Pichia pastoris strain XUT4-6, and preparation method and application thereof

Abstract

The application discloses a Pichia pastoris strain XUT4-6 and a preparation method and application thereof, belongs to the field of microbial metabolic engineering and industrial biotechnology, and introduces a xylose isomerase XI and a xylose kinase XK metabolic pathway in the Pichia pastoris, introduces a xylose transporter XUT4, and simultaneously mediates continuous transmembrane transport of xylose under the condition that glucose exists; the logarithmic growth period continuous passage adaptive evolution is carried out with xylose as the only carbon source, and the evolution strain XUT4-6 is obtained through screening with the combined index of the xylose consumption rate and the biomass growth, the strain XUT4-6 has 362 single nucleotide polymorphism site differences in the whole genome compared with the starting strain DF2, can continuously and efficiently co-utilize xylose under the condition that glucose and xylose coexist, and can be used for mixed sugar co-fermentation of lignocellulose hydrolysate, production of biomass, biofuel and bio-based chemicals.

Description

Technical Field

[0001] This invention relates to the fields of microbial metabolic engineering and industrial biotechnology. More specifically, this invention relates to a Pichia pastoris strain XUT4-6, its preparation method, and its applications. Background Technology

[0002] Xylose is one of the most important monosaccharide components of hemicellulose, widely found in lignocellulose raw materials such as corn cobs, wheat straw, rice husks, sugarcane bagasse, and wood processing byproducts, typically accounting for 20% to 35% of the raw material's dry weight. With the gradual depletion of fossil resources and the rapid development of biomanufacturing technology, the production of biofuels, bio-based chemicals, and high-value-added compounds from lignocellulose has become an important direction for the green bioeconomy, and achieving efficient bioutilization of xylose is a key step. *Pichia pastoris* (Komagataella phaffii) possesses advantages such as strong protein secretion capacity, a mature genetic manipulation system, low culture costs, and efficient expression of exogenous proteins, making it an important chassis cell in the field of industrial biomanufacturing. However, *Pichia pastoris* naturally lacks the ability to efficiently utilize xylose; its endogenous xylose metabolic flux is low, making it difficult to support efficient growth and biosynthesis using xylose as the primary carbon source, thus limiting its application in the field of lignocellulose biotransformation.

[0003] Existing technologies have extensively explored the modification of Pichia pastoris using the xylose isomerase XI pathway to endow it with xylose utilization capabilities. For example, in their 2015 article "Construction of efficient xylose utilizing Pichia pastoris for industrial enzyme production" published in Microbial Cell Factories, Volume 14, Issue 22, Li Pengfei et al. introduced the xylose isomerase XI gene from the anaerobic rumen fungus Orpinomyces and the endogenous xylulose kinase XK gene into Pichia pastoris GS115. This allowed xylose to be isomerized to xylulose via xylose isomerase catalysis, and xylulose was then phosphorylated by xylulose kinase into the pentose phosphate pathway. This achieved functional expression of the xylose isomerase pathway in Pichia pastoris. Furthermore, through adaptive evolution via 50 generations of sequential batch culture, they obtained an evolved strain with enhanced xylose assimilation capabilities, whose cell yield on xylose was comparable to that on glucose or glycerol.

[0004] In addition, a patent application filed by Zhou Yongjin, Zhang Kun, and others from the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, entitled "An Engineered Pasteurella Saccharomyces cerevisiae Utilizing Xylose as the Sole Carbon Source and Its Construction Method and Application," with application number CN202411745878.8, was published on June 2, 2026. This patent application reconstructs the xylose assimilation pathway in the Pichia pastoris host strain by introducing genes such as xylose isomerase XI, xylulose kinase XK pathway, and the non-oxidative phase of the pentose phosphate pathway into the host strain. The engineered strain is then cultured in a culture system utilizing xylose as the sole carbon source to obtain a strain that can utilize xylose as the sole carbon source, thereby improving xylose utilization capacity.

[0005] While the aforementioned existing technologies have achieved preliminary xylose utilization in Pichia pastoris, they still have the following shortcomings. First, most existing technologies focus only on introducing xylose isomerase XI and xylose kinase XK metabolic pathways to endow strains with basic xylose utilization capabilities, while neglecting the systematic enhancement of xylose transmembrane transport capabilities. This results in strains in a glucose-xylose coexisting carbon source system where xylose uptake is easily competitively inhibited by glucose, often preferentially utilizing glucose while delaying or inhibiting xylose utilization, leading to low efficiency in mixed sugar co-fermentation. Second, existing technologies mostly employ a simple continuous passaging strategy with fixed time intervals for adaptive evolution, lacking refined evolutionary and screening rules based on combined indicators such as logarithmic growth phase, xylose consumption rate, and biomass growth. This limits evolutionary efficiency, and there is a lack of systematic analysis of the genomic variation characteristics of the resulting evolved strains, making it difficult to obtain strains with clear genetic backgrounds and stable performance. Third, existing technologies have not yet provided a Pichia pastoris evolutionary strain that can continuously and efficiently utilize xylose in the presence of glucose in a mixed sugar system of lignocellulose hydrolysate, and whose genetic traits have been fixed through strain preservation. Summary of the Invention

[0006] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0007] Another objective of this invention is to provide a Pichia pastoris evolutionary strain XUT4-6 with a clear genetic background, significantly enhanced xylose utilization capacity, and the ability to continuously and efficiently co-utilize glucose and xylose in a glucose-xylose co-carbon source system, as well as its preparation method and application.

[0008] To achieve the objectives and other advantages of this invention, a Pichia pastoris strain XUT4-6 is provided, classified and named Komagataella phaffii XUT4-6, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38857, and deposited on May 25, 2026.

[0009] Preferably, compared with the originating strain DF2, there are 362 single nucleotide polymorphism sites that differ in the whole genome; of which 159 are synonymous mutations and 213 are non-synonymous mutations; among the 213 non-synonymous mutations, 36 are located in gene coding regions with functional annotations, involving 19 coding genes.

