Methanol-tolerant Eimerella lipenosa mutant strain as well as screening method and application thereof

Abstract

The invention provides a methanol-tolerant Eimenococcus sp. Mutant strain as well as a screening method and application thereof, the methanol-tolerant Eimenococcus sp. Mutant strain is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC M 20232483. The mutant strain can effectively utilize an industrial byproduct methanol as a carbon source to carry out efficient protein production and valuable carotenoid accumulation; the mutant strain shows excellent photosynthesis capability and nitrogen assimilation efficiency under a methanol culture condition. Effective technical support is provided for methanol-based industrial biological refining application of microalgae, and the method has remarkable environmental and economic benefits.

Description

Technical Field

[0001] The invention belongs to the field of microalgae biotechnology, and particularly relates to a methanol-resistant Lipococcus emersonii mutant strain, a screening method and an application thereof. Background Art

[0002] Global protein demand is projected to increase significantly in the coming decades as the global population grows and living standards improve. This growing demand poses significant challenges to sustainable food security, including increased agricultural land use, water depletion, greenhouse gas emissions, and competition for resources. These challenges are driving the search for alternative protein sources beyond traditional agriculture. Microalgae are promising candidates for sustainable protein production due to their high photosynthetic efficiency, rapid growth rate, limited land requirements, and ability to utilize non-arable land and various water sources, including wastewater.

[0003] Among various microalgae species, Graesiella emersonii shows great promise due to its robust growth characteristics, environmental adaptability, and naturally high protein content (40–60% of dry weight under optimal conditions). Its ability to efficiently remove nutrients from wastewater while simultaneously fixing carbon dioxide makes it a promising candidate for environmental remediation technologies. Furthermore, its high lipid content and valuable cellular components make it a versatile resource for biodiesel production, nutritional applications, and various industrial processes. However, its ability to utilize alternative carbon sources such as methanol remains relatively underexplored.

[0004] Traditional microalgae cultivation typically relies on carbon dioxide as the primary carbon source, but this has limitations, including low gas-liquid mass transfer efficiency, unstable pH, low energy conversion efficiency (<3%), suboptimal photobioreactor design, complex culture conditions, high cost, low RuBisCO affinity, and difficulty in scaling up production. Consequently, research is underway to explore alternative carbon sources to increase productivity while reducing production costs.

[0005] Methanol is a promising alternative carbon source for microbial cultivation. Methanol is a simple C1 compound produced as a byproduct of various industrial processes and offers several advantages, including high water solubility, ease of transportation, and relatively low cost. Using methanol in microalgae cultivation aligns with circular economy principles and has the potential to convert industrial byproducts into valuable protein biomass.

[0006] Methanol metabolism in microorganisms typically involves a stepwise oxidation process. First, methanol is oxidized to formaldehyde by methanol dehydrogenase, then converted to formic acid by formaldehyde dehydrogenase, and finally to carbon dioxide by formate dehydrogenase. These enzymes are often limiting factors in methanol utilization by microalgae. Some methylotrophic bacteria and yeasts possess efficient pathways for methanol assimilation through the ribulose monophosphate pathway or the serine pathway, but such pathways are generally underdeveloped in microalgae. Consequently, the natural ability of most microalgae to utilize methanol is limited by the toxic effects of high concentrations and a lack of efficient C1 carbon assimilation enzyme pathways. Currently, there is a lack of microalgae strains that can effectively metabolize methanol as a carbon source.

[0007] Mutagenesis offers a promising approach for improving microalgae's tolerance and utilization of unconventional carbon sources. Compared to traditional chemical or radiation mutagenesis methods, atmospheric pressure room temperature plasma (ARTP) technology offers advantages such as ambient operating conditions, rapid processing, high mutation efficiency, and minimal safety hazards, making it an effective tool for microbial mutagenesis. ARTP generates a variety of reactive species, including electrons, ions, and free radicals, which can penetrate cell membranes and induce DNA damage, leading to random mutations throughout the genome. Previous studies have successfully applied ARTP mutagenesis to improve various microbial traits in bacteria, yeast, and microalgae, including stress tolerance, growth rate, and carbon substrate utilization. However, its application in improving methanol utilization in microalgae remains underexplored.

[0008] Therefore, the present invention aims to develop a Lipococcus emersonii strain with enhanced methanol utilization ability through ARTP mutagenesis technology, so as to provide a more sustainable microalgae protein production method and an environmentally friendly approach for the utilization of industrial methanol. Summary of the Invention

[0009] The present invention aims to provide a methanol-tolerant Lipococcus emersonii mutant strain, its screening method, and its application, to address the prior art issue of the limited ability of microalgae to utilize methanol as a carbon source. This objective can be achieved through the following technical solutions:

[0010] The invention provides a methanol-resistant Lipococcus emersonii mutant strain. The methanol-resistant Lipococcus emersonii mutant strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 20232483.

[0011] A method for screening a methanol-resistant mutant of Lipococcus emersonii, comprising the following steps:

[0012] Step 1) subjecting a suspension containing Lipococcus emersonii cells to atmospheric pressure room temperature plasma (ARTP) mutagenesis to obtain a mutagenized suspension;

[0013] Step 2) inoculating the mutagenized suspension onto a methanol-containing culture medium for culturing, and screening for methanol-resistant Lipococcus emersonii mutants.

