Kluyveromyces marxianus and application thereof

Abstract

The invention discloses a Kluyveromyces marxianus strain and an application of the Kluyveromyces marxianus strain, and belongs to the field of synthetic biology and microorganism manufacturing. The strain has the characteristics of efficiently utilizing xylose and growing under a low-oxygen condition, and by taking the strain as a fermentation strain, the protein content of fermented straw powder is remarkably improved through the synergistic effect of the strain and cellulase under a straw powder high-solid fermentation condition. According to the yeast strain and the high-solid fermentation process thereof disclosed by the invention, the protein content of straw powder can be greatly increased, and an effective solution is provided for high-valued reutilization of straw agricultural wastes.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and microbial manufacturing, specifically involving a natural yeast strain with efficient xylose metabolism and low-oxygen adaptability and its application in high-solids fermentation of straw powder. Background Technology

[0002] Straw is an abundant agricultural waste and one of the most important renewable resources in nature. Its efficient utilization is crucial for the development of a sustainable bioeconomy. The main components of straw, cellulose and hemicellulose, can be hydrolyzed into fermentable sugars such as glucose and xylose, which can then be converted by microorganisms to produce high-value-added products such as fuel ethanol, single-cell proteins, or chemicals. However, existing industrial strains have limited xylose assimilation capacity and exhibit inhibitory effects on glucose catabolites, resulting in low microbial conversion efficiency of xylose. This limits its economic feasibility in biorefining processes, and achieving efficient co-utilization of glucose and xylose by microorganisms remains a significant challenge.

[0003] To address this challenge, traditional strategies typically involve genetically engineering conventional industrial strains to introduce or enhance their xylose metabolic pathways, thereby utilizing the monosaccharides produced after saccharification to convert them into the target product. This stepwise saccharification-fermentation process is not only complex and costly, but also suffers from inhibitors (such as furfural and phenolic compounds) generated during straw hydrolysis that suppress cellulase activity and hinder microbial growth, further increasing the process difficulty. Therefore, identifying microbial strains that can naturally and efficiently co-utilize glucose and xylose and tolerate inhibitors in straw hydrolysate is a key strategy for simplifying the process, reducing production costs, and achieving high-value reuse of straw. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Kluyveromyces marxianus that naturally possesses xylose utilization capabilities, can grow under low-oxygen conditions, and can be used for fermentation of straw powder, significantly increasing the protein content of the straw powder.

[0005] This invention provides a strain of Kluyveromyces marxianus Km-DYGE1. The Kluyveromyces marxianus Km-DYGE1 was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36000.

[0006] The Kluyveromyces martensii Km-DYGE1 provided by this invention can grow in a culture medium with xylose as the sole carbon source and tolerate low-oxygen environments.

[0007] The present invention provides a method for synthesizing products using the aforementioned Kluyveromyces martensii, including but not limited to Kluyveromyces martensii Km-DYGE1 and its metabolites and ferments.

[0008] The metabolites in the product include, but are not limited to, small molecule chemicals and biological macromolecule nutrients.

[0009] The small molecule chemicals include, but are not limited to, terpenoids, flavonoid polyphenols, non-flavonoid polyphenols, organic acids, and beet pigments;

[0010] The biological macromolecular nutrients are starch, lipids, and proteins;

[0011] Preferably, the terpene compound is β-carotene.

[0012] Preferably, the flavonoid polyphenol compound is any one of naringenin, quercetin, or taxanein.

[0013] Preferably, the non-flavonoid polyphenolic compound is resveratrol or polydipsia glycoside.

[0014] Preferably, the organic acids include, but are not limited to, coumaric acid, salicylic acid, mucoaceric acid, succinic acid, malic acid, itaconic acid, and lactic acid.

[0015] Preferably, the beet pigment is betaine.

[0016] The present invention further provides a straw microbial protein product obtained by high solids fermentation of Km-DYGE1 and straw powder and its fermentation method.

[0017] Specifically, the preparation method of the fermented straw powder includes the following steps:

[0018] 1) Pre-treat the straw powder by steam explosion, alkaline hydrolysis, and acid hydrolysis, and add 10%-50% (v / w) of pH adjuster to adjust the pH to 7;

[0019] 2) Add 50%-90% (v / w) straw powder bacterial enzyme mixture (containing yeast and cellulase), and let it ferment statically;

[0020] 3) Fermented straw powder can be obtained by freeze-drying the fermented material.