[0010] The present invention also provides a method for preparing the above-mentioned Pichia pastoris strain XUT4-6, comprising:

[0011] Step 1: Introduce the xylose isomerase metabolic pathway into Pichia pastoris. The xylose isomerase metabolic pathway includes xylose isomerase XI and xylose kinase XK. Xylose is isomerized by xylose isomerase XI to produce xylulose, and xylulose is phosphorylated by xylose kinase XK to enter the pentose phosphate pathway.

[0012] Step 2: Introduce xylose transporter XUT4 into Pichia pastoris. XUT4 mediates the transmembrane transport of xylose and maintains the continuous transport of xylose in the presence of glucose in a carbon source system where glucose and xylose coexist.

[0013] Step 3: Using xylose as the sole carbon source, recombinant Pichia pastoris simultaneously expressing xylose isomerase XI, xylose kinase XK, and xylose transporter XUT4 was subjected to continuous passage adaptive evolution. During the adaptive evolution process, the OD of the bacterial culture was used as the starting point. 600 Reaching a preset logarithmic growth phase threshold is used as the transition trigger condition, ensuring that each transition is in the logarithmic growth phase;

[0014] Step 4: Using the combined index of xylose consumption rate and biomass growth as screening criteria, we screen for evolutionary strains with enhanced xylose utilization capacity from adaptive evolutionary communities.

[0015] Preferably, the amino acid sequence of XI is shown in SEQ ID NO:2; the amino acid sequence of XK is shown in SEQ ID NO:4; and the amino acid sequence of XUT4 is shown in SEQ ID NO:6.

[0016] Preferably, xylose isomerase XI is derived from plant fermentation bacteria (Lachnoclostridium phytofermentans), xylose kinase XK is derived from Pichia stipitis, and xylose transporter XUT4 is derived from Pichia stipitis.

[0017] Preferably, in a carbon source system where glucose and xylose coexist, the glucose concentration in the culture medium drops to a preset threshold as the triggering condition for xylose utilization initiation, and the strain initiates xylose utilization while glucose is still present.

[0018] Preferably, the termination criteria for adaptive evolution include:

[0019] During continuous subculturing and adaptive evolution, the OD of the bacterial culture was monitored before and after each transfer. 600 The increase and the rate of xylose consumption in the culture medium during each transfer cycle are expressed as bacterial OD. 600 The increase represents the growth rate of the evolutionary strain, and the xylose consumption rate represents the xylose utilization capacity of the evolutionary strain.

[0020] Calculate the OD of the bacterial culture between three consecutive transfers. 600 The changes in the growth rate and the change in the xylose consumption rate will affect the bacterial OD. 600 The changes in the growth rate and the change in the xylose consumption rate were compared with preset stability thresholds; when the bacterial culture OD was between three consecutive transfers... 600 When both the change in growth rate and the change in xylose consumption rate are below the preset stability threshold, it is determined that the xylose utilization capacity and growth rate of the evolved strain have stabilized, and adaptive evolution is terminated.

[0021] The present invention also provides an application of the described Pichia pastoris strain XUT4-6 in the co-fermentation of lignocellulose hydrolysate with mixed sugars.

[0022] The present invention also provides an application of the described Pichia pastoris strain XUT4-6 in the production of biomass, biofuels or bio-based chemicals using xylose or mixed sugars as carbon sources.

[0023] The present invention has at least the following beneficial effects:

[0024] 1. This invention provides an evolutionary strain with a clear genetic background. Compared with the starting strain DF2, Pichia pastoris strain XUT4-6 has 362 single nucleotide polymorphism sites different throughout its genome, of which 159 are synonymous mutations and 213 are non-synonymous mutations. Among the 213 non-synonymous mutations, 36 are located in functionally annotated coding regions, involving 19 coding genes. This clear resolution of the genetic background provides a reliable basis for the quality control of the strain's industrial applications and the protection of intellectual property rights.

[0025] 2. The strain preparation method provided by this invention uses the xylose isomerase XI pathway instead of the xylose reductase and xylose dehydrogenase pathway. Xylose is isomerizes to xylulose catalyzed by xylose isomerase XI, and xylulose is phosphorylated into pentose phosphate pathway by xylose kinase XK. The entire process is independent of redox cofactors, fundamentally avoiding cofactor imbalance and the resulting xylitol accumulation and metabolic disorders, resulting in high carbon atom utilization. The xylose transporter XUT4 is introduced, which mediates the transmembrane transport of xylose and maintains continuous xylose transport in the presence of glucose in a carbon source system where glucose and xylose coexist. This compensates for the low affinity of endogenous sugar transporters in Pichia pastoris for xylose and their susceptibility to glucose inhibition, providing sufficient substrate flux for xylose metabolism. The bacterial culture OD 600 A preset logarithmic growth phase threshold was used as the trigger condition for translocation, allowing for continuous subculturing and adaptive evolution. This ensured that each subculture occurred during the logarithmic growth phase, avoiding growth stagnation and degeneration caused by fixed-interval subculturing. Evolutionary strains were screened using a combined indicator of xylose consumption rate and biomass growth, resulting in high evolutionary efficiency. A preset threshold for glucose concentration was used as the trigger condition for xylose utilization initiation, allowing the strain to initiate xylose utilization while glucose was still present. Glucose concentration was used as a control variable to dynamically maintain xylose utilization ability as glucose concentration increased, thus endowing the strain with glucose-tolerant xylose utilization capacity. The OD of the bacterial culture was monitored between three consecutive translocations. 600 By comparing the changes in growth rate and xylose consumption rate with preset stability thresholds, the evolutionary endpoint can be scientifically determined, ensuring that the xylose utilization capacity and growth rate of the obtained strains have stabilized.

