Construction method and application of an engineered strain for synthesizing astaxanthin through co-fermentation of straw pentose and hexose

Abstract

The application provides a construction method of an engineering strain for synthesizing astaxanthin through co-fermentation of straw C5 / C6 sugars and application thereof, and belongs to the technical field of synthetic biology and microbial genetic engineering. The recombinant vector for promoting astaxanthin synthesis through co-fermentation of straw C5 / C6 sugars comprises a recombinant expression vector X containing a xylose metabolic pathway and a recombinant expression vector Y containing an astaxanthin synthesis pathway. The recombinant vector can fully utilize the precursor supply capacity of the endogenous MVA pathway of Yarrowia lipolytica, and does not need to additionally introduce terpene precursor synthesis genes, thereby reducing the metabolic burden of heterologous expression. The engineering strain containing the recombinant vector constructed by the application can efficiently utilize glucose and xylose in a hydrolysate of corn straw in a fermentation medium with the corn straw hydrolysate as a carbon source, the utilization rate of xylose is greater than or equal to 90%, the astaxanthin yield in continuous feeding fermentation in a 5L tank can reach 331.89 mg / L, and the engineering strain has excellent fermentation performance and industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and microbial genetic engineering technology, and in particular relates to a method for constructing an engineered strain for synthesizing astaxanthin by co-fermentation of five- or six-carbon sugars from straw and its application. Background Technology

[0002] Straw is a widely available and abundant form of lignocellulose, containing a large amount of fermentable sugars. It has attracted significant attention in the field of green bioenergy and is currently one of the world's most promising renewable resources. The efficient and comprehensive utilization of straw is essential for addressing agricultural non-point source pollution and improving arable land quality, and it is also a core measure to promote the circular development of national agriculture and the economy.

[0003] Straw is mainly composed of cellulose (30%–40%), hemicellulose (20%–30%), and lignin (10%–20%). Cellulose hydrolysis products are primarily glucose (a hexose), while hemicellulose hydrolysis products are primarily xylose (a pentose). Both glucose and xylose can be used as carbon sources to be converted by microorganisms into higher-value metabolites, indicating a promising industrial prospect. Currently, although the comprehensive utilization of straw has made some progress, it is still in its initial stage. The main reasons limiting the large-scale industrial application of crop straw are the ineffective utilization of hemicellulose and the low added value of the products made from it. The main reason for the ineffective utilization of hemicellulose is that most existing microorganisms in nature lack xylose metabolic pathways, and even those that do have very low utilization efficiency. The main reason for the low added value of the products is that substances synthesized using existing microbial metabolic pathways often have short pathways and are easily synthesized, thus resulting in relatively low value.

[0004] Astaxanthin is a natural carotenoid with extremely strong antioxidant activity. Its antioxidant capacity is 10 times that of β-carotene and 500 times that of vitamin E. It has excellent physiological activities such as anti-oxidation, anti-inflammation, immune regulation, anti-tumor, protection of vision and skin, and improvement of body color in aquaculture. It is widely used in many fields such as food, medicine, cosmetics, and aquatic and livestock feed additives. The global market demand continues to grow rapidly, and there is a significant gap in the market supply of high-purity natural astaxanthin. In recent years, metabolic engineering and synthetic biology techniques have been used to construct astaxanthin engineered strains, but existing strains have obvious technical shortcomings: most engineered strains only optimize hexose metabolism and lack efficient pentose transport and metabolism modules, making it impossible to achieve simultaneous and efficient co-utilization of pentose and hexose in straw hydrolysate, with carbon source utilization rate of less than 60%; in heterologous synthesis pathways, insufficient supply of IPP precursors, low expression levels of key enzymes, and redox imbalance result in low astaxanthin yield (usually <50mg / L), far below the industrialization threshold; and they rely on pure sugars (glucose, sucrose) as carbon sources, with raw material costs accounting for more than 40%, making it difficult to achieve low-cost large-scale production.

[0005] The development of synthetic biology and green biomanufacturing has provided new ideas and solutions for the high-value utilization of crop straw. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a recombinant carrier for promoting astaxanthin synthesis by co-fermentation of straw penta- and hexa-carbon sugars.

[0007] The second objective of this invention is to provide an engineered strain that synthesizes astaxanthin through co-fermentation of five- or six-carbon sugars from straw.

[0008] A third objective of this invention is to provide a primer set for identifying the engineered bacteria.

[0009] The fourth objective of this invention is to provide the application of the recombinant vector, the engineered bacteria, or the primer set in the synthesis of astaxanthin through co-fermentation of straw penta- and hexa-carbon sugars.