[0014] Optionally, the step of subjecting the suspension containing Lipococcus emersonii cells to ARTP mutagenesis in step 1) specifically includes: using ultrapure helium as the plasma working gas, and the mutagenesis conditions are: RF power 100-150W, helium flow rate 8-12L·min-1, the distance between the plasma torch nozzle outlet and the sample plate 1-3mm; plasma jet temperature 25°C to 35°C, and treatment time 10 to 17 seconds.

[0015] Optionally, the methanol content of the culture medium containing methanol in step 2) is 8 g·L -1 ;

[0016] Preferably, the culture medium is Endo solid culture medium.

[0017] Optionally, in the step 2), after inoculation, the culture is carried out at 22-28° C., 30-60 μmol·m-2·s-1 light intensity and 12:12 hour light: dark cycle for 3-7 days to screen out methanol-resistant Lipococcus emersonii mutants.

[0018] Optionally, in step 2), methanol tolerance is assessed by measuring colony diameter and pigment intensity, 8 g·L -1 As the selection threshold, the mutant and wild-type (WT) algae were cultured in methanol gradient Endo culture dishes, with non-mutagenized algae as negative control, and the culture dishes were inverted and cultured for 5 days.

[0019] Optionally, the method further includes:

[0020] Step 3) inoculating the methanolic Emersonii Lipococcus mutants obtained by screening in step 2) onto a medium containing methanol for cultivation, measuring the growth rate of each mutant, and screening the methanolic Emersonii Lipococcus mutant with the highest growth rate;

[0021] Preferably, the culture medium is Endo liquid culture medium.

[0022] A preparation comprises the above-mentioned methanol-resistant Lipococcus emersonii mutant.

[0023] The methanol-resistant Lipococcus emersonii mutant or preparation is used in protein production, carotenoid production, industrial methanol bioconversion, high-protein microalgae feed production, wastewater resource treatment, and biorefining.

[0024] Optionally, the carotenoid comprises at least one of astaxanthin and canthaxanthin.

[0025] Optionally, methanol is used as a carbon source in the process of producing protein and carotenoids.

[0026] The beneficial effects of the present invention are:

[0027] The present invention provides a Lipococcus emersonii mutant strain with high methanol tolerance and utilization capacity, obtained through ARTP mutagenesis. This mutant strain can effectively utilize industrial byproduct methanol as a carbon source, resulting in efficient protein production and accumulation of valuable carotenoids. The mutant strain also exhibits excellent photosynthetic capacity and nitrogen assimilation efficiency under methanol culture conditions. This invention provides effective technical support for the industrial biorefining of microalgae using methanol, with significant environmental and economic benefits.

[0028] In Endo medium containing 8 g / L methanol, its specific growth rate is greater than 0.38 d-1, which is at least 30% higher than that of the wild-type algae strain; under 204 hours of culture conditions, the biomass accumulation exceeds 2.0 g / L, indicating that it has higher biomass accumulation, photosynthetic efficiency, nitrogen assimilation capacity and methanol utilization rate under methanol culture conditions; when methanol is used as the sole carbon source, the protein content is not less than 40%; its amino acid composition is balanced, and the essential amino acid content meets or exceeds the FAO / WHO nutritional standards, and the mutant strain is capable of producing high levels of high-value carotenoids such as astaxanthin and canthaxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 Figure 1 shows the methanol-resistant Lipococcolithophores emersonii mutant obtained in Example 1, where (a) is the isolated Lipococcolithophores emersonii cell; (b) is the phylogenetic tree; (c) is ... -1 Specific growth rates of wild-type and mutant algae strains in methanol-containing medium. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences (P < 0.05) based on one-way analysis of variance and LSD test.

[0031] Figure 2 The growth characteristics and photosynthetic performance of wild-type and mutant algae strains; (a) is based on OD 540Growth curve; (b) Chlorophyll a content; (c) Maximum quantum yield of photosystem II (Fv / Fm) during the cultivation period; (d) Biomass accumulation measured as dry weight. Data are presented as mean ± SD (n = 3). Significant differences between the mutant 11-3 + methanol group and the wild type (WT) + methanol group are indicated with asterisks (*: P < 0.05; **: P < 0.01).

[0032] Figure 3 Figure 3. Carbon and nitrogen utilization of wild-type and mutant algae strains. (a) Residual methanol concentration in the culture medium; (b) Residual total nitrogen concentration in the culture medium. Data are presented as mean ± SD (n = 3). Different lowercase superscript letters indicate significant differences between treatments (P < 0.05) based on one-way analysis of variance and LSD test.

[0033] Figure 4 Figure 1 shows the biochemical composition of wild-type and mutant algae strains under different culture conditions. (a) Carbohydrate content (percentage of dry weight); (b) Protein content (percentage of dry weight). Data are presented as mean ± SD (n = 3). Different letters at each time point indicate significant differences among treatments (P < 0.05) based on one-way analysis of variance and LSD test.

[0034] Figure 5 Changes in fatty acid profiles and pigmentation in wild-type and mutant strains. (a) Fatty acid content at different time points; (b) Changes in total saturated and unsaturated fatty acids during culture. Data are presented as mean ± SD (n = 3); (c) Phenotype and cell morphology of Lipococcoid algae on days 7 (left) and 15 (right) of methanol exposure; (d) Pigment accumulation (μg / g dry weight) in wild-type and mutant strains on days 7 and 15. Data are mean ± SD (n = 3). Different letters indicate significant differences (P < 0.05) as determined by one-way analysis of variance (ANOVA) and LSD test; (e) Carotenoid profile showing astaxanthin (peak a), zeaxanthin (peak b), and canthaxanthin (peak c).