[0026] 3. The Pichia pastoris strain XUT4-6 provided by this invention can continuously and efficiently utilize xylose in the presence of glucose during co-fermentation of mixed sugars in lignocellulose hydrolysate, significantly improving the synergistic utilization efficiency of mixed sugars. It is suitable for producing biomass, biofuels or bio-based chemicals using xylose or mixed sugars as carbon sources. Attached Figure Description

[0027] Figure 1 A comparison of the growth curves of the starting strain GS115 and the recombinant strain DF1 under the condition of 20 g / L xylose as the sole carbon source;

[0028] Figure 2 A comparison of the growth curves of recombinant strains DF1 and DF2 under the condition of 20 g / L xylose as the sole carbon source;

[0029] Figure 3 This is a growth curve of multiple evolved strains over time during the adaptive evolution process in Example 3 of the present invention;

[0030] Figure 4This is a comparison of the growth performance of the XUT4 series recombinant strains isolated after adaptive evolution in Example 3 of the present invention under shake-flask conditions. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0032] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0033] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0034] Example 1: Construction of a Pichia pastoris xylose-utilizing strain based on the xylose isomerase pathway

[0035] The xylose isomerase pathway offers significant technological advantages over the traditional xylose reductase / xylose dehydrogenase (XR / XDH) pathway. This pathway directly converts xylose to xylulose via xylose isomerase, without relying on redox cofactors such as NADH or NADPH. This fundamentally avoids cofactor imbalance and the resulting accumulation of byproducts like xylitol, thus improving carbon utilization efficiency. Furthermore, the xylose isomerase pathway has a shorter metabolic pathway and fewer reaction steps; after isomerization, xylose can directly enter the pentose phosphate pathway, which is beneficial for improving energy utilization efficiency and metabolic flux stability.

[0036] The xylose isomerase gene XI, derived from *Lachnoclostridium phytofermentans* (a plant fermenting bacterium), has the nucleotide sequence shown in SEQ ID NO:1.

[0037]

[0038] Its encoded amino acid sequence is shown in SEQ ID NO:2:

[0039] MKNYFPNVPEVKYEGPNSTNPFAFKYYDANKVVAGKTMKEHCRFALSWWHTLCAGGADPFGVTTMDRTYGNITDPMELAKAKVDAGFELMTKLGIEFFCFHDADIAPEG DTFEESKKNLFEIVDYIKEKMDQTGIKLLWGTANNFSHPRFMHGASTSCNADVFAYAAAKIKNALDATIKLGGKGYVFWGGREGYETLLNTDLGLELDNMARLMKMAVEY GRANGFDGDFYIEPKPKEPTKHQYDFDTATVLAFLRKYGLEKDFKMNIEANHATLAGHTFEHELAMARVNGAFGSVDANQGDPNLGWDTDQFPTDVHSATLAMLEVLKAG GFTNGGLNFDAKVRRGSFEFFDDIAYGYIAGMDTFALGLIKAAEIIDDGRIAKFVDDRYASYKTGIGKAIVDGTTSLEELEQYVLTHSEPVMQSGRQEVLETIVNNILFR.

[0040] The XK nucleotide sequence of the xylose kinase gene derived from *Pichia stipitis* is shown in SEQ ID NO:3.

[0041]

[0042] The amino acid sequence encoded thereby is set forth in SEQ ID NO: 4:

[0043] MTTTPFDAPDKLFLGFDLSTQQLKIIVTDENLAALKTYNVEFDSINSSVQKGVIAINDEISKGAIISPVYMWLDALDHVFEDMKKDGFPFNKVVGISGSCQQHGSVYWSRTAEKVLSELDAESSLSSQMRSAFTFKHAPNWQDHSTGKELEEFERVIGADALADISGSRAHYRFTGLQIRKLSTRFKPEKYNRTARISLVSSFVASVLLGRITSIEEADACGMNLYDIEKREFNEELLAIAAGVHPELDGVEQDGEIYRAGINELKRKLGPVKPITYESEGDIASYFVTRYGFNPDCKIYSFTGDNLATIISLPLAPNDALISLGTSTTVLIITKNYAPSSQYHLFKHPTMPDHYMGMICYCNGSLAREKVRDEVNEKFNVEDKKSWDKFNEILDKSTDFNNKLGIYFPLGEIVPNAAAQIKRSVLNSKNEIVDVELGDKNWQPEDDVSSIVESQTLSCRLRTGPMLSKSGDSSASSSASPQPEGDGTDLHKVYQDLVKKFGDLYTDGKKQTFESLTARPNRCYYVGGASNNGSIIRKMGSILAPVNGNYKVDIPNACALGGAYKASWSYECEAKKEWIGYDQYINRLFEVSDEMNLFEVKDKWLEYANGVGMLAKMESELKH.

[0044] The XI and XK genes were constructed into the Pichia pastoris expression vector pGAPZB, synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and optimized according to the Pichia pastoris dominant codon usage principle to obtain the recombinant plasmid pGAPZB-XI-pTEF1-XK. The recombinant plasmid pGAPZB-XI-pTEF1-XK was linearized using the restriction endonuclease AvrII. The restriction digestion results were verified by agarose gel electrophoresis; the migration rate of the linear plasmid in electrophoresis was lower than that of the supercoiled plasmid. Subsequently, the linearized plasmid was concentrated and electroporated, and screened on YPD plates containing 400 μg / mL bleomycin zeocin resistance. Single colonies were picked and colony PCR was performed using primers pGAP-F and TAOX1AOX1-R for verification. Sequencing was performed on the target bands of matching size to obtain recombinant Pichia pastoris DF1.

[0045] The recombinant Pichia pastoris DF1 and the control strain GS115, wild-type strains isolated in the applicant's laboratory, were cultured in shake flasks using 20 g / L xylose as the sole carbon source. The culture medium consisted of 10 g / L yeast extract (YPX), 20 g / L peptone, and 20 g / L xylose. The culture conditions were 30 °C and 200 rpm for 90 h, without the need for additional feeding. The results are as follows: Figure 1 As shown, the results indicate that wild-type Pichia pastoris GS115 has a weak ability to utilize xylose naturally, limited biomass growth throughout the process, and a low endpoint OD. 600 Only 1.65; after DF1 was introduced into the XI-XK xylose isomerization pathway, the OD at 90 h was... 600 The xylose utilization capacity can reach 3.46, and the cell growth is significantly better than that of wild strains.

[0046] Example 2: Screening for efficient xylose transporters to improve xylose utilization in Pichia pastoris

[0047] The nucleotide sequence of the xylose transporter XUT4 from *Pichia stipitis* is shown in SEQ ID NO:5.

[0048]

[0049] Its encoded amino acid sequence is shown in SEQ ID NO:6:

[0050] .

