R-3 Repository :: Browsing by Author "San, Ka-Yiu"

Browsing by Author "San, Ka-Yiu"

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A study of the fluid mechanics and the cultivation of mammalian cells in a magnetically stabilized fluidized bed bioreactor
(1995) Mathew, John; San, Ka-Yiu
The culture of anchorage dependent mammalian cells on microcarrier offers an attractive avenue for achieving high productivity of therapeutic and diagnostic proteins in bioreactors. Reducing production costs require high cell density that is accompanied by mass transfer limitations of nutrients and oxygen. High agitations required to overcome these limitations can cause considerable cell damage. In this work a novel magnetically stabilized fluidized bed (MSFB) bioreactor is developed to culture mammalian cells on microcarriers. A fluid mechanical study of the MSFB, using a laser light transmission technique showed that the local particle motion is reduced by increasing the applied magnetic field strength. This low turbulent behavior of particles in a MSFB allows for potential cultivation of cells in a three dimensional manner. Two types of magnetically susceptible microcarriers are developed for culturing cells in a MSFB. The performance of the MSFB and an ordinary fluidized bed is compared in terms of cell density, growth rate and death rate of baby hamster kidney (BHK-21) cells. Very high cell densities ($5\times10\sp7$ cells/ml) are obtained in both the modes of operation. Results indicate that cells grow at a faster rate in a MSFB as compared to an ordinary fluidized bed. To delineate the effects of flow and magnetic field on cell proliferation, BHK cells were cultured on nonmagnetically susceptible microcarriers in the presence and absence of a 80 gauss DC magnetic field. Five pairs of experiments showed that a uniform static magnetic field increased the growth of BHK-21 cells in a fluidized bed environment. The reduced local motion of particles and the high cell densities that can be attained in a MSFB makes it suitable as a 3-D cell culture system. Preliminary experiments showed the formation of 3-D cell aggregates when hepatoma cells were cultured to high cell densities in the MSFB. A linear stability analysis of the equations of motion describing a fluidized bed predicted that the stability of the state of uniform fluidization could be enhanced by applying a nonuniform magnetic field.
Aerobic succinate production in bacteria
(2007-07-17) San, Ka-Yiu; Bennett, George N.; Lin, Henry; Rice University; United States Patent and Trademark Office
Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.
Aerobic succinate production in bacteria
(2011-05-03) San, Ka-Yiu; Bennett, George N.; Lin, Henry; Rice University; United States Patent and Trademark Office
Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.
Aerobic succinate production in bacteria
(2007-08-28) San, Ka-Yiu; Bennett, George N.; Lin, Henry; Rice University; United States Patent and Trademark Office
Methods of increasing yields of succinate using aerobic culture methods and a multi-mutant E. coli strain are provided. Also provided is a mutant strain of E. coli that produces high amounts of succinic acid.
Application of back-propagation neural networks to system identification and process control
(1991) Broussard, Mark Randall; San, Ka-Yiu
Certain properties of the back-propagation neural network have been found to be potentially useful in structuring models for process control applications. The network's relative simplicity and its ability to learn by example are potentially important in the effort to develop automated continuous on-line system identification. The capacity of the network to form nonlinear mappings enhances research designed to advance nonlinear system identification techniques. Since most real processes are nonlinear, this prospect can have wide impact. The unstructured nature of the network model was found to be controllable by techniques developed in the study. Care must be taken to identify and train the network with consistent data that contains sufficient dynamical information. Model-based fine tuning of a controller using a network model that was identified with closed-loop data was successful for the linear and nonlinear systems examined. The utility of the model is a function of the dynamical history of the process. When the information content of the data is sufficient, the network can capture the most important features of system behavior so that fine tuning can be based on optimal parameters such as integral absolute error. This method offers a more complete picture of tuning options than that of other fine tuning techniques such as trial and error, which are not based on a system model. The techniques developed in the tuning effort may be extended to closed-loop model identification for the purpose of controller redesign. In this case, successful identification probably depends on the continuous on-line identification to correct for modeling error.
