CONSTRUCTION METHOD AND RECOMBINANT YEAST STAIN YARROWIA LIPOLYTICA FOR XYLITOL SYNTHESIS
20220259622 · 2022-08-18
Inventors
Cpc classification
C12Y106/01002
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention discloses a construction method and a recombinant yeast stain Yarrowia lipolytica for xylitol synthesis; Adopting Yarrowia lipolytica as the host, introducing genes into the host through metabolic engineering to enable the recombinant yeast to synthesize xylitol from glucose, fructose, glycerol and starch as carbon sources, block the synthesis pathway of by-products, so that it can synthesize xylitol from the aforesaid carbon sources by fermentation, thus obtain the engineered Yarrowia lipolytica strain to synthesize xylitol from glucose and other carbon sources. After fermentation, xylitol crystal is obtained by ion exchange, decolorization, concentration and crystallization of the clear and transparent fermentation liquor after isolation of the strains from the fermentation. This construction method of engineered Yarrowia lipolytica described in the invention, and the Yarrowia lipolytica strain obtained by this method can simplify the existing method for chemical synthesis of xylitol and have good application.
Claims
1. A construction method of recombinant Yarrowia lipolytica strain capable of synthesizing xylitol, comprising adopting the Yarrowia lipolytica strain capable of synthesizing erythritol as the host microorganism, by means of metabolic engineering or genetic engineering, to construct the recombinant Yarrowia lipolytica strain that synthesizes xylitol by fermentation with one carbon source or more carbon sources, including glucose, fructose, glycerol, or starch as carbon sources; the metabolic engineering or genetic engineering strategies include the expression of a gene encoding xylitol dehydrogenase and a gene encoding 5-p xylitol dehydrogenase in the cell of Yarrowia lipolytica from the host microorganisms, and the knockout or down-regulation of the transketolase gene in Yarrowia lipolytica.
2. The construction method as set forth in claim 1, wherein the host microorganism is the Yarrowia lipolytica strain whose genome contains DNA sequences with 97% or above identity with SEQ ID NO.3 sequence.
3. The construction method as set forth in claim 2, wherein the host microorganism is the Yarrowia lipolytica ery929 CGMCC No. 18478 that can synthesize erythritol.
4. The construction method as set forth in claim 1, wherein the expression of one or more of the following genes in the yeast Yarrowia lipolytica: (1) the gene encoding 5-p xylulose phosphatase; (2) the gene encoding xylitol transporter; and (3) the gene encoding NADP transhydrogenase.
5. The construction method as set forth in claim 4, wherein the knockout or down-regulation of one or more of the following genes in the yeast Yarrowia lipolytica: (1) mannitol dehydrogenase gene; (2) arabinitol dehydrogenase gene; (3) xylulose kinase gene; and (4) 5-p ribulose isomerase gene.
6. A recombinant Yarrowia lipolytica strain capable of synthesizing xylitol obtained by using the construction method of claim 1 for construction of a recombinant Yarrowia lipolytica strain capable of synthesizing xylitol.
7. The recombinant Yarrowia lipolytica strain capable of synthesizing xylitol as set forth in claim 6, wherein the strain is Yarrowia lipolytica ery959ΔTKLΔMDHΔArDHΔRPIΔXKS1 CGMCC No. 18479.
8. A method of fermentation for xylitol synthesis using a recombinant Yarrowia lipolytica strain capable of synthesizing xylitol as set forth in claim 6, comprising the following steps: S1. culturing the Yarrowia lipolytica strains in medium containing carbon, nitrogen, inorganic salts and water, and shaking or stirring, fermentation and culture at initial pH value of 3.0 ˜7.0 and temperature of 25 ˜35° C., then isolating the strains from the broth after fermentation to obtain xylitol-containing fermentation broth and yeast cells; and S2. proceeding isolation and purification to the xylitol-containing fermentation broth and yeast cells to obtain xylitol.
9. The method of synthesizing xylitol by fermentation with the recombinant Yarrowia lipolytica strains capable of synthesizing xylitol as set forth in claim 8, wherein, in step S1, the carbon source in the medium is one or a mixture of glucose, fructose, glycerol and starch, and the carbon source concentration in the medium is 50-350 g/L; the nitrogen source in the medium is one or a mixture of peptone, yeast cell powder, yeast extract, corn steep powder, diammonium hydrogen phosphate, ammonium citrate and amino acids; the inorganic salt in the medium is one or a mixture of magnesium sulfate, manganese chloride, copper chloride and zinc chloride.
10. The method of synthesizing xylitol by fermentation with the recombinant Yarrowia lipolytica strains capable of synthesizing xylitol as set forth in claim 8, wherein the isolation and purification mentioned in step S2 include the isolation of yeast cells from the broth to obtain the clear fermentation broth containing xylitol, the concentration to obtain the concentrated solution rich in xylitol, the primary crystallization to obtain crude products of xylitol, which would obtain the refined products of xylitol through redissolution, ion exchange removal of ions, decolorization, concentration and secondary crystallization to the crude products, as well as the drying procedure.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0086] Other features, objectives and advantages of the present invention will become more apparent by reading the detailed description of non-restrictive embodiments with reference to the following attached drawings:
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DETAILED DESCRIPTION
[0098] The present invention is hereby described in detail in combination with exemplary embodiments below. The following exemplary embodiments will help those skilled in the art to further understand the present invention, but shall not limit the present invention in any way. It should be noted that a number of adjustments and improvements can be made for those of ordinary skilled in the art without deviating from the concept of the present invention. These belong to the scope of protection of the present invention.
