Methanol dehydrogenase fusion proteins
Described herein are fusion proteins including methanol dehydrogenase (MeDH) and at least one other polypeptide such as 3-hexulose-6-phosphate dehydrogenase (HPS) or 6-phospho-3-hexuloisomerase (PHI), such as DHAS synthase or fructose-6-Phosphate aldolase or such as DHA synthase or DHA kinase. In a localized manner, the fusion protein can promote the conversion of methanol to formaldehyde and then to a ketose phosphate such as hexulose 6-phosphate or then to DHA and G3P. When expressed in cells, the fusion proteins can promote methanol uptake and rapid conversion to the ketose phosphate or to the DHA and D3P, which in turn can be used in a pathway for the production of a desired bioproduct. Beneficially, the rapid conversion to the ketose phosphate or to the DHA and G3P can avoid the undesirable accumulation of formaldehyde in the cell. Also described are engineered cells expressing the fusion protein, optionally include one or more additional metabolic pathway transgene(s), methanol metabolic pathway genes, target product pathway genes, cell culture compositions including the cells, methods for promoting production of the target product or intermediate thereof from the cells, compositions including the target product or intermediate, and products made from the target product or intermediate.
1 . A fusion protein which converts methanol to a ketose phosphate, the fusion protein comprising:
(1a) a first region comprising methanol dehydrogenase or an enzymatically active portion thereof, and (1b) a second region comprising 3-hexulose-6-phosphate dehydrogenase or an enzymatically active portion thereof;
wherein the fusion protein comprises one or more linker amino acid sequence(s) positioned between two or more of the first and second regions of the fusion protein and
wherein any of the methanol dehydrogenase, or the 3-hexulose-6-phosphate dehydrogenase is a bacterial sequence,
and
wherein the fusion protein has a structure as follows:
1a-L 1 -1b or 1b-L 1 -1a;
wherein L 1 is selected from (-) (a covalent bond) and a linker amino acid sequence.
2 . The fusion protein of claim 1 , wherein
one or more of the methanol dehydrogenase, or the 3-hexulose-6-phosphate dehydrogenase or enzymatically active portions thereof are from Bacillus sequences.
3 . The fusion protein of claim 1 wherein the methanol dehydrogenase (MeDH) or an enzymatically active portion thereof is from Bacillus methanolicus MGA3 MeDH (SEQ ID NO:1; 3EIJ77596.1); Bacillus methanolicus C1 MeDH (SEQ ID NO:13; AAA22593.1); Bacillus methanolicus PB1 MeDH (SEQ ID NO:14; EIJ77618.1); Bacillus methanolicus PB1 MeDH (SEQ ID NO:15; EIJ78790.1); Bacillus methanolicus MGA3 MeDH (SEQ ID NO:16; EIJ80770.1); Bacillus methanolicus PB1 MeDH (SEQ ID NO:17; EIJ78397.1); Bacillus methanolicus MGA3 MeDH (SEQ ID NO:18; EIJ83020.1); Lysinibacillus fusiformis MeDH (SEQ ID NO:19; EFI69743.1); Bacillus coagulans 36D1 MeDH (SEQ ID NO:20; YP_004860127.1); Lysinibacillus sphaericus MeDH (SEQ ID NO:21; YP_001699778.1); Bacillus azotoformans LMG 9581 MeDH (SEQ ID NO:22; ZP_11313277.1); Burkholderia thailandensis E264 MeDH (SEQ ID NO:23; ZP_05587334.1); Cupriavidus necator N-1 MeDH (SEQ ID NO:24; YP_004681552.1); uncultured organism MeDH (SEQ ID NO:25; AGF87161); Geobacter bemidjiensis Bem MeDH (SEQ ID NO:26; YP_002138168.1); Carboxydothermus hydrogenoformans Z-2901 MeDH (SEQ ID NO:27; YP_359772.1); Actinobacillus succinogenes 130Z MeDH (SEQ ID NO:28; YP_001343716.1); Acinetobacter baumannii Naval-82 MeDH (SEQ ID