[0035] Figure 6 qRT-PCR analysis of methanol-derived amino acid synthesis in wild-type and mutant strains. Significant differences between groups are indicated with asterisks (*: P < 0.05; ***: P < 0.001; ****: P < 0.0001). (a) Differential transcription levels of five key genes in the wild-type strain after 60 hours of treatment with 10 g / L methanol (compared to the control); (b) Differential transcription levels of five key genes in the mutant strain 11-3 after 60 hours of treatment with 10 g / L methanol (compared to the control); (c) Amino acid composition of the wild-type and mutant strains under different treatment groups on day 15 of culture. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0037] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intervening value in the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The present invention screened and obtained a methanol-resistant Lipococcus emersonii mutant strain, and the methanol-resistant Lipococcus emersonii mutant strain was deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 20232483.

[0041] The above-mentioned method for screening methanol-resistant Lipococcus emersonii mutants comprises the following steps:

[0042] Step 1) subjecting a suspension containing Lipococcus emersonii cells to mutagenesis using atmospheric pressure room temperature plasma (ARTP) technology to obtain a mutagenized suspension;

[0043] Step 2) inoculating the mutagenized suspension onto a methanol-containing culture medium for culturing, and screening for methanol-resistant mutants of Lipococcoid Emersonii.

[0044] Step 3) inoculating the methanolic Emersonii Lipococcus mutants obtained by screening in step 2) onto a medium containing methanol for cultivation, measuring the growth rate of each mutant, and screening the methanolic Emersonii Lipococcus mutant with the highest growth rate;

[0045] The step of subjecting the suspension containing Lipococcus emersonii cells to ARTP mutagenesis in step 1) specifically includes: using ultrapure helium as a plasma working gas, and the mutagenesis conditions are: radio frequency power of 100-150W, helium flow rate of 8-12L·min-1, the distance between the plasma torch nozzle outlet and the sample plate of 1-3mm; plasma jet temperature of 25°C to 35°C, and treatment time of 10 to 17 seconds.

[0046] The methanol content of the culture medium containing methanol in step 2) is 8 g·L -1 The culture medium is Endo solid medium. In the step 2), after inoculation, the culture is carried out at 22-28°C, 30-60 μmol·m-2·s-1 light intensity and 12:12 hour light: dark cycle for 3-7 days to screen out methanol-resistant Emersonia lipococcus mutants.

[0047] The culture medium is Endo liquid culture medium.

[0048] Example 1: ARTP mutagenesis and methanol-resistant mutant screening

[0049] Strain Source and Culture: Graesiella emersonii strain M7 was isolated from a shrimp aquaculture wastewater sample at the Longhua Biotechnology Innovation Institute of Shenzhen University. The microalgae was purified by continuous gradient dilution and then streaked onto solid culture medium. After screening using a single colony picking technique, the target strain was inoculated into Endo liquid medium in an Erlenmeyer flask. Culture purity was determined by repeated plating combined with light microscopy. The final strain was cultured in a constant temperature and illumination incubator at 28 ± 1°C, 30 μmol·m-2·s-1 white light intensity, and continuous illumination for 15 days.

[0050] The taxonomic identification of the algal strains was verified by GenBank BLAST alignment of 18S rRNA sequences using modified primers (forward: 5′-GCGGTAATTCCAGCTCCAA-3′, reverse: 5′-AATCCRAGAATTTCACCTCT-3′). Phylogenetic reconstruction was performed using MEGA 12.0, incorporating homologous green algae sequences. Clade stability was assessed using the neighbor-joining algorithm and 1000 bootstrap replicates. The screening method of the present invention specifically comprises the following steps:

[0051] Step 1) ARTP mutagenesis: Lipococcoid algae cells (3.30×10 cells·mL) in the logarithmic growth phase were cultured. -1ARTP mutagenesis was performed using a RT-PCR system. For each treatment, 10 μL of the mixture was applied to a stainless steel glass slide and placed in the chamber of an ARTP-M (Tianshu Biotechnology, China). The following operating parameters were used: RF power 120 W; helium flow rate 10 L min-1; distance between the plasma torch nozzle outlet and the sample plate 2 mm; plasma jet temperature 25°C–35°C; and treatment times of 0, 3, 5, 8, 10, 11, 12, 13, 14, 15, 16, and 17 seconds, respectively.

[0052] Step 2) Mutant screening: After mutagenesis, cells were diluted 10 3 times, and apply 0.1 mL of the dilution solution to the surfaces containing 0, 2, 4, 6, 8, and 10 g·L -1 The culture dishes were incubated at 25°C (50 μmol·m-2·s-1, 12:12 h light:dark cycle) for 5 days.

[0053] Methanol tolerance was assessed by measuring colony diameter and pigment intensity, and 8 g·L was determined based on preliminary inhibition tests. -1 The mutants and wild-type (WT) algae were then cultured in Endo culture dishes with a methanol gradient, with unmutated algae as a negative control, and the dishes were inverted for 5 days. Under the same culture conditions, in Endo liquid medium (5×10 4 Dominant colonies (characterized by larger diameter and darker pigment) were further verified in 24-well plates containing 10 cells / well, with three biological replicates for each treatment.