Embodiment 1 Acquisition of Yarrowia lipolytica ey929 (CGMCC No. 18478)
[0099] Take fresh bee hives from different sources and divide into several portions with 5 grams each. Use sterilized scissors to cut small pieces of each hive with the length less than 5 mm, soak them in 20 ml of sterile water containing 0.05% Tween 40, stir for 1 hour, centrifuge at 5000 rpm for 10 minutes, discard supernatant and hive fragments, suspend the precipitate with 1 ml of sterile water, and spread in sterilized hyperosmotic solid medium (composition: 400 g/L of anhydrous glucose, 12 g/L of yeast cell powder, 5 g/L of ammonium citrate, 3 g/L of peptone, 15 g/L of agar, pH5.5), spread 200 μl on each plate, then culture at 30° C. for 7 days. Select the yeast-like colony for pure culture, then take the pure cultured yeast colony and conduct the test of erythritol synthesis by fermentation. Liquid medium composition: 300 g/L of anhydrous glucose, 8 g/L of yeast cell powder, 5 g/L of ammonium citrate, 3 g/L of peptone, 0.02 g/L of copper chloride, 0.02 g/L of manganese chloride, 0.05 g/L of vitamin B1, initial pH5.5. After fermentation for 5 days in a 30° C. incubator shaker, use the HPLC to detect the fermentation broth and compare it with the standard erythritol. If the peak time is completely consistent with the standard erythritol, use the GC-MS to further detect the fermentation broth. HPLC and GC-MS results (
TABLE-US-00001 P.sub.26srDNA-F: (SEQ ID NO. 1) 5′-tagtgcagatcttggtggtagtagc-3′ P.sub.26srDNA-R: (SEQ ID NO. 2) 5′-ctgcttcggtatgataggaagagc-3′
[0100] The amplification conditions are as follows:
[0101] (1) Initial denaturation at 95° C. for 5 minutes
[0102] (2) Denaturation at 94° C. for 30 seconds
[0103] (3) Annealing at 55° C. for 30 seconds
[0104] (4) Elongation at 72° C. for 90 seconds
[0105] (5) Final elongation at 72° C. for 10 minutes
[0106] Step (2) to step (4) shall perform 30 cycles.
[0107] According to the above conditions, take the genome of the yeast with the highest erythritol production as template for PCR, 1.4 kb DNA could be amplified, then proceed sequencing, and coded as SEQ ID No.3 (part of 26S rDNA sequence).
[0108] Input the above sequences into NCBI database for sequence comparison, and the results show that it is 98% or higher identity with the 26S rDNA sequence of Yarrowia lipolytica E122, and 98% or higher identity with the 26S rDNA sequence of Yarrowia lipolytica W29 (CLIB89). Therefore, it can be determined that the yeast that can synthesize erythritol screened in the present invention is Yarrowia lipolytica or Candida lipolytica.
[0109] The inventor induce mutagenesis to the yeast with compound chemical reagents and in combination with adaptive evolution, raise the fermentation temperature from 30° C. to 35° C. The methods adopted are as follows:
[0110] Suspend the fresh yeast with 1.5% ethyl methyl sulfonate (EMS) and 0.5% diethyl sulfate (DES) for 1-10 hours, spread separately in hypertonic YPD culture (300 g/L of anhydrous glucose, 10 g/L of yeast cell powder, 5 g/L of ammonium citrate, 3 g/L of peptone, 15 g/L of agar, PH5.5), and culture at 35° C. for 10 days. Proceed pure culture to the newly grown colonies, and adaptive evolution at 35° C. After 180 days of high-temperature adaptive evolution, select a single colony with vigorous growth for testing of erythritol synthesis by fermentation at 35° C., composition of fermentation medium: 300 g/L of anhydrous glucose, 8 g/L of yeast cell powder, 5 g/L of ammonium citrate, 3 g/L of peptone, 0.02 g/L of copper chloride, 0.02 g/L of manganese chloride, 0.05 g/L of vitamin B1, initial pH5.5. Through fermentation test, it is found that one strain still keep the same efficiency of synthesizing erythritol with its wild-type at 35° C., and most of the other strains could grow at 35° C., but synthesize more mannitol. Name the new strain which can grow well at 35° C. and synthesize erythritol efficiently as ery929. The yield of erythritol synthesized from 300 g/L glucose have reached 174 g/L. The strain ery929 is now preserved at China General Microbiological Culture Collection Center (CGMCC), with the deposit number of CGMCC No. 18478.
Embodiment 2 Construct the Recombinant Yarrowia lipolytica Strain Capable of Synthesizing Xylitol
[0111] (1) Overexpress the Xylitol Dehydrogenase Gene in Yarrowia lipolytica.
[0112] Clone the xylitol dehydrogenase gene of the optimally synthsized Candida maltosa (SEQ ID NO. 9) to the integrative expression plasmid vector pSWV-Int (
[0113] Then, transform the plasmid pUB4-CRE containing Cre recombinase into mutants expressing xylitol dehydrogenase, and screen in YPD agar medium containing hygromycin as selective marker (10 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 15 g/L of agar, 300 μg/ml of hygromycin, pH 6.0). Transfer the resulting transformants to the minimal medium containing sucrose (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 15 g/L of agar powder, pH 6.0), and select mutants with sucrase gene loss (i.e., no reuse of sucrose). Then, culture the mutants that can not use sucrose in hygromycin free liquid YPD, and then apply gradient dilution and spread on the hygromycin free solid YPD. Select the mutants that can not resist hygromycin from the resulting transformants and transfer to YPD containing hygromycin, that is, the overexpression of xylitol dehydrogenase gene. In the meanwhile, screen mutants with loss of marker sucrase gene (Suc2), which can be used in hosts that overexpress other genes. Proceed total RNA isolation to the mutant and perform reverse transcription, and use reverse transcription products as templates for fluorescence quantitative PCR (RT-qPCR) to detect the expression level of xylitol dehydrogenase gene. Compared with the control strain ery929, it is found that the xylitol dehydrogenase gene of the mutant strain has obvious amplification curve, while the control strain has no amplification curve, indicating that xylitol dehydrogenase gene get expressed in the mutant strain.
[0114] Inoculate the mutants that overexpressed xylitol dehydrogenase gene and lost sucrase gene (Suc2) in fermentation medium for xylitol synthesis test. Composition of fermentation medium: 200 g/L of glucose, 8 g/L yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.05 g/L of zinc chloride, 0.01 g/L of manganese chloride, 0.05 g/L of vitamin B1, pH6.0. Take samples periodically for detection, 85 hours to run out of glucose, and the content of xylitol, erythritol and mannitol are 0.2 g/L, 96.4 g/L and 12 g/L, respectively. If the xylitol dehydrogenase gene of the above mentioned Candida maltosa are replaced by the xylitol dehydrogenase gene of Gluconobacter oxydans, and transformed into strain ery929, the fermentation test results show that the content of xylitol, erythritol and mannitol are 0.3 g/L, 90.2 g/L and 11 g/L, respectively. If the xylitol dehydrogenase gene of the above mentioned Candida maltosa are substituted by the xylitol dehydrogenase gene of Debaryomyces hansenii, and transformed into strain ery929, the fermentation test results show that the content of xylitol, erythritol and mannitol are 0.2 g/L, 98.6 g/L and 13 g/L, respectively. From the results of fermentation, it can be seen that the yield of synthesis of xylitol by Yarrowia lipolytica is very low if only xylitol dehydrogenase gene is overexpressed.
