Effect of Modulated Glucose Uptake on High‐Level Recombinant Protein Production in a Dense <i>Escherichia coli</i> Culture

Chou, C. Perry; Bennett, George N.; San, Ka‐Yiu

doi:10.1021/bp00030a009

Cited by 48 publications

(38 citation statements)

References 9 publications

(10 reference statements)

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“…It is argued that a properly designed new reaction pathway will perturb the existing pathway networks positively and will result in a better strain with the desired cellular characteristics . Two different approaches have been investigated to modulate the glucose uptake rate in E. coli (Chou et al, 1994a;Chou et al, 1994b). In the first study, methyl α-glucoside (α-MG), a glucose analog, which shares the same phosphotransferase system (PTS) was used to modulate the glucose uptake rate.…”

Section: An Example Of Metabolic Engineeringmentioning

confidence: 99%

“…This compound is a nontoxic, metabolically inert, competitive inhibitor to enzyme II glc of the PTS. The glucose analog addition resulted in a reduced acetate accumulation in the reactor and subsequently led to a significant enhancement in recombinant protein production (Chou et al, 1994a). Based on the results from this study, a metabolically engineered E coli strain with reduced acetate synthesis rate was constructed through the modification of glucose uptake rate (Chou et al, 1994b).…”

Section: An Example Of Metabolic Engineeringmentioning

confidence: 99%

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Genetic and metabolic engineering

Yang¹,

Bennett²,

San³

1998

Electron. J. Biotechnol.

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Recent advances in molecular biology techniques, analytical methods and mathematical tools have led to a growing interest in using metabolic engineering to redirect metabolic fluxes for industrial and medical purposes. Metabolic engineering is referred to as the directed improvement of cellular properties through the modification of specific biochemical reactions or the introduction of new ones, with the use of recombinant DNA technology (Stephanopoulos, 1999). This multidisciplinary field draws principles from chemical engineering, biochemistry, molecular and cell biology, and computational sciences. The aim of this article is to give an overview of the various strategies and tools available for metabolic engineers and to review some of the recent work that has been conducted in our laboratories in the metabolic engineering area.Metabolic engineering is generally referred to as the targeted and purposeful alteration of metabolic pathways found in an organism in order to better understand and utilize cellular pathways for chemical transformation, energy transduction, and supramolecular assembly (Lessard, 1996). This multidisciplinary field draws principles from chemical engineering, computational sciences, biochemistry, and molecular biology. In essence, metabolic engineering is the application of engineering principles of design and analysis to the metabolic pathways in order to achieve a particular goal. This goal may be to increase process productivity, as in the case in production of antibiotics, biosynthetic precursors or polymers, or to extend metabolic capability by the addition of extrinsic activities for chemical production or degradation. Previous strategies to attain these goals seem more of an art with experimentation by trial-and-error. Two papers in the early 1990s advocated the switch from this artistic approach to a more systematic and rational approach (Bailey, 1991, Stephanopoulos and Vanillo, 1991). This scientific approach would involve the use of recombinant DNA technology and a better understanding of cellular physiology to modify intermediary metabolism. Several reviews on metabolic engineering cover general

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Section: An Example Of Metabolic Engineeringmentioning

confidence: 99%

Section: An Example Of Metabolic Engineeringmentioning

confidence: 99%

Genetic and metabolic engineering

Yang¹,

Bennett²,

San³

1998

Electron. J. Biotechnol.

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“…Further, acid production was undetectable in the glucose-citrate cultures. Subsequent work showed that productivity of recombinant protein (units of protein/vol of culture-time) in glucose-citrate medium was 5-to 10-fold higher (26), further emphasizing the utility of reducing acid production.Although there have been several efforts to reduce acid formation in E. coli by metabolic engineering (6,8), there has been no report on metabolic engineering of B. subtilis. Based on measurements and theoretical analyses, we identified pyruvate kinase (PYK) as the activity most likely affected by growth in glucose-citrate medium (12,15).…”

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confidence: 99%

“…Although there have been several efforts to reduce acid formation in E. coli by metabolic engineering (6,8), there has been no report on metabolic engineering of B. subtilis. Based on measurements and theoretical analyses, we identified pyruvate kinase (PYK) as the activity most likely affected by growth in glucose-citrate medium (12,15).…”

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confidence: 99%

Characterization of Growth and Acid Formation in a Bacillus subtilis Pyruvate Kinase Mutant

Fry

Zhu

Domach

et al. 2000

Appl Environ Microbiol

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Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO 2 production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.Acid production is among the important factors that limit process stability and cell concentration and thus cell-based biotechnological processes (e.g., see references 14, 23, and 25). Numerous approaches have been used in an attempt to reduce acid formation. One mechanism involves maintaining low levels of glucose in fed batch reactors (24,27). While this can lead to increased cell mass and product formation, it is a capitalintensive method. Manipulation of the growth media might also be used to reduce acid formation relative to the amount of glucose consumed (13,24).Majewski and Domach (17) used a constrained network analysis of the main metabolic pathways in conjunction with reported measurements of enzymatic activity levels to explain acid production by bacterial cells. This analysis suggested that Escherichia coli and Bacillus subtilis have excess glycolytic capacity relative to the Krebs cycle. This idea is consistent with the notion that given all the anabolic and catabolic tasks that metabolic networks must perform, stoichiometric conflicts and other conflicts arise, leading to imperfect coordination of all tasks. It is thus an overflow or "spilling" of excess carbon that leads to acid production.In experiments using B. subtilis to test the overflow hypothesis, it was found that a small amount of citrate added to glucose minimal medium (0.1 mol of citrate/1 mol of glucose) caused the rate of glucose (or total carbon) use per cell to decline several-fold, while the maximal growth rate was not diminished (12). Further, acid production was undetectable in the glucose-citrate cultures. Subsequent work showed that productivity of recombinant protein (units o...

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“…This second potential site of flux regulation also provides a "safety valve" for checking triose pool increases in response to the PYK bottleneck. Furthermore, the elevation of glucose-6-phosphate suggests that precursor supply for NADPH production by the HMP pathway is not threatened, which could conceivably result if the PTS system were downregulated (Chou et al 1994).…”

Section: Discussionmentioning

confidence: 99%