[0054] Step 3) Growth evaluation and selection of mutants: The mutants screened were grown in a -1 The growth of the mutant algae strains obtained by the above screening was evaluated under the culture conditions of methanol. The mutant algae strains were named in the "AB" format, where A represents the exposure time of mutagenesis (seconds) and B represents the serial isolation number of the colony. The dry weight and OD of the cells were measured daily. 540 The growth was monitored by optical density.

[0055] The specific growth rate (μ) is calculated as follows: μ = ln (OD 540 _t / OD 540 _0) / Δt

[0056] Analysis of cell survival after ARTP treatment showed a typical sigmoidal relationship between exposure time and survival rate. An 11-second treatment resulted in approximately 90% lethality. The high frequency of positive mutants in the 11-second treatment groups (11-1 to 11-7) suggests that this exposure duration achieves an optimal balance between mutagenic efficacy and cell viability.

[0057] The results showed that under high methanol conditions (8 g·L -1 ), the specific growth rate of the wild type G.emersonii was 0.282 day-1, and several mutant strains (11-7, 15-1 and 16-2) had no significant improvement compared with the wild type (P>0.05). The growth rates of mutant strains 16-1 and 16-3 were slightly increased, which were 0.337 and 0.363 day-1, respectively, with increases of 19.6% and 28.9%, respectively.

[0058] Five mutants in the 11-second exposure group (11-1, 11-2, 11-4, 11-5, and 11-6) showed significant improvements, with growth rates of 0.389, 0.386, 0.380, 0.388, and 0.390 days-1, respectively, which were 37.9%, 36.9%, 34.8%, 37.6%, and 38.3% higher than the wild type, respectively. Notably, strain 11-3 showed the highest growth rate of 0.415 days-1, a 59.9% increase compared to the wild type, and was therefore selected for full characterization. This methanol-tolerant Emersonii Lipococcoidea mutant was deposited in the China Center for Type Culture Collection on December 7, 2023, with the deposit number CCTCC M20232483 and named Graesiella emersonii SZU-ZQ01.

[0059] Figure 1 Figure 1 shows the methanol-resistant Lipococcolithophores emersonii mutant obtained in Example 1, where (a) is the isolated Lipococcolithophores emersonii cell; (b) is the phylogenetic tree; (c) is ... -1 Specific growth rates of wild-type and mutant algae strains in methanol-containing medium. Data are presented as mean ± SD (n = 3). Different letters indicate significant differences (P < 0.05) based on one-way analysis of variance and LSD test.

[0060] Example 2 Comparison of growth characteristics and photosynthetic performance of wild-type and mutant algae strains

[0061] Large-scale culture conditions

[0062] Endo medium without carbon source was used to maintain G. emersonii cultures. For comparative analysis, wild type and selected mutant strain 11-3 were cultured in 500 mL of Endo medium without carbon source in 1000 mL Erlenmeyer flasks under the following culture conditions: 1) wild type alone (denoted as WT), 2) wild type with 10 g·L -1 Methanol (expressed as WT+MeOH), 3) strain 11-3 alone (expressed as 11-3), 4) strain 11-3 with 10 g·L -1 Methanol (expressed as 11-3+MeOH), and 5) culture medium with 10 g·L -1Methanol (no algae, control). All cultures were cultured at 3×10 5 cells·mL -1 The cells were started and maintained under optimized conditions: 40 μmol·m-2·s-1 light intensity (16:8 h light:dark cycle), 28±1°C, 200 rpm stirring and 1 vvm aeration with 5% CO2 in air.

[0063] Biomass and growth measurements

[0064] The biomass of microalgae was quantified by measuring the dry weight of algal cells after centrifugation. The absorbance at 540 nm (OD 540 Specific growth rate (μ) was calculated according to the formula: μ = ln (OD 540 _t / OD 540 _0) / Δt.

[0065] Photosynthetic parameter analysis

[0066] Chlorophyll fluorescence was measured using a PAM 2500 (Woltz, Germany). Samples (3 mL) were dark-adapted for 20 minutes, and then the maximum quantum yield of photosystem II (Fv / Fm) was measured to assess photosynthetic performance. Chlorophyll a content was determined using a PHYTO-PAM (Woltz, Germany).

[0067] Result Analysis

[0068] Figure 2 The growth characteristics and photosynthetic performance of wild-type and mutant algae strains; (a) is based on OD 540 Growth curve; (b) Chlorophyll a content; (c) Maximum quantum yield of photosystem II (Fv / Fm) during the cultivation period; (d) Biomass accumulation measured as dry weight. Data are presented as mean ± SD (n = 3). Significant differences between the mutant 11-3 + methanol group and the wild type (WT) + methanol group are indicated with asterisks (*: P < 0.05; **: P < 0.01).

[0069] The growth of the wild type and mutant (11-3) in Endo medium without adding carbon source and with adding 10 g / L methanol were compared. Figure 2 As shown, in the absence of exogenous carbon, there were no significant differences in growth parameters and photosynthetic efficiency (Fv / Fm) throughout the incubation period, except for an increase in biomass after 132 h.

[0070] When methanol was used as the sole exogenous carbon source, the growth of strain 11-3 was better than that of the wild type, and its biomass increased by 1.24 times (2.02 ± 0.087 g·L) after 204 h. -1 vs 1.63±0.033g·L-1 ), and absorbance also showed similar results. Significant differences were observed after 84 hours of culture (p < 0.01), indicating enhanced metabolic adaptability to high methanol concentrations.