[0115] When the above expression vector is used to transform the Yarrowia lipolytica CLIB122, which can not produce erythritol, under the same conditions for 90 hours, it is found that neither xylitol nor erythritol can be detected, but 6 g/L of mannitol and a large amount of glucose (153 g/L) is detected.
[0116] (2) Overexpress the 5-p Xylitol Dehydrogenase Gene (also known as 5-p Xylulose Reductase) in Yarrowia lipolytica.
[0117] Respectively replace the xylitol dehydrogenase gene of the integrated expression vector pSWV-CmXDH in step (1) with the 5-p xylulose reductase genes of Clostridioides difficile, Lactobacillus rhamnosus, and Lactobacillus plantarum (SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 17, SEQ ID NO. 20 et al. in the sequence table) to obtain the integrated expression vector containing 5-p xylulose reductase gene. Transform the Yarrowia lipolytica ery929 to obtain the transformant containing 5-p xylulose reductase gene. Fermentation under the same conditions as in step (1), the test results show that the contents of xylitol, erythritol and mannitol are 0.3-0.7 g/L, 92-98 g/L and 10-12 g/L, respectively. The results show that the yield of xylitol from glucose by Yarrowia lipolytica is still very low if only 5-p xylulose reductase gene is contained. In order to testify that the gene is expressed in the cell, perform total RNA isolation to the transformant and reverse transcription, and use reverse transcription products as templates for fluorescence quantitative PCR to detect the expression level of 5-p xylulose reductase gene. Compared with the control strain ery929, it is found that the 5-p xylulose reductase gene of the mutant strain has obvious amplification curve, while the control strain has no amplification curve, indicating that 5-p xylulose reductase gene get expressed in the transformant.
[0118] (3) Overexpress the 5-p Xylulose Phosphatase Gene in Yarrowia lipolytica.
[0119] Respectively replace the xylitol dehydrogenase gene of the integrated expression vector in step (1) with the genes that containing the activity 5-p xylulose reductase from Kluyveromyces marxianus, Saccharomyces cerevisiae, Komagataella phaffii, Lactobacillus plantarum and Bacillus subtilis (SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 24 et al. in the sequence table) to obtain the integrated expression vector containing 5-p xylulose phosphatase gene. Transform the Yarrowia lipolytica ery929 to obtain the transformant contaning 5-p xylulose phosphatase gene. Fermentation under the same conditions as in step (1), the test results show that no xylitol is detected by liquid chromatography, and the contents of erythritol and mannitol are 95-102 g/L and 10-12 g/L, respectively. The results show that Yarrowia lipolytica can not synthesize xylitol from glucose if only 5-p xylulose phosphatase gene is contained. In order to testify that the 5-p xylulose phosphatase gene is expressed in the transformant, the inventor performed quantitative PCR analysis. The specific operations are as follows: perform total RNA isolation to the transformant (use the Trizol for extraction), then reverse transcription (use the commercial reverse transcription kit), and take 2 microliter reverse transcription products for fluorescence quantitative PCR (use the 2 microliter reverse transcription products), operate in fluorescence quantitative PCR instrument as a 20 microliter reaction system. After the reaction, it is found that the transformant has amplification curve and the gene is amplified, while the control strain has no amplification, indicating that the gene get expressed in the transformant.
[0120] (4) Overexpress the Xylitol Transporter Gene or NADP Transhydrogenase Gene in Yarrowia lipolytica.
[0121] Respectively replace the xylitol dehydrogenase gene of the integrated expression vector in step (1) with the xylitol transporter gene or NADP transhydrogenase gene, to obtain the integrated expression vector containing xylitol transporter gene or NADP transhydrogenase gene. Transform the Yarrowia lipolytica ery929 to obtain the transformant contaning xylitol transporter gene or NADP transhydrogenase gene. Fermentation under the same conditions as in step (1), the test results show that no xylitol is detected, and the contents of erythritol and mannitol are 96-104 g/L and 9-12 g/L, respectively. The results show that Yarrowia lipolytica can not synthesize xylitol from glucose if only xylitol transporter gene or NADP transhydrogenase gene is contained. Fluorescence quantitative PCR detection shows that the transformant has amplification curve and the gene is amplified, while the control strain has no amplification, indicating that the NADP transhydrogenase gene get expressed in the transformant.
[0122] The above-mentioned results indicate that recombinant Yarrowia lipolytica can only produce a small amount of xylitol if it contains only xylitol dehydrogenase or 5-p xylulose reductase gene, however, xylitol synthesis can not be detected if the recombinant strain contains only 5-p xylulose phosphatase gene, xylitol transporter or NADP transhydrogenase gene. In order to verify the synergistic effect of these five genes, transfer the five genes into Yarrowia lipolytica to test whether the synthesis efficiency of xylitol is improved.
[0123] (5) Acquisition of Yarrowia lipolytica ery959 that can Simultaneously Express Five Genes: Xylitol Dehydrogenase Gene, 5-p Xylulose Reductase Gene, 5-p Xylulose Phosphatase Gene, Xylitol Transporter Gene and NADP Transhydrogenase Gene.
[0124] Using the recombinant yeast that overexpressed xylitol dehydrogenase gene of Gluconobacter oxidans in step (1) and recovered the sucrase maker (Suc2) as the host, transfer respectively the 5-p xylitol dehydrogenase gene (SEQ ID NO. 14), 5-p xylulose phosphatase gene (SEQ ID NO. 31), xylitol transporter gene (SEQ ID NO. 32) and NADP transhydrogenase gene (SEQ ID NO. 44) into Yarrowia lipolytica for expression. Refer to step (1) for the methods of transformation and recovery of selective markers. Obtain the recombinant Yarrowia lipolytica ery959 that can express the above five genes simultaneously. In order to verify that the five genes in ery959 are expressed, the inventor processed the total RNA isolaton, reverse transcription and fluorescence quantitative detection, and found that the five genes had typical amplification curves, indicating that the five introduced exogenous genes got expressed. The amplification curves are shown in
[0125] According to the above results, xylitol production cannot be greatly improved by expressing genes related to xylitol synthesis in Yarrowia lipolytica, and erythritol is still synthesized in large quantities. The reason may be that 5-p xylulose, the precursor of xylitol synthesis, still flows into the pathway of erythritol synthesis mainly through ketotransferase. Therefore, it is possible to significantly improve the synthesis of xylitol by further knocking out the transketolase gene and blocking the pathway of 5-p xylulose into the synthesis of erythritol.
