Evaluating Vibrio natriegens as a new host for efficient plasmid DNA production in biopharmaceutical applications
Abstract
The increasing demand for plasmid DNA (pDNA) in biopharmaceutical applications such as gene therapies, DNA vaccines, and mRNA-based treatments has highlighted the need for more efficient and scalable production systems. Traditionally, Escherichia coli has served as the preferred host due to its well-established genetic tools and high-density cultivation capability. However, the search for alternative hosts that offer faster growth, greater metabolic flexibility, and reduced production times has led to growing interest in Vibrio natriegens. This marine bacterium, known as the fastest-growing organism with a doubling time of less than 10 minutes, has shown great potential in synthetic biology, yet its role in large-scale pDNA production remains underexplored. In this study, the performance of Vibrio natriegens (Vmax) as a pDNA production host was evaluated and compared to E. coli DH5α, both carrying the peGFP plasmid. Cultures were grown over a 6-hour period, followed by plasmid isolation using the High Pure Plasmid Isolation Kit (Roche). Plasmid yields were quantified spectrophotometrically, and the quality was assessed via agarose gel electrophoresis to verify plasmid topology. The results demonstrated that V. natriegens achieved pDNA yields comparable to and even higher thanthose obtained with E. coli under the same cultivation conditions. Notably, V. natriegens displayed faster growth kinetics, reaching high optical densities within a significantly shorter time frame. This rapid growth could translate into shorter production cycles and higher overall productivity in industrial settings. Furthermore, the comparable plasmid topology observed in both hosts supports the potential of V. natriegensfor downstream applications in gene therapy and vaccine development. The findings presented in this study underscore the promise of Vibrio natriegens as a next-generation production host for pDNA manufacturing. Ongoing work aims to optimize culture conditions and genetic tools for this organism, ultimately contributing to the development of scalable and sustainable biopharmaceutical production platforms. Future research should explore the integration of V. natriegens into continuous manufacturing processes and assess its performance with different plasmid DNA relevant for clinical applications.
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DIG4BIO Digitalization of Biomanufacturing Evaluating Vibrio natriegens as a new host for efficient plasmid DNA production in biopharmaceutical applications Lara Möller, Simão P. Fonseca, Jorge João, A. Rita Silva-Santos, Duarte M. Prazeres 1
1. Introduction →DIG4BIO →Plasmid DNA Production → Vibrio natriegens 2. Hypotheses 3. Methods →Shake Flasks →HT Mini-Bioreactor System →Bench-Top Bioreactor 3. Results and Discussion 4. Conclusion and Outlook Agenda ”From Shake Flasks to Cultivation Strategy: Assessing Vibrio natriegens for Biotechnological Plasmid DNA Production”1
Introduction 2
DIG4BIO: Digitalization for Biomanufacturing –The Project WHAT is DIG4BIO? It is aTwinning action that aims to: •advance the research capacity and profile of IST/IST-ID in the field of digitalization of biomanufacturing •strengthen the research management and administrative skills of IST/IST-ID. HOW? Through a strategic alliance with experts in the fields of: •digital biotechnology from PROSYS, DTU (DK) and •lab automation from KIWI-biolab, TU Berlin (DE) 3
Therapeutics & Biomanufacturing: → DNA vaccines → Gene therapy plasmids →Template for mRNA vaccines or therapeutics Plasmid DNA –A Foundation of Genetic Engineering 4 Plasmid DNA Genetic Engineering & Cloning Protein Expression & Research Tools Synthetic Biology & Metabolic Engineering [1] System Transfer
Plasmid DNA –A Foundation of Genetic Engineering 5 Schematic representation of pDNA uptake and expression in host cells. Adapted from Martínez-Puente et al. (2022) Plasmid DNA Requirements for High-Quality pDNA in Biomanufacturing →High supercoiled fraction (≥ 90%) → stability, transfection efficiency, regulatory demand →Minimal isoforms (OC, linear, multimers) →Low impurities (hcDNA, RNA, proteins, endotoxins)