NO:29; ZP_16224338.1); Clostridium pasteurianum DSM 525 MeDH (SEQ ID NO: 30; AAC45651.1); Methanosarcina mazei Tuc01 MeDH (SEQ ID NO:31; YP_007491369.1); Desulfovibrio vulgaris str. ‘Miyazaki F’ MeDH (SEQ ID NO:32; YP_002434746); Desulfovibrio africanus str. Walvis Bay MeDH (SEQ ID NO:33; YP_005052855); Clostridium perfringens str. 13 MeDH (SEQ ID NO:34; NP_561852.1); Vibrio campbellii ATCC BAA-1116 MeDH (SEQ ID NO:35; YP_001447544); Desulfotomaculum reducens MI-1 MeDH (SEQ ID NO:36; YP_001113612.1); Desulfovibrio vulgaris str. Hildenborough MeDH (SEQ ID NO:37; YP_011618); Photobacterium profundum 3TCK MeDH (SEQ ID NO:38; ZP_01220157.1); Geobacillus sp. Y4.1MC1 MeDH (SEQ ID NO: 39; YP_003990729.1); Desulfovibrio fructosovorans JJ MeDH (SEQ ID NO:40; ZP_07335453.1); Shewanella oneidensis MR-1 MeDH (SEQ ID NO:41; NP_717107); Sebaldella termitidis ATCC 33386 MeDH (SEQ ID NO:42; YP_003310546.1); Paenibacillus peoriae KCTC 3763 MeDH (SEQ ID NO:43; ZP_10241531.1); Klebsiella pneumoniae subsp. pneumoniae MGH 78578 MeDH (SEQ ID NO:44; YP_001337153.1); Escherichia coli MeDH (SEQ ID NO:45; YP_026233.1); Clostridium perfringens ATCC 13124 MeDH (SEQ ID NO:46; YP_694908); Ralstonia eutropha H16 MeDH (SEQ ID NO:47; YP_725376.1); Thermoanaerobacter sp. X514 MeDH (SEQ ID NO:48; YP_001663549); human gut metagenome MeDH (SEQ ID NO:49; EKC54576); or Geobacillus themodenitrificans NG80-2 MeDH (SEQ ID NO:50; YP_001126968.1).
4 . The fusion protein of claim 3 wherein the methanol dehydrogenase or an enzymatically active portion thereof is a polypeptide having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to Bacillus methanolicus MGA3MeDH (SEQ ID NO:1; 3EIJ77596.1) or to any other MeDH according to claim 3 .
5 . The fusion protein of claim 1 wherein the region comprising 3-hexulose-6-phosphate dehydrogenase or an enzymatically active portion thereof is from Bacillus methanolicus MGA3 HPS (SEQ ID NO: 3; AAR39392.1); Bacillus methanolicus PB1 HPS (SEQ ID NO: 51; EIJ81375.1); Methylobacillus flagellatus HPS (SEQ ID NO: 52; YP_544362.1; WP_011478618.1); Bacillus subtilis HPS (SEQ ID NO: 53; NP_388228.1); Methylophilus methylotrophus HPS (SEQ ID NO: 54; WP_018986666.1); or Methylophilus methylotrophus ATCC 53528 HPS (SEQ ID NO: 55; WP_018985298.1).
6 . The fusion protein of claim 5 wherein 3-hexulose-6-phosphate dehydrogenase or an enzymatically active portion thereof is a polypeptide having 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity to Bacillus methanolicus MGA3 HPS (SEQ ID NO: 4; AAR39393.1) or to any other HPS according to claim 5 .
7 . The fusion protein of claim 1 having one linker amino acid sequence.
8 . The fusion protein of claim 7 wherein the one linker amino acid sequence comprises one or more amino acids selected from the group consisting of glycine, alanine, serine, and threonine.
9 . The fusion protein of claim 7 , wherein the one or two linker amino acid sequence has a length in the range of 1-50 amino acid residues, or 1-20 amino acid residues.
10 . The fusion protein of claim 7 wherein the one linker amino acid sequence is [(GGS) x (GS) y (GGS) z ] n where x is 1-6, y is 1-6, z is 0-3, and n is 1-4.
11 . The fusion protein of claim 1 , wherein one or more of the methanol dehydrogenase, or the 3-hexulose-6-phosphate dehydrogenase, or enzymatically active portions thereof, are from Bacillus methanolicus MGA3 or Bacillus methanolicus PB1.
12 . The fusion protein of claim 7 wherein the one linker amino acid sequence is [(GGS) x (GS) y (GGS) z ] n where x is 1-3, y is 1-3, z is 1-2, and n is 1-2.