Application of back-propagation neural networks to the modeling and control of multiple-input, multiple-output processes
(1991) Takasu, Shinji; San, Ka-Yiu
Certain properties of back-propagation neural networks have been found to be useful in structuring models for multiple-input, multiple-output (MIMO) processes. The network's simplicity and its ability to identify the non-linearity can have wide impacts on the construction of model-based control system. Care must be taken to train the network with consistent data that contains sufficient dynamic information. A predictive control system based on the network model was proposed. Although the controller is relatively simple in terms of concept and computation, it shows excellent performances both in servo and regulator problems. Model prediction error sometimes causes a cyclic behavior in process responses; however, it can be stabilized by imposing certain constraints of controller action. The constraints are also effective for noisy measurements. Use of neural networks for modeling and control of MIMO system appears to be very promising with its ability to treat non-linearity and process interactions.
Application of metabolic and biochemical engineering techniques for the enhancement of recombinant protein production in Escherichia coli
(1995) Aristidou, Aristos Andrea; San, Ka-Yiu
This study focuses on the application of metabolic engineering techniques to diminish acetate excretion, as a means to enhance recombinant protein productivities. The recombinant model system is based on the formation of plasmid encoded cadA-LacZ fusion protein with $\beta$-Galactosidase activity under the control of the pH-regulated promoter. This recently developed expression system has the potential for very high gene expression, of 30 to 40% of the total soluble cell protein. Three approaches are presented. E. coli acetate mutants were isolated and characterized as recombinant hosts in relatively dense cultures. An acetate kinase mutant (ack) was found to excrete less acetate and in the meantime produce significantly higher amounts of recombinant protein, compared with the parent strain. In addition, this strain was observed to be less susceptible to fermentor dissolved oxygen deficiencies. The possibility of replacing the common carbon source glucose with its isomer fructose was further investigated. Fructose is a feasible alternative since its uptake is more tightly regulated, and also because it is a less effective catabolic repressor. Comparative fermentation studies indicate that acetate levels were reduced to less than 6 mM from more than 90 mM, while in the meantime a 35% improvement in biomass yields was achieved. High cell density batch fermentations using fructose resulted in volumetric $\beta$-Galactosidase activities of 2.2 million U/ml, which represents a 65% improvement compared with similar glucose cultures. The third method focuses on minimizing the metabolic imbalances through the genetic manipulation of the host organism. The alsS gene from B. Subtilis encoding the acetolactate synthase enzyme was successfully expressed in E. coli. This enzyme redirects pyruvate away from acetate, towards a non-inhibitory byproduct, acetoin. Acetate excretion can be maintained below 20 mM even in dense cultures employing rich glucose media. Moreover the engineered strain is a more efficient host for the production of recombinant proteins. The volumetric expression of $\beta$-Galactosidase was found to increase by about 50% in batch cultivations and by about 220% in high cell density fed-batch cultivations.
Application of metabolic and biochemical engineering techniques to enhance metabolic flux throughput and improve succinate production in Escherichia coli
(2001) Chhiber, Aasheesh; San, Ka-Yiu
Using biochemical engineering principles and metabolic engineering tools in tandem can considerably improve process productivity by manipulation of the throughput of certain pathways. Escherichia coli was chosen as the model system because of its vast available genetic information, rapid growth and the practice of standardized experimental protocols. The pathway leading to the production of succinic acid was examined for the effects of level of carbon substrate available to the system and overexpression of the phosphoenolpyruvate carboxylase (PEPC) enzyme system coupled with pathway manipulations and use of different carbon sources. Incorporation of the glcU gene from Staphylococcus xylosus encoding a novel glucose uptake protein resulted in enhancement of metabolic flux throughput in E. coli. This was further utilized to increase the productivity of a specific metabolite, acetoin, encouraging the use of this system to improve succinate levels. Further, overexpression of the feedback resistant Sorghum PEPC in the host was coupled to three pathway manipulations: ackA-pta mutant, ackA-pta-nuo mutant, and ackA-pta-nuo-ldh mutant, with a high yield value of 0.65 mole succinate per mole of glucose utilized obtained with the ackA-pta-nuo-ldh mutant. Using a reduced carbon source such as sorbitol increased the succinate production to approximately 16 g/l.
