KR101827822B1 - Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) with improved xylitol production activity and method for production of xylitol thereof - Google Patents

Abstract

The present invention relates to a method for producing xylitol using Cluyveromyces marcianus 36907-FMEL1 (KCTC18459P) improved in xylitol production ability through mutation and capable of mass-producing xylitol at a relatively high temperature.

Description

(Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) with improved xylitol production activity and method for production of xylitol), which has improved production ability of xylitol,

The present invention relates to Kluyveromyces marcans 36907-FMEL1 (KCTC18459P) having improved xylitol production ability through mutation and a production method of xylitol using the same.

Xylitol is a pentose alcohol, which has a low calorie content and is used as a natural sweetener substitute for sugar because it does not form an acid which causes tooth decay. It is also actively used in chewing gum, confectionery, medicine and oral hygiene.

In addition, xylitol does not interfere with insulin action in cells, and promotes insulin secretion, so that it does not affect blood glucose. Because of this characteristic, it can be used as a substitute for glucose in diabetic patients and is actively used for medical use.

On the other hand, xylitol has been produced through a chemical dehydrogenation process using xylose, which is not only low in production yield of xylitol but also produces a large amount of by-products, which is very inefficient. Therefore, production of xylitol using biological methods rather than chemical methods is the best method in terms of high production yield and environmental safety.

However, most studies on xylitol production using conventional biological methods have used non-thermostable yeast or bacteria that are not resistant to heat, and researches at a temperature as low as 30 to 37 ° C which is suitable for strain growth rather than optimal temperature for enzyme activity It is a fact that it is performing.

On the other hand, Kluyveromyces marxianus ) are widely used to produce biofuels from plant fiber since they have a variety of substrates, are fast growing, and have heat resistance characteristics. The heat-resistant strain Kluyveromyces marxianus ) has advantages such as low cost for cooling, efficient enzymatic hydrolysis, simultaneous saccharification fermentation and minimization of contamination.

To date, the introduction of the xylose metabolism pathway has led to the development of recombinant Kluyveromyces marjans ( Kluyveromyces marxianus ), but there was a limit to the efficient production of xylitol.

Korean Patent No. 10-1246780 (registered on March 31, 2013) discloses a method for producing xylitol in a mixed medium of xylose and arabinose using a xylitol producing strain newly introduced with an Arabidopsis pathway, (AraA), L-ribulokinase (araB), and L-ribulokinase (araB). The present invention relates to a method for efficiently producing xylitol by inhibiting the production of arabitol, In order to efficiently express the L-ribulose-5-phosphate 4-epimerase (araD) enzymes in the Candida tropicalis strain, codon optimization was performed, and then each gene was subjected to glyceraldehyde- Was inserted into a cassette containing a promoter of glyceraldehyde-3-phosphate dehydrogenase and URA3 as a selectable marker and introduced into a Candida sp. Strain (Candida sp.) To obtain xylitol Xylitol production method of inhibiting arabitol produced to interfere with Shaanxi purification and crystallization process have been described. Korean Registered Patent No. 10-1194752 (Registration Date: Oct. 1, 2012) discloses a method for producing xylitol in a medium containing glucose as a carbon source using a xylitol producing strain in which xylose reductase is constitutively expressed More particularly, the present invention relates to a method of producing codon optimization of a gene encoding Neurospora crassa, which expresses xylose reductase from Candida tropicalis with high activity, To obtain optimized xylose reductase; In order to express the optimized gene, the promoter and terminator portion of the glyceraldehyde-3-phosphate dehydrogenase of the cloned Candida tropicalis; And a selection marker URA3, a xylitol producing strain in which the xylose reductase produced by introducing the expression vector into Candida tropicallis inactivated with xylitol dehydrogenase, and a strain producing the xylitol dehydrogenase Discloses a method for producing xylitol without inhibition of glucose as a carbon source through fed-batch culture.

In the present invention, the productivity of xylitol, Kluyveromyces marcescans marxianus ) To develop and provide strains.

The present invention also provides a method for mass-producing xylitol using the strain.

The present invention provides Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) with improved xylitol production ability.

On the other hand, a method for producing xylitol is provided, wherein Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) is inoculated and cultured in a medium supplemented with xylose.

On the other hand, in the present invention, the production method of xylitol is preferably selected from the group consisting of Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) at 30 to 40 DEG C and 350 to 450 rpm.

In the present invention, a mutant strain capable of effectively producing xylitol from xylose was selected after random mutagenesis of Kluyveromyces marxianus strain 36907, a thermostable yeast strain, was selected.

The finally selected as Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus) for 36907-FMEL1 (KCTC18459P) strains, the parental strain is Vero Cluj My process Marcia Taunus (Kluyveromyces marxianus ) Compared to 36907, xylitol production was increased by 120% and yield was increased by 39%.

Meanwhile, in the present invention, in order to improve xylitol productivity at high temperature, Kluyveromyces marcans marxianus ) 36907-FMEL1 (KCTC18459P), and it was confirmed that the productivity of xylitol was increased at 40 ° C and 400rpm.