[0010] The fifth objective of this invention is to provide a method for synthesizing astaxanthin by co-fermentation of five- or six-carbon sugars from straw.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A recombinant vector for promoting astaxanthin synthesis by co-fermentation of five- or six-carbon sugars from straw, the recombinant vector comprising a recombinant expression vector X containing the xylose metabolism pathway and a recombinant expression vector Y containing the astaxanthin synthesis pathway; The recombinant expression vector X containing the xylose metabolism pathway includes XYL1 Gene expression unit - XYL2 Gene expression unit - XKS1 A multi-gene expression cassette X consisting of gene expression units tandemly; The recombinant expression vector Y containing the astaxanthin synthesis pathway includes CarRP Gene expression unit - CarB Gene expression unit - CrtW Gene expression unit - CrtZ Y is a multi-gene expression cassette with gene expression units tandemly.

[0012] Preferred, XYL1 The structure of the gene expression unit is the promoter TEF1- XYL1 Gene terminator XPR2; XYL2 The structure of the gene expression unit is the promoter TEF1- XYL2 Gene terminator XPR2; XKS1 The structure of the gene expression unit is the promoter TEF1- XKS1 Gene terminator XPR2; XYL1The nucleotide sequence of the gene is shown in SEQ ID NO.1; XYL2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; XKS1 The nucleotide sequence of the gene is shown in SEQ ID NO.3; the nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO.15; and the nucleotide sequence of the terminator XPR2 is shown in SEQ ID NO.16.

[0013] Preferred, CarRP The structure of the gene expression unit is the promoter GPD1- CarRP Gene-terminator LIP2; CarB The structure of the gene expression unit is the promoter GPD1- CarB Gene-terminator LIP2; CrtW The structure of the gene expression unit is the promoter GPD1- CrtW Gene-terminator LIP2; CrtZ The structure of the gene expression unit is the promoter GPD1- CrtZ Gene-terminator LIP2; CarRP The nucleotide sequence of the gene is shown in SEQ ID NO.4; CarB The nucleotide sequence of the gene is shown in SEQ ID NO. 5; CrtW The nucleotide sequence of the gene is shown in SEQ ID NO.6; CrtZ The nucleotide sequence of the gene is shown in SEQ ID NO.7; the nucleotide sequence of the promoter GPD1 is shown in SEQ ID NO.17; and the nucleotide sequence of the terminator LIP2 is shown in SEQ ID NO.18.

[0014] The present invention also provides an engineered strain for synthesizing astaxanthin by co-fermentation of five- and six-carbon sugars from straw. The engineered strain is introduced into the recombinant vector using Yersinia lipolytica strain Po1f as the starting strain to obtain an engineered strain that produces high levels of astaxanthin.

[0015] Preferably, the recombinant vector is introduced using a lithium acetate / PEG-mediated chemical conversion method.

[0016] The present invention also provides a primer set for identifying the engineered bacteria, the primer set comprising primers with nucleotide sequences as shown in SEQ ID NO.19~SEQ ID NO.32.

[0017] The present invention also provides the application of the recombinant vector, the engineered bacteria, or the primer set in the synthesis of astaxanthin by co-fermentation of straw penta- and hexa-carbon sugars.

[0018] This invention also provides a method for synthesizing astaxanthin by co-fermentation of five- and six-carbon sugars from straw. After the engineered bacteria are activated into a seed liquid, the seed liquid is inoculated into a fermentation medium for continuous fed-batch fermentation. The fermentation medium is a straw hydrolysate containing 40 g / L peptone and 20 g / L yeast extract. The total content of glucose and xylose in the straw hydrolysate is 40 g / L.

[0019] Preferably, the feeding is to maintain the total sugar concentration in the fermentation medium below 3 g / L.

[0020] Preferably, the feeding medium is concentrated corn stalk hydrolysate, and the total content of glucose and xylose in the concentrated corn stalk hydrolysate is 600 g / L.

[0021] Preferably, the preparation method of straw hydrolysate includes: after straw is crushed, acid hydrolysis is performed using dilute sulfuric acid, followed by enzymatic hydrolysis using a complex enzyme of cellulase and hemicellulase, centrifugation after enzymatic hydrolysis, collection of supernatant, addition of activated carbon for decolorization and impurity removal, filtration to remove activated carbon, and obtaining straw hydrolysate.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a recombinant vector for promoting astaxanthin synthesis through co-fermentation of straw pentose and hexaose sugars. It constructs a complete pathway for astaxanthin synthesis through co-fermentation of straw pentose and hexaose sugars containing only 7 core genes, systematically integrating the two major modules of xylose assimilation and astaxanthin synthesis. This recombinant vector can fully utilize the precursor supply capacity of the endogenous MVA pathway of Yersinia lipolyticis without the need to introduce additional terpene precursor synthesis genes, greatly simplifying the genetic operation process, reducing the metabolic burden of heterologous expression, and solving the problems of excessive gene number, complex construction, and poor strain stability in the prior art.

[0023] This invention is achieved through XYL1 + XYL2 + XKS1 The three-gene combination was used to construct an efficient xylose metabolism pathway adapted to Yersinia lipolytica. Through codon optimization and coenzyme preference modification, the redox imbalance in the xylose metabolism process was effectively alleviated, the accumulation of xylitol byproducts was reduced, and efficient assimilation of xylose was achieved. This enabled Yersinia lipolytica to simultaneously utilize glucose and xylose in straw hydrolysate, significantly improving the utilization rate of straw carbon source and reducing the cost of fermentation substrate.