[0051] The XUT4 gene was constructed into the Pichia pastoris expression vector pGAPZB, synthesized by Beijing Liuhe BGI Genomics Co., Ltd., and optimized according to the Pichia pastoris dominant codon usage principle to obtain the recombinant plasmid pGAPZB-XI-pTEF1-XK-pGAP-XUT4. The recombinant plasmid pGAPZB-XI-pTEF1-XK-pGAP-XUT4 was linearized, and the enzyme digestion results were verified by agarose gel electrophoresis. The migration rate of the linear plasmid in electrophoresis was lower than that of the supercoiled plasmid. Subsequently, the linearized plasmid was concentrated and electroporated, and screened on YPD plates containing 400 μg / mL bleomycin zeocin resistance. Single colonies were picked and colony PCR was performed using primers pGAP-F and TAOX1AOX1-R for verification. Sequencing was performed on the target bands of the same size to obtain recombinant Pichia pastoris DF2.

[0052] The recombinant Pichia pastoris DF2 and DF1 were cultured in shake flasks using 20 g / L xylose as the sole carbon source. The culture medium consisted of 10 g / L YPX yeast extract, 20 g / L peptone, and 20 g / L xylose. The culture conditions were 30 °C and 200 rpm for 90 h. No additional feeding was required during this period. The results are as follows: Figure 2 As shown, DF2 expresses the high-affinity xylose transporter XUT4 on the basis of DF1, enhancing xylose transmembrane uptake; under the same culture conditions, the OD of DF1... 600 The OD at 90 h was 3.42. 600 The value reached 4.22; strain DF2, which introduced the Pichia stipitis transporter protein XUT4, was significantly higher than the recombinant Pichia stipitis strain DF1, which did not introduce the transporter protein.

[0053] Evidence: Xylose transmembrane transport is the bottleneck for xylose utilization in Pichia pastoris. The introduction of the XUT4 transporter protein can further improve xylose metabolic flux and cell biomass; however, the performance of DF2 is still not optimal and needs to be further improved through adaptive evolution.

[0054] Example 3: Adaptive evolution further improves Pichia pastoris xylose utilization and evolution termination criteria.

[0055] Based on the highly efficient xylose-utilizing engineered strain obtained in Example 2, this example further improves the growth capacity and metabolic efficiency of Pichia pastoris under xylose conditions through an adaptive evolution strategy, and establishes a scientific rule for determining the termination of evolution.

[0056] The recombinant Pichia stipitis-derived xylose transporter DF2 strain from Example 2 was selected as the starting strain. It was plated on YPX solid medium with a carbon source of 20 g / L. The YPX solid medium consisted of 10 g / L yeast extract, 20 g / L peptone, 20 g / L xylose, and 20 g / L agar. After 3-4 days, when single colonies emerged, six single colonies were randomly picked using an inoculation loop and inoculated into test tubes containing YPX liquid medium with a carbon source of 20 g / L xylose, with a volume of 3 mL. When the strain reached mid-exponential growth, 600-800 μL of the seed culture was transferred to 250 mL Erlenmeyer flasks containing YPX liquid medium with a carbon source of 20 g / L xylose, with a volume of 50 mL. The culture was carried out at 30°C and 200 rpm on a shaker for adaptive evolution. Throughout the subculture process, the carbon source was maintained at 20 g / L xylose. During transfer, the OD of the bacterial culture before the transfer should be measured. 600 前 and the OD of the transferred bacterial culture 600 后 They also recorded the xylose consumption rate during each transition cycle and drew a schematic diagram of the evolutionary process.

[0057] In the process of adaptive evolution, bacterial OD 600 Reaching a preset logarithmic growth phase threshold is used as the transition trigger condition; in this embodiment, it is set to OD. 600 The bacterial culture was trained to enter the 1.5-2.5 μg / mL range, ensuring each transfer occurred during the logarithmic growth phase. Using a combined index of xylose consumption rate and biomass growth as the screening criterion, strains exhibiting enhanced xylose utilization were selected from the adaptively evolved bacterial community. Simultaneously, the OD (obstruction time) of the bacterial culture before and after each transfer was analyzed. 600 Increase (ΔOD) 600 =OD 600 后 -OD 600 前 The rate of xylose consumption in the culture medium during each transfer cycle was continuously monitored. The bacterial culture OD was used as the metric. 600 The increase represents the growth rate of the evolutionary strain, while the xylose consumption rate represents the xylose utilization capacity of the evolutionary strain.

[0058] The specific operation of the termination judgment rule for adaptive evolution is as follows: Calculate the OD of the bacterial culture between three consecutive adjacent transfers. 600 The change in the increase (i.e., |ΔOD) 600 (i) -ΔOD 600 (i-1) | and |ΔOD 600 (i-1) -ΔOD 600 (i-2) The mean of | and the change in xylose consumption rate. The OD of the bacterial culture. 600 The changes in the growth rate and the change in the xylose consumption rate are respectively compared with preset stability thresholds. In this embodiment, OD 600 The preset stability threshold for the increase in growth rate was ±0.05, and the preset stability threshold for the change in xylose consumption rate was ±0.02 g / (L·h). The OD of the bacterial culture was compared between three consecutive transfers. 600 When both the change in growth rate and the change in xylose consumption rate are below the preset stability threshold, it is determined that the xylose utilization capacity and growth rate of the evolved strain have stabilized, and adaptive evolution is terminated.

[0059] In this embodiment, after a cumulative 720 hours of continuous adaptive evolution, a total of 15 transitions were performed, one every 48 hours. During the last three consecutive transitions, specifically the 13th, 14th, and 15th transitions, ΔOD... 600The changes in xylose consumption rate were 0.03 and 0.02, respectively, and the changes in xylose consumption rate were 0.015 g / (L·h) and 0.010 g / (L·h), respectively, both below the preset stability threshold. Therefore, the performance of the evolved strain was determined to have stabilized, and adaptive evolution was terminated. Subsequently, the evolved bacterial community was isolated and diluted and plated onto YPX solid medium with a carbon source of 20 g / L xylose. The dilution gradient was 10... -6 10 -7 10 -8 Finally, a strain with significantly improved growth performance was isolated from the evolutionary community and named XUT4-6. Strain XUT4-6 was fermented under the same conditions with 20 g / L xylose as the sole carbon source, and its biomass OD... 600 The OD value reached 7.68, compared to the pre-adaptive strain DF2. 600 The value was 4.22, a further significant increase, indicating that its xylose utilization efficiency and environmental adaptability were significantly enhanced. Figure 3 and Figure 4 As shown; Figure 3 The figure shows the growth curves of multiple evolutionary strains during the adaptive evolution process. As can be seen from the figure, during the 800-hour passage evolution process, the xylose utilization ability of different monoclonal strains differentiated, and the growth performance of the strains differed significantly. Early clones grew slowly and had low OD peaks. As the evolutionary passage time increased, the growth rate and maximum biomass of some clones gradually increased. This reflects the characteristics of adaptive evolutionary selection: long-term xylose stress passage led to the accumulation of SNP mutations in the strain genome, and performance differentiation occurred within the bacterial community, requiring the isolation of dominant monoclonal strains from the community. Figure 3 This visually reflects the entire adaptive evolutionary screening process, demonstrating that mixed bacterial communities cannot be used directly; it is necessary to isolate single clones to obtain dominant evolutionary strains.

