Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production
Abstract Background We previously developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain’s performance has been optimized during growth with glucose. However, lignocellulosic biomass...
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| Format: | Article |
| Language: | English |
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BMC
2018-01-01
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| Series: | Microbial Cell Factories |
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| Online Access: | http://link.springer.com/article/10.1186/s12934-018-0862-6 |
| _version_ | 1811303977454665728 |
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| author | Xi Wang Ee-Been Goh Harry R. Beller |
| author_facet | Xi Wang Ee-Been Goh Harry R. Beller |
| author_sort | Xi Wang |
| collection | DOAJ |
| description | Abstract Background We previously developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain’s performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. Results In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our most efficient methyl ketone-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose–xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. Conclusions This work demonstrated a strategy for engineering simultaneous utilization of C6 and C5 sugars in E. coli without sacrificing production of fatty acid-derived compounds. |
| first_indexed | 2024-04-13T07:58:46Z |
| format | Article |
| id | doaj.art-24ee70dc773a4f62a08982ea942aa0cd |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-13T07:58:46Z |
| publishDate | 2018-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-24ee70dc773a4f62a08982ea942aa0cd2022-12-22T02:55:20ZengBMCMicrobial Cell Factories1475-28592018-01-0117111110.1186/s12934-018-0862-6Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone productionXi Wang0Ee-Been Goh1Harry R. Beller2Joint BioEnergy Institute (JBEI)Joint BioEnergy Institute (JBEI)Joint BioEnergy Institute (JBEI)Abstract Background We previously developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain’s performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. Results In this study, genetic manipulations were performed to alleviate carbon catabolite repression in our most efficient methyl ketone-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose–xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks. Conclusions This work demonstrated a strategy for engineering simultaneous utilization of C6 and C5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.http://link.springer.com/article/10.1186/s12934-018-0862-6Carbon catabolite repressionMethyl ketonesNADPHcAMP receptor proteinMetabolic engineering |
| spellingShingle | Xi Wang Ee-Been Goh Harry R. Beller Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production Microbial Cell Factories Carbon catabolite repression Methyl ketones NADPH cAMP receptor protein Metabolic engineering |
| title | Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production |
| title_full | Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production |
| title_fullStr | Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production |
| title_full_unstemmed | Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production |
| title_short | Engineering E. coli for simultaneous glucose–xylose utilization during methyl ketone production |
| title_sort | engineering e coli for simultaneous glucose xylose utilization during methyl ketone production |
| topic | Carbon catabolite repression Methyl ketones NADPH cAMP receptor protein Metabolic engineering |
| url | http://link.springer.com/article/10.1186/s12934-018-0862-6 |
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