Biochemical and genetic engineering strategies to enhance recombinant protein production in Escherichia coli
(1995) Chou, Chih-Hsiung; San, Ka-Yiu
Escherichia coli has been widely used to produce high-value recombinant proteins for years. Although high recombinant protein productivity can be attained, perhaps the most important goal for such processes, that of achieving both high gene expression and high cell density simultaneously, is still challenging to both biochemists and biochemical engineers. A series of approaches to overcome this problem are evaluated in this study. First, a novel pH-inducible gene expression system, in which the expression of foreign gene products is directed by a pH down-shift, was chosen and characterized. This system was shown to have many attractive features, including high-level expression (40% of total cellular protein), fast response, and easy manipulation. It thus can serve as a proper model system for studying the fundamental mechanism of high-level gene expression in E. coli. Second, several factors limiting the culture performance were identified by systematically optimizing the culture conditions. Among those, acetate overproduction was shown to be critically involved. Various approaches on the basis of biochemical and genetic engineering techniques were successfully exploited to bypass such a cultivation bottleneck. Finally, the effects of various genetic elements, including the genes responsible for carbohydrate uptake and several stationary-phase genes, on recombinant protein production were investigated by genetic manipulation of the host strain. Several strategies were then developed to genetically construct more potent strains for recombinant protein production. The information is important not only for the modification of several structural models developed recently, but also for economic interest in terms of improving bioprocesses without further investment in equipment.
Biosynthesis of Medium-Chain ω-Hydroxy Fatty Acids by AlkBGT of Pseudomonas putida GPo1 With Native FadL in Engineered Escherichia coli
(Frontiers, 2019) He, Qiaofei; Bennett, George N.; San, Ka-Yiu; Wu, Hui
Hydroxy fatty acids (HFAs) are valuable compounds that are widely used in medical, cosmetic and food fields. Production of ω-HFAs via bioconversion by engineered Escherichia coli has received a lot of attention because this process is environmentally friendly. In this study, a whole-cell bio-catalysis strategy was established to synthesize medium-chain ω-HFAs based on the AlkBGT hydroxylation system from Pseudomonas putida GPo1. The effects of blocking the β-oxidation of fatty acids (FAs) and enhancing the transportation of FAs on ω-HFAs bio-production were also investigated. When fadE and fadD were deleted, the consumption of decanoic acid decreased, and the yield of ω-hydroxydecanoic acid was enhanced remarkably. Additionally, the co-expression of the FA transporter protein, FadL, played an important role in increasing the conversion rate of ω-hydroxydecanoic acid. As a result, the concentration and yield of ω-hydroxydecanoic acid in NH03(pBGT-fadL) increased to 309 mg/L and 0.86 mol/mol, respectively. This whole-cell bio-catalysis system was further applied to the biosynthesis of ω-hydroxyoctanoic acid and ω-hydroxydodecanoic acid using octanoic acid and dodecanoic acid as substrates, respectively. The concentrations of ω-hydroxyoctanoic acid and ω-hydroxydodecanoic acid reached 275.48 and 249.03 mg/L, with yields of 0.63 and 0.56 mol/mol, respectively. This study demonstrated that the overexpression of AlkBGT coupled with native FadL is an efficient strategy to synthesize medium-chain ω-HFAs from medium-chain FAs in fadE and fadD mutant E. coli strains.