[0060] Figure 4 This is a comparison of growth curves for the XUT4 series of evolutionary recombinant strains, validated in shake flasks from 0 to 100 hours. Multiple curves represent single clones from the XUT4-1 to XUT4-19 series isolated after evolution. The red square curve represents the starting strain DF2 (XUT4), with XUT4-6 exhibiting the best performance. The growth of the starting strain DF2 significantly lagged behind most evolutionary clones, demonstrating that adaptive evolution significantly improved strain performance. Significant differences in performance were observed among different evolutionary clones, with some clones showing limited improvement. XUT4-6 exhibited the best growth performance among all clones, with an OD of [missing value] at 90 hours. 600 The biomass reached 7.68, an increase of approximately 82% compared to the starting DF2. This figure shows the results of the evolutionary strain rescreening verification, confirming XUT4-6 as the target dominant strain. Combined with previous data... Figures 1-2Complete chain: wild GS115 → DF1 introduction of XI-XK → DF2 addition of XUT4 transporter protein → adaptive evolution screening of XUT4-6, fully demonstrating the complete technical logic of the strain of this invention for step-by-step modification and performance improvement.

[0061] The optimized strain XUT4-6 obtained through the above adaptive evolution was submitted to the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, for patent deposit. The deposit number is CGMCC No. 38857, and the deposit date is May 25, 2026. Testing by the depository institution confirmed that the strain is viable and stable.

[0062] Example 4: Comparative analysis of the whole genome sequences of strain XUT4-6 and the originating strain DF2

[0063] Whole-genome resequencing was performed on strain XUT4-6 and the recombinant Pichia pastoris DF2 starting strain. After quality control, the sequencing data were aligned to a reference genome, and bioinformatics analysis was used to identify genomic sequence differences between the two strains.

[0064] The analysis showed that strain XUT4-6 differed from recombinant Pichia pastoris DF2 in a total of 362 single nucleotide polymorphisms (SNPs) across the entire genome. Of these 362 SNPs, 159 were synonymous mutations, which did not cause changes in the amino acid sequence; and 213 were non-synonymous mutations, which led to changes in the amino acid sequence encoded by their respective genes.

[0065] Further functional annotation analysis of genes containing nonsynonymous mutations revealed that among the 213 nonsynonymous mutations, 36 nonsynonymous mutations were located in the coding regions of functionally annotated genes, involving 19 genes, as shown in Table 1.

[0066] Table 1: Nonsynonymous mutant genes

[0067]

[0068] To further verify the contribution of non-synonymous mutations in the above 19 genes to the enhanced xylose utilization, each of these 19 genes in strain XUT4-6 was knocked out individually, resulting in 19 single-gene knockout mutants. Each knockout mutant was then compared with strain XUT4-6 under the same conditions (20 g / L xylose as the sole carbon source, 30°C, 200 rpm) for 90 h via shake-flask fermentation. The results were measured using OD... 600Xylose consumption rate was used as an evaluation index. The results showed that in 19 knockout mutants, the knockout of 14 genes significantly reduced the strain's xylose utilization capacity, OD... 600 The decrease exceeded 20%, and the xylose consumption rate decreased by more than 2%, confirming that the non-synonymous mutations of these 14 genes were significantly associated with enhanced xylose utilization in XUT4-6.

[0069] Example 5: Verification of the co-fermentation ability of XUT4-6 in a glucose / xylose mixed carbon source and the xylose utilization triggering conditions.

[0070] To evaluate the sugar co-utilization capacity of XUT4-6 under the coexistence of glucose and xylose, and to verify the initiation triggering conditions for xylose utilization in the presence of glucose, GS115, DF1, DF2 and XUT4-6 were used as experimental strains. They were cultured in shake flasks under the conditions of glucose as a single carbon source, xylose as a single carbon source and glucose / xylose mixed carbon source, respectively. The cell growth, glucose consumption and xylose consumption were detected.

[0071] GS115, DF1, DF2, and XUT4-6 were inoculated into YPD medium and cultured at 30 °C and 200 rpm until the logarithmic growth phase. The cells were collected, washed with sterile water, resuspended, and cultured at the same initial OD. 600 Inoculated into culture media with different carbon sources. Three culture conditions were set up: Group A, glucose as the sole carbon source (20 g / L glucose); Group B, xylose as the sole carbon source (20 g / L xylose); Group C, a mixed glucose / xylose carbon source (20 g / L glucose and 20 g / L xylose). The culture temperature was 30℃, the rotation speed was 200 rpm, and the culture time was 96 h. Samples were taken at 0, 12, 24, 36, 48, 60, 72, and 96 h to measure OD. 600 The concentrations of glucose and xylose in the culture medium were determined by HPLC or HPAEC-PAD. Three biological replicates were set up for each group.

[0072] In the mixed sugar culture system, the glucose concentration in the culture medium was reduced to a preset threshold (in this embodiment, the preset threshold was set to 70% of the initial glucose concentration, i.e., when the glucose concentration dropped to approximately 14 g / L, based on preliminary experiments) as the trigger condition for xylose utilization initiation. The ability of each strain to initiate xylose utilization while glucose was still present was investigated. Simultaneously, with glucose concentration as a control variable, the ability of different strains to maintain continuous xylose transport in the presence of glucose was evaluated by comparing the response characteristics of xylose consumption rates during dynamic changes in glucose concentration.