[0126] (6) Knocking out the Transketolase Gene on the Basis of ery959 to Obtain the Mutant ery959 ΔTKL12.
[0127] Construct and synthesize respectively the gene disruption cassettes of transketolase gene 1 (YlTKL1) and transketolase gene 2 (YlTKL2), and transform the Yarrowia lipolytica strain obtained in step (5), then knock out the two transketolase genes. Gene disruption cassettes successively contains 1 kb-1.5 kb bases upstream of the transketolase gene, retrievable selective markers (sucrase gene, with 1oxP sites at both ends of the gene, facilitating the recovery of selective markers), and 1 kb-1.5 kb bases downstream of the transketolase gene. After synthesis, the transketolase gene disruption cassettes are used to transform the Yarrowia lipolytica obtained in step (5), and screen in minimal medium supplemented with sucrose and ammonium sulfate (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 0.05 g/L each of phenylalanine, tyrosine and tryptophan, 15 g/L of agar powder, pH 6.0). Since the Yarrowia lipolytica strain obtained in step (5) cannot utilize sucrose, transformants that can grow in minimal medium containing sucrose do contain sucrose gene (Suc2), which hydrolyzes sucrose into glucose and fructose, and thus can grow. Extract the genome of the mutant and amplify by PCR with primers of P.sub.TKL1-F/P.sub.TKL1-R and P.sub.TKL2-F/P.sub.TKL2-R (primer sequences: SEQ ID NO. 46-49). The results show that both transketolase gene fragments of the control strain can be amplified (about 1100 bp DNA fragment), while the mutant can not, indicating that the two transketolase gene are knocked out (
[0128] Primer sequences used to amplify YlTKL1 gene fragment:
TABLE-US-00002 P.sub.TKL1-F: (SEQ ID NO. 46) 5′-tgaataggagacttgacagtctggc-3′ P.sub.TKL1-R: (SEQ ID NO. 47) 5′-ctctgagatcatccgagcattcaag-3
[0129] Primer sequences used to amplify YlTKL2 gene fragment:
TABLE-US-00003 P.sub.TKL2-F: (SEQ ID NO. 48) 5′-atgccccctttcaccctggcagacac-3′ P.sub.TKL2-R: (SEQ ID NO. 49) 5′-ctataacccggcacagagccttggcg-3′
[0130] Then, transform the plasmid pUB4-CRE containing Cre recombinase into mutant with both YlArDH1 and YlArDH2 knocked out, and screen in YPD agar medium containing hygromycin as selective marker (10 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 0.05 g/L each of phenylalanine, tyrosine and tryptophan, 15 g/L of agar, 300 μg/ml of hygromycin, pH 6.0). Transfer the resulting transformants to the minimal medium containing sucrose (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 0.05 g/L each of phenylalanine, tyrosine and tryptophan, 15 g/L of agar powder, pH 6.0), and select mutants with sucrase gene loss (i.e., no reuse of sucrose). Then, culture the mutants that can not use sucrose in hygromycin free liquid YPD, and then apply gradient dilution and spread on the hygromycin free solid YPD. Select the mutants that can not resist hygromycin from the resulting transformants and transfer to YPD containing hygromycin, that is, the mutant with the transketolase gene being knocked out and the sucrase gene has lost. The mutant can simultaneously express xylitol dehydrogenase gene, 5-p xylulose reductase gene, 5-p xylulose phosphatase gene, xylitol transporter gene and NADP transhydrogenase gene, in the meanwhile, the transketolase gene is knocked out. It can be used as host for other gene knockout. The sequence codes of gene disruption cassettes of transketolase genes 1 and 2 are SEQ ID NO. 50 and SEQ ID NO. 51, respectively.
[0131] Conduct the test of xylitol synthesis from glucose by fermentation with mutant ery959ΔTKL12 in the fermentation medium same to the fermentation medium in step (1), and supplemented with 0.05 g/L each of phenylalanine, tyrosine and tryptophan. Periodically take samples to detect glucose and product production, and it is found that the glucose utilization rate decreased significantly, the control strain ery959 can run out of glucose within 90 hours, and the cell OD.sub.600 is 22.5, while the mutant strain ery959ΔTKL12 still can not use up glucose at 220 hours (sterile water is added during the period to compensate for volatile water). The contents of xylitol, mannitol, arabitol, ribitol and residual glucose are 23 g/L, 36 g/L, 3 g/L, 3 g/L and 84 g/L, respectively. The OD.sub.600 is 22.5, and no erythritol are detected, indicating that the knocking out of transketolase gene plays a very important role in the synthesis of xylitol and erythritol. It also indicates that knocking out of TKL gene can inhibit cell growth, and the addition of three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) could not completely restore the density of control strain ery929. The known literature also demonstrates that, transketolase is a key enzyme in the synthesis of erythritol, and its activity is very high (Sawada et al. 2009. Key roles in transketolase activity in erythritol production by Trichosporonoides megachiliensis SN-G42. Journal of Bioscience and Bioengineering, 108: 385-390) Since cell growth is inhibited after the transketolase gene is knocked out and glucose use is significantly slower, therefore, in order to increase the cell growth and glucose utilization rate appropriately, transfer in the transketolase gene YlTKL1 with weakened promoter on the basis of strain ery959ΔTKL12 whose transketolase gene is knocked out, to partially restore the expression of transketolase gene 1. Conduct gene fusion between the weak promoter sequence (SEQ ID NO. 78) and the 5′ end of the transketolase YlTKL1 gene (SEQ ID NO. 74), to form a new sequence coded as SEQ ID NO. 79, and transform to ery959ΔTKL12, then screen on minimal medium (composition: 6 g/L of yeast nitrogen base, 10 g/L of glucose, 5 g/L of ammonium sulfate, 15 g/L of agar powder, pH6.5, without phenylalanine, tyrosine and tryptophan). Since the ery959ΔTKL12 cannot grow on the minimal medium without phenylalanine, tyrosine, and tryptophan, hence the newly grown transformant contains SEQ ID NO. 79 (down-regulate the transketolase gene), which is named ery959ΔTKL.