[0071] At the beginning of the culture period, the chlorophyll a content of each group did not change much, ranging from 0.07 to 0.1 g·L at 12 h. -1 The concentration reached its peak at 132 hours, and the group with methanol addition had the highest content, which was the wild type (0.233±0.013mg·L -1 ), wild type + methanol (0.413 ± 0.013 mg·L -1 )、11-3(0.277±0.007mg·L -1 ) and 11-3 + methanol (0.500 mg·L -1 After 132 hours, the chlorophyll a content in each group decreased. The group without methanol addition recovered to a near-initial value at 204 hours, while the group with methanol addition maintained a higher level.

[0072] Under methanol stress, the maximum photochemical quantum yield (Fv / Fm) of both strains decreased. However, at the end of the experiment, strain 11-3 maintained a significantly higher photosynthetic capacity (0.40±0.02 vs. 0.30±0.02). This increased photosynthetic resilience may be due to enhanced protective mechanisms against methanol-induced stress, potentially involving alterations in thylakoid membrane integrity, electron transport efficiency, or antioxidant capacity.

[0073] Example 3 Comparison of carbon and nitrogen utilization by wild-type and mutant algae strains

[0074] Nitrogen utilization analysis

[0075] During the cultivation process of each treatment in Example 2, the total nitrogen content in the culture medium was measured using a multi N / C 3100 total organic carbon analyzer (Analytical Instruments Jena, Germany) to evaluate nitrogen utilization capacity. The supernatant of the centrifuged sample was filtered (0.22 μm), and 200 μL was taken for three analyses.

[0076] Methanol content analysis

[0077] Methanol quantification was performed using gas chromatography (GC; Agilent 7890B, USA) equipped with a flame ionization detector and a capillary column. Nitrogen was used as the carrier gas, and the injection volume was 1 μl. Operating parameters included oven temperature (150°C) and injector / detector temperature (180°C). A 3 mL culture sample was centrifuged (6000 rpm, 5 minutes), filtered (0.22 μm), and residual methanol in the supernatant was quantified by comparison with a standard. To assess methanol volatilization, control flasks containing culture medium and methanol were maintained under the same conditions without inoculation of algal cells.

[0078] Result Analysis

[0079] Figure 3 Figure 3. Carbon and nitrogen utilization of wild-type and mutant algae strains. (a) Residual methanol concentration in the culture medium; (b) Residual total nitrogen concentration in the culture medium. Data are presented as mean ± SD (n = 3). Different lowercase superscript letters indicate significant differences between treatments (P < 0.05) based on one-way analysis of variance and LSD test.

[0080] The results are as follows Figure 3 As shown, total nitrogen (TN) analysis at an initial concentration of 494.12 mg / L indicated that nitrogen assimilation and methanol metabolism were synergistically enhanced. The 11-3 + methanol group maintained the lowest residual TN throughout the incubation period. Terminal TN values ​​(204 hours) were: 190.3 ± 4.6 mg / L (wild type), 168.35 ± 1.85 mg / L (wild type + methanol), 190.3 ± 4.6 mg / L (11-3), and 110.9 ± 8.9 mg / L (11-3 + methanol). Although nitrogen consumption was similar between the wild type and unsupplemented 11-3 (both 303.82 ± 5.4 mg / L), methanol addition significantly enhanced nitrogen assimilation in the mutant strain. Nitrogen consumption in the 11-3 + methanol group was 17.60% greater than in the wild type + methanol group and 26.09% greater than in the 11-3 group without methanol, indicating metabolic coupling between C1 metabolism and nitrogen assimilation pathways.

[0081] Methanol residual analysis showed that strain 11-3 had significantly improved methanol utilization compared to the wild-type strain. Starting from the early stages of incubation (36 hours), the methanol concentration in the 11-3 group was consistently lower than that in the wild-type strain and the methanol control group. By the end of incubation (204 hours), the residual methanol concentrations were 6.23 g / L (control), 4.92 ± 0.19 g / L (wild-type strain), and 3.23 ± 0.17 g / L (11-3), respectively. The methanol consumption of the mutant strain was 34.32% higher than that of the wild-type strain and 48.19% higher than that of the control, indicating that ARTP mutagenesis significantly enhanced C1 metabolic capacity.

[0082] Example 4 Comparison of Carbohydrate Content and Protein Content

[0083] Cell component analysis methods

[0084] During the cultivation process for each treatment in Example 2, carbohydrates were quantified using the anthrone-sulfuric acid method, with absorbance measured at 625 nm. Yield and productivity were calculated based on a glucose standard curve. Protein was determined using the Bradford method. An equal volume (0.5 mL) of Bradford reagent and diluted sample were mixed, incubated at room temperature for 5 minutes, and absorbance was measured at 595 nm. Concentrations were calculated using a bovine serum albumin standard curve.

[0085] Result Analysis

[0086] Figure 4 Figure 2 shows the biochemical composition of wild-type and mutant algae strains under different culture conditions. (a) shows carbohydrate content (percentage of dry weight); (b) shows protein content (percentage of dry weight). Data are presented as mean ± SD (n = 3). Different letters at each time point indicate significant differences between treatments (P < 0.05) based on one-way analysis of variance and LSD test.