[0024] This invention uses fungal-derived... CarRP + CarB A simplified dual-gene β-carotene synthesis pathway, combined with paracoccal-derived... CrtW and pantothenic origin CrtZThe astaxanthin functionalization module significantly reduces the number of genes compared to the traditional bacterial CRT multi-gene cluster, and has extremely high codon compatibility with the Yersinia lipolyticis chassis. It has high soluble expression efficiency and strong catalytic activity. Only 4 genes are needed to complete the synthesis of astaxanthin from GGPP (geranyl pyrophosphate), resulting in high product conversion efficiency.

[0025] This invention constructs an engineered strain containing the recombinant vector. In a fermentation medium with straw hydrolysate as the carbon source, it can efficiently utilize glucose and xylose in the hydrolysate, with a xylose utilization rate of ≥90%. In a 5L tank continuous fed fermentation, the astaxanthin yield can reach 331.89 mg / L, demonstrating excellent fermentation performance and industrial application potential.

[0026] This invention uses agricultural waste straw as a fermentation carbon source to achieve low-cost, green, and sustainable production of astaxanthin, while solving the environmental pollution problem caused by straw burning. It has both economic and environmental benefits and is suitable for large-scale industrial application. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the astaxanthin synthesis pathway constructed in this invention; Figure 2 Diagram of the astaxanthin carrier structure for synthesis; Figure 3 This is a plate screening diagram of engineered strains; Figure 4 The results of PCR amplification and identification of exogenous genes in engineered strains; Figure 5 The UPLC-MS / MS chromatogram for astaxanthin detection; Figure 6 This is a graph showing the changes in the content of various substances during tank fermentation. Detailed Implementation

[0028] This invention provides a recombinant vector for promoting astaxanthin synthesis through co-fermentation of five- and six-carbon sugars from straw. The recombinant vector comprises a recombinant expression vector X containing the xylose metabolism pathway and a recombinant expression vector Y containing the astaxanthin synthesis pathway. The recombinant expression vector X containing the xylose metabolism pathway includes... XYL1 Gene expression unit - XYL2 Gene expression unit - XKS1 A multi-gene expression cassette X containing tandem gene expression units; the recombinant expression vector Y containing the astaxanthin synthesis pathway includes... CarRP Gene expression unit - CarB Gene expression unit - CrtW Gene expression unit - CrtZ Y is a multi-gene expression cassette with gene expression units tandemly.

[0029] In this invention, XYL1, XYL2 and XKS1 Genes related to the xylose metabolism pathway. CarRP , CarB, CrtW and CrtZ These are genes involved in the astaxanthin synthesis pathway.

[0030] Among them, the XYL1 The gene is derived from *Saccharomyces cerevisiae* and encodes xylose reductase. It has been optimized and modified according to the codon preference of *Yarrowia lipolytica* and can catalyze the reduction of D-xylose to xylitol, which is the first key reaction in xylose metabolism. XYL1 The codon-optimized nucleotide sequence of the exogenous gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.8. XYL2 Genes and XYL1 Homologous pairing, derived from the tree trunk Schaefflera, encodes the xylitol dehydrogenase gene. After codon optimization, it can catalyze the oxidation of xylitol to D-xylitol, completing the core transformation of xylose into the central carbon metabolic precursor. XYL2 The codon-optimized nucleotide sequence of the exogenous gene is shown in SEQ ID NO.2, and the encoded amino acid sequence is shown in SEQ ID NO.9. XKS1 The gene, derived from *Saccharomyces cerevisiae*, encodes xylulose kinase, which catalyzes the phosphorylation of D-xylulose to D-xylulose-5-phosphate. This seamlessly connects xylose metabolism to the endogenous pentose phosphate (PPP) pathway, representing a key rate-limiting step for efficient xylose utilization and preventing the excessive accumulation of xylitol byproducts. XKS1 The nucleotide sequence of the exogenous gene after codon optimization is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.10.

