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Carbon catabolite repression in Thermoanaerobacterium saccharolyticum.

Tsakraklides V, Shaw AJ, Miller BB, Hogsett DA, Herring CD - Biotechnol Biofuels (2012)

Bottom Line: We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum.Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mascoma Corporation, 67 Etna Road, Suite 300, New Hampshire, 03766, Lebanon. cherring@mascoma.com.

ABSTRACT

Background: The thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.

Results: We report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.

Conclusion: Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

No MeSH data available.


Related in: MedlinePlus

T. saccharolyticum CCR is relieved by HPr His15Asp. Cultures of strains M2476 (HPr, solid lines) and M2907 (HPr His15Asp, dashed lines) supplemented with the indicated sugars were grown in the absence (black lines) or presence (red lines) of 5 g/L 2-deoxyglucose.
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Figure 2: T. saccharolyticum CCR is relieved by HPr His15Asp. Cultures of strains M2476 (HPr, solid lines) and M2907 (HPr His15Asp, dashed lines) supplemented with the indicated sugars were grown in the absence (black lines) or presence (red lines) of 5 g/L 2-deoxyglucose.

Mentions: A BLAST search of the T. saccharolyticum genome with B. subtilis CCR genes showed significant matches for HPr (E-value: 3 × 10-17), HPrK (1 × 10-85), Crh (9 × 10-24) and CcpA (16 matches with E-values: 3x10-19-1 × 10-85) suggesting that T. saccharolyticum uses the same elements of CCR as B. subtilis. To study the role of HPr in T. saccharolyticum CCR, we replaced the endogenous HPr gene in strain M2476 with a modified copy encoding the histidine phosphomimetic HPr His15Asp to create strain M2907. We reasoned that if HPr mediates PTS import and CCR, the mutant strain would be compromised for PTS sugar import and exhibit resistance to CCR. We evaluated growth of strains M2476 and M2907 on glucose, xylose, mannose, galactose, arabinose or cellobiose as the primary carbon source. Growth of the mutant strain M2907 in all sugars tested (Figure 2, dashed black lines) indicates that these sugars can be imported independently of HPr as a PTS phosphate donor. M2907 grew more slowly than M2476 on glucose, mannose and cellobiose, likely due to reduced PTS function in the mutant strain. M2907 grew faster than its parent on xylose, galactose, arabinose and cellobiose in the presence of 2-deoxyglucose (Figure 2, red lines) suggesting that CCR of these sugars is relieved by the HPr His15Asp mutation. While other ethanologen strains of T. saccharolyticum grow well on galactose, M2476 does not, which was confirmed in bottle cultures. The HPr His15Asp mutation slightly improved growth in galactose.


Carbon catabolite repression in Thermoanaerobacterium saccharolyticum.

Tsakraklides V, Shaw AJ, Miller BB, Hogsett DA, Herring CD - Biotechnol Biofuels (2012)

T. saccharolyticum CCR is relieved by HPr His15Asp. Cultures of strains M2476 (HPr, solid lines) and M2907 (HPr His15Asp, dashed lines) supplemented with the indicated sugars were grown in the absence (black lines) or presence (red lines) of 5 g/L 2-deoxyglucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526391&req=5

Figure 2: T. saccharolyticum CCR is relieved by HPr His15Asp. Cultures of strains M2476 (HPr, solid lines) and M2907 (HPr His15Asp, dashed lines) supplemented with the indicated sugars were grown in the absence (black lines) or presence (red lines) of 5 g/L 2-deoxyglucose.
Mentions: A BLAST search of the T. saccharolyticum genome with B. subtilis CCR genes showed significant matches for HPr (E-value: 3 × 10-17), HPrK (1 × 10-85), Crh (9 × 10-24) and CcpA (16 matches with E-values: 3x10-19-1 × 10-85) suggesting that T. saccharolyticum uses the same elements of CCR as B. subtilis. To study the role of HPr in T. saccharolyticum CCR, we replaced the endogenous HPr gene in strain M2476 with a modified copy encoding the histidine phosphomimetic HPr His15Asp to create strain M2907. We reasoned that if HPr mediates PTS import and CCR, the mutant strain would be compromised for PTS sugar import and exhibit resistance to CCR. We evaluated growth of strains M2476 and M2907 on glucose, xylose, mannose, galactose, arabinose or cellobiose as the primary carbon source. Growth of the mutant strain M2907 in all sugars tested (Figure 2, dashed black lines) indicates that these sugars can be imported independently of HPr as a PTS phosphate donor. M2907 grew more slowly than M2476 on glucose, mannose and cellobiose, likely due to reduced PTS function in the mutant strain. M2907 grew faster than its parent on xylose, galactose, arabinose and cellobiose in the presence of 2-deoxyglucose (Figure 2, red lines) suggesting that CCR of these sugars is relieved by the HPr His15Asp mutation. While other ethanologen strains of T. saccharolyticum grow well on galactose, M2476 does not, which was confirmed in bottle cultures. The HPr His15Asp mutation slightly improved growth in galactose.

Bottom Line: We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum.Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mascoma Corporation, 67 Etna Road, Suite 300, New Hampshire, 03766, Lebanon. cherring@mascoma.com.

ABSTRACT

Background: The thermophilic anaerobe Thermoanaerobacterium saccharolyticum is capable of directly fermenting xylan and the biomass-derived sugars glucose, cellobiose, xylose, mannose, galactose and arabinose. It has been metabolically engineered and developed as a biocatalyst for the production of ethanol.

Results: We report the initial characterization of the carbon catabolite repression system in this organism. We find that sugar metabolism in T. saccharolyticum is regulated by histidine-containing protein HPr. We describe a mutation in HPr, His15Asp, that leads to derepression of less-favored carbon source utilization.

Conclusion: Co-utilization of sugars can be achieved by mutation of HPr in T. saccharolyticum. Further manipulation of CCR in this organism will be instrumental in achieving complete and rapid conversion of all available sugars to ethanol.

No MeSH data available.


Related in: MedlinePlus