[0087] like Figure 4 As shown, since the air was not isolated, the algal cells were still able to use CO2 in the air for photosynthesis and accumulate carbon sources during the culture process. In the absence of a carbon source, both algal strains accumulated carbohydrates (wild type: 25.66% ± 0.98% at 180 hours, 11-3: 29.93% ± 1.67% at 180 hours) and protein (wild type: 30.13% ± 0.32% at 180 hours, 11-3: 30.27% ± 0.47% at 180 hours) over time, with no significant difference in protein.

[0088] Under the action of methanol, both showed higher accumulation: at 180 hours, carbohydrates reached 30.20% ± 0.09% (wild type + methanol) and 39.90% ± 0.28% (11-3 + methanol), increases of 17.71% and 33.31% respectively compared to the carbon-free control, while protein reached 32.82% ± 1.45% (wild type + methanol) and 40.09% ± 0.27% (11-3 + methanol), increases of 8.93% and 32.45%, respectively. Notably, the 11-3 + methanol group showed better methanol carbon assimilation compared to the wild type + methanol group, with a 32.10% higher carbohydrate content and a 22.15% higher protein synthesis. The simultaneous enhancement of both biomass components indicates a comprehensive improvement in carbon fixation efficiency, rather than simply a change in the distribution of metabolic flux between cellular components.

[0089] Example 5: Changes in fatty acid profiles of wild-type and mutant algae strains

[0090] Lipid extraction and analysis methods

[0091] During the cultivation process for each treatment in Example 2, lipids were extracted using the chloroform-methanol method. Approximately 10 mg of freeze-dried algal biomass was homogenized with 2 mL of chloroform / methanol (2:1, v / v), centrifuged (4000 rpm, 10 minutes), and the lower phase was collected. After multiple extractions, the extracts were combined and evaporated under nitrogen before gravimetric determination.

[0092] Fatty acid profile analysis was performed using gas chromatography-mass spectrometry (GC-MS) (Agilent 7890B-5977A, USA) using a DB-23 capillary column (60 m × 0.25 mm × 0.25 μm). The extracted lipids were transesterified with 1% H2SO4 in methanol (85°C, 2.5 hours). FAMEs were extracted with hexane and analyzed using helium as a carrier gas (1 mL / min). Temperature program: 50°C (1 minute), increased to 175°C (25°C / minute), then increased to 230°C (4°C / minute) and maintained for 5 minutes. FAMEs were identified by comparison with authentic standards (Sigma-Aldrich, USA).

[0093] Lipid content was calculated as follows: Lipid content (mg / g) = crude lipid weight / cell dry weight

[0094] Result Analysis

[0095] Figure 5 Figure 4. Changes in fatty acid profiles and pigmentation in wild-type and mutant strains. (a) Fatty acid content at different time points; (b) Changes in total saturated and unsaturated fatty acids during cultivation. Data are presented as mean ± SD (n = 3).

[0096] Fatty acid composition analysis revealed significant metabolic adaptability in the methanol-supplemented microalgae strains. Both the wild-type and mutant strain 11-3 exhibited dynamic fatty acid profile changes, with the major fatty acids including C16:0, C18:0, C18:1, C18:2, and C18:3. Methanol supplementation significantly increased the proportions of C18:1 and C18:2 in both strains. By day 10, strain 11-3 supplemented with methanol accumulated significantly more C18:1 (23.5 ± 1.2% of total fatty acids) than the wild-type strain supplemented with methanol (18.7 ± 0.9%).

[0097] Unsaturation analysis showed that lipid unsaturation was enhanced in cultures supplemented with methanol. By day 10, the UFA / SFA ratio in 11-3 + methanol reached 1.82 ± 0.11, significantly exceeding the ratios in wild-type + methanol (1.52 ± 0.08) and cultures without methanol supplementation (wild-type: 1.36 ± 0.07; 11-3: 1.42 ± 0.05).

[0098] This enhanced lipid unsaturation represents a key adaptive mechanism for maintaining membrane integrity against methanol-induced damage. The preferential accumulation of C18:1 and C18:2 in strain 11-3 suggests alterations in the fatty acid biosynthesis pathway, likely involving upregulated desaturase activity. These membrane lipid changes may contribute to the enhanced photosynthetic performance of strain 11-3, as unsaturated fatty acids maintain thylakoid membrane functionality under stress conditions, consistent with the observed elevated Fv / Fm ratio.

[0099] Example 6 Comparison of amino acid composition and carotenoids

[0100] Key gene expression analysis

[0101] Total RNA extraction: TRIzol reagent was used to extract total algal RNA, and PrimeScript TM The RT Reagent Kit (including gDNA Eraser) was used to remove genomic DNA contamination. Reverse transcription and cDNA synthesis were completed using the RT Primer Mix as the primer system and incubated at 37°C for 15 minutes.

[0102] qRT-PCR quantitative analysis: Actin gene was used as an internal reference, and the target gene transcription level was detected on the QuantStudio 6Flex real-time fluorescence quantitative PCR instrument (Applied Biosystems, USA) using SYBR Premix Ex Taq Kit (TakaraBio). The reaction conditions are as follows: 1) pre-denaturation: 95℃ for 30 seconds; 2) cyclic amplification (40 cycles): 95℃ for 10 seconds → 60℃ for 30 seconds; 3) melting curve analysis: continuous heating from 60-95℃ (0.3℃ / second). All experimental samples were set up for three biological replicates, and the relative expression of genes was measured by 2 -ΔΔ The Ct method was used for calculation. The primer sequences used are shown in Table 1.

