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Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an alpha-Gal epitope.

Ruffing AM, Chen RR - Microb. Cell Fact. (2010)

Bottom Line: The difficulty associated with synthesizing the alpha-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the alpha-Gal epitope.Agrobacterium sp.Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332-0100, USA.

ABSTRACT

Background: Oligosaccharides containing a terminal Gal-alpha1,3-Gal moiety are collectively known as alpha-Gal epitopes. alpha-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the alpha-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the alpha-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the alpha-Gal epitope, Gal-alpha1,3-Lac.

Results: Agrobacterium sp. ATCC 31749 was engineered to produce Gal-alpha1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:alpha1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the alpha-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting alpha-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-alpha1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-alpha1,3-Lac.

Conclusions: The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of alpha-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the alpha-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.

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Comparison of Gal-α1,3-Lac synthesis by ATCC 31749/pBQET and ATCC 31749/pBQETY. Synthesis of Gal-α1,3-Lac after 150 hours by ATCC 31749/pBQET and ATCC 31749/pBQETY without and with rifampicin. Data are averages of three independent experiments with the standard deviation indicated by error bars.
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Figure 4: Comparison of Gal-α1,3-Lac synthesis by ATCC 31749/pBQET and ATCC 31749/pBQETY. Synthesis of Gal-α1,3-Lac after 150 hours by ATCC 31749/pBQET and ATCC 31749/pBQETY without and with rifampicin. Data are averages of three independent experiments with the standard deviation indicated by error bars.

Mentions: ATCC 31749/pBQETY was employed to determine the effect of increased lactose availability on α-Gal epitope synthesis. The LacY-expressing strain synthesized 0.65 g/L of Gal-α1,3-Lac, a 67% improvement over the initial engineered strain, ATCC 31749/pBQET (Figure 4). Surprisingly, the activity of the GalE:α1,3-GalT fusion enzyme was 4.7-fold lower in ATCC 31749/pBQETY compared to ATCC 31749/pBQET. The lower activity in the LacY-expressing strain is presumably due to lower expression of the fusion enzyme. Unlike ATCC 31749/pBQET, the expression exhibited weak dependence on IPTG concentration (Table 1). To determine if the lower fusion enzyme activity of ATCC 31749/pBQETY restricts Gal-α1,3-Lac production, 0.05 mM of IPTG was used for α-Gal epitope synthesis, as both ATCC 31749/pBQET and ATCC 31749/pBQETY have similar fusion enzyme activities at this IPTG concentration. The amount of Gal-α1,3-Lac synthesized by the LacY-expressing strain was similar to that produced with 1 mM IPTG; this was expected as the enzyme activity levels are very similar at both IPTG concentrations. Unexpectedly, the amount of Gal-α1,3-Lac produced by ATCC 31749/pBQET was similar for both 0.05 mM and 1 mM IPTG despite over a 5-fold reduction in fusion enzyme activity (Table 1). This suggests that the activity of the fusion enzyme is not a limiting factor in Gal-α1,3-Lac synthesis in the wild type background.


Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an alpha-Gal epitope.

Ruffing AM, Chen RR - Microb. Cell Fact. (2010)

Comparison of Gal-α1,3-Lac synthesis by ATCC 31749/pBQET and ATCC 31749/pBQETY. Synthesis of Gal-α1,3-Lac after 150 hours by ATCC 31749/pBQET and ATCC 31749/pBQETY without and with rifampicin. Data are averages of three independent experiments with the standard deviation indicated by error bars.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818619&req=5

Figure 4: Comparison of Gal-α1,3-Lac synthesis by ATCC 31749/pBQET and ATCC 31749/pBQETY. Synthesis of Gal-α1,3-Lac after 150 hours by ATCC 31749/pBQET and ATCC 31749/pBQETY without and with rifampicin. Data are averages of three independent experiments with the standard deviation indicated by error bars.
Mentions: ATCC 31749/pBQETY was employed to determine the effect of increased lactose availability on α-Gal epitope synthesis. The LacY-expressing strain synthesized 0.65 g/L of Gal-α1,3-Lac, a 67% improvement over the initial engineered strain, ATCC 31749/pBQET (Figure 4). Surprisingly, the activity of the GalE:α1,3-GalT fusion enzyme was 4.7-fold lower in ATCC 31749/pBQETY compared to ATCC 31749/pBQET. The lower activity in the LacY-expressing strain is presumably due to lower expression of the fusion enzyme. Unlike ATCC 31749/pBQET, the expression exhibited weak dependence on IPTG concentration (Table 1). To determine if the lower fusion enzyme activity of ATCC 31749/pBQETY restricts Gal-α1,3-Lac production, 0.05 mM of IPTG was used for α-Gal epitope synthesis, as both ATCC 31749/pBQET and ATCC 31749/pBQETY have similar fusion enzyme activities at this IPTG concentration. The amount of Gal-α1,3-Lac synthesized by the LacY-expressing strain was similar to that produced with 1 mM IPTG; this was expected as the enzyme activity levels are very similar at both IPTG concentrations. Unexpectedly, the amount of Gal-α1,3-Lac produced by ATCC 31749/pBQET was similar for both 0.05 mM and 1 mM IPTG despite over a 5-fold reduction in fusion enzyme activity (Table 1). This suggests that the activity of the fusion enzyme is not a limiting factor in Gal-α1,3-Lac synthesis in the wild type background.

Bottom Line: The difficulty associated with synthesizing the alpha-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the alpha-Gal epitope.Agrobacterium sp.Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332-0100, USA.

ABSTRACT

Background: Oligosaccharides containing a terminal Gal-alpha1,3-Gal moiety are collectively known as alpha-Gal epitopes. alpha-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the alpha-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the alpha-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the alpha-Gal epitope, Gal-alpha1,3-Lac.

Results: Agrobacterium sp. ATCC 31749 was engineered to produce Gal-alpha1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:alpha1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the alpha-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting alpha-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-alpha1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-alpha1,3-Lac.

Conclusions: The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of alpha-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the alpha-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.

Show MeSH
Related in: MedlinePlus