[0031] The CarRP The gene is derived from Mucor and encodes a bifunctional phytoene synthase / lycopene β-cyclase. After codon optimization by Yersinia lipolytica, it possesses dual activities of phytoene synthase and lycopene β-cyclase. A single gene can complete two key catalytic steps: first, catalyze the condensation of 2 GGPP molecules to generate phytoene; second, catalyze the β-ionone cyclization reaction at both ends of lycopene, which greatly reduces the metabolic burden of heterologous expression. CarRP The codon-optimized nucleotide sequence of the exogenous gene is shown in SEQ ID NO.4, and the encoded amino acid sequence is shown in SEQ ID NO.11. CarB The gene, derived from *Blanctomyces trispora*, encodes phytorepinephrine dehydrogenase. After codon optimization, it catalyzes a four-step dehydrogenation reaction of phytorepinephrine to produce lycopene. CarRPThe complete synthetic pathway from GGPP to β-carotene can be completed with only 2 genes, making full use of the terpene precursors provided by the endogenous MVA pathway of Yersinia lipolytica, without the need to introduce other precursor synthesis genes; CarB The codon-optimized nucleotide sequence of the exogenous gene is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.12. CrtW The gene is derived from Paracoccus and encodes β-carotene ketolase. After codon optimization by Yersinia lipolytica, it can efficiently catalyze the β-ionone ring C4 ketylation reaction of β-carotene and its hydroxylated derivatives, which is one of the key rate-limiting steps in astaxanthin synthesis. CrtW The codon-optimized nucleotide sequence of the exogenous gene is shown in SEQ ID NO. 6, and the encoded amino acid sequence is shown in SEQ ID NO. 13. CrtZ The gene is derived from pan-bacteria and encodes β-carotene hydroxylase. After codon optimization, it can efficiently catalyze the β-ionone ring C3-hydroxylation reaction of β-carotene and its ketoyl derivatives. It works synergistically with CrtW to complete the complete conversion from β-carotene to astaxanthin without the accumulation of intermediate byproducts. CrtZ The nucleotide sequence of the exogenous gene after codon optimization is shown in SEQ ID NO.7, and the encoded amino acid sequence is shown in SEQ ID NO.14.

[0032] In this invention, each gene is placed under the transcriptional regulation of a constitutive strong promoter and terminator; a single-gene expression unit is constructed in the order of promoter-target gene-terminator, ensuring that each gene can be independently and efficiently transcribed and expressed in Yersinia lipophila. XYL1 The structure of the gene expression unit is the promoter TEF1- XYL1 Gene terminator XPR2; XYL2 The structure of the gene expression unit is the promoter TEF1- XYL2 Gene terminator XPR2; XKS1 The structure of the gene expression unit is the promoter TEF1- XKS1 The gene terminator XPR2; the nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO.15; the nucleotide sequence of the terminator XPR2 is shown in SEQ ID NO.16. CarRP The structure of the gene expression unit is the promoter GPD1- CarRP Gene-terminator LIP2; CarB The structure of the gene expression unit is the promoter GPD1- CarB Gene-terminator LIP2; CrtW The structure of the gene expression unit is the promoter GPD1- CrtW Gene-terminator LIP2; CrtZ The structure of the gene expression unit is the promoter GPD1- CrtZ The gene terminator LIP2; the nucleotide sequence of promoter GPD1 is shown in SEQ ID NO.17; the nucleotide sequence of terminator LIP2 is shown in SEQ ID NO.18.

[0033] In this invention, three gene expression units of the xylose metabolism pathway are involved. XYL1 - XYL2 - XKS1 A multi-gene expression cassette X was formed by tandem gene expression, with NotI and SacI restriction sites at both ends. The multi-gene expression cassette X was then ligated with the Yersinia lipolyticis integrative expression vector pYL2 (GenBank: KU378203.1) after digestion with NotI and SacI restriction endonucleases. The ligation product was transformed into E. coli DH5α competent cells to obtain a recombinant expression vector X containing the xylose metabolism pathway (e.g., pYL2). Figure 2 As shown): pYL2- XYL1 - XYL2 - XKS1 The recombinant expression vector X selected URA3 (uracil) auxotroph as a screening marker.

[0034] In this invention, four single-gene expression units of the astaxanthin synthesis pathway are used. CarRP - CarB - CrtW - CrtZ A multi-gene expression cassette Y was formed by tandemly connecting the gene expression cassettes, with SpeI and XhoI restriction sites at both ends. The multi-gene expression cassette Y was then ligated with the Yersinia lipolyticis integrative expression vector pYL2 after digestion with SpeI and XhoI restriction endonucleases. The ligation product was transformed into E. coli DH5α competent cells to obtain a recombinant expression vector Y containing the astaxanthin synthesis pathway (e.g., pYL2). Figure 2 As shown): pYL2- CarRP - CarB - CrtW - CrtZ The recombinant expression vector Y selects LEU2 (leucine) auxotroph as the screening marker.

[0035] This invention also provides an engineered strain for synthesizing astaxanthin through co-fermentation of five- and six-carbon sugars from straw. The engineered strain uses *Yersinia lipolytica* strain Po1f as the starting strain, and preferably employs a lithium acetate / PEG-mediated chemical transformation method to introduce the recombinant vector, resulting in an engineered strain that produces high levels of astaxanthin. The starting strain of this invention is *Yersinia lipolytica* strain URA3. - LEU2 -Dual auxotrophic strain. In a specific embodiment of this invention, the *Yarrowia lipophila* (Po1f) strain is designated ATCC-MYA2613. The astaxanthin synthesis pathway in the engineered strain constructed in this invention, which utilizes straw pentose and hexaose co-fermentation to synthesize astaxanthin, is as follows: Figure 1 As shown.