Characterization and application of a periplasmic protein releasing system for extracellular recombinant protein production in Escherichia coli
(1993) Yu, Peng; San, Ka-Yiu
Escherichia coli is commonly used as a host in the production of high value therapeutic proteins. However, one major drawback associated with E. coli being a host is the intracellular location of product proteins. As a result, mechanical cell disruption is commonly employed in biotechnology industry for protein release. Unfortunately, mechanical disintegration always contaminates the target protein with other cellular substances. On the other hand, progress in the study of protein translocation across membrane often allows the transport of proteins from the cytoplasm to the periplasm. However, the outer membrane still presents a barrier for their extracellular release. The objective of this work is to bridge the gap between protein translocation and extracellular protein release. The main focus is to characterize a periplasmic protein release system and to apply the system to different processes for extracellular protein production. Bacteriocin Release Protein (BRP) and glycine were the two systems studied using a-amylase as the model protein. Both agents were shown to be effective in periplasmic protein release. When used alone in a batch fermentor, BRP released 41% of the periplasmic a-amylase extracellularly without causing substantial cell lysis. Similarly, glycine achieved a 45% protein release. Synergistic combination of BRP and glycine resulted in further increase in a-amylase release. At an optimal combination, 78% of a-amylase was released. Application of the release system for continuous extracellular protein production was successful. A maximum productivity of 8.3 units/ml/hr was obtained, of which one third was in the extracellular broth. Mathematical simulation suggested that higher expression levels of a-amylase at slower growth rates is mainly due to a higher plasmid copy number. Incorporation of hollow fiber cartridges into a chemostat process allowed continuous production of cell-free proteins as well as retention of recombinant cells. At an optimal dilution rate, 42% of a-amylase was produced in a cell-free form. Substitution of the Amp$\sp{\rm r}$ gene in pBR322-amy with a Km$\sp{\rm r}$ gene in an attempt to improve the performance of the release system resulted in a higher accumulation of a-amylase in the cytoplasm. The observation demonstrated that antibiotic markers are important parameters in a-amylase translocation and distribution.
Characterization and modeling of the pH-regulated expression vector, pSM-10
(1992) Tolentino, Gerard Joaquin; San, Ka-Yiu
A new type of plasmid vector has been developed for Escherichia coli. Foreign gene expression can be altered by external pH changes. pSM-10 is the first generation of these plasmid constructs that utilizes the cadA operon for gene regulation. The growth conditions necessary to maximize this response were characterized. GJT-001, a $cadR\sp-$ strain of E. coli, was isolated that allowed study of pH regulation without requiring high lysine concentrations. Up to a 200-fold increase in specific activity of $\beta$-galactosidase was observed upon lowering the pH of the growth media from pH 8.0 to 5.5. Anaerobic growth conditions can independently induce high gene expression. Maximum activities are observed under a combination of low pH and anaerobic conditions. The pH-induction effect is fully reversible and provides a high degree of expression control. The highest levels of induction were observed in batch culture when pH- and anaerobic-step changes were introduced at low optical densities. Rich media is also necessary for highest expression. Further investigation revealed that a possible induction factor may be present in yeast extract. In addition, pH induction is limited at high optical densities due to an apparent inhibition by accumulated waste products, specifically acetate. A mathematical model was developed for $\beta$-galactosidase expression, incorporating the effects of growth rate, yeast extract consumption and acetate inhibition. From this model maximal batch productivities are best achieved under aerobic growth conditions that maximize growth rate and optical density and also minimizes the inhibition effect of acetate.
Cofactor engineering for advancing chemical biotechnology
(Elsevier, 2013) Wang, Yipeng; San, Ka-Yiu; Bennett, George N.
Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.