[0021] Specifically, in step 2), the cellulase concentration in the bacterial-enzyme mixture is 15-25 FPU / mL, and the Kluyveromyces martensii concentration is 0.8-1.2 OD. 600 / mL, static fermentation temperature is 25-35℃, fermentation time is 36-60 h.

[0022] Preferably, in step 2), the cellulase concentration is 20 FPU / mL and the Kluyveromyces concentration is 1 OD. 600 / mL, static fermentation temperature is 30℃, fermentation time is 48 h.

[0023] More specifically, each liter of the bacterial enzyme mixture contains 7.5 g ammonium sulfate, 14.4 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 1 mL vitamin solution, and 2 mL Trace solution.

[0024] In practice, the protein content in the fermented straw powder is not less than 3.7%.

[0025] This invention provides a strain of *Kluyveromyces martensii* that can efficiently utilize xylose and is tolerant of low oxygen conditions. Co-fermentation of this strain with straw powder under high-solids conditions can significantly increase the protein content of the straw powder, making it suitable for the industrial utilization of straw waste. Attached Figure Description

[0026] Figure 1 Biomass of different yeast strains when xylose is the sole carbon source. Sc: Saccharomyces cerevisiae; Yl: Yersinia lipolytica; Pp: Pichia pastoris; Km-DYGE0: Kluyveromyces martensii; Km-DYGE1: Kluyveromyces martensii.

[0027] Figure 2 Maximum biomass of different yeast strains under hypoxic conditions.

[0028] Figure 3 Growth curves of strain Km-DYGE1 in different lignocellulose hydrolysate media.

[0029] Figure 4 Changes in monosaccharide content in hydrolysate of strain Km-DYGE1 before and after culture.

[0030] Figure 5 Changes in monosaccharide content in straw powder before and after high-solids co-fermentation.

[0031] Figure 6 Changes in protein content in straw powder before and after high-solids co-fermentation.

[0032] Information on the preservation of biological materials:

[0033] The Kluyveromyces marxianus of this invention was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China), with accession number CGMCC No. 36000 and classification name Kluyveromyces marxianus. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0035] Example 1: Evaluation of the growth performance of different yeast strains under xylose as the sole carbon source

[0036] Carbon-free mineral salt culture medium: Each liter contains 7.5 g ammonium sulfate, 14.4 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 1 mL vitamin solution and 2 mL Trace solution.

[0037] Different yeast strains were activated on YPD plates and then pre-cultured in a mineral salt medium with glucose as the sole carbon source, using OD... 600 =0.05 was transferred to a mineral salt medium with xylose as the sole carbon source and cultured for 70 h. The absorbance at 600 nm was measured (OD). 600 ), the result is as follows Figure 1 As shown.

[0038] Experimental results showed that *Saccharomyces cerevisiae*, *Yersinia lipolytica*, and *Pichia pastoris* could not grow when xylose was the sole carbon source, while *Kluyveromyces martensii* could grow. Among them, strain Km-DYGE0, after 70 hours of cultivation, showed a high OD... 600 The OD value of strain Km-DYGE1 was 5.8. 600 The result of 9.2 indicates that Km-DYGE1 has better xylose utilization.

[0039] The Km-DYGE1 strain was obtained as follows: Fruit samples from Tianjin were mixed and appropriately diluted using carbon-free mineral salt medium. The diluted bacterial solution was spread on YPD solid medium and incubated at 30℃ for 24-48 hours. After single, independent colonies appeared on the plates, milky white, round, well-defined, opaque, and smooth yeast colonies were selected for repeated streak purification. Single colonies were picked and observed under an inverted microscope. Genomic DNA was extracted from oval or round colonies with a size of approximately 3-5 μm. The ITS rDNA region was amplified and sequenced using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The nucleic acid sequence was compared with existing sequences in the GenBank database, and the strain was identified as Kluyveromyces marxianus, and named Kluyveromyces marxianus Km-DYGE1.

[0040] The nucleotide sequence of the amplified product is shown in SEQ ID NO.1.