[0036] The present invention also provides a primer set for identifying the engineered bacteria, the primer set comprising primers with nucleotide sequences as shown in SEQ ID NO.19~SEQ ID NO.32.

[0037] The present invention also provides the application of the recombinant vector, the engineered bacteria, or the primer set in the preparation of astaxanthin.

[0038] This invention also provides a method for preparing astaxanthin, wherein the engineered bacteria are activated into a seed culture, and the seed culture is inoculated into a fermentation medium for continuous fed-batch fermentation; the fermentation medium is a straw hydrolysate containing 40 g / L peptone and 20 g / L yeast extract; the total glucose and xylose content in the straw hydrolysate is 40 g / L; the feeding is to maintain the total sugar concentration in the fermentation medium below 3 g / L.

[0039] The present invention preferably uses YPD medium (composed of 20 g / L glucose, 20 g / L peptone, and 10 g / L yeast extract) to activate the engineered bacteria; the preferred activation conditions are incubation at 28°C for 24 hours, with shaking at 220 rpm during incubation. The activated seed culture is preferably inoculated into the fermentation medium at an inoculation rate of 5% v / v. The fermentation medium is a diluted corn stalk hydrolysate containing 40 g / L peptone and 20 g / L yeast extract. The total glucose and xylose content in the diluted corn stalk hydrolysate is 40 g / L. The fed-batch medium is a concentrated corn stalk hydrolysate with a total glucose and xylose content of 600 g / L.

[0040] In this invention, the method for preparing straw hydrolysate includes: crushing the straw, acid hydrolysis with dilute sulfuric acid, enzymatic hydrolysis with cellulase and hemicellulase, centrifuging after enzymatic hydrolysis, collecting the supernatant, adding activated carbon for decolorization and impurity removal, filtering to remove activated carbon, and obtaining straw hydrolysate.

[0041] In the preparation method of the straw hydrolysate of the present invention, the straw is any one of corn straw, rice straw, and wheat straw, preferably corn straw; the straw is preferably air-dried and then pulverized, preferably pulverized to pass through a 40-mesh sieve; the pulverized straw is preferably acid-hydrolyzed with sulfuric acid at a mass fraction of 2.0%, the acid hydrolysis temperature is preferably 100℃, and the acid hydrolysis time is preferably 2h; after the acid hydrolysis is completed, the system is cooled, and the pH of the system is adjusted with Ca(OH)2, preferably to pH 4.8; the enzymatic hydrolysis is preferably performed by adding 20 FPU of cellulase and 10 IU of hemicellulase per gram of corn straw, the enzymatic hydrolysis temperature is preferably 45℃, and the enzymatic hydrolysis time is preferably 72h; after the enzymatic hydrolysis is completed, the pH is adjusted to 7.0, and the insoluble solids are removed by centrifugation; the amount of activated carbon added is preferably 0.5% (w / v), and the decolorization and impurity removal treatment is performed at 45℃ for a time of 60min, and the activated carbon is removed by filtration to obtain the straw hydrolysate. The present invention dilutes and / or concentrates the obtained straw hydrolysate for use in the co-fermentation synthesis of astaxanthin using straw penta- and hexa-carbon sugars.

[0042] This invention utilizes synthetic biology techniques to create engineered microorganisms, introducing exogenous xylose metabolic pathways and astaxanthin synthesis pathways into the strains, endowing them with the ability to co-fermentate high-value-added astaxanthin using glucose and xylose, hydrolysates from straw. This invention not only develops an effective utilization method for hemicellulose resources in straw, increasing usable resources by 20%–30% and solving the problem of insufficient resource utilization, but also opens up a new model for high-value utilization of straw, addressing the issue of low added value in products prepared from straw raw materials at present, and promoting the industrial-scale application of straw. Ultimately, it changes the pattern of comprehensive straw utilization, which is mainly focused on fertilizer, fuel, and feed, thereby solving problems such as low straw utilization rate, low economic benefits, and serious environmental pollution, and has good social, economic, and ecological benefits.

[0043] In specific embodiments of the present invention, restriction endonucleases, seamless cloning kits, and DNA polymerases were all purchased from Novizan Biosciences Co., Ltd.; gene synthesis, primer synthesis, and sequencing services were all provided by Sangon Biotech (Shanghai) Co., Ltd.; astaxanthin standards were purchased from Sigma-Aldrich; and other routine reagents were purchased from Shanghai Sinopharm Group Co., Ltd.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1 An engineered bacterium that synthesizes astaxanthin through co-fermentation of five- and six-carbon sugars from straw.

[0046] 1. Optimization and synthesis of genes related to astaxanthin synthesis and xylose utilization: Derived from tree trunk Schafer yeast XYL1 , XYL2 brewer's yeast XKS1 The source of Mucor CarRP Origin of Trispora brasiliensis CarB Paracoccus source CrtW Pantothenic origin CrtZ Using the original encoded sequence as a template, sequence optimization is performed according to the following principles: Optimize according to the codon preference of Yersinia lipophila to improve gene translation efficiency; Eliminate inverted repeat sequences, stem-loop structures, and transcription termination signals within genes, balance GC / AT content, and improve mRNA stability; Optimize the N-terminal sequence of the gene-encoded protein to conform to the N-terminal principle and improve the stability of the translated protein; Optimize the mRNA secondary structure free energy to further improve gene expression efficiency.