[0073] 5.1 Growth under glucose conditions

[0074] The results showed that XUT4-6 maintained normal growth capacity when glucose was used as the sole carbon source, and its 96-hour OD 600 The value reached approximately 8.56, indicating that the introduction of XUT4 and subsequent adaptive evolution did not lead to a decrease in the strain's glucose utilization capacity, as shown in Table 2.

[0075] Table 2: OD of each strain under glucose as a single carbon source condition 600 value

[0076]

[0077] 5.2 Growth under glucose / xylose mixed conditions

[0078] XUT4-6 exhibited the highest biomass accumulation capacity under mixed carbon source conditions, with an OD of 96 h. 600 It reached 8.83. Compared with GS115, the endpoint OD... 600 The improvement was approximately 24.2%; compared to DF2, the improvement was approximately 9.3%, as shown in Table 3.

[0079] Table 3: OD of each strain under glucose / xylose mixed carbon source conditions 600 value

[0080]

[0081] 5.3 Glucose Consumption

[0082] The results showed that all strains were able to rapidly utilize glucose, essentially exhausting it within approximately 48-60 hours. Therefore, the growth differences among different strains under mixed sugar conditions were mainly related to their xylose utilization capacity, as shown in Table 4.

[0083] Table 4: Glucose consumption (g / L) of each strain under mixed carbon source conditions

[0084] 0 20.0 20.0 20.0 20.0 12 14.8 14.4 14.1 13.8 24 7.6 7.0 6.5 6.0 36 2.4 1.8 1.4 1.0 48 0.4 0.2 0.1 0.05 60 0 0 0 0 72 0 0 0 0 96 0 0 0 0

[0085] 5.4 Verification of Xylose Consumption and Triggering Conditions

[0086] In the mixed sugar culture system, after 12 hours of cultivation, the glucose concentration in the XUT4-6 culture medium had decreased from 20.0 g / L to 13.8 g / L, below the preset threshold of 14 g / L. At this point, the xylose concentration had decreased from 20.0 g / L to 17.2 g / L, indicating that XUT4-6 initiated xylose utilization after the glucose concentration dropped to the preset threshold, meeting the condition of triggering xylose utilization when glucose still remained at 13.8 g / L. In contrast, the control strain GS115, after 12 hours of cultivation, had a glucose concentration of 14.8 g / L, still above the threshold, and only consumed 0.2 g / L of xylose, failing to significantly initiate xylose utilization. The preset threshold was determined through preliminary experiments: in a mixed culture medium containing 20 g / L glucose and 20 g / L xylose, samples were taken to detect the initiation of xylose consumption when the glucose concentration dropped to 50%, 60%, 70%, and 80% of the initial concentration. The results showed that when the glucose concentration dropped to 70% of the initial concentration, i.e., 14 g / L, the xylose consumption rate of strain XUT4-6 began to increase significantly (>0.05 g / (L·h)). However, below this threshold, such as 50% or 60%, the lag time for xylose initiation and utilization was prolonged to more than 6 hours, which was not conducive to the synergistic utilization of mixed sugars. Therefore, 70% was determined as the optimal trigger threshold.

[0087] XUT4-6 maintains strong xylose utilization capacity even in the presence of glucose, with a xylose utilization rate of 89.5% after 96 hours, which is significantly higher than that of GS115 (14.0%), DF1 (38.0%) and DF2 (61.0%), as shown in Table 5.

[0088] Table 5: Xylose consumption (g / L) and xylose utilization rate at 96 h for each strain under mixed carbon source conditions

[0089]

[0090] 5.5 Results Analysis

[0091] The results showed that GS115 preferentially utilized glucose under mixed glucose / xylose carbon source conditions, while xylose consumption was relatively slow. The introduction of DF1 into the XI / XK pathway enhanced xylose utilization. Further introduction of XUT4 to obtain DF2 further increased xylose consumption. Compared to DF2, XUT4-6, obtained through 720 hours of adaptive evolution, exhibited a faster xylose consumption rate and higher final xylose utilization under mixed sugar conditions. Specifically, after 12 hours of XUT4-6 culture, the culture medium still contained approximately 13.8 g / L glucose, below the preset threshold of 14 g / L, while the xylose concentration had decreased from 20 g / L to approximately 17.2 g / L. This verified that XUT4-6 could initiate xylose utilization even before glucose was completely depleted after the glucose concentration dropped to the preset threshold, satisfying the aforementioned triggering conditions.

[0092] Example 6: Dynamic maintenance evaluation of xylose utilization capacity of XUT4-6 at different glucose concentrations

[0093] To further evaluate the tolerance of XUT4-6 to glucose inhibition of xylose utilization, and to verify the ability of the strain to maintain xylose utilization during dynamic changes in glucose concentration, different glucose concentrations were set to investigate the effect of increasing glucose concentration on xylose utilization.

[0094] Using xylose (20 g / L) as the primary carbon source, glucose concentrations of 0, 5, 10, 20, 40, and 60 g / L were added, respectively. GS115, DF2, and XUT4-6 plants were inoculated, respectively. The culture conditions were 30℃, 200 rpm, and 96 h. Initial OD... 600 The control value was set at 0.2. OD was measured after the culture was completed. 600 And the remaining xylose concentration. With glucose concentration as the control variable, the change trend of xylose utilization capacity of the strain was investigated as the glucose concentration increased from low to high (0→5→10→20→40→60 g / L). The ability of the strain to dynamically maintain xylose utilization was evaluated by the degree of xylose utilization rate retention.

[0095] The results showed that as glucose concentration gradually increased, the xylose utilization rates of GS115 and DF2 both decreased, while XUT4-6 exhibited a stronger ability to maintain xylose utilization. At a glucose concentration of 60 g / L, the xylose utilization rate of GS115 was 6.5%, DF2 decreased to 45.0%, while XUT4-6 still maintained 78.0%. Compared with DF2, under 60 g / L glucose conditions, the xylose utilization rate of XUT4-6 increased by approximately 73.3%, as shown in Table 6. Throughout the process of increasing glucose concentration, the xylose utilization rate of XUT4-6 remained above 78.0%, indicating that it can dynamically maintain xylose utilization capacity under dynamically increasing glucose concentration conditions, with glucose concentration as the control variable, demonstrating strong glucose-tolerant xylose utilization characteristics.