Plasmid DNA Production Bioprocess overview of plasmid DNA production (Prather et al., 2003). 6 1. Plasmid Design & Strain Selection 2. Transformation & Clonal Selection 3. Plasmid Production 4. Plasmid Purification Plasmid DNA Production – Key Upstream Parameters 1. Physicochemical conditions: →pH →Temperature →Dissolved xygen 3. Process mode: →Batch →Fed-batch 2. Bioreactor parameters: →Agitation →Aeration →Medium composition
Plasmid DNA Production –The Current Host of Choice Standard host for plasmid DNA production and cloning: E. Coli DH5α (Invitrogen, USA) E. coli DH5α carries key knockouts that for plasmid DNA production: recA1 prevents recombination → improves plasmid stability endA1 increases DNA purity during extraction hsdR17 disables restriction enzymes → enhances transformation efficiency 7 +What makes E. coli the standard host? E. coli DH5α has its limitations within: Slow growth rate long process time →low volumetric productivity Endotoxins challenge for therapeutic-grade pDNA Metabolic burden plasmid loss and instability at high µ Sensitivity to DO/pH difficulties with scale-up –What are the limitations of this system? These limitations highlight the need for more efficient and robust hosts for pDNA production
E. coli V. natriegens 8 64 1 hour Vibrio natriegens –A Potential Host for Plasmid DNA Production The key advantage of V. natriegens is its extremely rapid doubling time of approximately 10 minutes under optimized conditions –compared to ~25 minutes for E. coli Vibrio natriegens is: •Halophilic bacterium •Gram-negative •Marine •Non-toxic •Highly metabolically versatile Microscope image of V. natriegens (The Microbial Menagerie, 2019) 8
Results Part 1: Shake Flasks Experiments 15 •Vmax outperformed DH5α in biomass production under both tested media conditions (p< 0.05) •Significantly higher pDNA yields achieved with Vmax within 6–8h (p< 0.05) •Agarose gels show multiple plasmid isoforms in Vmax, while DH5α mainly produced supercoiled pDNA 0 2 4 6 0 2 4 6 8 Time (h) Biomass [g L-1] DH5a 2xYT Vmax 2xYT(+V2) DH5a MSM Vmax MSM(+V2) 0 5 10 15 20 2 4 6 8 Time (h) pDNA [mg L-1] DH5a 2xYT Vmax 2xYT(+V2) DH5a MSM Vmax MSM(+V2) Biomass Accumulation in Shake Flask Cultivation pDNA Yields in Shake Flask Cultivation Agarose Gel of Isolated pDNA 2000 – 10000 – 4000 – 1000 – 200 – 2000 – 10000 – 4000 – 1000 – 200 – LVmax 6 h Vmax 8 h LDH5α 6 h DH5α 8 h a b c a b c a b c a b c L: NZYDNA Ladder III (200–10000 bp) Vmax 6 h/ 8 h: V. natriegens Vmax peGFP pDNA isolated after 6 and 8 h cultivation in MSM(+V2) medium DH5α 6 h / 8 h: E. coli DH5α peGFP pDNA isolated after and 6 and 8 h cultivation in MSM medium *lanes a–c = biological replicates Band size (bp) 2000 – 10000 – 4000 – 1000 – 200 – 2000 – 10000 – 4000 – 1000 – 200 – LVmax 6 h Vmax 8 h LDH5α 6 h DH5α 8 h a b c a b c a b c a b c L: NZYDNA Ladder III (200–10000 bp) Vmax 6 h/ 8 h: V. natriegens Vmax peGFP pDNA isolated after 6 and 8 h cultivation in MSM(+V2) medium DH5α 6 h / 8 h: E. coli DH5α peGFP pDNA isolated after and 6 and 8 h cultivation in MSM medium *lanes a–c = biological replicates Band size (bp) Goal → Compare biomass accumulation and pDNA production of V. natriegens and E. coli
•Plasmid DNA yields showed high distribution between reactors •Biomass up to 40 g/L reached within 12 h in 2xYT(+V2) with μ-set = 0.4 Results Part 2: HT Screening in Mini-bioreactors 16 •DO was not oscillating according to the feeding pulses at higher µ-set, system reached OTR limit •Acetate accumulation stayed moderate (< 5 g L-1), even at higher µ-set of 0.4 0 10 20 30 40 2.5 5.0 7.5 10.0 12.5 Time (h) Biomass [g L-1] m setpoint 2xYT(+V2) (m = 0.2 h-1) 2xYT(+V2) (m = 0.4 h-1) 0 100 200 300 400 500 2.5 5.0 7.5 10.0 12.5 Time (h) pDNA [mg L-1] m setpoint 2xYT(+V2) (m = 0.2 h-1) 2xYT(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint 2×YT(+V2) (m = 0.2 h-1) 2×YT(+V2) (m = 0.4 h-1) Biomass and pDNA Yields in 2xYT(+V2) Mini-bioreactors 0 50 100 150 200 0.0 2.5 5.0 7.5 10.0 12.5 Time (h) DO (%) m setpoint 2xYT(+V2) (m = 0.2 h-1) 2xYT(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint 2×YT(+V2) (m = 0.2 h-1) 2×YT(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint 2×YT(+V2) (m = 0.2 h-1) 2×YT(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint 2×YT(+V2) (m = 0.2 h-1) 2×YT(+V2) (m = 0.4 h-1) DO and Metabolites Profiles in 2xYT(+V2) Mini-bioreactors