[0041] Example 2: Growth of different yeast strains under hypoxic conditions

[0042] Different types of *Yersinia lipolyticis* and *Kluyveromyces martensii* were inoculated into a mineral salt medium with glucose as the sole carbon source. 1 mL of liquid paraffin was added to the medium, and the mixture was incubated at 30°C for 24 h. After centrifugation at 2000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in mineral salt medium according to OD... 600 The inoculum was transferred again to a mineral salt medium with glucose as the sole carbon source at an inoculum concentration of 0.05, and 1 mL of liquid paraffin was added. After static incubation at 30°C for 2-3 days, the OD was measured.600 (Results as follows) Figure 2 (As shown).

[0043] The results showed that after culturing under hypoxic conditions for 2-3 days, the OD of *Yarrowia lipolyticis* increased. 600 There was no significant increase compared to the inoculation time, while the OD of the two Kluyveromyces martensii strains... 600 Exceeding 0.5. Among them, the OD of Km-DYGE1... 600 The value was significantly greater than that of Km-DYGE0, indicating that strain Km-DYGE1 possesses the special ability to grow efficiently under low-oxygen conditions.

[0044] Example 3: Growth of strain Km-DYGE1 in different hydrolysates

[0045] Three types of corn stalk hydrolysates were prepared: 1) Hydrolysate 1: obtained by enzymatic hydrolysis after acid treatment; 2) Hydrolysate 2: obtained by direct enzymatic hydrolysis after steam explosion; 3) Hydrolysate 3: obtained by enzymatic hydrolysis after steam explosion and water washing. All three hydrolysates were prepared at 50℃ for 48 hours. The cellulase concentration used in hydrolysate 1 was 10 FPU / g, while the cellulase concentration used in hydrolysates 2 and 3 was 19.65 IU / g.

[0046] Preparation of hydrolysate culture medium: Add ammonium sulfate (7.5 g / L), potassium dihydrogen phosphate (14.4 g / L), magnesium sulfate heptahydrate (0.5 g / L), vitamin solution (1 mL / L) and Trace solution (2 mL / L) to the hydrolysate. After filtration through a 0.22 μm filter membrane for sterilization, the hydrolysate culture medium is obtained.

[0047] Strain strain Km-DYGE1 was inoculated into three different hydrolysate culture media and cultured at 30℃ with shaking at 220 rpm for 5 days. OD was measured periodically. 600 (Results as follows) Figure 3 As shown), and the changes in monosaccharide concentration before and after culture were analyzed (results are shown in Figure 1). Figure 4 (As shown).

[0048] The results showed that Km-DYGE1 could grow in different hydrolysate media, and the OD in hydrolysate-1 medium was [missing information]. 600 ≥8, OD in hydrolysate-2 and hydrolysate-3 media 600≥10. Km-DYGE1 was able to fully utilize glucose and xylose in hydrolysate-1 and hydrolysate-3, with glucose and xylose consumption rates in hydrolysate-2 reaching 99.7% and 96.0%, respectively. Conventional yeast strains exhibit a strict carbon metabolite repression effect; when glucose and xylose are present simultaneously, xylose utilization is significantly delayed and inefficient. These results indicate that Km-DYGE1 strain differs from conventional yeast strains, possessing the ability to simultaneously and efficiently utilize both glucose and xylose, and has high application potential in the efficient bioconversion of straw.

[0049] Example 4: Synergistic utilization of monosaccharides from straw hydrolysis by bacteria and enzymes

[0050] Enzyme solution: A mineral salt culture medium solution without a carbon source, containing 20 FPU / mL of cellulase.

[0051] Strain Km-DYGE1 was inoculated into a mineral salt medium with glucose as the sole carbon source and cultured for 12 h to obtain a seed culture. The seed culture was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded to obtain bacterial cells. The bacterial cells were resuspended in an enzyme solution to obtain a bacterial-enzyme mixture. The concentration of cellulase in the mixture was 20 FPU / mL, and the concentration of Km-DYGE1 was 1 OD. 600 / mL. The straw powder was pretreated by steam explosion, alkaline hydrolysis, and acid hydrolysis, and the pH was adjusted to 7 by adding 10%-50% (v / w) of pH adjuster. Then, 50%-90% (v / w) of the initial straw powder mass (specifically 50% (v / w)) of the bacterial enzyme mixture was added, and the mixture was allowed to ferment at 30℃ for 48 h. The fermented straw powder was washed three times with deionized water, and then freeze-dried to obtain fermented straw powder. The monosaccharide concentration in the first wash water and the protein content of the straw powder were determined (results are shown in the figure). Figure 5 and Figure 6 ).