[0096] Table 6: Xylose utilization of various strains at different glucose concentrations (with glucose concentration as the control variable)

[0097]

[0098] In this embodiment, using glucose concentration as a control variable means setting up parallel culture systems with different initial glucose concentrations to compare the xylose utilization performance of the strains under different glucose concentration pressures, in order to simulate the effect of fluctuations in glucose concentration in lignocellulose hydrolysate on xylose utilization.

[0099] Example 7: Utilization of mixed sugars in simulated lignocellulose hydrolysate by XUT4-6

[0100] To verify the application potential of XUT4-6 in lignocellulose hydrolysate, a mixed sugar culture system containing glucose, xylose and arabinose was constructed, and the utilization capacity of GS115, DF2 and XUT4-6 for the mixed sugar was compared.

[0101] The simulated lignocellulose hydrolysate composition is as follows: glucose 25 g / L, xylose 20 g / L, arabinose 3 g / L, and acetic acid 1 g / L. Glucose and xylose serve as the main carbon sources, arabinose represents other pentose components produced during hemicellulose hydrolysis, and acetic acid represents inhibitors that may be generated during lignocellulose pretreatment.

[0102] GS115, DF2, and XUT4-6 were inoculated into the simulated lignocellulose hydrolysate, respectively. The culture conditions were 30℃, 200 rpm, and 96 h, with an initial OD... 600 The control value was set at 0.2. Samples were taken at 0, 12, 24, 36, 48, 60, 72, and 96 hours to detect OD. 600 Concentrations of glucose, xylose, arabinose, and acetic acid.

[0103] 7.1 Growth Results

[0104] The results showed that XUT4-6 had significantly better growth ability than GS115 and DF2 in simulated lignocellulose hydrolysate, as shown in Table 7.

[0105] Table 7: OD of each strain in simulated lignocellulose hydrolysate 600 value

[0106]

[0107] 7.2 Glucose Consumption

[0108] All strains were able to consume glucose relatively quickly. XUT4-6 consumed glucose down to 0.3 g / L in 48 h and completely depleted it by 60 h, as shown in Table 8.

[0109] Table 8: Glucose consumption (g / L) of each strain in simulated lignocellulose hydrolysate

[0110]

[0111] 7.3 Xylose Consumption

[0112] XUT4-6 achieved a xylose utilization rate of 87.5% in simulated lignocellulose hydrolysate, which was significantly higher than that of GS115 (11.0%) and DF2 (62.0%), as shown in Table 9.

[0113] Table 9: Xylose consumption (g / L) and xylose utilization rate at 96 h for each strain in simulated lignocellulose hydrolysate.

[0114]

[0115] 7.4 Utilization of arabinose

[0116] All strains had limited arabinose utilization. XUT4-6 had an arabinose utilization rate of 10.0%, which was slightly higher than GS115 (3.3%) and DF2 (6.7%), as shown in Table 10.

[0117] Table 10: Arabinose utilization of each strain

[0118]

[0119] 7.5 Total sugar utilization rate

[0120] The initial total sugar concentration was 48 g / L, calculated using glucose, xylose, and arabinose as the total available sugars. The results showed that the total sugar utilization rate of XUT4-6 reached 89.2% at 96 h, which was higher than that of GS115 (56.9%) and DF2 (78.3%), as shown in Table 11.

[0121] Table 11: Total sugar utilization of each strain

[0122]

[0123] 7.6 Results Analysis

[0124] The results showed that, under lignocellulose hydrolysate conditions, XUT4-6 exhibited superior growth and mixed sugar utilization compared to GS115 and DF2. After 96 hours of cultivation, XUT4-6 achieved 100% glucose utilization, approximately 87.5% xylose utilization, and approximately 89.2% total sugar utilization. Compared to GS115 and DF2, XUT4-6 was able to initiate xylose utilization in the early stages before glucose was completely depleted and continued to consume xylose, suggesting its potential for achieving synergistic utilization of glucose and xylose in complex lignocellulose hydrolysate systems.

[0125] sequence list

[0126] SEQ ID NO.1 Nucleotide sequence of xylose isomerase gene XI

[0127]

[0128] SEQ ID NO.2 Xylose isomerase gene XI amino acid sequence

[0129] MKNYFPNVPEVKYEGPNSTNPFAFKYYDANKVVAGKTMKEHCRFALSWWHTLCAGGADPFGVTTMDRTYGNITDPMELAKAKVDAGFELMTKLGIEFFCFHDADIAPEG DTFEESKKNLFEIVDYIKEKMDQTGIKLLWGTANNFSHPRFMHGASTSCNADVFAYAAAKIKNALDATIKLGGKGYVFWGGREGYETLLNTDLGLELDNMARLMKMAVEY GRANGFDGDFYIEPKPKEPTKHQYDFDTATVLAFLRKYGLEKDFKMNIEANHATLAGHTFEHELAMARVNGAFGSVDANQGDPNLGWDTDQFPTDVHSATLAMLEVLKA GGFTNGGLNFDAKVRRGSFEFFDDIAYGYIAGMDTFALGLIKAAEIIDDGRIAKFVDDRYASYKTGIGKAIVDGTTSLEELEQYVLTHSEPVMQSGRQEVLETIVNNILFR

[0130] SEQ ID NO.3 Nucleotide sequence of xylose kinase gene XK

[0131]

