Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol
Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the ef...
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description | Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to D-lactic acid (D-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for D-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of D-lactate from pyruvate (D-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic D-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of D-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of D-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for D-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of D-lactate and is redox balanced, thus representing a viable metabolic pathway. |
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We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to D-lactic acid (D-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for D-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of D-lactate from pyruvate (D-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic D-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of D-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of D-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for D-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of D-lactate and is redox balanced, thus representing a viable metabolic pathway.</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>EISSN: 1098-6596</identifier><identifier>DOI: 10.1128/AEM.00664-10</identifier><identifier>PMID: 20472739</identifier><identifier>CODEN: AEMIDF</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Alcohol ; Anaerobiosis ; ATP ; Biotechnology ; Biotechnology - methods ; Chemical synthesis ; Culture Media ; E coli ; Enzymes ; Escherichia coli ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Escherichia coli - growth & development ; Escherichia coli - metabolism ; Escherichia coli Proteins - genetics ; Escherichia coli Proteins - metabolism ; Fermentation ; Genetic Engineering - methods ; Glycerol - metabolism ; Lactic Acid - biosynthesis ; Metabolic Networks and Pathways ; Metabolism ; Raw materials</subject><ispartof>Applied and Environmental Microbiology, 2010-07, Vol.76 (13), p.4327-4336</ispartof><rights>Copyright American Society for Microbiology Jul 2010</rights><rights>Copyright © 2010, American Society for Microbiology 2010</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c525t-154b9f37e51bf9f7a61e52b3b11d42fbe867fd672e9fe1497be27afe465d03ee3</citedby><cites>FETCH-LOGICAL-c525t-154b9f37e51bf9f7a61e52b3b11d42fbe867fd672e9fe1497be27afe465d03ee3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897450/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897450/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,315,728,781,785,886,3189,3190,27929,27930,53796,53798</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20472739$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Mazumdar, Suman</creatorcontrib><creatorcontrib>Clomburg, James M</creatorcontrib><creatorcontrib>Gonzalez, Ramon</creatorcontrib><title>Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol</title><title>Applied and Environmental Microbiology</title><addtitle>Appl Environ Microbiol</addtitle><description>Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to D-lactic acid (D-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for D-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of D-lactate from pyruvate (D-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic D-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of D-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of D-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for D-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of D-lactate and is redox balanced, thus representing a viable metabolic pathway.</description><subject>Alcohol</subject><subject>Anaerobiosis</subject><subject>ATP</subject><subject>Biotechnology</subject><subject>Biotechnology - methods</subject><subject>Chemical synthesis</subject><subject>Culture Media</subject><subject>E coli</subject><subject>Enzymes</subject><subject>Escherichia coli</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - growth & development</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Fermentation</subject><subject>Genetic Engineering - methods</subject><subject>Glycerol - metabolism</subject><subject>Lactic Acid - biosynthesis</subject><subject>Metabolic Networks and Pathways</subject><subject>Metabolism</subject><subject>Raw materials</subject><issn>0099-2240</issn><issn>1098-5336</issn><issn>1098-6596</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1v1DAQxS0EosvCjTNEXLiQ4m_HF6RVWVqkRSCVni3HGW9cJXGxk6L-97hsKR8nTtbYv3kzfg-h5wQfE0Kbt5vtp2OMpeQ1wQ_QimDd1IIx-RCtMNa6ppTjI_Qk50uMMceyeYyOKOaKKqZXqN1m10MKrg-2cnEI1fmcbJhytZ32YQJI0FU-puosjtFDGmGa7RyuofqSYre4OcSpir56X-9sKVy1caE0pDhWp8ONgxSHp-iRt0OGZ3fnGl182H49Oat3n08_nmx2tRNUzDURvNWeKRCk9dorKwkI2rKWkI5T30Ijle-koqA9EK5VC1RZD1yKDjMAtkbvDrpXSztC58qmyQ7mKoXRphsTbTB_v0yhN_t4bWijFRe4CLy-E0jx2wJ5NmPIDobBThCXbJTgjdTFu_8hBSte60K--oe8jEuaig9GUKWbQt5Cbw6QSzHnBP5-aYLNbcimhGx-hlxuCv7iz4_ew79S_T20D_v-e0hgbB6NhdEoaQgznBVsjV4eIG-jsfsUsrk4p5gwTBqhqcbsB1ztt3U</recordid><startdate>20100701</startdate><enddate>20100701</enddate><creator>Mazumdar, Suman</creator><creator>Clomburg, James M</creator><creator>Gonzalez, Ramon</creator><general>American Society for Microbiology</general><general>American Society for Microbiology (ASM)</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7SN</scope><scope>7SS</scope><scope>7ST</scope><scope>7T7</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>SOI</scope><scope>5PM</scope></search><sort><creationdate>20100701</creationdate><title>Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol</title><author>Mazumdar, Suman ; Clomburg, James M ; Gonzalez, Ramon</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c525t-154b9f37e51bf9f7a61e52b3b11d42fbe867fd672e9fe1497be27afe465d03ee3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Alcohol</topic><topic>Anaerobiosis</topic><topic>ATP</topic><topic>Biotechnology</topic><topic>Biotechnology - methods</topic><topic>Chemical synthesis</topic><topic>Culture Media</topic><topic>E coli</topic><topic>Enzymes</topic><topic>Escherichia coli</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - growth & development</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Fermentation</topic><topic>Genetic Engineering - methods</topic><topic>Glycerol - metabolism</topic><topic>Lactic Acid - biosynthesis</topic><topic>Metabolic Networks and Pathways</topic><topic>Metabolism</topic><topic>Raw materials</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Mazumdar, Suman</creatorcontrib><creatorcontrib>Clomburg, James M</creatorcontrib><creatorcontrib>Gonzalez, Ramon</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Environment Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>Environment Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Applied and Environmental Microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Mazumdar, Suman</au><au>Clomburg, James M</au><au>Gonzalez, Ramon</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol</atitle><jtitle>Applied and Environmental Microbiology</jtitle><addtitle>Appl Environ Microbiol</addtitle><date>2010-07-01</date><risdate>2010</risdate><volume>76</volume><issue>13</issue><spage>4327</spage><epage>4336</epage><pages>4327-4336</pages><issn>0099-2240</issn><eissn>1098-5336</eissn><eissn>1098-6596</eissn><coden>AEMIDF</coden><abstract>Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to D-lactic acid (D-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for D-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of D-lactate from pyruvate (D-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic D-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of D-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of D-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for D-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of D-lactate and is redox balanced, thus representing a viable metabolic pathway.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>20472739</pmid><doi>10.1128/AEM.00664-10</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Alcohol Anaerobiosis ATP Biotechnology Biotechnology - methods Chemical synthesis Culture Media E coli Enzymes Escherichia coli Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli - growth & development Escherichia coli - metabolism Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Fermentation Genetic Engineering - methods Glycerol - metabolism Lactic Acid - biosynthesis Metabolic Networks and Pathways Metabolism Raw materials |
title | Escherichia coli Strains Engineered for Homofermentative Production of D-Lactic Acid from Glycerol |
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