[0052] like Figure 5 The results showed that, under high-solids conditions, compared with the control group, the glucose concentration of straw powder co-fermented with strain Km-DYGE1 decreased from 2.15 g / L to 0.09 g / L (consumption rate 95.8%), and the xylose concentration decreased from 2.92 g / L to 0.21 g / L (consumption rate 92.7%). Figure 6 The results showed that under high-solids conditions, the protein content of the fermented straw powder increased from 3.13% to 3.99%, a significant increase of 27.5%. This indicates that strain Km-DYGE1 and cellulase have a significant synergistic effect under high-solids conditions, making it an effective method to increase the protein content of straw.

Claims

1. A strain of Kluyveromyces marxianus naturally capable of utilizing xylose and growing under low oxygen conditions, characterized in that, Its preservation number is CGMCC No. 36000.

2. A method of using the synthetic product of Kluyveromyces marxianus according to claim 1, characterized in that, The product includes but is not limited to Kluyveromyces marxianus cells and / or metabolites.

3. The method of claim 2, wherein, The metabolites include but are not limited to small molecule chemicals and biological macromolecular nutrients.

4. The method of claim 3, wherein, The small molecule chemicals include but are not limited to terpenoids, flavonoid polyphenol compounds, non-flavonoid polyphenol compounds, organic acids, betalains; The biological macromolecular nutrients are starch, lipids and proteins.

5. The method of claim 4, wherein, The terpenoids are beta-carotene; The flavonoid polyphenol compounds are any one of naringenin, quercetin or taxifolin; The non-flavonoid polyphenol compounds are resveratrol and polydatin; The organic acids include but are not limited to p-coumaric acid, salicylic acid, muconic acid, succinic acid, malic acid, itaconic acid, lactic acid; The betalains are betacyanins.

6. The method of claim 2, wherein, The product is a straw microbial protein product obtained by fermentation of the Kluyveromyces marxianus mixed with straw powder.

7. The method of claim 6, wherein, The protein content of the fermented straw powder is not less than 3.7%.

8. The method of claim 6, wherein, The preparation method of the fermented straw powder comprises the following steps: 1) steam explosion, alkaline hydrolysis and acid hydrolysis pretreatment of the straw powder, and adding 10%-50% (v / w) of pH adjuster to adjust the pH to 7; 2) adding 50%-90% (v / w) of a mixture of yeast and cellulase to ferment; 3) drying the fermentation product to obtain the fermented straw powder; The mixture of yeast and cellulase contains 7.5 g of ammonium sulfate, 14.4 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 1 mL of vitamin solution and 2 mL of Trace solution per liter.

9. The method of claim 8, wherein, Step 2) the concentration of cellulase in the mixture is 15-25 FPU / mL, the concentration of K. marxianus is 0.8-1.2 OD / mL, the temperature of static fermentation is 25-35 °C, and the fermentation time is 36-60 h; preferably, in step 2) the concentration of cellulase is 20 FPU / mL, the concentration of K. marxianus is 1 OD / mL, the temperature of static fermentation is 30 °C, and the fermentation time is 48 h. 600 Step 2) the concentration of cellulase in the mixture is 15-25 FPU / mL, the concentration of K. marxianus is 0.8-1.2 OD / mL, the temperature of static fermentation is 25-35 °C, and the fermentation time is 36-60 h; preferably, in step 2) the concentration of cellulase is 20 FPU / mL, the concentration of K. marxianus is 1 OD / mL, the temperature of static fermentation is 30 °C, and the fermentation time is 48 h. 600 Step 2) the concentration of cellulase in the mixture is 15-25 FPU / mL, the concentration of K. marxianus is 0.8-1.

10. The product obtained by the method of any one of claims 2 to 9.

Images