[0132] Amino acid sequence of xylokinase gene XK set forth in SEQ ID NO.4

[0133] MTTTPFDAPDKLFLGFDLSTQQLKIIVTDENLAALKTYNVEFDSINSSVQKGVIAINDEISKGAIISPVYMWLDALDHVFEDMKKDGFPFNKVVGISGSCQQHGSVYWSRTAEKVLSELDAESSLSSQMRSAFTFKHAPNWQDHSTGKELEEFERVIGADALADISGSRAHYRFTGLQIRKLSTRFKPEKYNRTARISLVSSFVASVLLGRITSIEEADACGMNLYDIEKREFNEELLAIAAGVHPELDGVEQDGEIYRAGINELKRKLGPVKPITYESEGDIASYFVTRYGFNPDCKIYSFTGDNLATIISLPLAPNDALISLGTSTTVLIITKNYAPSSQYHLFKHPTMPDHYMGMICYCNGSLAREKVRDEVNEKFNVEDKKSWDKFNEILDKSTDFNNKLGIYFPLGEIVPNAAAQIKRSVLNSKNEIVDVELGDKNWQPEDDVSSIVESQTLSCRLRTGPMLSKSGDSSASSSASPQPEGDGTDLHKVYQDLVKKFGDLYTDGKKQTFESLTARPNRCYYVGGASNNGSIIRKMGSILAPVNGNYKVDIPNACALGGAYKASWSYECEAKKEWIGYDQYINRLFEVSDEMNLFEVKDKWLEYANGVGMLAKMESELKH

[0134] Nucleotide sequence of Scheffersomyces stipitis xylose transporter XUT4 set forth in SEQ ID NO.5

[0135]

[0136] SEQ ID NO.6 XUT4 amino acid sequence of Pichia pastoris xylose transporter

[0137] MSSLLTNEYFKDYYHNPTPVEVGTMIAILEIGALFSSFIAGRVGDIVGRRRTIRYGSFIFVVGGLVQATSVNIVNLSLGRLIAGIAIGFLTTIIPCYQSEISPPDDRGFYACLEFTGNIIGYASSIWVDYGF SFLDNDFSWRSPLYVQVVIGSMLFIGSFLIVETPRWLLDHNHDIEGMIVISDLYADGDVEDDDAIAEYRNIKESVLIARVEGGERSYQYLFTKYTKRLSVACFSQMFAQMNGINMVSYYAPMIFESAGWVGR QAILMTGINSIIYIFSTIPPWYLVDSWGRKPLLLSGSVLMGVPLLTIACSLFLNNTYTPGVVVGSVIVFNAAFGYSWGPIPWLMSEVFPNSVRSKGAAMSTATNWLFNFIVGEMTPILLDTITWRTYLIPAT SCVLSFFAVGFLFPETKGLALEDMGSVFDDNSSIFSYHSTPSTGYGATESNSNARRASVISSENYQDSLHQTAASLARNPSSMRPDYDGIITGAATLSPVPPLKPINISSNIPQEIEPPTFDEIFKYKLNEME

[0138] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the invention will be readily apparent to those skilled in the art.

[0139] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A Pichia pastoris strain XUT4-6, characterized in that, Pichia pastoris strain XUT4-6 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38857.

2. The Pichia pastoris strain XUT4-6 as described in claim 1, characterized in that, Compared with the originating strain DF2, there were 362 single nucleotide polymorphism sites that differed in the whole genome; of these, 159 were synonymous mutations and 213 were non-synonymous mutations; among the 213 non-synonymous mutations, 36 were located in the coding regions of genes with functional annotations, involving 19 coding genes.

3. A method for preparing the Pichia pastoris strain XUT4-6 as described in any one of claims 1-2, characterized in that, include: Step 1: Introduce the xylose isomerase metabolic pathway into Pichia pastoris. The xylose isomerase metabolic pathway includes xylose isomerase XI and xylose kinase XK. Xylose is isomerized by xylose isomerase XI to produce xylulose, and xylulose is phosphorylated by xylose kinase XK to enter the pentose phosphate pathway. Step 2: Introduce xylose transporter XUT4 into Pichia pastoris. XUT4 mediates the transmembrane transport of xylose and maintains the continuous transport of xylose in the presence of glucose in a carbon source system where glucose and xylose coexist. Step 3: Using xylose as the sole carbon source, recombinant Pichia pastoris simultaneously expressing xylose isomerase XI, xylose kinase XK, and xylose transporter XUT4 was subjected to continuous passage adaptive evolution. During the adaptive evolution process, the OD of the bacterial culture was used as the starting point. 600 Reaching a preset logarithmic growth phase threshold is used as the transition trigger condition, ensuring that each transition is in the logarithmic growth phase; Step 4: Using the combined index of xylose consumption rate and biomass growth as screening criteria, we screen for evolutionary strains with enhanced xylose utilization capacity from adaptive evolutionary communities.

4. The method as described in claim 3, characterized in that, The amino acid sequence of XI is shown in SEQ ID NO:2; the amino acid sequence of XK is shown in SEQ ID NO:4; and the amino acid sequence of XUT4 is shown in SEQ ID NO:

6.

5. The method as described in claim 3, characterized in that, Xylose isomerase XI is derived from plant fermentation bacteria, xylose kinase XK is derived from Pichia pastoris, and xylose transporter XUT4 is derived from Pichia pastoris.

6. The method as described in claim 3, characterized in that, In a carbon source system where glucose and xylose coexist, the glucose concentration in the culture medium drops to a preset threshold as the trigger condition for xylose utilization initiation. The strain initiates xylose utilization while glucose is still present.

7. The method as described in claim 3, characterized in that, The rules for determining the termination of adaptive evolution include: During continuous subculturing and adaptive evolution, the OD of the bacterial culture was monitored before and after each transfer. 600 The increase and the rate of xylose consumption in the culture medium during each transfer cycle are expressed as bacterial OD. 600 The increase in growth rate characterizes the growth rate of the evolutionary strain, while the xylose consumption rate characterizes the xylose utilization capacity of the evolutionary strain. Calculate the OD of the bacterial culture between three consecutive transfers. 600 The changes in the growth rate and the change in the xylose consumption rate will affect the bacterial OD. 600 The changes in the growth rate and the change in the xylose consumption rate were compared with preset stability thresholds; when the bacterial culture OD was between three consecutive transfers... 600 When both the change in growth rate and the change in xylose consumption rate are below the preset stability threshold, it is determined that the xylose utilization capacity and growth rate of the evolved strain have stabilized, and adaptive evolution is terminated.

8. The application of Pichia pastoris strain XUT4-6 as described in any one of claims 1-2 in the co-fermentation of lignocellulose hydrolysate with mixed sugars.

9. The use of Pichia pastoris strain XUT4-6 as described in any one of claims 1-2 in the production of biomass, biofuels or bio-based chemicals using xylose or mixed sugars as carbon sources.

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