Optimization of recombinant bacterial fermentations for pharmaceutical production

Two computer programs were developed and used to determine the optimum operating parameters of a fedbatch and a continuous two-stage process for fermentation of recombinant bacteria. The study was conducted in three phases: (a) developing two computer programs for simulation and optimization of the above processes, (b) conducting batch culture fermentations to verify the performance of the biokinetic model, and (c) conducting fedbatch and two-stage continuous fermentation experiments to closely examine the simulation and optimization results. The Miao and Kompala (1992) biokinetic model was used for simulation of the bacterial growth and cloned gene expression. The Pattern-Search method, developed by Hooke and Jeeves (1962), was incorporated in the programs to determine the optimum values of the parameters. Extensive studies of the optimization results showed 30-40% higher productivities for the two stage continuous process over the fedbatch process when using the same media in both processes. In addition, increasing the number of stages in the continuous two-stage process resulted in very limited improvement in the productivity of the process (10-12%). The information from the process optimization was then used to design batch, fedbatch nd two stage continuous experiments. Recombinant E. coli (strain BL21DE3) with an inducible gene (sensitive to IPTG, isopropyl-â-D-thiogalactopyranoside) was used throughout the experiments. The experimental results from the fedbatch and two stage continuous processes clearly showed good agreement with the simulation and optimization results $(\cong$15% deviation). The experiments also revealed that the maintenance of plasmid harboring cells over the long-term operation could be an important barrier in achieving the predicted high productivity in the two stage continuous process. Finally, in addition to computer programs for optimization of genetically modified microorganisms, a new computer program with a generic algorithm for optimization of multiple CFSTR fermentation with any kind of biokinetic model was developed. The program was used to optimize multiple CFSTRs with the cybernetic biokinetic model for the first time. Besides finding the optimum residence times for multiple CFSTRs operation, the effect of inaccuracies in different cybernetic model parameters on the overall productivity of the process was investigated. The simulation results illustrated that, a single CFSTR was more sensitive in its operation to inaccuracies in the biokinetic constants as compared to optimized CFSTRs in series (2-8 times more sensitive)

Multi-Scale Host-Aware Modeling for Analysis and Tuning of Synthetic Gene Circuits for Bioproduction

