EP-4735580-A1 - REGULATION OF CELLULAR ENERGY METABOLISM BY LIPOATE PROTEIN LIGASE AND ITS APPLICATIONS

Abstract

Provided are methods for regulating cellular energy metabolism by lipoate protein ligase, wherein cell growth or production of molecules of interest in a cell is improved by overexpressing a lipoate protein ligase in the cell.

Inventors

• ZENG, Anping
• HU, Zhijuan
• YANG, Peiguo
• YANG, Runqing
• WANG, YINGYING

Assignees

• Westlake University

Dates

Publication Date

20260506

Application Date

20240628

Claims (20)

1. A recombinant host cell that is engineered to overexpress a lipoate protein ligase.
2. The recombinant host cell of claim 1, wherein the host cell is prokaryote or a eukaryote, preferably a cyanobacterium, a plant cell, an algal cell or a mammalian cell.
3. The recombinant host cell of claim 1 or 2, wherein the host cell is a cyanobacterium, a diatom cell, a U2OS cell or a CHO cell.
4. The recombinant host cell of any one of claims 1-3, wherein the overexpressed lipoate protein ligase is located in the cytoplasm or the mitochondrion of the host cell.
5. The recombinant host cell of any one of claims 1-4, wherein the lipoate protein ligase is expressed in fusion with a mitochondrial target signal (MTS) .
6. The recombinant host cell of any one of claims 1-5, wherein the MTS is SOD2 MTS, OTC MTS, Cox8a MTS or any combination thereof, or the MTS is MTS of TPI-GapC3.
7. The recombinant host cell of any one of claims 1-6, wherein the lipoate protein ligase is LplA, LplJ, LplB, LipL1, LipL2 or a functional variant thereof.
8. The recombinant host cell of any one of claims 1-7, wherein the host cell is capable of producing a molecule of interest.
9. The recombinant host cell of claim 8, wherein the host cell is engineered to produce the molecule of interest.
10. The recombinant host cell of claim 9, wherein the molecule of interest comprises biological or product and/or byproduct of biochemical or biological process in the recombinant host cells.
11. The recombinant host cell of any one of claims 8-10, wherein the molecule of interest comprises vaccine, protein, organic acid, amino acid, nucleotide and nucleoside, lipid and fatty acid, diol, carbohydrate, aromatic compound, vitamin or antibiotic.
12. The recombinant host cell of any one of claims 8-11, wherein the molecule of interest comprise antibody, enzyme, polyunsaturated fatty acid, glycosylated protein or carotenoid.
13. A method of producing molecules of interest in a host cell, the method comprising: culturing the recombinant host cell of any one of claims 8-12 under suitable conditions to produce the molecules of interest.
14. Use of the recombinant host cell of any one of claims 1-12 in producing molecules of interest.
15. A method of increasing production of molecules of interest in a cell, the method comprising: culturing the recombinant host cell of any one of claims 8-12 under suitable conditions to produce the molecules of interest.
16. A method of increasing production of molecules of interest in a cell, the method comprising: engineering a cell that is capable of producing the molecules of interest to overexpress a lipoate protein ligase.
17. Use of the recombinant host cell of any one of claims 1-12 in increasing production of molecules of interest in a cell.
18. A method of producing biomass, the method comprising culturing the recombinant host cell of any one of claims 1-12 under suitable conditions.
19. The method of claim 18, wherein the biomass is the recombinant host cells.
20. Use of the recombinant host cell of any one of claims 1-12 in producing biomass.

