Yeast having high rubisco carboxylase activity and method for constructing the same

Abstract

This invention aims to create a spatially isolated local reaction microenvironment for Rubisco by introducing a complete β-carboxysome microcompartment system. This design goal avoids oxygen inhibition, laying the foundation for ultimately achieving efficient and specific CO2 fixation. It provides a novel approach to overcome the fundamental problem of low efficiency of carbon-fixing enzymes in cytosols due to intense oxygen competition. By introducing the complete carboxysome system as a module, engineered yeast strains with high carboxylase activity and significantly improved ethanol production were obtained, providing a solid foundation for building next-generation yeast cell factories that produce high-yield ethanol using CO2 as a raw material.

Description

Technical Field

[0001] This invention belongs to the field of biological carbon fixation and metabolic engineering, and specifically relates to a yeast strain with high Rubisco carboxylase activity. Background Technology

[0002] With the development of synthetic biology and metabolic engineering, utilizing microbial cell factories to convert one-carbon compounds such as carbon dioxide (CO2) into high-value-added chemicals has become an important strategy for achieving green biomanufacturing and carbon neutrality goals. (Saccharomyces cerevisiae) Saccharomyces cerevisiae With its clear genetic background and mature operating system, *Saccharomyces cerevisiae* is an ideal eukaryotic model organism for constructing such engineered cell factories. However, as a typical heterotrophic organism, *Saccharomyces cerevisiae* lacks an efficient natural CO2 fixation pathway, and its growth and product synthesis heavily rely on organic carbon sources such as glucose, which limits its carbon utilization efficiency and the sustainability of its processes.

[0003] To overcome this limitation, the main strategy of existing technologies is to introduce key enzymes of the heterologous Calvin cycle into yeast, especially ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) and phosphoribulose kinase (PRK), and express them heterologously in the cytoplasm of yeast cells. The technical logic is that by reconstructing carbon fixation in the cytoplasm, yeast can assimilate CO2, thereby enabling yeast cells to achieve autotrophic growth.

[0004] Although the aforementioned existing technologies have achieved some success in brewing yeast, they still have some drawbacks.

[0005] First, cytoplasmic expression of exogenous enzyme systems leads to cross-linking between these systems and the yeast's endogenous central carbon metabolism network. These factors influence yeast's own metabolism and also affect the correct folding and assembly of Rubisco in the cytoplasmic environment. Furthermore, yeast lacks its own mechanism for concentrating substrate CO2, resulting in low Rubisco carboxylation efficiency.

[0006] Studies have shown that the carbon fixation efficiency of Rubisco enzymes is severely limited in the oxygen-rich yeast cytosol environment. Specifically, Rubisco's carboxylase activity requires an anaerobic environment. Rubisco possesses dual catalytic properties, catalyzing not only the carboxylation of CO2 with ribulose-1,5-bisphosphate (RuBP) but also the oxygenation reaction of O2 with RuBP. Dissolved gases in the cytoplasm (including O2 and CO2) are directly balanced with the atmosphere without a specific concentration mechanism; the concentration of O2 (~21% of atmospheric concentration) is much higher than that of CO2 (approximately 0.04% of atmospheric concentration). Therefore, when Rubisco is expressed in the yeast cytoplasm, O2 acts as a competitive substrate, strongly competing with CO2 for Rubisco's active site. This leads to the activation of oxygenase activity, diverting the carbon substrate RuBP and reducing the net flux of CO2 fixation. Furthermore, a drawback of existing technologies is that the expression of exogenous enzyme systems promotes the production of toxic metabolites, potentially initiating or mimicking the photorespiration pathway, consuming ATP and reducing power, resulting in energy loss. Therefore, even with successful expression of active Rubisco and PRK in the "cytoplasmic expression" mode, the constructed Calvin cycle operates in an unfavorable environment of intense oxygen competition and extremely low substrate CO2 concentration. This results in low efficiency and economy (carbon atoms / energy consumption) of carbon fixation, failing to provide sufficient net carbon gain for cell growth. These reasons explain why yeast strains modified with existing technologies often cannot grow without organic carbon sources, and why the introduction of the entire pathway may affect their normal physiological and biochemical responses due to the additional burden of energy metabolism. Summary of the Invention

[0007] The core technical problem to be solved by this invention is to provide an innovative engineering solution to the problem that the carbon fixation activity of heterologous carbon fixation enzymes (such as Rubisco) is severely inhibited due to high oxygen partial pressure when expressed in yeast cytoplasm, resulting in low carbon fixation efficiency.