Results Part 2: HT Screening in Mini-bioreactors 17 0 10 20 30 40 2.5 5.0 7.5 10.0 12.5 Time (h) Biomass [g L-1] m setpoint MSM(+V2) (m = 0.2 h-1) MSM(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint MSM(+V2) (m = 0.2 h-1) MSM(+V2) (m = 0.4 h-1) 0 100 200 300 400 500 2.5 5.0 7.5 10.0 12.5 Time (h) pDNA [mg L-1] m setpoint MSM(+V2) (m = 0.2 h-1) MSM(+V2) (m = 0.4 h-1) Biomass and pDNA Yields in MSM(+V2) Mini-bioreactors 0 50 100 150 200 0.0 2.5 5.0 7.5 10.0 12.5 Time (h) DO (%) m setpoint MSM(+V2) (m = 0.2 h-1) MSM(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint MSM(+V2) (m = 0.2 h-1) MSM(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint MSM(+V2) (m = 0.2 h-1) MSM(+V2) (m = 0.4 h-1) 0 20 40 60 0 5 10 15 2.5 5.0 7.5 10.0 12.5 Time (h) Glucose [g L-1] Acetate [g L-1] Metabolite Acetate Glucose m setpoint 2×YT(+V2) (m = 0.2 h-1) 2×YT(+V2) (m = 0.4 h-1) DO and Metabolites Profiles in 2xYT(+V2) Mini-bioreactors •Plasmid DNA yields showed high distribution between reactors •Biomass up to 20 g/L reached within 11 h in MSM(+V2) with μ-set = 0.4 •DO was oscillating according to the feeding pulses at higher µ-set •High glucose and acetate accumulation at the end of cultivation: indicator for limitation, over-feeding
Results Part 3: Upscaling in 1.5L Bench-Top Reactor 18 •Process scaled to 1.5 L with stable growth in both media •MSM(+V2) yielded up to 100 mg L-1 •Smearing in MSM(+V2) gel likely due to unprocessed biomass, not multimer formation RUN ID 1 0 2 4 6 8 010 20 Time (h) Biomass [g L-1] Run ID 1 2xYT(+V2) MSM(+V2) L L L: NZYDNA Ladder III (200–10000 bp) V. natriegens Vmax peGFP pDNA isolated after different time points of cultivation in 2xYT(+V2) (left) and MSM(+V2) medium (right) in Run 1. MSM(+V2) reactor2xYT(+V2) reactor 2.8 h 4 h 5 h 26 h6 h 5 h 6 h 26 h 10000 – 2000 – 4000 – 1000 – 200 – Band size (bp) Biomass in 1.5 L Bench-top Reactors (Run ID 1) pDNA Yields in 1.5 L Bench-top Reactors (Run ID 1) Agarose Gel of Isolated pDNA (Run ID 1)
Results Part 3: Upscaling in 1.5L Bench-Top Reactor 19 •Process scaled to 1.5 L with stable growth in both media •MSM(+V2) yielded up to 200 mg L-1 •Smearing in MSM(+V2) gel likely due to unprocessed biomass, not multimer formation RUN ID 2 0 2 4 6 8 010 20 Time (h) Biomass [g L-1] Run ID 2 2xYT(+V2) MSM(+V2) 10000 – 2000 – 4000 – 1000 – 200 – 10000 – 2000 – 4000 – 1000 – 200 – 2xYT(+V2) reactor MSM(+V2) reactor L L 2 h 3 h 4.5 h 9 h 26 h 3.5 h 4.5 h 6 h 8 h 26 h L: NZYDNA Ladder III (200–10000 bp) V. natriegens Vmax peGFP pDNA isolated after different time points of cultivation in 2xYT(+V2)(top) and MSM(+V2) medium (bottom) in Run 2. Band size (bp) 10000 – 2000 – 4000 – 1000 – 200 – 10000 – 2000 – 4000 – 1000 – 200 – 2xYT(+V2) reactor MSM(+V2) reactor L L 2 h 3 h 4.5 h 9 h 26 h 3.5 h 4.5 h 6 h 8 h 26 h L: NZYDNA Ladder III (200–10000 bp) V. natriegens Vmax peGFP pDNA isolated after different time points of cultivation in 2xYT(+V2)(top) and MSM(+V2) medium (bottom) in Run 2. Band size (bp) Biomass in 1.5 L Bench-top Reactors (Run ID 2) pDNA Yields in 1.5 L Bench-top Reactors (Run ID 2) Agarose Gel of isolated pDNA (Run ID 1)
Results Part 4: Upstream Process Scale Comparison 20 27 Doubling Time (min) K-value [g L-1 DCW] Maximum pDNA Yield [mg L-1] pDNA Topology Shake Flask MiniBioreactor Bench-top Bioreactor 29 65 (µ=0.2) & 80 (µ=0.4) 75 (µ=0.2) & 56 (µ=0.4) 33 (Run 1) & 32 (Run 2) 28 (Run 1) & 18 (Run 2) 5 5 17 (µ=0.2) & 42 (µ=0.4) 12 (µ=0.2) & 14 (µ=0.4) 6 (Run 1) & 6 (Run 2) 4 (Run 1) & 4 (Run 2) 20 11 60 (µ=0.2) & 105 (µ=0.4) 118 (µ=0.2) & 228 (µ=0.4) 7 (Run 1) & 55 (Run 2) 92 (Run 1) & 195 (Run 2) 2xYT(+V2) MSM(+V2) pDNA topology was similar across all cultivation conditions, with a dominant supercoiled fraction and additional isoforms appearing at higher molecular weights.