[ES] Esta Tesis ha sido dedicada al modelado multiescala considerando al anfitrión celular para el análisis y ajuste de circuitos genéticos sintéticos para bioproducción. Los objetivos principales fueron: 1. El desarrollo de un modelo que considere el anfitrión celular de tamaño reducido enfocado para simulación y análisis. 2. El desarrollo de herramientas de programación para el modelado y la simulación, orientada a la biología sintética. 3. La implementación de un modelo multiescala que considere las escalas relevantes para la bioproducción (biorreactor, célula y circuito sintético). 4. El análisis del controlador antitético considerando las interacciones célula-circuito, como ejemplo de aplicación de las herramientas desarrolladas. 5. El desarrollo y la validación experimental de leyes de control robusto para biorreactores continuos. El trabajo presentado en esta Tesis cubre las tres escalas del proceso de bioproducción. La primera escala es el biorreactor: esta escala considera la dinámica macroscópica del sustrato y la biomasa, y como estas dinámica se conecta con el estado interno de las células. La segunda escala es la célula anfitriona: esta escala considera la dinámica interna de la célula y la competencia por los recursos limitados compartidos para la expresión de proteínas. La tercera escala es el circuito genético sintético: esta escala considera la dinámica de expresión de los circuitos sintéticos exógenos y la carga que inducen en la célula anfitriona. Por último, como > escala, parte de la Tesis se ha dedicado a desarrollar herramientas de software para el modelado y la simulación. Este documento se divide en siete capítulos. El Capítulo 1 es una introducción general al trabajo de la Tesis y su justificación; también presenta un mapa visual de la Tesis y enumera las principales contribuciones. El Capítulo 2 muestra el desarrollo del modelo del anfitrión celular (los Capítulos 4 y 5 hacen uso de este modelo para sus simulaciones). El Capítulo 3 presenta OneModel: una herramienta de software desarrollada en la Tesis que facilita el modelado y la simulación en biología sintética, en particular, facilita el uso del modelo del anfitrión celular. El Capítulo 4 utiliza el modelo del anfitrión celular para montar el modelo multiescala que considera el biorreactor y analiza el título, la productividad y el rendimiento en la expresión de una proteína exógena. El Capítulo 5 analiza un circuito más complejo, el recientemente propuesto y muy citado controlador biomolecular antitético, utilizando el modelo del anfitrión celular. El Capítulo 6 muestra el diseño de estrategias de control no lineal que permiten controlar la concentración de biomasa en un biorreactor continuo de forma robusta. El Capítulo 7 resume y presenta las principales conclusiones de la Tesis. En el Apéndice A se muestra el desarrollo teórico del modelo del anfitrión celular. Esta Tesis destaca la importancia de estudiar la carga celular en los sistemas biológicos, ya que estos efectos son muy notables y generan interacciones entre circuitos aparentemente independientes. La Tesis proporciona herramientas para modelar, simular y diseñar circuitos genéticos sintéticos teniendo en cuenta estos efectos de carga y permite el desarrollo de modelos que conecten estos fenómenos en los circuitos genéticos sintéticos, que van desde la dinámica intracelular de la expresión génica hasta la dinámica macroscópica de la población de células dentro del biorreactor.[CA] Aquesta Tesi tracta del modelat multiescala considerant l'amfitrió ce\lgem ular per a l'anàlisi i ajust de circuits genètics sintètics per a bioproducció. Els objectius principals van ser: 1. El desenvolupament d'un model de grandària reduïda que considere l'amfitrió ce\lgem ular, enfocat al seu ús en simulació i anàlisi. 2. El desenvolupament d'eines de programari per al modelatge i la simulació, orientada a la biologia sintètica. 3. La implementació d'un model multiescala que considere les escales rellevants per a la bioproducció (bioreactor, cè\lgem ula i circuit sintètic). 4. L'anàlisi del controlador antitètic considerant les interacciones cè\lgem ula-circuit, com a exemple d'aplicació de les eines desenvolupades. 5. El desenvolupament i la validació experimental de lleis de control robust per a bioreactors continus. El treball presentat en aquesta Tesi cobreix les tres escales del procés de bioproducció. La primera escala és el bioreactor: aquesta escala considera la dinàmica macroscòpica del substrat i la biomassa, i com aquestes dinàmiques es connecten amb l'estat intern de les cè\lgem ules. La segona escala és la cè\lgem ula amfitriona: aquesta escala considera la dinàmica interna de la cè\lgem ula i la competència pels recursos limitats compartits per a l'expressió de proteïnes. La tercera escala és la del circuit genètic sintètic: aquesta escala considera la dinàmica d'expressió de circuits sintètics exógens i la càrrega que indueixen en la cè\lgem ula amfitriona. Finalment, com a > escala, part de la Tesi s'ha dedicat a desenvolupar eines de programari per al modelatge i la simulació. Aquest document es divideix en set capítols. El Capítol 1 és una introducció general al treball de la Tesi i la seua justificació; també presenta un mapa visual de la Tesi i enumera les principals contribucions. El Capítol 2 mostra el desenvolupament del model de l'amfitrió ce\lgem ular (els Capítols 4 i 5 fan ús d'aquest model per a les seues simulacions). El Capítol 3 