D printed single-use plastics, coupled with negligible capital costs for implementation, positions sterile 3D printing as a practical solution aligned with the 2030 Green Deal goals. Moreover, ongoing improvements in bioplastic production underscore the feasibility of meeting the 2050 targets of carbon neutrality for single-use plastics in life science research and development.

Cultivation optimization promotes ginsenoside and universal triterpenoid production by engineered yeast

Ginseng, a cornerstone of traditional herbal medicine in Asia, garnered significant attention for its therapeutic potential. Central to its pharmacological effects are ginsenosides, the primary active metabolites, many of which fall within the dammarane-type and share protopanaxadiol as a common precursor. Challenges in extracting protopanaxadiol and ginsenosides from ginseng arise due to their low concentrations in the roots. Emerging solutions involve leveraging microbial cell factories employing genetically engineered yeasts. Here, we optimized the fermentation conditions via the Design of Experiment, realizing 1.2 g/L protopanaxadiol in simple shake flask cultivations. Extrapolating the optimized setup to complex ginsenosides, like compound K, achieved 7.3-fold (0.22 g/L) titer improvements. Our adaptable fermentation conditions enable the production of high-value products, such as sustainable triterpenoids synthesis. Through synthetic biology, microbial engineering, and formulation studies, we pave the way for a scalable and sustainable production of bioactive compounds from ginseng.

Novel Approaches to Mortierella alpina Identification and Arachidonic Acid Production Optimization.

Arachidonic acid (ARA) is an integral constituent of cell structures and is instrumental for the nervous, muscular, and immune systems' functions. The sore need for this nutrient may be fulfilled via production based on the fungus Mortierella alpina. The identity of the M. alpina culture obtained from Assiut University, Egypt, was confirmed based on internal transcribed spacer DNA barcoding and elongation enzyme RNA sequencing. Liquid media glucose and peptone as carbon and nitrogen sources, respectively, and diverse micronutritional factors were adjusted for optimal biomass and ARA production. Shake flask cultivation at 25 °C for 7 days produced around 0.570 g of ARA per liter of culture. M. alpina treatment using mutagen 5-fluorouracil and octyl gallate-supplemented glucose-yeast-agar screening plates and shake-flask incubation at 25 °C, then at 20 °C, followed by aging at 10 °C, led to >3 g ARA/liter culture, a yield considered suitable for potential commercial production.

Optimization of Phycocyanobilin Synthesis in E. coli BL21: Biotechnological Insights and Challenges for Scalable Production.

Phycocyanobilin (PCB) is a small chromophore found in certain phycobiliproteins, such as phycocyanins (PCs) and allophycocyanins (APCs). PCB, along with other phycobilins (PBs) and intermediates such as biliverdin (BV) or phycoerythrobilin (PEB), is attracting increasing biotechnological interest due to its fluorescent and medicinal properties that allow potential applications in biomedicine and the food industry. This study aims to optimize PCB synthesis in Escherichia coli BL21 (DE3) and scale the process to a pre-industrial level. Parameters such as optimal temperature, inducer concentration, initial OD600, and stirring speed were analyzed in shake flask cultures to maximize PCB production. The best results were obtained at a temperature of 28 °C, an IPTG concentration of 0.1 mM, an initial OD600 of 0.5, and an orbital shaking speed of 260 rpm. Furthermore, the optimized protocol was scaled up into a 2 L bioreactor batch, achieving a maximum PCB concentration of 3.8 mg/L. Analysis of the results revealed that biosynthesis of exogenous PBs in Escherichia coli BL21 (DE3) is highly dependent on the metabolic burden of the host. Several scenarios, such as too rapid growth, high inducer concentration, or mechanical stress, can advance entry into the stationary phase. That progressively halts pigment synthesis, leading, in some cases, to its excretion into the growth media and ultimately triggering rapid degradation by the host. These conclusions provide a promising protocol for scalable PCB production and highlight the main biotechnological challenges to increase the yields of the process.

Foam fractionation studies of recombinant human apolipoprotein A-I

Apolipoprotein A-I (apoA-I), the primary protein component of plasma high-density lipoproteins (HDL), is comprised of two structural regions, an N-terminal amphipathic α-helix bundle domain (residues 1–184) and a hydrophobic C-terminal domain (residues 185–243). When a recombinant fusion protein construct [bacterial pelB leader sequence – human apoA-I (1–243)] was expressed in Escherichia coli shaker flask cultures, apoA-I was recovered in the cell lysate. By contrast, when the C-terminal domain was deleted from the construct, large amounts of the truncated protein, apoA-I (1–184), were recovered in the culture medium. Consequently, following pelB leader sequence cleavage in the E. coli periplasmic space, apoA-I (1–184) was secreted from the bacteria. When the pelB-apoA-I (1–184) fusion construct was expressed in a 5 L bioreactor, substantial foam production (~30 L) occurred. Upon foam collection and collapse into a liquid foamate, SDS-PAGE revealed that apoA-I (1–184) was the sole major protein present. Incubation of apoA-I (1–184) with phospholipid vesicles yielded reconstituted HDL (rHDL) particles that were similar in size and cholesterol efflux capacity to those generated with full-length apoA-I. Mass spectrometry analysis confirmed that pelB leader sequence cleavage occurred and that foam fractionation did not result in unwanted protein modifications. The facile nature and scalability of bioreactor-based apolipoprotein foam fractionation provide a novel means to generate a versatile rHDL scaffold protein.

Biosynthesis of a Functional Fragment of Human Collagen II in Pichia pastoris.

Collagen II (COL2) is the major component of cartilage tissue and is widely applied in pharmaceuticals, food, and cosmetics. In this study, COL fragments were extracted from human COL2 for secretory expression in Pichia pastoris. Three variants were successfully secreted by shake flask cultivation with a yield of 73.3-100.7 mg/L. The three COL2 variants were shown to self-assemble into triple-helix at 4 °C and capable of forming higher order assembly of nanofiber and hydrogel. The bioactivities of the COL2 variants were validated, showing that sample 205 exhibited the best performance for inducing fibroblast differentiation and cell migration. Meanwhile, sample 205 and 209 exhibited higher capacity for inducing in vitro blood clotting than commercial mouse COL1. To overexpress sample 205, the expression cassettes were constructed with different promoters and signal peptides, and the fermentation condition was optimized, obtaining a yield of 172 mg/L for sample 205. Fed-batch fermentation was carried out using a 5 L bioreactor, and the secretory protease Pep4 was knocked out to avoid sample degradation, finally obtaining a yield of 3.04 g/L. Here, a bioactive COL2 fragment was successfully identified and can be overexpressed in P. pastoris; the variant may become a potential biomaterial for skin care.

Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.