[0047] Optimized XYL1 , XYL2 , XKS1 , CarRP , CarB , CrtW and CrtZ The nucleotide sequences of the gene are shown in SEQ ID NO.1 to SEQ ID NO.7, and the corresponding encoded amino acid sequences are shown in SEQ ID NO.8 to SEQ ID NO.14. The optimized gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0048] 2. Construction of single-gene expression units and multi-gene recombinant expression vectors: 2.1 Construction of single-gene expression units: Optimized XYL1 , XYL2 and XKS1 Using TEF1 as the promoter (SEQ ID NO.15) and XPR2 as the terminator (SEQ ID NO.16), three independent single-gene expression units were constructed using seamless cloning technology. Each expression unit has a structure of "TEF1 promoter - target gene - XPR2 terminator". The optimized... CarRP C arB , CrtW and CrtZ Four genes were used as promoters (SEQ ID NO.17) and terminators (SEQ ID NO.18). Through seamless cloning technology, four independent single-gene expression units were constructed, each with the structure of "GPD1 promoter-target gene-LIP2 terminator".

[0049] 2.2 Construction of multi-gene tandem expression cassettes: The three single-gene expression units were sequentially linked, and the linking order of the xylose metabolism pathway expression cassette was as follows: XYL1 Gene expression unit - XYL2 Gene expression unit - XKS1 Gene expression units were constructed, and simultaneously, URA3 (uracil) selection marker expression units were constructed. NotI and SacI restriction sites were introduced at both ends of the expression cassette to obtain multi-gene expression cassette X; the astaxanthin synthesis pathway expression cassette connection order is as follows: CarRP Gene expression unit - CarB Gene expression unit - CrtW Gene expression unit - CrtZ Gene expression units were constructed, and a LEU2 (leucine) selection marker expression unit was also constructed. SpeI and XhoI restriction sites were introduced at both ends of the expression cassette to obtain the multi-gene expression cassette Y. The full sequence was synthesized and sequenced by Sangon Biotech (Shanghai) Co., Ltd. to ensure complete sequence accuracy.

[0050] 2.3 Construction of recombinant expression vectors: The multi-gene expression cassette X was digested with NotI and SacI restriction endonucleases, and the target fragment was recovered. Simultaneously, the integrative expression vector pYL2 from *Yersinia lipolytica* was digested with the same restriction endonucleases, and the vector backbone was recovered. The target fragment and the vector backbone were ligated using Gibson seamless cloning (one-step homologous recombination). The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar containing kanamycin, and incubated upside down at 37°C for 12 h. Single colonies were picked for PCR identification and double enzyme digestion verification. Positive clones were sent for sequencing verification. Those with correct sequencing were identified as the recombinant expression vector X: pYL2- using URA3 auxotrophic phenotype as a selection marker. XYL1 - XYL2 - XKS1 .

[0051] The multi-gene expression cassette Y was digested with SpeI and XhoI restriction endonucleases, and the target fragment was recovered. Simultaneously, the integrative expression vector pYL2 from *Yersinia lipolytica* was digested with the same restriction endonucleases, and the vector backbone was recovered. The target fragment and the vector backbone were ligated using Gibson seamless cloning (one-step homologous recombination). The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar containing kanamycin, and incubated upside down at 37°C for 12 h. Single colonies were picked for PCR identification and double enzyme digestion verification. Positive clones were sent for sequencing verification. Those with correct sequencing were identified as the recombinant expression vector Y: pYL2- using the LEU2 auxotrophic phenotype as a selection marker. CarRP - CarB - CrtW - CrtZ .

[0052] 3. Genetic transformation and screening of engineered strains of Yersinia lipophila: 3.1 Vector linearization: Based on the vector backbone sequence, a restriction endonuclease with a unique cleavage site in the vector integration guide region that does not destroy the target gene expression cassette and selection markers was selected to linearize the recombinant expression vector, and the linearized vector fragment was recovered and purified.

[0053] 3.2 Yeast Transformation and Screening: Mix 10 μL of linearized vector X fragment with 100 μL of Yersinia lipophila competent cells and transform them according to the lithium acetate / PEG-mediated chemical transformation method. Spread the transformed bacterial culture on uracil-deficient YNB solid medium and incubate at 28°C upside down for 2-3 days until single colonies grow.

[0054] Select healthy single colonies from the above plates to make competent cells, and then transform vector Y according to the above method. Spread the transformed bacterial solution on uracil-deficient and leucine-deficient YNB solid medium and incubate at 28°C upside down for 2-3 days until single colonies grow.