[0132] Conduct the test of xylitol synthesis from glucose by fermentation with mutant ery959 ΔTKL in the fermentation medium same to the fermentation medium in step (1) without phenylalanine, tyrosine and tryptophan. Periodically take samples to detect the composition of fermentation broth, and it is found that the glucose utilization rate becomes significantly faster, and the chromatographic analysis shows that the contents of xylitol, mannitol, arabitol, ribitol and erythritol are 58 g/L, 23 g/L, 3 g/L, 3 g/L and 5 g/L, respectively, and the cell OD.sub.600 is 18.4.
[0133] Although knocking out or down-regulating the expression of transketolase gene can result in a significant decrease in the content of erythritol, more mannitol and arabitol are synthesized. Therefore, further knockout of mannitol dehydrogenase and arabinol dehydrogenase genes can theoretically reduce or block the synthesis of mannitol and arabinol.
[0134] (7) Knock out the Mannitol Dehydrogenase Gene of Mutant ery959ΔTKL to Obtain the Strain ery959ΔTKLΔMDH with the mannitol dehydrogenase gene knocked out.
[0135] Construct and synthesize respectively the gene disruption cassettes of mannitol dehydrogenase gene 1 (YlMDH1) and mannitol dehydrogenase gene 2 (YlMDH2), and transform the Yarrowia lipolytica strain ery959ΔTKL, then knock out the two mannitol dehydrogenase genes. Gene disruption cassettes successively contains 1 kb-1.5 kb bases upstream of the gene, retrievable selective markers (such as aminocyclitol phoshotransferase gene, sucrase gene, with 1oxP sites at both ends of the gene, facilitating the recovery of selective markers), and 1 kb-1.5 kb bases downstream of the gene. After synthesis, it is used to transform Yarrowia lipolytica ery959ΔTKL, then screen in the minimal medium supplemented with sucrose and ammonium sulfate (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 15 g/L of agar powder, pH 6.0). Since the Yarrowia lipolytica ery959ΔTKL cannot utilize sucrose, transformants that can grow in minimal medium containing sucrose do contain sucrase, which hydrolyzes sucrose into glucose and fructose, and thus can grow. Since the sucrase gene is located in the middle of the upstream and downstream homologous sequence of the mannitol dehydrogenase gene in the gene disruption cassette, there are mutants with mannitol dehydrogenase gene knocked out in the transformants, and the mannitol dehydrogenase gene is replaced by sucrase gene in the mutant. Extract the genome of the mutant and perform PCR with the primers of the two mannitol dehydrogenase genes (sequences of peimers are SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55). The mannitol dehydrogenase gene of the control strain can be amplified (about 900 bp target DNA fragment), while that of the mutant can not, indicating that the mannitol dehydrogenase gene is indeed knocked out (
[0136] Primer sequences used to amplify YlMDH1 gene fragment:
TABLE-US-00004 P.sub.MDH1-F: (SEQ ID NO. 52) 5′-ctatctccacaacaatgcctgcaccag-3′ P.sub.MDH1-R: (SEQ ID NO. 53) 5′-ccggttacacatgactgtaggaaac-3
[0137] Primer sequences used to amplify YlMDH2 gene fragment:
TABLE-US-00005 P.sub.MDH2-F: (SEQ ID NO. 54) 5′-ccatacacagcaccacctcaatc-3′ P.sub.MDH2-R: (SEQ ID NO. 55) 5′-tctatatacatcctctaaggagc-3′
[0138] Then, transform the plasmid containing Cre recombinase (pUB4-CRE, from the following references: Fickers et al. 2003. New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia Methods, 55, 727-737) into mutants that YlMDH1 and YlMDH2 have been knockout, and recover sucrase selective markers. Screen in YPD agar medium containing hygromycin as selective marker (10 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 15 g/L of agar, 300 μg/ml of hygromycin, pH 6.0). Transfer the resulting transformants to the minimal medium containing sucrose (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of saccharose, 15 g/L of agar powder, pH 6.0), and select mutants with sucrase gene loss (i.e., no reuse of sucrose). Then, culture the mutants that can not use sucrose in hygromycin free liquid YPD, and then apply gradient dilution and spread on the hygromycin free solid YPD. Select the mutants that can not resist hygromycin from the resulting transformants and transfer to YPD containing hygromycin, that is, the knocking out of mannitol dehydrogenase gene. In the meanwhile, mutants with loss of sucrase gene, can be used as host for other gene knockout. The sequence codes of gene disruption cassettes of mannitol dehydrogenase genes 1 and 2 are shown in SEQ ID NO. 56 and SEQ ID NO. 57, respectively.
[0139] Conduct the test of xylitol synthesis from glucose by fermentation with mutant ery959 ΔTKLΔMDH in the fermentation medium same to the fermentation medium in step (1). Take samples periodically for detection, 104 hours to run out of glucose, and the content of xylitol, erythritol and ribitol are 86 g/L, 5 g/L and 3 g/L, respectively, as well as no mannitol and arabitol are detected. It can be seen that knockout of the mannitol dehydrogenase gene can eliminate both mannitol and arabitol, the by products, but ribitol is still produced. In order to eliminate ribitol, the inventor carried out an experiment to knock out the arabinol dehydrogenase gene.
[0140] (8) Knock out the Arabinitol Dehydrogenase Gene of Mutant ery959ΔTKLΔMDH to Obtain the Strain ery959ΔTKLΔMDHΔArDH with the Arabinitol Dehydrogenase Gene Knocked out.