Results Part 5: Testing of Different Plasmids 21 0 2 4 0 2 4 6 Time (h) Biomass [g L-1] Plasmid pCEP4 pUC18 pUC57 0 5 10 15 20 25 pCEP4 pUC18 pUC57 Plasmid pDNA [mg L-1] a b c 10000 – 2000 – 4000 – 1000 – 200 – LpUC18 pCEP4 pUC57 a b c a b c L: NZYDNA Ladder III (200–10000 bp) V. natriegens Vmax pUC18, pCEP4 and pUC57 pDNA isolated after 6 h of cultivation in 2xYT(+V2) medium. *lanes a–c = biological replicates Band size (bp) •Comparable biomass accumulation across all plasmidbearing strains •No significant differences in volumetric pDNA yields (p > 0.05) Biomass Accumulation in Shake Flask Cultivation pDNA Yields in Shake Flasks Cultivation Agarose Gel of Different Isolated pDNA •Agarose gel confirmed the plasmid-specific topology
pDNA: pVAX1GFP Application: Transfection of mammalian cells with plasmid DNA Purpose: Test plasmid DNA from V. natriegens for direct use in eukaryotic gene expression Result: GFP fluorescence observed in mammalian cells → confirms pDNA transfection efficiency pDNA: peGFP Application: In vitro protein expression using E. colibased cell-free system Purpose: Rapid test to evaluate plasmid DNA integrity and expression potential of V. natriegens-derived plasmids Result: GFP expression bands observed on Western blot → confirms functional plasmid DNA from V. natriegens Results Part 6: Plasmid Functionality Assessment 23.10.2024 12:34:57 ht t ps: //b enc hl ing .co m /s/se qv LaLf OPsnFlRSHRP9GmQ/ ed it 1/ 1 12. FINAL_peGFP (2967 bp) 12. FINAL_peGFP 2967 bp 2 5 0 5 0 0 7 5 0 1 0 0 0 1 2 5 0 1 5 0 0 1 7 5 0 2 0 0 0 2 2 5 0 2 5 0 0 2 7 5 0 A m p R e G F P o r i A m p R p r o m o t e r A m p R T 7 t e r m i n a t o r R B S T 7 p r o m o t e r e G F P e G F P T 7 t a g ( g e n e 1 0 l e a d e r ) NheI BmtI,AseI,XbaI,BarI,NdeI,BseRI,+1 BtgZI BsrFI PfoI +2 +1 +1 +9 +8 +5 BmtI TspMI,+4 BlpI StyI EcoO109I BspEI HindIII PshAI BsrBI TfiI AflIII PciI,NspI DrdI BciVI MmeI,HaeII,BseYI,ApaLI MmeI AlwNI PfoI BspHI BanI AhdI BmrI +4 BsrFI BglI AseI FspI AclI BsrDI PvuI ScaI BsaHI AclI XmnI ApaLI EarI,SspI,BspHI,BciVI,BcoDI,BsmAI,BsrBI 1. Cell-Free Protein Expression 2. mRNA Transfection pDNA: pUC57-Amp-GFP Application: mRNA synthesis in vitro and transfection into mammalian cells Purpose: Evaluate suitability of V. natriegens plasmid DNA as template for therapeutic mRNA Result: GFP fluorescence observed in mammalian cells → confirms mRNA transfection efficiency 3. pDNA Transfection E. coli E. coli E. coli V. natriegens V. natriegens V. natriegens 27 kDa 22
Conclusion and Outlook 23
Conclusion 24 H1 H3 H4 V. natriegens can achieve pDNA yields comparable to or exceeding E. coli in batch cultivation → Up to ~4× higher volumetric pDNA yield than E. coli DH5α in 6–8 h → Stable plasmid maintenance in complex and defined media Fed-batch cultivation of V. natriegens is feasible and maintains pDNA presence → Robust µ-controlled growth in HT mini-bioreactors → Biomass up to 40 g/L; maintained pDNA presence H2 V. natriegens can be scaled up to 1.5 L bench-top reactors without performance loss → Successful operation at 1.5 L bench-top scale → Comparable physiology across scales pDNA produced in V. natriegens remains structurally intact and functionally active → pDNA is transcriptionally active (mRNA), CFPS-functional, and transfection-capable