[0008] This invention explores a novel technical approach distinct from traditional cytoplasmic expression strategies: it aims to heterologously reconstruct and assemble highly efficient carbon-fixing microcompartments (carboxysomes) derived from cyanobacteria in eukaryotic Saccharomyces cerevisiae, and investigate their impact on yeast ethanol production. Carboxysomes are protein microcompartments that encapsulate Rubisco and carbonic anhydrase through a capsid protein, creating a locally high-concentration CO2 environment within the microcompartment. This significantly enhances carbon fixation efficiency and inhibits the energy-consuming oxygenation reaction of Rubisco. This invention belongs to the fields of synthetic biology, metabolic engineering, and microbial technology.

[0009] Specifically, this invention aims to create a low-oxygen, high-CO2 local microenvironment for carbon fixation enzymes by reconstructing a β-carboxysome, a multi-protein complex derived from cyanobacteria and possessing spatial separation characteristics, within the cells of *Saccharomyces cerevisiae*. This maximizes the carboxylation efficiency of Rubisco, reduces energy loss due to its oxygenase activity, and ultimately achieves effective regulation and optimization of yeast metabolic flux, thereby improving the yeast's ability to produce ethanol.

[0010] Based on the above research, the present invention provides a method for constructing a yeast strain with high Rubisco carboxylase activity, including the steps of transferring a core gene expression cassette of β-carboxysome and an expression cassette of helper genes into the yeast strain.

[0011] In one specific implementation, the core gene includes RbcL , RbcS and ccaA .

[0012] In one specific implementation, the helper gene includes one or more combinations of capsid protein genes, scaffold protein genes, and other helper genes.

[0013] In one specific implementation, the outer shell protein gene is selected from... ccmK2 , ccmK3 , ccmK4 , ccmL , ccmO , ccmP One or more combinations thereof.

[0014] In one specific implementation, the scaffold protein gene is selected from... ccmM35 , ccmM58 , ccmN , rbcX One or more combinations thereof.

[0015] In one specific implementation, the other accessory genes are selected from... PRK , HSP60 , HSP10 and Raf1 One or more combinations thereof.

[0016] In one specific implementation, the Rubisco gene expression cassette, the capsid protein gene expression cassette, and the scaffold protein gene expression cassette are integrated into the chromosome of the Saccharomyces cerevisiae, and the expression cassettes of the other auxiliary genes are integrated into independently replicated plasmids.

[0017] In one specific implementation, the promoters in the Rubisco gene expression cassette and the helper gene expression cassette are selected from: pPGK1, pPDC1, pRPL8B, pTEF2, pRPL3, pYEF3, pENO2, pTDH3, pTPI1, pPYK1, pPGI1, pRPL4A, pRPL15A, pADH1, pPFK1, pCCW12, pTEF1, pRPL1BIn one specific implementation plan, the promoter used is derived from Saccharomyces cerevisiae.

[0018] In one specific implementation, the expressed β-carboxysome-related gene is derived from Synechococcus PCC 7942. RbcL , RbcS , ccaA , ccmK2 , ccmK3 , ccmK4 , ccmL , ccmO , ccmP , ccmM35 , ccmM58 , ccmN , rbcX , PRK , HSP60 , HSP10 and Raf1 The encoded protein sequences are shown in SEQ ID NO:1-17.

[0019] The present invention also provides an engineered brewing yeast constructed by the above method.

[0020] The present invention also provides the application of the above-mentioned engineered brewing yeast in CO2 fixation or ethanol production.

[0021] Compared to existing technologies that limit the expression of a few carbon-fixing enzymes in yeast cytoplasm, this invention improves the activity of Rubisco carboxylase by systematically reconstructing complete β-carboxysome microcompartments.

[0022] First, this invention provides a novel approach to address the fundamental problem of low efficiency of carbon-fixing enzymes in cytosols due to intense oxygen competition. Existing technologies, when expressing enzymes such as Rubisco in a hyperotropic cytoplasmic environment, strongly activate their harmful oxygenase activity, severely restricting carbon fixation. This invention, by introducing a complete β-carboxysome microcompartment system, aims to create a spatially relatively isolated local reaction microenvironment for Rubisco. This design goal circumvents oxygen inhibition, laying the foundation for ultimately achieving efficient and specific CO2 fixation.