presenta OneModel: una eina de programari desenvolupada en la Tesi que facilita el modelatge i la simulació en biologia sintètica, en particular, facilita l'ús del model de l'amfitrió ce\lgem ular. El Capítol 4 utilitza el model de l'amfitrió ce\lgem ular per a muntar el model multiescala que considera el bioreactor i analitza el títol, la productivitat i el rendiment en l'expressió d'una proteïna exògena. El Capítol 5 analitza un circuit més complex, el recentment proposat i molt citat controlador biomolecular antitètic, utilitzant el model de l'amfitrió ce\lgem ular. El Capítol 6 mostra el disseny d'estratègies de control no lineal que permeten controlar la concentració de biomassa en un bioreactor continu de manera robusta. El Capítol 7 resumeix i presenta les principals conclusions de la Tesi. En l'Apèndix A es mostra el desenvolupament teòric del model de l'amfitrió ce\lgem ular. Aquesta Tesi destaca la importància d'estudiar la càrrega ce\lgem ular en els sistemes biològics, ja que aquests efectes són molt notables i generen interaccions entre circuits aparentment independents. La Tesi proporciona eines per a modelar, simular i dissenyar circuits genètics sintètics tenint en compte aquests efectes de càrrega i permet el desenvolupament de models que connecten aquests fenòmens en els circuits genètics sintètics, que van des de la dinàmica intrace\lgem ular de l'expressió gènica fins a la dinàmica macroscòpica de la població de cè\lgem ules dins del bioreactor.[EN] This Thesis was devoted to the multi-scale host-aware analysis and tuning of synthetic gene circuits for bioproduction. The main objectives were: 1. The development of a reduced-size host-aware model for simulation and analysis purposes. 2. The development of a software toolbox for modeling and simulation, oriented to synthetic biology. 3. The implementation of a multi-scale model that considers the scales relevant to bioproduction (bioreactor, cell, and synthetic circuit). 4. The host-aware analysis of the antithetic controller, as an example of the application of the developed tools. 5. The development and experimental validation of robust control laws for continuous bioreactors. The work presented in this Thesis covers the three scales of the bioproduction process. The first scale is the bioreactor: this scale considers the macroscopic substrate and biomass dynamics and how these dynamics connect to the internal state of the cells. The second scale is the host cell: this scale considers the internal dynamics of the cell and the competition for limited shared resources for protein expression. The third scale is the synthetic genetic circuit: this scale considers the dynamics of expressing exogenous synthetic circuits and the burden they induce on the host cell. Finally, as a > scale, part of the Thesis was devoted to developing software tools for modeling and simulation. This document is divided into seven chapters. Chapter 1 is an overall introduction to the Thesis work and its justification; it also presents a visual map of the Thesis and lists the main contributions. Chapter 2 shows the development of the host-aware model (Chapters 4 and 5 make use of this model for their simulations). Chapter 3 presents OneModel: a software tool developed in the Thesis that facilitates modeling and simulation for synthetic biology---in particular, it facilitates the use of the host-aware model---. Chapter 4 uses the host-aware model to assemble the multi-scale model considering the bioreactor and analyzes the titer, productivity (rate), and yield in expressing an exogenous protein. Chapter 5 analyzes a more complex circuit, the recently proposed and highly cited antithetic biomolecular controller, using the host-aware model. Chapter 6 shows the design of nonlinear control strategies that allow controlling the concentration of biomass in a continuous bioreactor in a robust way. Chapter 7 summarizes and presents the main conclusions of the Thesis. Appendix A shows the theoretical development of the host-aware model. This Thesis emphasizes the importance of studying cell burden in biological systems since these effects are very noticeable and generate interactions between seemingly unconnected circuits. The Thesis provides tools to model, simulate and design synthetic genetic circuits taking into account these burden effects and allowing the development of models that connect phenomena in synthetic genetic circuits, ranging from the intracelullar dynamics of gene expression to the macroscopic dynamics of the population of cells inside the bioreactor.This research was funded by MCIN/AEI/10.13039/501100011033 grant number PID2020-117271RB-C21, and MINECO/AEI, EU grant number DPI2017-82896- C2-1-R. The author was recipient of the grant “Programa para la Formación de Personal Investigador (FPI) de la Universitat Politècnica de València — Subprograma 1 (PAID-01-2017)”. The author was also a grantee of the predoctoral stay “Ayudas para Movilidad de Estudiantes de Doctorado de la Universitat Politècnica de València 2019”. The Control Theory and Systems Biology Lab of the ETH Zürich is acknowledged for accepting the author in their facilities as predoctoral stay and their valuable collaboration sharing knowledge.Santos Navarro, FN. (2022). Multi-Scale Host-Aware Modeling for Analysis and Tuning of Synthetic Gene Circuits for Bioproduction [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/183473Premios Extraordinarios de tesis doctorale