Cofactor engineering of intracellular CoA/acetyl-CoA and its subsequent effect on isoamyl acetate production in Escherichia coli
(2004) Vadali, Ravishankar V.; San, Ka-Yiu
Traditional metabolic engineering focused on pathway manipulation strategies like amplification addition or deletion of pathway to manipulate fluxes. However, cofactors play an essential role in cellular metabolism and their manipulation has the potential to be used, as an additional tool to achieve desired metabolic engineering goals. Coenzyme A and its derivative acetyl-CoA are important cofactors involved in many biosynthetic pathways and precursors for many industrially useful compounds. Our study focused on increasing the intracellular level/fluxes of CoA and acetyl-CoA. This was accomplished by overexpression of key rate controlling enzyme panK in the CoA biosynthesis pathway along with simultaneous supplementation of precursor pantothenic acid. The effect of such precise alteration of CoA metabolism on extracellular metabolite formation was studied. The utility of CoA manipulation system in enhancing production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA was demonstrated. This novel approach of cofactor manipulation was combined with the more traditional approach of competing pathway deletion, acetate production pathway in this case, to further enhance isoamyl acetate productivity. Overexpression of panK led to a significant increase in CoA levels. Acetyl-CoA levels also increased but not as much as CoA leaving much of it in an unacetylated form. The central carbon flux was enhanced, either by overexpression of pdh or pps to increase acetyl-CoA, under elevated CoA levels. The flux through the acetyl-CoA node increased under such conditions. This enhanced carbon flux was efficiently channeled to isoamyl acetate production pathway by inactivating the acetate production pathway. The combination of these metabolic engineering strategies led to a significant increase in isoamyl acetate production. We used production of isoamyl acetate as a model system to demonstrate the beneficial effects of CoA/acetyl-CoA manipulations in enhancing product productivity. This methodology can be easily extended to any other production systems involving the cofactors CoA/acetyl-CoA. Additional studies in our lab have shown that CoA/acetyl-CoA manipulation system is useful in improving productivities of succinate and lycopene.
Design and implementation of metabolic networks for the improvement of product yields in cofactor-limiting systems in Escherichia coli
(2006) Sanchez Molina, Ailen M.; San, Ka-Yiu
Metabolic engineering is the rational alteration of the genetic structure of an organism to make this organism achieve a desired goal. One important aspect of metabolic engineering is the manipulation of metabolic pathways in microorganisms to increase the yield and productivity of cofactor dependent products. When designing a metabolic network to maximize product formation from a substrate, it is crucial to take into consideration cofactor constraint and maintain a proper balance between yield and productivity. The purpose of this study is to design and optimize a metabolic network to increase the yield and productivity of cofactor dependent products taking into consideration cofactor constraint. The production of succinate, a valuable specialty chemical, was used as a model system to explore the effect of manipulating NADH in vivo as well as to study the effect of alleviating cofactor constraint through pathway engineering. Additionally the production of the biodegradable polymer poly-beta-hydroxybutyrate was used as a model system to explore the effect of manipulating NADPH availability in vivo. Currently, the maximum theoretical succinate yield under strictly anaerobic conditions through the fermentative succinate biosynthesis pathway is limited to one mole per mole of glucose due to NADH limitation. In order to surpass the maximum anaerobic theoretical succinate yield from glucose, a genetically engineered E. coli strain was constructed to meet the NADH requirement and carbon demand to produce high quantities and yield of succinate. The implemented strategic design involves a dual succinate synthesis route, which diverts required quantities of NADH through the traditional fermentative pathway and maximizes the carbon converted to succinate by balancing the carbon flux through the fermentative pathway and the glyoxylate pathway (which has a lower NADH requirement). The implementation of this metabolic network to produce succinate in E. coli increases the succinate yield from glucose to 1.6 mol/mol with an average anaerobic productivity rate of 10 mM/h. The final strain demonstrated to be stable and robust in performance. Based on the proposed stoichiometric model, the experimental estimated metabolic fluxes of this strain were in excellent agreement with theoretical optimized fluxes (Cox et al. 2005).