[0103] Table 1. Primer sequences used for qRT-PCR analysis of gene expression in Lipococcus

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[0106] Amino acid composition analysis method

[0107] Amino acid composition was determined by HPLC (SHIMADZU LC-2030C 3D, Japan). Lyophilized biomass (10 mg) was hydrolyzed with 6 M HCl (110° C., 24 h, vacuum), filtered, evaporated, and redissolved in 0.02 M HCl. Amino acids were derivatized with o-phthalaldehyde and separated using a sodium acetate buffer / acetonitrile gradient on a C18 column (250 mm × 4.6 mm, 5 μm) and quantified using standards.

[0108] Carotenoid analysis methods

[0109] Carotenoids were extracted from freeze-dried biomass (20 mg) using acetone containing 0.1% butylated hydroxytoluene (BHT), sonicated (30 minutes, 4°C), and centrifuged (10,000 rpm, 10 minutes). The filtered supernatant was analyzed by high performance liquid chromatography (LC-2030C 3D, SHIMADZU, Japan) using a diode array detector on a C30 column and a methanol / methyl tert-butyl ether / water gradient. Carotenoids were identified by comparing their retention time and absorption spectra with authentic standards (astaxanthin, canthaxanthin, and zeaxanthin from Sigma-Aldrich) and quantified using a calibration curve.

[0110] Result Analysis

[0111] Figure 5 Figure 2. Changes in fatty acid profiles and pigmentation in wild-type and mutant strains. (c) Phenotype and cell morphology of Lipococcoid algae on days 7 (left) and 15 (right) of methanol exposure. (d) Pigment accumulation (μg / g dry weight) in wild-type and mutant strains on days 7 and 15. Data: mean ± SD (n = 3). Different letters indicate significant differences (P < 0.05) as determined by one-way analysis of variance (ANOVA) and LSD tests. (e) Carotenoid profile showing astaxanthin (peak a), zeaxanthin (peak b), and canthaxanthin (peak c).

[0112] Pigment synthesis in microalgae represents a key physiological adaptation mechanism with multiple functions, including photosynthetic light harvesting, photoprotection, and cellular defense against oxidative stress. Comparative analysis of the pigment profiles of wild-type and strain 11-3 under methanol supplementation revealed distinct biphasic response patterns. During the early culture period (day 7), zeaxanthin dominated in both strains, with concentrations significantly elevated in methanol-supplemented cultures. This xanthophyll cycle carotenoid acts as a photoprotectant, alleviating photoinhibition by enhancing PSII repair mechanisms and singlet oxygen reduction. Methanol-induced zeaxanthin accumulation indicates activation of photoprotective mechanisms against potential oxidative stress, with zeaxanthin promoting thermal dissipation of excitation energy via a non-photochemical quenching pathway.

[0113] By day 15, a distinct shift in pigment composition occurred, characterized by a decrease in zeaxanthin concentration, coinciding with the accumulation of ketocarotenoids (astaxanthin and canthaxanthin) under all experimental conditions. This biochemical shift corresponded to a visible change in culture color from green to orange. Strain 11-3 exhibited higher astaxanthin production than the wild type under both methanol-free (0.916±0.053 vs. 0.733±0.048 mg / g) and methanol-supplemented conditions (0.803±0.049 vs. 0.591±0.041 mg / g). Notably, methanol supplementation reduced astaxanthin accumulation in both strains while differentially affecting canthaxanthin synthesis—a decrease in the wild type and an increase in strain 11-3.

[0114] The contrasting effects of zeaxanthin and astaxanthin suggest a complex, biphasic response to methanol supplementation in cells. The initial increase in zeaxanthin may represent an adaptive photoprotective mechanism against methanol-induced mild oxidative stress. The subsequent decrease in astaxanthin accumulation under sustained methanol exposure may reflect a reduction in cellular stress through efficient methanol assimilation or a shift in carbon flux away from secondary carotenoid synthesis and toward primary metabolism.

[0115] To elucidate the molecular mechanism of enhanced methanol utilization, this study used qRT-PCR to analyze the transcript levels of five key enzymes in central carbon metabolism: fructose-1,6-bisphosphatase (FBP), phosphoglycerate kinase (PGK), glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICD), and malate dehydrogenase (MDH). These enzymes represent key regulatory nodes in glycolysis (FBP, PGK), the pentose phosphate pathway (G6PDH), and the tricarboxylic acid cycle (ICD, MDH), respectively.

[0116] Figure 6 qRT-PCR analysis of methanol-derived amino acid synthesis in wild-type and mutant strains. Significant differences between groups are indicated with asterisks (*: P < 0.05; ***: P < 0.001; ****: P < 0.0001). (a) Differential transcription levels of five key genes in the wild-type strain after 60 hours of treatment with 10 g / L methanol (compared to the control); (b) Differential transcription levels of five key genes in the mutant strain 11-3 after 60 hours of treatment with 10 g / L methanol (compared to the control); (c) Amino acid composition of the wild-type and mutant strains under different treatment groups on day 15 of culture.