Whey Lactose as a Raw Material for Microbial Production of Biodegradable Polyesters

Fossil fuel technologie

Engineering of Aspergillus niger for the production of secondary metabolites

Background: Filamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers. To overcome these limitations, we report here the engineering of the industrial fungus Aspergillus niger to produce high titers (up to 4,500 mg • l-1) of secondary metabolites belonging to the class of nonribosomal peptides. Results: For a proof-of-concept study, we heterologously expressed the 351 kDa nonribosomal peptide synthetase ESYN from Fusarium oxysporum in A. niger. ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene esyn1 was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of A. niger cultures. The enniatins were isolated and purified by means of reverse phase chromatography and their identity and purity proven by tandem MS, NMR spectroscopy and X-ray crystallography. The initial yields of 1 mg • l-1 of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of A. niger in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating A. niger in 5 l fed batch fermentations. Finally, an autonomous A. niger expression host was established, which was independent from feeding with the enniatin precursor D-2-hydroxyvaleric acid D-Hiv. This was achieved by constitutively expressing a fungal D-Hiv dehydrogenase in the esyn1-expressing A. niger strain, which used the intracellular ɑ-ketovaleric acid pool to generate D-Hiv. Conclusions: This is the first report demonstrating that A. niger is a potent and promising expression host for nonribosomal peptides with titers high enough to become industrially attractive. Application of the Tet-on system in A. niger allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced.EC/FP7/607332/EU/Quantitative Biology for Fungal Secondary Metabolite Producers/QuantFungDFG, EXC 314, Unifying Concepts in Catalysi

Optimal control of fed-batch fermentation processes

Optimisation of a fed-batch fermentation process typically uses the calculus of variations or Pontryagin's maximum principle to determine an optimal feed rate profile. This often results in a singular control problem and an open loop control structure. The singular feed rate is the optimal feed rate during the singular control period and is used to control the substrate concentration in the fermenter at an optimal level. This approach is supported by biological knowledge that biochemical reaction rates are controlled by the environmental conditions in the fermenter; in this case, the substrate concentration. Since an accurate neural net-based on-line estimation of the substrate concentration has recently become available and is currently employed in industry, we are therefore able to propose a method which makes use of this estimation. The proposed method divides the optimisation problem into two parts. First, an optimal substrate concentration profile which governs the biochemical reactions in the fermentation process is determined. Then a controller is designed to track the obtained optimal profile. Since the proposed method determines the optimal substrate concentration profile, the singular control problem is therefore avoided because the substrate concentration appears nonlinearly in the system equations. Also, the process is then operated in closed loop control of the substrate concentration. The proposed method is then called "closed loop optimal control". The proposed closed loop optimal control method is then compared with the open loop optimal feed rate profile method. The comparison simulations from both primary and secondary metabolite production processes show that both methods give similar performance in a case of perfect model while the closed loop optimal control provides better performance than the open loop method in a case of plant/model mismatch. The better performance of the closed loop optimal control is due to an ability to compensate for the modelling errors using feedback

Simultaneous clarification and purification of recombinant penicillin G acylase using tangential flow filtration anion-exchange membrane chromatography

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins. Conventional purification processes are lengthy, technically complicated, product specific and time-consuming. To address this issue, herein we develop a one step purification system that due to the nature of the non-selective secretion system and the versatility of ion-exchange membrane chromatography can be widely applied to the production of many recombinant proteins. This was achieved through the integration of the intrinsically coupled upstream, midstream and downstream processes, a connection that is rarely exploited. A bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography medium while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange medium, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level of 487 U/L/OD600, with more than 90% was localized in the extracellular medium. Both, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. Furthermore, formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. iv At harvest, the whole non-clarified culture broth was applied directly to a tangential flow filtration anion-exchange membrane chromatography system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry). Due to their size, cells remained in the retentate while the extracellular medium penetrated the membrane. Most contaminate proteins were captured by the anion-exchange membrane, whereas the purified PAC was collected in the filtrate. The batch time for both cultivation and purification was less than 24 h and recombinant PAC with high purity (19 U/mg), process yield (74%), and productivity (41 mg/L) was obtained