Dynamics of plasmid-containing cells in a chemostat under transient conditions
(1990) Weber, Allison Elizabeth; San, Ka-Yiu
One of the major concerns in the utilization of plasmid-containing organisms in fermentation processes is the stability of the plasmid. In general, it is observed that two parameters, the probability of plasmid loss due to faulty segregation and the difference in the specific growth rate between the recombinant and reverted cells, govern the dynamics of the reactor. These parameters are affected by a number of factors: the genetic make-up of the host cells and the plasmid and the reactor operating parameters such as temperature, pH, dilution rate, and growth medium. It is speculated that by manipulating these factors it may be possible to enhance the stability of the plasmid-containing population. In this work the effects of well-defined perturbations in reactor conditions on plasmid maintenance and expression of a population of Escherichia coli K12 strain containing the plasmid pBR322 grown in a chemostat with a non-selective medium were studied. In a set of experiments the cultures were exposed to square-wave oscillations in the dilution rate. Under these conditions the cultures were capable of maintaining a mixed population of plasmid-containing and plasmid-free cells for a longer period of time than a culture grown under a constant dilution rate. The effect of the perturbations on plasmid copy number and expression were also studied. The data was dependent on the amplitude and frequency of the oscillations. In addition, the experiments indicate that adaptation of the culture to the transient conditions will reduce or totally eliminate any advantage created by the perturbations. An unstructured model, evaluating the probability of plasmid loss and the differences in the specific growth rate between the recombinant and reverted cells grown under dilution rate oscillations, was developed to describe the experimental data. The results indicate that the transient conditions provide a favorable environment for the plasmid-containing population. It was further observed, through the use of minimum inhibitory concentration experiments, that the plasmid-containing cells existed as a heterogeneous population with respect to the plasmid copy number. This population distribution could play an important role in plasmid stability and expression.
Engineered bacteria produce succinate from sucrose
(2015-02-24) San, Ka-Yiu; Bennett, George N.; Wang, Jian; Rice University; United States Patent and Trademark Office
Bacteria optimized to produce succinate and other feedstocks by growing on low cost carbon sources, such as sucrose.
Ester production and metabolic flux redistribution in Escherichia coli under aerobic conditions
(2005) Singh, Randeep; San, Ka-Yiu
Two alcohol acetyltransferases from Saccharomyces cerevisiae (ATF1 and ATF2) can catalyze the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA). The respective genes were cloned from and expressed in an appropriate ack-pta strain of Escherichia coli. The new genetically engineered strains of E. coli produce isoamyl acetate, an ester, when isoamyl alcohol is added externally to the cell culture medium. Since acetyl-CoA is a substrate for this esterification reaction, the competing ackA-pta pathway at the acetyl-CoA node was inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Experiments were carried out in aerobic shake flasks to investigate isoamyl acetate production over long periods of growth at various temperatures and starting optical densities. The ackA-pta mutant strain containing the pBAD-ATF1 plasmid exhibited the highest molar ester yield from glucose (1.13) after 48 hours of aerobic growth at 25°C.
High molar succinate yield bacteria by increasing the intracellular NADH availability
(2011-04-19) San, Ka-Yiu; Bennett, George N.; Sánchez, Ailen; Rice University; United States Patent and Trademark Office
The invention relates to increasing the yield of succinate in bacteria by increasing the intracellular availability of cofactors such as NADH.
Improvement of NADPH bioavailability in Escherichia coli through the use of phosphofructokinase deficient strains
(Springer-Verlag, 2013) Wang, Yipeng; San, Ka-Yiu; Bennett, George N.
NADPH-dependent reactions play important roles in production of industrially valuable compounds. In this study, we used phosphofructokinase (PFK)-deficient strains to direct fructose-6-phosphate to be oxidized through the pentose phosphate pathway (PPP) to increase NADPH generation. pfkA or pfkB single deletion and double-deletion strains were tested for their ability to produce lycopene. Since lycopene biosynthesis requires many NADPH, levels of lycopene were compared in a set of isogenic strains, with the pfkA single deletion strain showing the highest lycopene yield. Using another NADPH-requiring process, a one-step reduction reaction of 2-chloroacrylate to 2-chloropropionic acid by 2- haloacrylate reductase, the pfkA pfkB double-deletion strain showed the highest yield of 2-chloropropionic acid product. The combined effect of glucose-6-phosphate dehydrogenase overexpression or lactate dehydrogenase deletion with PFK deficiency on NADPH bioavailability was also studied. The results indicated that the flux distribution of fructose-6- phosphate between glycolysis and the pentose phosphate pathway determines the amount of NAPDH available for reductive biosynthesis.

Browsing by Author "San, Ka-Yiu"

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