[0141] Construct and synthesize respectively the gene disruption cassettes of arabinitol dehydrogenase gene 1 (YlArDH1) and arabinitol dehydrogenase gene 2 (YlArDH2), and transform the Yarrowia lipolytica strain ery959ΔTKLΔMDH, then knock out the two arabinitol dehydrogenase genes. Gene disruption cassettes successively contains 1 kb-1.5 kb bases upstream of the gene, retrievable selective markers (sucrase gene, with 1oxP sites at both ends of the gene, facilitating the recovery of selective markers), and 1 kb-1.5 kb bases downstream of the gene. After synthesis, the arabinitol gene disruption cassettes are used to transform the Yarrowia lipolytica ery959ΔTKLΔMDH with the mannitol dehydrogenase gene knocked out, and screen in minimal medium supplemented with sucrose and ammonium sulfate (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 15 g/L of agar powder, pH 6.0). Since the Yarrowia lipolytica strain with the arabinitol dehydrogenase gene knocked out cannot utilize sucrose, transformants that can grow in minimal medium containing sucrose do contain sucrase, which hydrolyzes sucrose into glucose and fructose, and thus can grow. Extract the genome of the transformant in the mutant and amplify by PCR with primers of P.sub.ArDH1-F/P.sub.ArDH1-R and P.sub.ARDH2-F/P.sub.ArDH2-R (primer sequences: SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61). The results show that the arabinitol dehydrogenase gene of the control strain can be amplified (about 900 bp DNA fragment), while the mutant can not, indicating that the two arabinitol dehydrogenase genes are knocked out (
[0142] Primer sequences used to amplify YlArDH1 gene fragment:
TABLE-US-00006 P.sub.ArDH1-F: (SEQ ID NO. 58) 5′- accagatggtgtaacctccatcgac-3′ P.sub.ArDH1-R: (SEQ ID NO. 59) 5′-ggaagtggtggtctgggtatcgcag-3
[0143] Primer sequences used to amplify YlArDH2 gene fragment:
TABLE-US-00007 P.sub.ArDH2-F: (SEQ ID NO. 60) 5′-cacatacaccacaacacacacaaaatc-3′ P.sub.ArDH2-R: (SEQ ID NO. 61) 5′-ttcctctgagacaatcgcgtcggatc-3′
[0144] Then, transform the plasmid pUB4-CRE containing Cre recombinase into mutant with both YlArDH1 and YlArDH2 knocked out, to recover sucrase selective markers. Screen in YPD agar medium containing hygromycin as selective marker (10 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 15 g/L of agar, 300 μg/ml of hygromycin, pH 6.0). Transfer the resulting transformants to the minimal medium containing sucrose (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 15 g/L of agar powder, pH 6.0), and select mutants with sucrase gene loss (i.e., no reuse of sucrose). Then, culture the mutants that can not use sucrose in hygromycin free liquid YPD, and then apply gradient dilution and spread on the hygromycin free solid YPD. Select the mutant ery959ΔTKLΔMDHΔArDH that can not resist hygromycin from the resulting transformants and transfer to YPD containing hygromycin, that is, the knocking out of arabinitol dehydrogenase gene. In the meanwhile, mutants with loss of sucrase gene, can be used as host for other gene knockout. The sequence codes of gene disruption cassettes of arabinitol dehydrogenase genes 1 and 2 are SEQ ID NO. 62 and SEQ ID NO. 63.
[0145] Conduct the test of xylitol synthesis from glucose by fermentation with mutant ery959ΔTKLΔMDHΔArDH in the fermentation medium same to the fermentation medium in step (1). Take samples periodically for detection, 106 hours to run out of glucose, and the content of xylitol and erythritol are 87 g/L and 6 g/L respectively, and no mannitol, arabitol and ribitol are detected.
[0146] (9) Knock out the 5-p Ribulose Isomerase Gene of the Mutant ery959ΔTKLΔMDHΔArDH to Obtain the Yarrowia lipolytica ery959ΔTKLΔMDHΔArDHΔRPI with the 5-p Ribulose Isomerase Gene Knocked out
[0147] Construct and synthesize the gene disruption cassette of 5-p ribulose isomerase gene (RPI), and transform the Yarrowia lipolytica strain ery959ΔTKLΔMDHΔArDH, then knock out the RPI. Gene disruption cassettes successively contains 1 kb-1.5 kb bases upstream of the 5-p ribulose isomerase gene, retrievable selective markers (sucrase gene, with 1oxP sites at both ends of the gene, facilitating the recovery of selective markers), and 1 kb-1.5 kb bases downstream of the 5-p ribulose isomerase gene. After synthesis, the 5-p ribulose isomerase gene disruption cassette is used to transform the Yarrowia lipolytica ery959ΔTKLΔMDHΔArDH, and screen in minimal medium supplemented with sucrose and ammonium sulfate (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 15 g/L of agar powder, pH 6.0). Since the above-mentioned Yarrowia lipolytica strain with the transketolase gene, mannitol dehydrogenase gen, arabinitol dehydrogenase gene knocked out cannot utilize sucrose, hence transformants that can grow in minimal medium containing sucrose do contain sucrase, which hydrolyzes sucrose into glucose and fructose, and thus can grow. Extract the genome of the mutant and amplify by PCR with primer of P.sub.RPI-F/P.sub.RPI-R (primer sequence: SEQ ID NO. 64-65). The results show that the 5-p ribulose isomerase gene fragments of the control strain can be amplified (about 600 bp DNA fragment), while the mutant can not, indicating that the 5-p ribulose isomerase gene is knocked out (
[0148] Primer sequences used to amplify YlRPI gene fragment:
TABLE-US-00008 P.sub.RPI-F: (SEQ ID NO. 64) 5′-aactgcctcctcttgagcaggccaag-3′ P.sub.RPI-R: (SEQ ID NO. 65) 5′-ggaacagcagcttgatcttgatgtgc-3
[0149] Transform the plasmid pUB4-CRE containing Cre recombinase into the mutant with RPI gene knocked out. Refer to the method described above or retrieving sucrase selective markers. Sequence of 5-p ribulose isomerase gene disruption cassette is SEQ ID NO. 66.
[0150] Conduct the test of xylitol synthesis from glucose by fermentation with mutant ery959 ΔTKLΔMDHΔArDHΔRPI in the fermentation medium same to the fermentation medium in step (1). Take samples periodically for detection, 102 hours to run out of glucose, and the content of xylitol and erythritol are 92.3 g/L and 6.4 g/L respectively, and no mannitol, arabitol and ribitol are detected.
[0151] (10) Knock out the Xylulose Kinase Gene of the Mutant ery959ΔTKLΔMDHΔArDHΔRPI to obtain the Yarrowia lipolytica ery959 ΔTKLΔMDHΔArDHΔRPIΔXKS1 with the Xylulose Kinase Gene Knocked out.