[0055] Select healthy, single red colonies (characteristics of astaxanthin synthesis-positive colonies), transfer them to YPD solid medium, and incubate at 28°C upside down for 2 days to obtain resistant positive transformants (e.g., Figure 3 (As shown).

[0056] 4. Molecular identification of engineered *Yarrowia lipophila* strains: Genomic DNA was extracted from the positive transformants obtained above. Using wild-type Yersinia lipolyticis Po1f as a negative control, the seven exogenous target genes were identified by PCR amplification using the specific primer pairs shown in Table 1.

[0057] PCR reaction system (50 μL): 10×PCR Buffer 5.0 μL; dNTPs (2.5 mmol / L) 4 μL; Genomic DNA template 1 μL (20~50 ng); Upstream primer 1 μL; Downstream primer 1 μL; High-fidelity Taq enzyme 0.3 μL; Sterile water to 50 μL.

[0058] PCR reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min.

[0059] Table 1 PCR Amplification Specific Primers

[0060] PCR results ( Figure 4 The results showed that the engineered bacteria constructed in this invention could amplify specific bands of the same size as the seven target genes, while the wild-type control strain did not have corresponding bands, proving that the seven exogenous target genes have been successfully integrated into the genome of Yersinia lipophila.

[0061] Example 2 Example 1: Verification of the fermentation performance of engineered bacteria in straw hydrolysate.

[0062] 1. Preparation of straw hydrolysate: Corn stalks were collected, air-dried, and passed through a 40-mesh sieve. Acid hydrolysis was performed using 2.0% sulfuric acid at 100℃ for 2 hours. After cooling, the pH of the system was adjusted to 4.8 with Ca(OH)₂. The pretreated biomass was then enzymatically hydrolyzed by adding 20 FPU of cellulase and 10 IU of hemicellulase per gram of corn stalks. The reaction was carried out at 45℃ and 150 rpm with shaking for 72 hours. After adjusting the pH to 7.0, the system was centrifuged at 9000 rpm for 10 minutes to remove insoluble solids. 0.5% (w / v) activated carbon was added to the resulting hydrolysate, and the mixture was stirred at 45℃ and 150 rpm for 60 minutes to decolorize and remove impurities such as proteins and phenols. The activated carbon was then removed by centrifugation or filtration, ultimately yielding a total corn stalk hydrolysate containing 46.5 g / L glucose and 22.3 g / L xylose.

[0063] The total hydrolysate of corn stalks was treated as follows: diluted to a total glucose and xylose content of 40 g / L; and concentrated to a total glucose and xylose content of 600 g / L. It was then stored at 4℃ for later use.

[0064] 2. Fermentation experiment: YPD medium (seed culture medium): glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L.

[0065] Fermentation medium: Straw hydrolysate containing 40 g / L peptone and 20 g / L yeast extract, wherein the total content of glucose and xylose in the straw hydrolysate is 40 g / L.

[0066] Feeding medium: concentrated straw hydrolysate, with a total glucose and xylose content of 600 g / L.

[0067] Astaxanthin content detection: The fermentation broth was centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The cells were washed twice with sterile water and freeze-dried to obtain dried bacterial cells. 0.1 g of dried bacterial cells were accurately weighed, and the cell walls were broken up using the acid-heat method. Acetone was added and extracted three times in the dark. The extracts were combined, concentrated by nitrogen blowing, and then diluted to 10 mL with methanol. The solution was filtered through a 0.22 μm organic filter membrane, and the astaxanthin content was detected by high performance liquid chromatography-MS / MS. A standard curve was plotted using astaxanthin standards for quantification.

[0068] UPLC-MS / MS detection conditions: The chromatographic column was a CORTECS UPLC C18, the column temperature was 35℃, the flow rate was 0.45 mL / min, the injection volume was 2 µL, and the mobile phase was phase A: 0.1% formic acid aqueous solution; phase B: methanol. Gradient elution was performed, and the elution program and mass spectrometry conditions are shown in Tables 2-4. Table 2 Gradient elution program

[0069] Table 3 Mass Spectrometry Parameters

[0070] Table 4 Multistage Reaction Monitoring Parameters

[0071] The engineered bacteria obtained in Example 1 were inoculated into seed culture medium and cultured at 28°C and 220 rpm for 24 h with shaking to obtain seed culture. The seed culture was inoculated into a 5L fully automated mechanically stirred fermenter at an inoculation rate of 5% v / v, with an initial volume of 2L (fermentation medium). The entire process was carried out in the dark at 30°C with a constant aeration rate of 1.0 vvm. The dissolved oxygen concentration was maintained at 30% by adjusting the stirring speed (200~800 rpm) in conjunction with the aeration rate. The feeding rate was maintained to keep the total sugar concentration in the fermentation broth below 3 g / L. Fermentation lasted for 96 h. Results showed that the astaxanthin content (…) was high as detected by UPLC-MS / MS. Figure 5 The astaxanthin concentration reached 331.89 mg / L, the intracellular astaxanthin content was 9.86 mg / g DCW, and the xylose utilization rate exceeded 90%. Figure 6 ).