[0023] Secondly, this invention demonstrates the powerful ability to functionally integrate complex multi-protein systems into a eukaryotic host. Existing modifications typically involve only a few genes, while this invention employs an efficient assembly strategy to successfully introduce a complete carboxysome system as a module into yeast. This not only achieves detectable expression of all key component proteins but also endows Rubisco with carboxylase activity, proving the feasibility of functional co-expression of such a large heterologous multi-gene system in yeast and providing a crucial paradigm for the construction of complex modules in synthetic biology.

[0024] Finally, this invention achieves a powerful reprogramming of the host metabolic network and significantly enhances the synthetic capacity of the target product. Existing modifications often fail to fundamentally shift carbon flow. However, this invention intervenes in central carbon metabolism by systematically introducing a carboxysome module. High-performance liquid chromatography (HPLC) analysis shows that the engineered strain achieved a several-fold increase in ethanol production, while the synthesis of the byproduct glycerol was effectively inhibited. This phenotype of "increased ethanol production and reduced glycerol consumption" conclusively demonstrates that this invention can efficiently redirect carbon resources to the target product synthesis pathway, providing a solid foundation for constructing a next-generation yeast cell factory with high ethanol production using CO2 as a raw material. Attached Figure Description

[0025] Figure 1 Construction of engineered strain β1. This includes the development of the coat protein gene. ccmK2 , ccmK3 , ccmK4 , ccmL , ccmO , ccmP Kernel protein genes rbcL , rbcS , ccaA and scaffold protein genes ccmM , ccmN , rbcX The expression cassette is integrated onto chromosome IV. PRK , HSP60 , HSP10 and Raf1 The expression cassette was integrated into plasmid pRS424-4TU.

[0026] Figure 2 This assay is for the detection of β-carboxysosome protein expression. CBB refers to Coomassie Brilliant Blue staining, which serves as a control for sample loading. #1, #2, and #3 are three independent yeast strains.

[0027] Figure 3 Rubisco enzyme activity assay for Saccharomyces cerevisiae strain β1. CK was strain JDY52-URR. Data represent the mean ± standard deviation of three biological replicates.

[0028] Figure 4 HPLC was used to determine the ethanol and glycerol content in yeast. (A) shows the ethanol yield of the yeast strain; (B) shows the glycerol yield of the yeast strain. CK is strain JDY52-URR. Data represent three biological replicates. Error bars represent standard deviations. Student's t-test was used for statistical significance analysis (***p<0.001). Detailed Implementation

[0029] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0030] 1. Chassis Cells and Modular Assembly Strategy This invention provides a technical solution for systematically constructing and evaluating artificial carbon-fixing microcompartments in Saccharomyces cerevisiae. The core of this solution lies in integrating a complex multi-gene system into the yeast genome in one step, attempting to form subcellular structures conducive to carbon fixation, using materials from Synechococcus (…). Synechococcus elongatus Thirteen carboxysosome proteins from PCC7942 were modularly assembled and co-expressed in Saccharomyces cerevisiae JDY52-URR. This not only introduced carboxysosome structural proteins but also co-expressed functionally optimized elements, including PRK, the molecular chaperone system (HSP60 / HSP10), and the Rubisco assembly factor Raf1.

[0031] A *Saccharomyces cerevisiae* strain with a clear genetic background was selected as the host. Using the Golden Gate-based standardized assembly system, the target gene was combined with yeast promoters and terminators to form a standard transcription unit. The correspondence between the target gene and its corresponding promoter and terminator in the gene expression cassette is shown in Table 1. RbcL , RbcS , ccaA , ccmK2 , ccmK3 , ccmK4 , ccmL , ccmO , ccmP , ccmM35 , ccmM58 , ccmN , rbcX , PRK , HSP60 , HSP10 and Raf1 The encoded protein sequences are shown in SEQ ID NO:1-17.

[0032] Table 1. Core genes, corresponding promoters, and terminators for constructing β-carboxysosome yeast engineered strains.

[0033] 2. Integration of multi-gene transcription units like Figure 1 As shown, this is to achieve the core genes of the β-carboxysome (including the coat protein gene) ccmK2 , ccmK3 , ccmK4 , ccmL , ccmO , ccmP Kernel protein gene rbcL , rbcS ,ccaA and scaffold protein genes ccmM , ccmN , rbcX To achieve stable co-expression of these genes in yeast, this invention utilizes Golden Gate homologous recombination technology. The DNA coding frames of the aforementioned genes, along with their corresponding promoters and terminators (Table 1), are amplified by PCR, and then recombined into a single transcription unit. These different transcription units are then recombined into a large DNA fragment containing multiple tandem transcription units using the Golden Gate method. This large DNA fragment is then transformed into yeast cells. In yeast, this large DNA fragment is integrated into the yeast genome. HO Site.