[0152] Construct and synthesize the gene disruption cassette of xylulose kinase gene (Y/XKS1), and transform the Yarrowia lipolytica strain ery959ΔTKLΔMDHΔArDHΔRPI, then knock out the YlXKS1. Gene disruption cassette successively contains 1 kb-1.5 kb bases upstream of the xylulose kinase gene, retrievable selective markers (sucrase gene, with 1oxP sites at both ends of the gene, facilitating the recovery of selective markers), and 1 kb-1.5 kb bases downstream of the xylulose kinase gene. After synthesis, the xylulose kinase gene disruption cassette is used to transform the Yarrowia lipolytica ery959ΔTKLΔMDHΔArDHΔRPI, and screen in minimal medium supplemented with sucrose and ammonium sulfate (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 15 g/L of agar powder, pH 6.0). Since the Yarrowia lipolytica strain with the transketolase gene knocked out cannot utilize sucrose, transformants that can grow in minimal medium containing sucrose do contain sucrase, which hydrolyzes sucrose into glucose and fructose, and thus can grow. Extract the genome of the mutant and amplify by PCR with primer of P.sub.XKS1-F/P.sub.XKS1-R (primer sequence: SEQ ID NO. 67-68). The results show that the xylulose kinase gene fragments of the control strain can be amplified (about 800 bp DNA fragment), while the mutant can not, indicating that the xylulose kinase gene is knocked out (
[0153] Primer sequences used to amplify YlXKS1 gene fragment (the amplified product is 0.8 kb):
TABLE-US-00009 P.sub.XKS1-F: (SEQ ID NO. 67) 5′-gactggatctttcgactcaacagctc-3′ P.sub.XKS1-R: (SEQ ID NO. 68) 5′-ccaaagacacaatcacgtcattggcc-3
[0154] Then, transform the plasmid pUB4-CRE containing Cre recombinase into the mutant with YlXKS1 gene knocked out, and screen in YPD agar medium containing hygromycin as selective marker (10 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 15 g/L of agar, 300 μg/ml of hygromycin, pH 6.0). Transfer the resulting transformants to the minimal medium containing sucrose (6 g/L of yeast nitrogen base, 5 g/L of ammonium sulfate, 10 g/L of sucrose, 15 g/L of agar powder, pH 6.0), and select mutants with sucrase gene loss (i.e., no reuse of sucrose). Then, culture the mutants that can not utilize sucrose in hygromycin free liquid YPD, and then apply gradient dilution and spread on the hygromycin free solid YPD. Select the mutants that can not resist hygromycin from the resulting transformants and transfer to YPD containing hygromycin, that is, the mutant ery959ΔTKLΔMDHΔArDHΔRPIΔXKS1 with the xylulose kinase gene knocked out, and in the meanwhile, with loss of sucrase gene. Sequence of xylulose kinase gene disruption cassette is SEQ ID NO. 69.
[0155] Conduct the test of xylitol synthesis from glucose by fermentation with mutant ery959ΔTKLΔMDHΔArDHΔRPIΔXKS1 in the fermentation medium same to the fermentation medium in step (1). Take samples periodically for detection, 104 hours to run out of glucose, and the content of xylitol and erythritol are 98 g/L and 6.5 g/L, respectively.
[0156] As can be seen from the results of the above ten embodiments, the mutant ery959ΔTKLΔMDHΔArDHΔRPIΔXKS1 that overexpress five enzyme genes (xylitol dehydrogenase gene, 5-p xylulose reductase gene, 5-p xylulose phosphatase gene, xylitol transporter gene and NADP transhydrogenase gene), simultaneously with five enzyme genes (transketolase gene, mannitol dehydrogenase gene, arabinitol dehydrogenase gene, 5-p ribulose isomerase gene and xylulose kinase gene) knocked out and weak express transketolase gene 1 have the best effect on the synthesis of xylitol by fermentation. After 104 hours of fermentation from 200 g of anhydrous glucose, the fermentation broth contains 98 g of xylitol. Deposit this representative strain with the deposit number of CGMCC No.18479. The following steps are optimization tests that using the representative strain as an example to synthesize xylitol by fermentation.
[0157] (11) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 25° C. and 50 g/L of Glucose.
[0158] Inoculate the recombinant yeast strains CGMCC No.18479 in a 2 L flask (using baffled flask to increase the effect of stirring dissolved oxygen) containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 50 g/L of glucose, 2 g/L of yeast cell powder, 3 g/L of peptone, 1 g/L of hydrogen diamine phosphate, initial pH5.5, fermentation at 25° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 75 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 12 g/L, and the conversion rate is 24%.
[0159] (12) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 25° C. and 200 g/L of Glucose.
[0160] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 200 g/L of glucose, 5 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of hydrogen diamine phosphate, 0.01 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.01 g/L of zinc chloride, 0.02 g/L of magnesium sulfate, initial pH5.5, fermentation at 25° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 115 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 96 g/L, and the conversion rate is 48%.
[0161] (13) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 28° C. and 300 g/L of Glucose.
[0162] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 300 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.01 g/L of zinc chloride, 0.02 g/L of magnesium sulfate, initial pH5.5, fermentation at 28° C. at 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 140 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 145 g/L, and the conversion rate is 48.3%.
[0163] (14) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 30° C. and 300 g/L of Glucose.
[0164] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 300 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.01 g/L of zinc chloride, 0.02 g/L of magnesium sulfate, initial pH5.5, fermentation at 30° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 110 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 148 g/L, and the conversion rate is 49.3%.
[0165] (15) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 30° C. and 350 g/L of Glucose.
[0166] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 350 g/L of glucose, 12 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.02 g/L of copper chloride, 0.04 g/L of magnesium sulfate, initial pH5.5, fermentation at 30° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 138 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 158 g/L, and the conversion rate is 45.1%.
[0167] (16) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 35° C. and 300 g/L of Glucose.
[0168] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 300 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.02 g/L of magnesium sulfate, initial pH5.5, fermentation at 35° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 135 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 122 g/L, and the conversion rate is 40.7%.
[0169] (17) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 32° C. and 300 g/L of Glucose at Initial pH3.0.
[0170] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 300 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.02 g/L of magnesium sulfate, prepare the initial pH3.0 with citric acid, fermentation at 32° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 115 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 142 g/L, and the conversion rate is 47.3%.
[0171] (18) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 33° C. and 250 g/L of Glucose.
[0172] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 250 g/L of glucose, 10 g/L of yeast extract, 5 g/L of steep powder, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.01 g/L of zinc chloride, 0.02 g/L of magnesium sulfate, initial pH5.5, fermentation at 33° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 108 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 121 g/L, and the conversion rate is 48.4%.
[0173] (19) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 30° C. and 300 g/L of Glucose at Initial pH7.0.
[0174] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 300 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.02 g/L of magnesium sulfate, prepare the initial pH7.0 with sodium hydroxide, fermentation at 30° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 112 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 132 g/L, and the conversion rate is 44%.
[0175] (20) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 30° C. and 100 g/L of Fructose.