Description

REGULATION OF CELLULAR ENERGY METABOLISM BY LIPOATE PROTEIN LIGASE AND ITS APPLICATIONS FIELD OF THE INVENTION The present invention relates to genetic recombinant technology, especially methods for regulating cellular energy metabolism by lipoate protein ligase. BACKGROUND OF THE INVENTION Protein lipoylation represents a highly conserved posttranslational modification (PTM) that is essential for the function of key enzymes involved in cellular energy metabolism. Four mitochondrial enzyme complexes are known to have lipoylation modification, including pyruvate dehydrogenase (PDH) , alpha-ketoglutarate dehydrogenase (OGDH) , branched-chain alpha-keto acid dehydrogenase (BCKDH) , and glycine cleavage system (GCS) 1. While PDH and OGDH regulate distinct carbon entries into TCA cycle, BCKDH and GCS catalyze the catabolism of branched-chain amino acids and glycine, all of which represent key metabolic pathways. The conserved lipoylation reaction consists of two distinct pathways. The first pathway is the de novo biosynthetic pathway initiated from octanoyl-ACP derived from fatty acid biosynthesis. In E. coli, the octanoyl transferase (LipB) catalyzes the transfer of octanoyl group to the lipoylated protein and two sulfur atoms are then inserted into the C6 and C8 positions by the lipoyl synthase (LipA) . In the second salvage pathway, the exogenous lipoic acid is activated and transferred to the lipoylated proteins catalyzed by lipoate protein ligase A (LplA) . The de novo pathway of lipoylation in human cells is different from that in E. coli. First, octanoyl transferase (LIPT2) transfers octanoyl group to H protein of glycine cleavage system. Second, LIAS inserts sulfur atoms into the octanoyl group of GCSH to form lipoyl group. Last, lipoyl transferase (LIPT1) transfers lipoyl group from GCSH to the E2 subunits of other lipoylated proteins (Figure 1) 2. The de novo pathway is well conserved across all the organisms while the salvage pathway has only been revealed in bacteria, for example, LplJ from Bacillus subtilis and LplA from E. coli. Cellular energy metabolism is necessary for all the bioprocesses in living organisms. Mitochondrial respiration is the central pathway of cellular energy metabolism. For synthetic biology, energy supply is a longstanding bottleneck in metabolic engineering, causing inefficiency in metabolic flux and affecting broad cellular functions3. Given that lipoylation orchestrates key mitochondrial catabolic pathways that are primary sources of NADH, FADH2 and acetyl-CoA for energy production, we herein hypothesize whether introduction of a salvage lipoylation pathway catalyzed by lipoate protein ligase can regulate cell energy metabolism in eukaryotic cells or not. Regulation of cellular energy metabolism achieved by lipoate protein ligase may offer novel solutions to solve the metabolic burden and enhance the productivity of commodity chemicals and therapeutic proteins in synthetic biology industry. BRIEF SUMMARY OF THE INVENTION In first aspect, the present invention provides a recombinant host cell that is engineered to overexpress a lipoate protein ligase. In some embodiments, the host cell is prokaryote or a eukaryote, preferably a cyanobacterium, a plant cell, an algal cell, or an animal cell, e.g., a mammalian cell. In some embodiments, the host cell is a cyanobacterium, a diatom cell, a U2OS cell or a CHO cell. In some embodiments, the overexpressed lipoate protein ligase is located in the cytoplasm or the mitochondrion of the host cell. In some embodiments, the lipoate protein ligase is expressed with or without being in fusion with a mitochondrial target signal (MTS) . In some embodiments, the MTS is SOD2 MTS, OTC MTS, Cox8a MTS or any combination thereof, or the MTS is MTS of TPI-GapC3. In some embodiments, the lipoate protein ligase is LplA or LplJ, LplB, LipL1, LipL2 or a functional variant thereof. In some embodiments, the host cell is capable of producing molecules of interest. In some embodiments, the host cell is engineered to produce the molecules of interest. In some embodiments, the molecule of interest comprises biological or product and/or byproduct of biochemical or biological process in the recombinant host cells. In some embodiments, the molecule of interest comprises vaccine, protein, organic acid, amino acid, nucleotide and nucleoside, lipid and fatty acid, diol, carbohydrate, aromatic compound, vitamin or antibiotic. In some embodiments, the molecule of interest comprise antibody, enzyme, polyunsaturated fatty acid, glycosylated protein or carotenoid. In second aspect, the present invention provides a method of producing molecules of interest in a host cell, the method comprising: culturing any one of the aforementioned recombinant host cells under suitable conditions to produce the molecules of interest. In third aspect, the present invention provides use of any one of the aforementioned recombinant host cells in producing molecules of interest. In fourt