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Probing the heterologous metabolism supporting 6-deoxyerythronolide B biosynthesis in Escherichia coli.

Zhang H, Wang Y, Boghigian B, Pfeifer BA - Microb Biotechnol (2009)

Bottom Line: More specifically, the heterologous proteins responsible for 6dEB production were varied by adjusting their respective gene dosage levels.In this way, heterologous components required for posttranslational modification, 6dEB biosynthesis, and substrate provision were adjusted in expression levels to observe the relative effect each has on final heterologous biosynthesis.The results indicate that both the biosynthetic and substrate provision heterologous proteins impact 6dEB formation to a greater extent when compared with posttranslational modification and suggest these components for future protein and metabolic engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Tufts University, Medford, MA 02155, USA.

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Related in: MedlinePlus

SDS‐PAGE analysis of Sfp, PCC and DEBS through different combinations of E. coli strains and plasmids. The size and location of the Sfp, DEBS and PCC enzymes have been denoted by black‐filled dots within the molecular weight marker notations. The Sfp, PCC and DEBS labels above the indicated lanes represent samples exhibiting enhanced protein levels as a result of enhanced gene dosage. Culture conditions were identical to those described for 6dEB product formation (Fig. 2). Post culture, cells were washed and resuspended in TE buffer. Cell densities were equalized between samples and sonication was performed with a Fisher Scientific Sonic Dismembrator Model 100 at maximum setting for a tip probe for three 10 s intervals. Lysates were then centrifuged and the whole cell lysate and soluble fractions were analysed by SDS‐PAGE. Only the soluble fraction is presented. The reagents and chemicals used in this study were purchased from Fisher Scientific and Sigma. Below the gel is a semi‐quantitative densitometry analysis of protein level differences assessed using ImageJ software version 1.40g (http://rsb.info.nih.gov/ij/). The analysis provides the ratio of densitometry measured band densities between respective Sfp, PCC and DEBS strain pairs (as defined in Table 2); n = 3 and numbers in parentheses are standard deviations.
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f1: SDS‐PAGE analysis of Sfp, PCC and DEBS through different combinations of E. coli strains and plasmids. The size and location of the Sfp, DEBS and PCC enzymes have been denoted by black‐filled dots within the molecular weight marker notations. The Sfp, PCC and DEBS labels above the indicated lanes represent samples exhibiting enhanced protein levels as a result of enhanced gene dosage. Culture conditions were identical to those described for 6dEB product formation (Fig. 2). Post culture, cells were washed and resuspended in TE buffer. Cell densities were equalized between samples and sonication was performed with a Fisher Scientific Sonic Dismembrator Model 100 at maximum setting for a tip probe for three 10 s intervals. Lysates were then centrifuged and the whole cell lysate and soluble fractions were analysed by SDS‐PAGE. Only the soluble fraction is presented. The reagents and chemicals used in this study were purchased from Fisher Scientific and Sigma. Below the gel is a semi‐quantitative densitometry analysis of protein level differences assessed using ImageJ software version 1.40g (http://rsb.info.nih.gov/ij/). The analysis provides the ratio of densitometry measured band densities between respective Sfp, PCC and DEBS strain pairs (as defined in Table 2); n = 3 and numbers in parentheses are standard deviations.

Mentions: To verify gene expression design, the strains used to vary heterologous gene dosage were first analysed by SDS‐PAGE. As seen in Fig. 1, protein levels correlate with gene dosage design. For example, the DEBS genes are observed when expressed from the pET expression plasmids but are not observed when expressed from low copy number within the chromosome. Figure 1 shows the same trend for the PCC and Sfp enzyme levels. Densitometry analysis was further used to assess and confirm expression differences within the SDS‐PAGE data.


Probing the heterologous metabolism supporting 6-deoxyerythronolide B biosynthesis in Escherichia coli.

Zhang H, Wang Y, Boghigian B, Pfeifer BA - Microb Biotechnol (2009)

SDS‐PAGE analysis of Sfp, PCC and DEBS through different combinations of E. coli strains and plasmids. The size and location of the Sfp, DEBS and PCC enzymes have been denoted by black‐filled dots within the molecular weight marker notations. The Sfp, PCC and DEBS labels above the indicated lanes represent samples exhibiting enhanced protein levels as a result of enhanced gene dosage. Culture conditions were identical to those described for 6dEB product formation (Fig. 2). Post culture, cells were washed and resuspended in TE buffer. Cell densities were equalized between samples and sonication was performed with a Fisher Scientific Sonic Dismembrator Model 100 at maximum setting for a tip probe for three 10 s intervals. Lysates were then centrifuged and the whole cell lysate and soluble fractions were analysed by SDS‐PAGE. Only the soluble fraction is presented. The reagents and chemicals used in this study were purchased from Fisher Scientific and Sigma. Below the gel is a semi‐quantitative densitometry analysis of protein level differences assessed using ImageJ software version 1.40g (http://rsb.info.nih.gov/ij/). The analysis provides the ratio of densitometry measured band densities between respective Sfp, PCC and DEBS strain pairs (as defined in Table 2); n = 3 and numbers in parentheses are standard deviations.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815759&req=5

f1: SDS‐PAGE analysis of Sfp, PCC and DEBS through different combinations of E. coli strains and plasmids. The size and location of the Sfp, DEBS and PCC enzymes have been denoted by black‐filled dots within the molecular weight marker notations. The Sfp, PCC and DEBS labels above the indicated lanes represent samples exhibiting enhanced protein levels as a result of enhanced gene dosage. Culture conditions were identical to those described for 6dEB product formation (Fig. 2). Post culture, cells were washed and resuspended in TE buffer. Cell densities were equalized between samples and sonication was performed with a Fisher Scientific Sonic Dismembrator Model 100 at maximum setting for a tip probe for three 10 s intervals. Lysates were then centrifuged and the whole cell lysate and soluble fractions were analysed by SDS‐PAGE. Only the soluble fraction is presented. The reagents and chemicals used in this study were purchased from Fisher Scientific and Sigma. Below the gel is a semi‐quantitative densitometry analysis of protein level differences assessed using ImageJ software version 1.40g (http://rsb.info.nih.gov/ij/). The analysis provides the ratio of densitometry measured band densities between respective Sfp, PCC and DEBS strain pairs (as defined in Table 2); n = 3 and numbers in parentheses are standard deviations.
Mentions: To verify gene expression design, the strains used to vary heterologous gene dosage were first analysed by SDS‐PAGE. As seen in Fig. 1, protein levels correlate with gene dosage design. For example, the DEBS genes are observed when expressed from the pET expression plasmids but are not observed when expressed from low copy number within the chromosome. Figure 1 shows the same trend for the PCC and Sfp enzyme levels. Densitometry analysis was further used to assess and confirm expression differences within the SDS‐PAGE data.

Bottom Line: More specifically, the heterologous proteins responsible for 6dEB production were varied by adjusting their respective gene dosage levels.In this way, heterologous components required for posttranslational modification, 6dEB biosynthesis, and substrate provision were adjusted in expression levels to observe the relative effect each has on final heterologous biosynthesis.The results indicate that both the biosynthetic and substrate provision heterologous proteins impact 6dEB formation to a greater extent when compared with posttranslational modification and suggest these components for future protein and metabolic engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Tufts University, Medford, MA 02155, USA.

Show MeSH
Related in: MedlinePlus