[0117] According to the test results, (1) the metabolic inhibition of the wild-type strain was found. After exposure to methanol, the wild-type strain significantly downregulated the expression of key metabolic genes: FBP (0.36 times), G6PDH (0.42 times), and MDH (0.31 times). Figure 6a); ICD is the only enzyme with stable expression; this phenomenon is consistent with the global transcriptional repression pattern induced by methanol stress in methylotrophic bacteria (Stepanov and Zolotareva, 2015). (2) Metabolic remodeling of mutant 11-3. The mutant showed significant gene activation under methanol conditions: FBP (1.11 times), PGK (0.55 times), G6PDH (1.03 times), and MDH (1.59 times) were all upregulated ( Figure 6 b) Enhanced expression of the key initial oxidase, methanol dehydrogenase (MDH), promotes the improvement of formaldehyde production efficiency;

[0118] The mutant assimilates the methanol carbon skeleton through two pathways: an oxidative decarboxylation pathway: formaldehyde → formate → CO₂ (catalyzed by formate dehydrogenase), followed by refixation via the Calvin cycle; and a one-carbon assimilation pathway: formaldehyde is incorporated into serine metabolism via tetrahydrofolate. This is supported by the significant increase in serine content in the experimental data. References: The methanol oxidation pathway is based on a metabolic model of methylotrophic bacteria (Stepanov and Zolotareva, 2015); the carbon flow allocation mechanism is based on a metabolic remodeling strategy for engineered cyanobacteria (Xiong et al., 2015).

[0119] Amino acid profile analysis ( Figure 6 c) shows significant qualitative differences between wild-type and strain 11-3 of the present invention under methanol supplementation. Both strains synthesized a complete essential amino acid profile (38.2-45.6% of total protein), exceeding the FAO / WHO nutritional recommendation of 33.9% for human nutrition. However, the strains exhibited distinct metabolic patterns in response to methanol supplementation.

[0120] Wild-type cultures showed preferential enhancement of branched-chain amino acid biosynthesis, while lysine and methionine levels decreased. In contrast, strain 11-3 of the present invention maintained elevated levels of all essential amino acids, with particularly significant increases in lysine (6.8% vs. 5.2% in wild-type + methanol), methionine (3.1% vs. 2.2% in wild-type + methanol), and tryptophan (1.8% vs. 1.2% in wild-type + methanol).

[0121] Under all experimental conditions, glutamate and aspartate dominated the non-essential amino acids (accounting for 20-25% of the total amino acid content). Addition of methanol to strain 11-3 of the present invention resulted in increased accumulation of proline and alanine, while simultaneously decreasing glycine. The increased methionine content in 11-3 + methanol (41% higher than the wild-type + methanol) suggests an altered sulfur assimilation pathway in the mutant strain.

[0122] From a nutritional perspective, the amino acid profile of the present algal strain 11-3 under methanol supplementation is advantageous compared to traditional protein sources, exhibiting a balanced essential amino acid composition and having sufficient levels of commonly limiting amino acids such as lysine and methionine, enhancing its potential for producing high-quality protein in food and feed applications.

[0123] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A methanol-resistant mutant of Lipococcus emersonii, characterized in that: The methanol-resistant Lipococcus emersonii mutant strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCM 20232483.

2. A method for screening methanol-resistant Lipococcus emersonii mutants, characterized in that: The steps include: Step 1) subjecting a suspension containing Lipococcus emersonii cells to atmospheric pressure and room temperature plasma mutagenesis to obtain a mutagenized suspension; Step 2) inoculating the mutagenized suspension onto a methanol-containing culture medium for culturing, and screening for methanol-resistant Lipococcus emersonii mutants.

3. The method for screening a methanol-resistant Lipococcus emersonii mutant according to claim 2, wherein: In the step 1), a suspension containing Lipococcus emersonii cells is subjected to atmospheric pressure and room temperature plasma mutagenesis. The treatment steps specifically include: using helium as the plasma working gas, and the mutagenesis conditions are: radio frequency power of 100-150W, helium flow rate of 8-12L·min-1, the distance between the plasma torch nozzle outlet and the sample plate is 1-3mm; the plasma jet temperature is 25°C to 35°C, and the treatment time is 10 to 17 seconds.

4. The method for screening a methanol-resistant Lipococcus emersonii mutant according to claim 2, wherein: The methanol content of the culture medium containing methanol in step 2) is 8 g·L-1; Preferably, the culture medium is Endo solid culture medium.

5. The method for screening a methanol-resistant Lipococcus emersonii mutant according to claim 2, wherein: In the step 2), after inoculation, the culture is carried out at 22-28° C., 30-60 μmol·m-2·s-1 light intensity and 12:12 hour light: dark cycle for 3-7 days to screen and obtain methanol-resistant Lipococcus emersonii mutants.

6. The method for screening a methanol-resistant Lipococcus emersonii mutant according to claim 2, wherein: The method further comprises: Step 3) The methanol-resistant Emersonii Lipococcus mutants screened in step 2) are inoculated into a medium containing methanol for cultivation, the growth rate of each mutant is measured, and the methanol-resistant Emersonii Lipococcus mutant with the highest growth rate is screened; preferably, the medium is Endo liquid medium.

7. A preparation, characterized in that The invention comprises the methanol-resistant Lipococcus emersonii mutant strain according to claim 1.

8. Use of the methanol-resistant Lipococcus emersonii mutant according to claim 1 or the preparation according to claim 7 in protein production, carotenoid production, industrial methanol bioconversion, high-protein microalgae feed production, wastewater resource treatment, and biorefining.

9. The use according to claim 8, characterized in that The carotenoids include at least one of astaxanthin and canthaxanthin.

10. The use according to claim 8, characterized in that In the process of producing protein and carotenoid, methanol is used as carbon source.

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