[0072] In summary, this invention, by introducing and optimizing a complete core exogenous gene system for xylose metabolism and astaxanthin synthesis in *Yersinia lipolytica*, successfully achieves the simultaneous utilization and efficient synthesis of astaxanthin from straw penta- and hexa-carbon sugars in *Yersinia lipolytica*. This provides a novel and efficient technical solution for the low-cost, high-resource-utilization, and environmentally friendly production of high-value functional active substance astaxanthin using agricultural waste straw as raw material in a microbial fermentation system.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant vector for promoting astaxanthin synthesis through co-fermentation of straw's five- and six-carbon sugars, characterized in that, The recombinant vectors include recombinant expression vector X containing the xylose metabolism pathway and recombinant expression vector Y containing the astaxanthin synthesis pathway; The recombinant expression vector X containing the xylose metabolism pathway includes XYL1 Gene expression unit - XYL2 Gene expression unit - XKS1 A multi-gene expression cassette X consisting of gene expression units tandemly; The recombinant expression vector Y containing the astaxanthin synthesis pathway includes CarRP Gene expression unit - CarB Gene expression unit - CrtW Gene expression unit - CrtZ Y is a multi-gene expression cassette with gene expression units tandemly.

2. The recombinant vector according to claim 1, characterized in that, XYL1 The structure of the gene expression unit is the promoter TEF1- XYL1 Gene-terminator XPR2; XYL2 The structure of the gene expression unit is the promoter TEF1- XYL2 Gene-terminator XPR2; XKS1 The structure of the gene expression unit is the promoter TEF1- XKS1 Gene-terminator XPR2; XYL1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; XYL2 The nucleotide sequence of the gene is shown in SEQ ID NO.2; XKS1 The nucleotide sequence of the gene is shown in SEQ ID NO.3; the nucleotide sequence of the promoter TEF1 is shown in SEQ ID NO.15; and the nucleotide sequence of the terminator XPR2 is shown in SEQ ID NO.

16.

3. The recombinant vector according to claim 1, characterized in that, CarRP The structure of the gene expression unit is the promoter GPD1 -CarRP Gene-terminator LIP2; CarB The structure of the gene expression unit is the promoter GPD1- CarB Gene-terminator LIP2; CrtW The structure of the gene expression unit is the promoter GPD1- CrtW Gene-terminator LIP2; CrtZ The structure of the gene expression unit is the promoter GPD1- CrtZ Gene-terminator LIP2; CarRP The nucleotide sequence of the gene is shown in SEQ ID NO.4; CarB The nucleotide sequence of the gene is shown in SEQ ID NO.5; CrtW The nucleotide sequence of the gene is shown in SEQ ID NO.6; CrtZ The nucleotide sequence of the gene is shown in SEQ ID NO.7; the nucleotide sequence of the promoter GPD1 is shown in SEQ ID NO.17; and the nucleotide sequence of the terminator LIP2 is shown in SEQ ID NO.

18.

4. An engineered strain for synthesizing astaxanthin through co-fermentation of five- and six-carbon sugars from straw, characterized in that, The engineered bacteria are derived from Yersinia lipophila strain Po1f, which is introduced into the recombinant vector described in any one of claims 1 to 3 to obtain engineered bacteria that produce high levels of astaxanthin.

5. The engineered bacteria according to claim 4, characterized in that, The recombinant vector was introduced using a lithium acetate / PEG-mediated chemical transformation method.

6. A primer set for identifying the engineered bacteria of claim 4 or 5, characterized in that, The primer set includes primers with nucleotide sequences as shown in SEQ ID NO.19~SEQ ID NO.

32.

7. The application of the recombinant vector according to any one of claims 1 to 3, the engineered bacteria according to any one of claims 4 to 5, or the primer set according to claim 6 in the synthesis of astaxanthin by co-fermentation of straw penta- and hexa-carbon sugars.

8. A method for synthesizing astaxanthin through co-fermentation of five- and six-carbon sugars from straw, characterized in that, After activating the engineered bacteria according to any one of claims 4 to 5 into a seed liquid, the seed liquid is inoculated into a fermentation medium for continuous fed-batch fermentation; the fermentation medium is a straw hydrolysate containing 40 g / L peptone and 20 g / L yeast extract; the total content of glucose and xylose in the straw hydrolysate is 40 g / L.

9. The method according to claim 8, characterized in that, The feeding was to maintain the total sugar concentration in the fermentation medium below 3 g / L.

10. The method according to claim 9, characterized in that, The preparation method of straw hydrolysate includes: after straw is crushed, acid hydrolysis is performed using dilute sulfuric acid, followed by enzymatic hydrolysis using a complex enzyme of cellulase and hemicellulase. After enzymatic hydrolysis, the straw is centrifuged, the supernatant is collected, activated carbon is added for decolorization and impurity removal, and the activated carbon is removed by filtration to obtain straw hydrolysate.

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