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Yeast expressing a synthetic calvin cycle

A Calvin cycle, yeast technology, applied in biosynthesis, fermentation, enzymes, etc.

Inactive Publication Date: 2020-05-08
UNIV FUR BODENKULTUR WIEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this system it is not possible to decouple carbon assimilation from energy supply in the form of NADH

Method used

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  • Yeast expressing a synthetic calvin cycle
  • Yeast expressing a synthetic calvin cycle
  • Yeast expressing a synthetic calvin cycle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0442] Embodiment 1 prepares culture medium

[0443] LB medium is used for the cultivation of Escherichia coli DH 10B, and the operation steps are as follows.

[0444] Prepare LB medium (10.0g*L -1 Soy peptone (Quest), 5.0g*L -1 Yeast extract (MERCK) and 5.0g*L -1 NaCl, and adjusted the pH value to 7.4-7.6 with 4N NaOH), and distributed in 500mL Schott bottles. The sterilization method is autoclaving at 121°C for 20 minutes.

[0445] Yeast peptone (YP) medium was used to cultivate Pichia pastoris CBS7435 wt in shake flasks, and the operation steps were as follows.

[0446] Before adding carbon source, YP-medium (20.0g*L -1 Soy peptone (Quest), 10.0g*L -1 Yeast extract (MERCK, pH adjusted to 7.4–7.6 with 4N NaOH) was autoclaved. Prepare 10 times glucose stock solution (220g*L -1 D(+)-glucose monohydrate) and sterilized by autoclaving. Add 10 times the glucose stock solution to the YP medium at a ratio of 1 / 10 to obtain a YPD medium.

[0447] Glycerol-containing batch ...

Embodiment 2

[0459] Example 2 Constructing plasmids and linear DNA fragments

[0460] All expression cassettes (promoter, CDS, terminator) were constructed by Golden Gate cloning (Engler et al.PloSOne 4(5):e5553.doi:10.1371 / journal.pone.0005553) and flanked by the corresponding integration sites point to replace the above three genes, namely aox1, das1 and das2. Follow the plasmid DNA construct operating procedure published in (Sarkari, et al.2017BioresourceTechnology.doi:10.1016 / j.biortech.2017.05.004) to realize the operation for constructing all linear DNA fragments and guide RNA (gRNA) / hCas9 plasmid process.

[0461] The coding sequences (CDS) of the genes described in Table 5 were combined with the methanol-inducible promoter and terminator sequences of Pichia pastoris CBS7435 wt (Table 6).

[0462] Table 5: Creation of the synthetic Calvin cycle in Pichia pastoris (Centraalbureau voorSchimmelcultures, NL, genome sequenced by (Küberl et al., 2011; Valli et al., 2016)) according to e...

Embodiment 3

[0476] Example 3 Construction of a Pichia pastoris strain expressing a functional Calvin cycle targeting peroxisomes

[0477] To create GaT_pp_10 and control Pichia pastoris strains, three genes in the Pichia pastoris genome were deleted, namely AOX1 (ORF ID: PP7435_Chr4-0130), DAS1 (ORF ID: PP7435_Chr3-0352) and DAS2 ( ORF ID: PP7435_Chr3-0350), and 8 genes PGK1, TDH3, TPI1, PRK, TKL, GroEL, GroES and RuBisCO (Table 5 and Table 6) were integrated into the genome.

[0478] 3.1 Transformation of Pichia pastoris

[0479] Pichia pastoris transformation was performed on chemically competent cells by electroporation as described below. A single colony of Pichia pastoris (CBS7435 wt or the corresponding clone) was inoculated into 10 mL of YPD pre-culture, and cultured overnight ( o ver n ight) (o / n; ~16h) (shaker; 180rpm; 28°C). The next day, 100 mL of the main culture was inoculated. The inoculum volume of the preculture was calculated as follows so that the main culture reach...

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Abstract

A yeast comprising a nucleotide sequence expression system expressing a synthetic Calvin cycle comprising heterologous genes, which include at least a) a gene encoding an enzyme from the class of theribulose-bisphosphate carboxylases (EC number: 4.1.1.39) (RuBisCO gene); and b) a gene encoding an enzyme from the class of the ribulose phosphate kinases (EC number: 2.7.1.19) (PRK gene), which is expressing; wherein the yeast optionally comprises a heterologous expression construct expressing a gene of interest (GOI) and / or wherein each of said RuBisCO gene and said PRK gene, is fused with a nucleotide sequence encoding a peroxisomal targeting signal (PTS).

Description

technical field [0001] The present invention relates to yeast comprising a heterologous gene expressing a synthetic Calvin cycle, and to a method for fixing carbon dioxide while culturing the yeast. Background technique [0002] Greenhouse gas emissions and related climate change are among the most pressing issues of our society. Use CO 2 Instead of fossil resources as a carbon source for industrial production processes, greenhouse gas emissions can be significantly limited. Biotechnology is one of the key technologies of bio-economy. Many feed and food applications, as well as basic chemical and pharmaceutical production, started with microorganisms as catalysts. These processes are mostly based on plant-derived resources, such as sugars, but are rarely based directly on atmospheric CO 2 . However, increased use of plant-derived carbon has been linked to changes in land use and other detrimental effects on the planet. Therefore, it would be ideal to produce organisms ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N9/88C12N15/81
CPCC12N1/16C12N9/88C12N15/81Y02E50/10C12Y102/01012C12Y202/01001C12Y207/01001C12Y207/02003C12Y401/01039C12Y503/01001C07K2319/01C12N2800/22C07K14/245C07K14/765C12N9/0008C12N9/1022C12N9/1217C12N9/1294C12N9/485C12N9/90C12N15/815C12P7/46C12P7/56C12Y207/01019C12Y304/17002
Inventor D·马塔诺维赫M·绍尔M·施泰格尔T·加斯勒B·加塞尔
Owner UNIV FUR BODENKULTUR WIEN
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