Synthetic methylotrophs

• US 10,640,746 B2
• Filed: 01/13/2017
• Issued: 05/05/2020
• Est. Priority Date: 01/15/2016
• Status: Active Grant

• Current Assignee: University of Delaware
• Original Assignee: University of Delaware
• Inventors: Papoutsakis, Eleftherios T., Whitaker, William Brian, Bennett, Robert Kyle
• Primary Examiner(s): Raghu, Ganapathirama
• Application Number:
  US16/070,318
• Publication Number:
  US 20190024040A1
• Time in Patent Office: 1,208 Days
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 1/32 Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12R 2001/15 Corynebacterium
• CPC Class Codes: C12R 2001/19 Escherichia coli
• CPC Class Codes: C12Y 101/01244 Methanol dehydrogenase (1.1.1.244)

Abstract

The present invention provides a non-naturally occurring microbe capable of growing in a medium comprising methanol, comprising a heterologous polynucleotide encoding a heterologous methanol dehydrogenase (MDH) derived from a Corynebacterium organism (Cor), wherein the MDH is expressed in the microbe, and wherein the MDH exhibits a Km of no more than 3 mM for methanol. Also provided are uses of the non-naturally occurring microbe for oxidizing methanol and producing a metabolite as well as the preparation of the non-naturally occurring microbe.

Claims

1. A non-naturally occurring microbe capable of growing in a medium comprising methanol, comprising a heterologous polynucleotide encoding a heterologous methanol dehydrogenase (MDH) from a Corynebacterium organism (Cor), wherein the microbe expresses the heterologous MDH and one or more heterologous ribulose monophosphate (RuMP) pathway enzymes, wherein the heterologous MDH consists of the amino acid sequence of SEQ ID NO:

1, wherein the one or more RuMP pathway enzymes comprise heterologous 3-hexulose-6-phosphate synthase (HPS) and heterologous 3-hexulose-6-phosphate isomerase (PHI), and wherein the non-naturally occurring microbe is E. coli.

2. The non-naturally occurring microbe of claim 1, wherein the Cor is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium sp., Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium lubricantis, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium casei, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium sp. HMSC064E07, Corynebacterium lactis, Corynebacterium sp. HMSC077G07, Corynebacterium sp. HMSC074C05, Corynebacterium humireducens, Corynebacterium resistens, Corynebacterium vitaeruminis, Corynebacterium durum, Corynebacterium ulcerans, Corynebacterium ulcerans FRC11, Corynebacterium ulcerans NCTC 12077, Corynebacterium ulcerans FRC58, Corynebacterium efficiens, Corynebacterium ulcerans 0102, Corynebacterium terpenotabidum, Corynebacterium lipophiloflavum, Corynebacterium diphtheria, Corynebacterium sp. HMSC034A01, Corynebacterium sp. HMSC034B08, Corynebacterium sp. HMSC05H05, Corynebacterium mustelae, Corynebacterium sp. HMSC04H06, Corynebacterium pseudotuberculosis, Corynebacterium sp. HMSC070H05, Corynebacterium sp. HMSC29G08, Corynebacterium sp. HMSC11D10, Corynebacterium kutscheri, Corynebacterium freiburgense, Corynebacterium pseudotuberculosis FRC41, Corynebacterium sp. HMSC067D03, Corynebacterium sp. HMSC036E10, Corynebacterium jeddahense, Corynebacterium ciconiae, Corynebacterium coyleae, Corynebacterium sputi, Corynebacterium tuscaniense, Corynebacterium sp. HMSC074A01, Corynebacterium variabile, Corynebacterium nuruki, Corynebacterium testudinoris, Corynebacterium striatum, Corynebacterium pseudodiphtheriticum, Corynebacterium sp. EPI-003-04-2554_SCH2473622, Corynebacterium sp. HMSC06C06, Corynebacterium sp. SN15, Corynebacterium propinquum, Corynebacterium sp. KPL1818, Corynebacterium sp. KPL1824, Corynebacterium timonense, Corynebacterium sp. KPL1824, Cryobacterium flavum, Cryobacterium luteum, Cryobacterium levicorallinum, Arthrobacter sp. 162MFSha1.1, Rothia sp. ND6WE1A, and Arthrobacter sp. Soil761.

3. The non-naturally occurring microbe of claim 1, wherein the Cor is a Corynebacterium glutamicum (Cgl).

4. The non-naturally occurring microbe of claim 1, wherein the methanol contributes to at least 40% of the carbon source for the non-naturally occurring microbe.

5. The non-naturally occurring microbe of claim 1, wherein the expression of the one or more RuMP pathway enzymes is under control of a formaldehyde responsive promoter.

6. The non-naturally occurring microbe of claim 1, further expressing one or more heterologous pentose-phosphate pathway (PPP) enzymes.

7. The non-naturally occurring microbe of claim 6, wherein the expression of the one or more heterologous PPP enzymes is under control of a formaldehyde responsive promoter.

8. The non-naturally occurring microbe of claim 6, wherein the one or more heterologous PPP enzymes comprise heterologous phosphofructokinase (PFK), heterologous fructose bisphosphate aldolase (FBA), heterologous transketolase (TKT), heterologous fructose/sedoheptulose biphosphatase (GLPX), heterologous transaldolase (TAL), heterologous ribose-5-phosphate isomerase (RPI) and heterologous ribulose phosphate epimerase (RPE).

9. The non-naturally occurring microbe of claim 1, wherein the non-naturally occurring microbe comprises a deletion of a frmRAB operon.

10. A method for oxidizing methanol, comprising growing the non-naturally occurring microbe of claim 1 in a medium comprising methanol, whereby the methanol is oxidized.

11. The method of claim 10, further comprising producing a metabolite.

12. The method of claim 11, wherein the metabolite is n-butanol.

13. The method of claim 11, wherein the metabolite is selected from the group consisting of 4-carbon chemicals, diacids, 3-carbon chemicals, higher carboxylic acids, alcohols of higher carboxylic acids, polyhydroxyalkanoates, and specialty chemicals.

14. The method of claim 13, wherein the 4-carbon chemicals are selected from the group consisting of butyrate, n-butanol, i-butanol, 2-butanol, 2,3-butanediol, and 1,4-butanediol.

15. The method of claim 13, wherein the diacids are selected from the group consisting of oxalic, malonic, succinic, glutaric, adipic, pimelic, phthalic, isophthalic, and terephthalic.

16. The method of claim 13, wherein the 3-carbon chemicals are selected from the group consisting of propanol, propanediol, lactate, and acrylate.

17. The method of claim 13, wherein the higher carboxylic acids are selected from the group consisting of pentanoic acids and hexanoic acids.

18. The method of claim 13, wherein the specialty chemicals are selected artemisinin, vanillin, anthocyanins and resveratrol.