[0034] In addition, the plasmid pRS424-4TU, which expresses PRK, HSP60, HSP10 and Raf1, was constructed and introduced into yeast, and the engineered strain β1 was successfully constructed.

[0035] This strategy ensures the stable inheritance and co-expression of all components at the genome level.

[0036] 3. Protein expression validation The detection was performed using Western blotting, which utilizes specific antibodies against different carboxysome components.

[0037] The results are as follows Figure 2 As shown, multiple target protein bands, including the Rubisco large subunit (RbcL), Rubisco small subunit (RbcS), and the key shell protein (CcmK2), were successfully detected in three independent strains, confirming the effective expression of the multi-gene system in yeast.

[0038] 4. Assay of Rubisco's carboxylase activity The carboxylase activity of Rubisco in crude cell extract was determined using a coupled enzyme reaction method. The carboxylase activity of Rubisco in the crude cell extract was quantitatively detected by monitoring the change in NADH absorbance at 340 nm using a spectrophotometer.

[0039] The results are as follows Figure 3 As shown, the Rubisco activity of the engineered strain β1 was significantly higher than that of the wild-type control that did not express the enzyme, demonstrating that the introduced carbon fixation module possesses the expected catalytic functional basis.

[0040] 5. Changes in ethanol and glycerol content of engineered strain β1 To assess the substantial impact of this artificial carbon fixation system on the host metabolic network, high performance liquid chromatography (HPLC) was used to quantitatively analyze key metabolites in the fermentation broth.

[0041] Yeast strains were cultured in SD medium at 28°C for 96 hours until the plateau phase was reached. Ethanol and glycerol content were analyzed using a high-performance liquid chromatography (LC-20AD, Shimadzu) system equipped with a differential refractive index detector (RID-10A). Separation was performed on an organic acid analysis column (Bio-Rad Aminex HPX-87), with the column temperature maintained at 60°C and the detector temperature at 40°C. The mobile phase was 5 mM sulfuric acid, and the flow rate was 0.5 mL / min. The initial flow rate was set at 0.2 mL / min, and after the column temperature and pressure stabilized, the flow rate was gradually increased to 0.5 mL / min in increments of 0.2 mL / min, with an injection volume of 20 μL.

[0042] The results are as follows Figure 4 As shown, compared with the wild-type strain, β1 exhibited significantly increased ethanol production, while the production of the byproduct glycerol was markedly reduced. This altered metabolic profile suggests that, although the complete carboxysome structure may not yet be formed, the introduced multi-component carbon fixation system has successfully integrated into and reshaped the central carbon metabolic flux of yeast, directing more carbon flux towards the synthesis pathway of the target product, ethanol.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a yeast strain with high Rubisco carboxylase activity, characterized in that, This includes the steps of transferring the core gene expression cassette of the β-carboxysome and the expression cassette of the helper gene into yeast.

2. The method according to claim 1, characterized in that, The core genes include RbcL , RbcS and ccaA .

3. The method according to claim 1, characterized in that, The accessory genes include one or more combinations of capsid protein genes, scaffold protein genes, and other accessory genes.

4. The method according to claim 3, characterized in that, The outer shell protein gene is selected from ccmK2 , ccmK3 , ccmK4 , ccmL , ccmO , ccmP One or more combinations thereof.

5. The method according to claim 3, characterized in that, The scaffold protein gene is selected from ccmM35 , ccmM58 , ccmN , rbcX One or more combinations thereof.

6. The method according to claim 3, characterized in that, The accessory gene is selected from PRK , HSP60 , HSP10 and Raf1 One or more combinations thereof.

7. The method according to claim 6, characterized in that, The Rubisco gene expression cassette, the capsid protein gene expression cassette, and the scaffold protein gene expression cassette are integrated into the chromosome of the Saccharomyces cerevisiae, and the expression cassettes of the other auxiliary genes are integrated into independently replicated plasmids.

8. The method according to any one of claims 1-7, characterized in that, The promoters in the Rubisco gene expression cassette and the helper gene expression cassette are selected from: pPGK1, pPDC1, pRPL8B, pTEF2, pRPL3, pYEF3, pENO2, pTDH3, pTPI1, pPYK1, pPGI1, pRPL4A, pRPL15A, pADH1, pPFK1, pCCW12, pTEF1, pRPL1B .

9. An engineered brewing yeast, characterized in that, It is constructed by the method of any one of claims 1-8.

10. The application of the engineered brewing yeast of claim 9 in CO2 fixation or ethanol production.

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