Heterotrophic Culture of Microalgae Using Biodiesel Derived Glycerol

Algae-based technologies are fast growing and the growing demand for sustainable technologies is evident from the growing energy demand and global warming. Microalgae culturing for higher lipid contents have been a hot topic of intense discussion in the past years. Crude glycerol, a byproduct from biodiesel production seems to be an attractive feedstock for microbial cultivation and crude glycerol has been proven as a good alternative feedstock for cultivation of Chlorella protothecoides. The effect of impurities present in the crude glycerol is important to develop a method for high-density cultivation of microalgae to increase the commercialization potential of algae systems. Through this study, a method to partially refine crude glycerol was developed to increase the suitability of biodiesel-derived glycerol for high-density cultivations. C. protothecoides grew best at an initial glycerol concentration of 90 g/L and a maximum biomass and lipid productivity of 4.45 and 2.28 g/L-day was achieved at an initial glycerol concentration of 120 g/L. Fed-batch studies increased the biomass and lipid concentrations and productivities. A maximum biomass and lipid concentration of 95.3 and 49.5 g/L-day was achieved while using PRG as a carbon source with a maximum productivity of 10.6 g/L-day. Yield biomass per substrate in the fed batch mode was observed to be 0.53. Comparing the data to published literature, these are the best results. Fatty acid profiles were observed to be very comparable to data published by other researches on C.protothecoides. Further studies on the effect of salinity on the growth of C.protothecoides, yielded no statistical significance in the biomass concentration and lipid content at a KCl concentration of 10 and 20 g/L, and a NaCl concentration of 10 g/L. Further increase in NaCl concentration to 20 g/L decreased the maximum biomass concentration. No growth was observed at salt concentrations of 40 g/L. Increasing salt concentrations had no impact on the relative fatty acid percentage of oleic acid (most abundant fatty acid produced by Chlorella protothecoides). Biomass productivities were significantly lower in the presence of salts, indicating that the present of salts decreases the biomass productivity. Increasing methanol concentrations were evaluated, and the results proved that methanol was not significantly consumed but evaporated by this species of algae. A methanol concentration 1 % (v/v) yielded similar biomass and lipid concentrations, Yx/sand Yp/s. The biomass productivity, however, was significantly lower with increase in methanol concentrations. Xylose proved to be detrimental to the growth of C. protothecoides as increasing xylose concentrations decreased biomass concentration. No growth was observed at a xylose concentration of 30 g/L. In summary, the effect of some impurities present in crude glycerol was evaluated and a method to refine crude glycerol proved successful. A method for high-density cultivation of C. protothecoides for increased productivities while using a waste stream is presented

Production of B-galactosidase using lactic acid bacteria and optimisation of fermentation parametters

Thesis (Master)--Izmir Institute of Technology, Biotechnology, Izmir, 2007Includes bibliographical references (leaves: 90-97)Text in English; Abstract: Turkish and Englishxiii, 120 leavesFood grade thermostable B -galactosidase preparations are always in demand for a number of industrial applications. Thermostable -galactosidases from LAB having a neutral pH-optimum can be safely used to reduce the lactose content of milk for the lactose intolerant people. In this study, -galactosidase was produced with high productivities by novel Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional Turkish yogurt samples in Toros mountain region. A full factorial statistical design was used separately for Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains in screening experiments. Among the strains, Lactobacillus delbrueckii subsp. bulgaricus 77 and Streptococcus thermophilus 95/2 were found to have high potential for B-galactosidase and lactic acid production, therefore these were used in the further optimisation studies.The efficiency of different cell disruption methods was investigated on the extraction of -galactosidase. Among these, lysozyme enzyme treatment was determined as the most effective method. Optimisation studies were carried out using response surface methodology to optimize fermentation conditions for pure strains as well as for mixed ones. Therefore, symbiotic relationship between St 95/2 and Lb 77 were investigated as well. Symbiotic relationship provided 39% and 6.1 % more -galactosidase activity and 44 % and 9.73 % more lactic acid production when compared to the optimisation results of pure strains Lb 77 and St 95/2, respectively.Overall, characterization studies showed that enzymes obtained from these strains can be considered as food grade and thermostable since they are obtained from thermophile, food originated novel LAB of local microflora