# US 8,771,979 B2 · Process for production of biomass from a carbon source > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,771,979 B2 - Document ID: US8771979B2 - Status: Granted - Kind: B2 - Application number: 11/991,652 - Filed: 2006-09-07 - Published: 2009-05-14 - Granted: 2014-07-08 - Earliest claimed: 2005-09-09 - Assignees: Dsm IP Assets B.V. - Applicants: Masako Shinjoh - Inventors: Masako Shinjoh - Web page: https://patentgrep.com/patent/US8771979B2 ## CPC - C12N 1/20: Bacteria; Culture media therefor - C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7) - C12N 15/52: Genes encoding for enzymes or proenzymes - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to microorganisms genetically engineered to increase yield and/or efficiency of biomass production from a carbon source, such as e.g. glucose. Also included are processes of using the polynucleotides and modified polynucleotide sequences to transform host microorganisms leading to a microorganism with reduced carbon source diversion, i.e. higher yield and/or efficiency of biomass production from a carbon source such as e.g. glucose. ## Claims ### Claim 1 A process for production of biomass from a carbon source, the process comprising cultivating a Gluconobacter microorganism in an aqueous nutrient medium under conditions that allow growth on said carbon source, said Gluconobacter microorganism being genetically engineered by disruption of a gene encoding a PQQ-dependent glucose dehydrogenase having at least 90% identity to the PQQ-dependent glucose dehydrogenase encoded by SEQ ID NO: 11 and introduction of a polynucleotide comprising a nucleotide sequence encoding a gluconate kinase, wherein the nucleotide sequence is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising SEQ ID NO:2; (b) a nucleotide sequence comprising SEQ ID NO:1; (c) a nucleotide sequence obtainable by polymerase chain reaction using genomic DNA from Gluconobacter as a template and the primer set according to SEQ ID NO:3 and SEQ ID NO:4 with an annealing step at 60° C. for 30 sec; and (d) a nucleotide sequence, selected from Gluconobacter, Gluconacetobacter or Acetobacter, the complementary strand of which hybridizes under highly stringent conditions to the nucleotide sequence as defined in (a), said highly stringent conditions comprising the steps of incubation of hybridization filters for 2 h to 4 days at 42° C. in a solution comprising 50% formamide followed by washing the filters twice for 5 to 15 minutes in 2×SSC/0.1% SDS at room temperature and twice for 5 to 30 minutes in 0.1×SSC/0.1% SDS at 65 to 68° C.; wherein a yield of at least 0.2 g biomass per gram glucose is produced. ### Claim 2 A process for production of biomass from a carbon source, the process comprising cultivating a Gluconobacter microorganism genetically engineered by disruption of a gene encoding a PQQ-dependent glucose dehydrogenase having at least 90% identity to the PQQ-dependent glucose dehydrogenase encoded by SEQ ID NO: 11 and overexpressing an endogenous gene comprising a polynucleotide leading to an improved yield and/or production of biomass from a carbon source produced by said microorganism, wherein said nucleotide sequence encodes a gluconate kinase and is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising SEQ ID NO:2; (b) a nucleotide sequence comprising SEQ ID NO:1; (c) a nucleotide sequence obtainable by polymerase chain reaction using genomic DNA from Gluconobacter as a template and the primer set according to SEQ ID NO:3 and SEQ ID NO:4 with an annealing step at 60° C. for 30 sec; and (d) a nucleotide sequence, selected from Gluconobacter, Gluconacetobacter or Acetobacter, the complementary strand of which hybridizes under highly stringent conditions to the nucleotide sequence as defined in (a), said highly stringent conditions comprising the steps of incubation of hybridization filters for 2 h to 4 days at 42° C. in a solution comprising 50% formamide followed by washing the filters twice for 5 to 15 minutes in 2×SSC/0.1% SDS at room temperature and twice for 5 to 30 minutes in 0.1×SSC/0.1% SDS at 65 to 68° C.; wherein a yield of at least 0.2 g biomass per gram glucose is produced. ### Claim 3 A process for production of biomass from a carbon source, the process comprising cultivating a Gluconobacter microorganism genetically engineered by disruption of a gene comprising the nucleotide sequence according to SEQ ID NO:11 and introduction of a polynucleotide comprising a nucleotide sequence encoding a gluconate kinase, wherein the nucleotide sequence is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising SEQ ID NO:2; (b) a nucleotide sequence comprising SEQ ID NO:1; (c) a nucleotide sequence obtainable by polymerase chain reaction using genomic DNA from Gluconobacter as a template and the primer set according to SEQ ID NO:3 and SEQ ID NO:4 with an annealing step at 60° C. for 30 sec; and (d) a nucleotide sequence, selected from Gluconobacter, Gluconacetobacter or Acetobacter, the complementary strand of which hybridizes under highly stringent conditions to the nucleotide sequence as defined in (a), said highly stringent conditions comprising the steps of incubation of hybridization filters for 2 h to 4 days at 42° C. in a solution comprising 50% formamide followed by washing the filters twice for 5 to 15 minutes in 2×SSC/0.1% SDS at room temperature and twice for 5 to 30 minutes in 0.1×SSC/0.1% SDS at 65 to 68° C.; wherein a yield of at least 0.2 g biomass per gram glucose is produced. ### Claim 4 (depends on 3) The process according to claim 3, wherein said polynucleotide sequence is present in the form of a vector. ### Claim 5 (depends on 3) The process according to claim 3, wherein said microorganism is genetically altered in such a way that it leads to an improved yield and/or efficiency of biomass produced from a carbon source by said microorganism. ### Claim 6 (depends on 3) The process according to claim 3 which is capable of producing biomass from glucose in a yield of at least about 0.2 g biomass/g glucose. ### Claim 7 (depends on 6) The process according to claim 6, wherein the yield is at least about 0.3 g biomass/g glucose. ### Claim 8 (depends on 3) The process according to claim 3, wherein the polynucleotide is operatively linked to expression control sequences and transferred into the microorganism. ### Claim 9 (depends on 1) The process according to claim 1, wherein the genetically engineered microorganism has increased and/or improved gluconate kinase activity compared to the non-genetically engineered microorganism. ### Claim 10 (depends on 9) The process according to claim 9, wherein the polynucleotide is overexpressed in the genetically engineered microorganism. ### Claim 11 (depends on 1) The process according to claim 1, wherein the carbon source is selected from the group consisting of glucose, sucrose, maltose, starch, cellulose, cellobiose, lactose, isomaltose, dextran, trehalose, and mixtures thereof. ### Claim 12 (depends on 2) The process according to claim 2, wherein the carbon source is selected from the group consisting of glucose, sucrose, maltose, starch, cellulose, cellobiose, lactose, isomaltose, dextran, trehalose, and mixtures thereof. ### Claim 13 (depends on 3) The process according to claim 3, wherein the carbon source is selected from the group consisting of glucose, sucrose, maltose, starch, cellulose, cellobiose, lactose, isomaltose, dextran, trehalose, and mixtures thereof. ### Claim 14 (depends on 1) The process according to claim 1, wherein the microorganism is selected from the group consisting of Gluconobacter frateurii, Gluconobacter cerinus, Gluconobacter thailandicus, and Gluconobacter oxydans. ### Claim 15 (depends on 1) The process according to claim 1, wherein the microorganism to be genetically engineered is Gluconobacter oxydans DSM 17078. ### Claim 16 (depends on 2) The process according to claim 2 which is capable of producing biomass from glucose in a yield of at least about 0.2 g biomass/g glucose.