[0176] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 100 g/L of fructose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.02 g/L of magnesium sulfate, initial pH5.5 with citric acid, fermentation at 30° C. and 250 rpm. Take samples periodically to determine the content of fructose and xylitol. After 120 hours of fermentation, fructose is still not completely consumed, the content of xylitol is determined to be 13 g/L, and the conversion rate is 13%.
[0177] (21) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 30° C., 200 g/L of Glucose and 100 g/L of Fructose.
[0178] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 200 g/L of glucose, 100 g/L of fructose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.02 g/L of magnesium sulfate, initial pH6.5, fermentation at 30° C. and 250 rpm. Take samples periodically to determine the content of glucose, fructose and xylitol. After 125 hours of fermentation, both glucose and fructose are completely consumed, the content of xylitol is determined to be 126.6 g/L, and the conversion rate of carbon sources, 300 g/L of glucose and fructose, is 42.2%.
[0179] (22) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 at 30° C. and 100 g/L Glycerol.
[0180] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 2 L baffled flask containing 50 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 100 g/L of glycerol, 5 g/L of yeast cell powder, 3 g/L of peptone, 2 g/L of ammonium citrate, 0.02 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.02 g/L of magnesium sulfate, initial pH5.5 with citric acid, fermentation at 30° C. and 250 rpm. Take samples periodically to determine the content of glycerol and xylitol. After 130 hours of fermentation, glycerol is still not completely consumed, the content of xylitol is determined to be 4.5 g/L, which may be due to that the transketolase gene is down-regulated by weak expression, and glycerol utilization efficiency is slowed down. In addition, the strain ery959ΔTKL12, whose transketolase gene is completely knocked out, can not synthesize xylitol from glycerol. Due to lack of transketone, the strain could not synthesize 5-p xylulose from glycerol, which is a precursor of xylitol, and thus no xylitol synthesized.
[0181] (23) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 Utilizing Starch as Carbon Source.
[0182] Add 100 g of starch (from corncob) to 350 mL of cold water while agitating the water until it becomes starch milk, thus obtain 415 mL of starch milk (mass volume percentage of starch is 24%, i.e. 240 g/L). Heat to 90° C., then add 0.2 grams of thermoresistant α-amylase and stir until the starch is liquefied and clear. After cooling to 60 degrees, add 0.2 grams of medium temperature β-amylase and 0.1 grams of Pullulanase for saccharification, hold for 5 hours, then use for fermentation materials, add 3.5 grams of yeast cell powder, 2 grams of steep powder, 1.5 grams of ammonium citrate, 0.1 grams of magnesium sulfate, sterilize at 108° C. for 30 minutes, then allow to cool. Inoculate the strains of recombinant yeast CGMCC 18479 in this medium with an initial opitical density (OD.sub.600) of 0.8, initial pH5.5, fermentation at 30° C. and 250 rpm. Take samples periodically to determine the content of glucose and xylitol. After 106 hours of fermentation, glucose in the fermentation medium is completely consumed, and the content of xylitol is determined to be 86 g/L, equivalent to the conversion rate of xylitol synthesized from starch is 35.8%.
[0183] In the above embodiments of xylitol synthesis by fermentation, the evaporated water must be replenished regularly to the initial weight during the fermentation process. Note down the weight of the fermentation bottle containing the fermentation liquid at the beginning of fermentation, and note down the weight every time you take a sample, and replenish the water with sterile water to the initial weight. The sample volume taken each time is 0.2 ml and diluted tenfold for HPLC determination of the content of carbon source materials (e.g. glucose, glycerol, fructose, etc.) and xylitol. The analytical column is SP0810 HPLC column of Shodex, refractive differential detector, pure water as mobile phase, flow rate is 1 mL/min, column temperature is 70 degrees.
[0184] (24) Synthesis of Xylitol using Yeast Strain CGMCC No.18479 in Fermenter.
[0185] Inoculate the strains of recombinant yeast CGMCC No.18479 in a 5 L-fermenter containing 3500 ml of fermentation medium with an initial opitical density (OD.sub.600) of 0.8. Fermentation medium components: 300 g/L of glucose, 10 g/L of yeast cell powder, 5 g/L of peptone, 3 g/L of ammonium citrate, 0.01 g/L of manganese chloride, 0.01 g/L of copper chloride, 0.01 g/L of magnesium sulfate, 0.02 g/L of zinc chloride, initial pH6.5, fermentation at 30° C. with the initial speed of agitation is 300 rpm, and increase to 450 rpm when the cells growth reach to OD.sub.600 above 3.0, and to 550 rpm when the OD.sub.600 surpass 10.0. Take samples periodically to determine the content of glucose and xylitol. Steriled water should be added to compensate for water evaporated during fermentation. After 110 hours of fermentation, glucose is completely consumed, the content of xylitol is determined to be 152 g/L, and the conversion rate is 50.7%.
[0186] In each of the above steps, the fermentation medium is sterilized and cooled to room temperature before inoculating yeast strains.
[0187] (25) Purification of Xylitol from Fermentation Broth.
[0188] After fermentation, place the fermentation broth into a 500 ml-centrifuge tube and centrifuge at 6000×g for 20 minutes to obtain the clear xylitol-containing supernatant. Use 200 ml of pure water to suspend and wash the precipitated yeast cells to release the xylitol in the cells, and obtain the supernatant by centrifugation again. Collection of all fermentation supernatant, then transfer to a rotatory evaporating flask for evaporation and concentration. Measure the refractive index during this period, and stop evaporating when refractive index (soluble solid content) reached 68. Transfer the concentrated solution into a spherical flask and stir slowly with a magnetic agitator in a gradient cooler at 50 rpm. When the temperature decreased to 22° C., fine granular crystals began to appear. With the gradual decrease of temperature, the amount of crystallization gradually increased. At this time, increase the stirring speed to 80 rpm. Stop stirring when the amount of crystallization does not increase, and separate the crystals by centrifugation to obtain the crude products of xylitol. Redissolve in distilled water until refractive index is 45, then process successively the steps of ion exchange, decolorization, removal of ions and pigments, reconcentration, crystallization, centrifugation and drying, to obtain the refined products of xylitol. Analysis through GC-Mass to identify the xylitol isolated and purified from fermentation broth and the standard xylitol.
[0189] The above description has described in detail certain exemplary embodiments. It is to be understood that the embodiments of the present invention are not to be limited to the above specific exemplary embodiments, that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments within the scope of the claim, which does not affect the substance of the invention.