In the present invention, Kluyveromyces marcescans ( Kluyveromyces marcescans) having improved xylitol productivity through mutation marxianus ) 36907-FMEL1 (KCTC18459P).

In addition, when this strain is used, xylitol can be mass-produced even at a relatively high temperature.

FIG. 1 shows the results of confirming the production of xylitol by fermenting Kluyveromyces marxianus strain 36907, a parent strain, in a medium supplemented with xylose. (○: OD, ◆: xylitol, ■: xylose, △: ethanol)
Figure 1B shows the results of a random mutagenesis of Kluyveromyces < RTI ID = 0.0 > ( Kluyveromyces < / RTI > marxianus ) 36907-FMEL1 (KCTC18459P) strain was fermented in a medium containing xylose to confirm the production of xylitol. (○: OD, ◆: xylitol, ■: xylose, △: ethanol)
Figures 2 A and B are the results of fermentation with Kluyveromyces marxianus 36907 supplied at 30 ° C and 40 ° C, respectively, with glucose. (?: Glucose,?: OD,?: Ethanol)
2 C and D show results of fermentation of Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) by feeding glucose at 30 ° C and 40 ° C, respectively. (?: Glucose,?: OD,?: Ethanol)
FIG. 3A shows the results of confirming the production of xylitol by fermenting Kluyveromyces marxianus strain 36907 at 40 DEG C in a medium supplemented with xylose. (○: OD, ◆: xylitol, ■: xylose, △: ethanol)
FIG. 3B shows the results of confirming the production of xylitol by fermenting Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strain at 40 ° C in a medium supplemented with xylose. (○: OD, ◆: xylitol, ■: xylose, △: ethanol)
Figure 4 shows that Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strains were tested at 300 (A), 350 (B), 400 (C), 450 (D) and 500 , And the amount of xylitol produced was measured. (■: xylose, ◇: xylitol, ●: OD, △: ethanol)
FIG. 5 shows the results of a strain of Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strain at 300 (A), 350 (B), 400 (C), 450 (D) and 500 , And the amount of xylitol produced was measured. (■: xylose, ◇: xylitol, ●: OD, △: ethanol)
FIG. 6 shows the results of comparing the amounts of xylitol produced by fermentation of Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strain at 30 and 40 ° C using a flask and a fermenter, respectively.

Kluyveromyces marxianus ) are used for the production of biofuels from plant fiber because they can be used in a variety of substrates, are fast growing, and have heat resistance. The advantages of heat-resistant strains include low cost of cooling, efficient hydrolysis of enzymes, simultaneous saccharification and fermentation, and minimization of contamination.

The present invention was to improve Kluyveromyces marxianus , a heat-resistant strain, to select strains having excellent xylitol production ability. Experimental candidates Among Kluyveromyces marxianus strains of seven , Kluyveromyces marxianus 36907 showed high yield and yield of xylitol.

Then, Cluj through random mutagenesis Vero My process Marcia Taunus (Kluyveromyces marxianus) the more xylitol production increased 36 907 mutant strain Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P). Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus) 36907-FMEL1 ( KCTC18459P) is the parental strain of Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus ) 36907, the yield of xylitol was 120%, the yield of production was 39%, and the production of xylitol was doubled.

In the present invention, ethylmethanesulfonate (EMS) was used to generate a random mutation, which caused an alkylation reaction to the guanine and thymine bases of the DNA to distort normal complementary binding, And the alkylated thymine are each bound to guanine.

After the mutation is induced by the characteristics of EMS, once the DNA replication occurs, a point mutation is caused in which the G-C base pair is changed to A-T and the T-A base pair is changed to C-G. These mutations have the effect of altering one nucleotide sequence, resulting in a change in the amino acid cipher code consisting of three bases. This leads to mutations that have lost function or acquired new functions.

On the other hand, Kluyveromyces marxianus ) 36907 and the Kluyveromyces marxianus ( Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) strain is a heat-resistant strain. Therefore, in the present invention, a fermentation experiment for producing xylitol from xylose at 45 DEG C for 120 hours was conducted at a relatively high temperature. However, both of the Kluyveromyces marxianus strains consumed 22 to 29 g / L of xylose and produced very poor yields of 10 to 12 g / L of xylitol.

Therefore, in the present invention, in order to further confirm the heat resistance of two strains of Kluyveromyces marxianus , YP medium supplemented with 80 g / L of glucose at 30 ° C and 45 ° C was used for glucose fermentation The experiment was carried out. As a result, Kluyveromyces marxianus ) 36907 and the Kluyveromyces marxianus ( Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) strain exhibited excellent heat resistance when glucose was used as a carbon source. However, the glucose consumption rate and the ethanol production rate at 30 ° C were slightly or almost similar to those at 45 ° C. From the above results, it can be concluded that two strains of Kluyveromyces marxianus according to the present invention have a very low ATP biosynthesis to maintain heat resistance when xylose is used as a carbon source.

Therefore, we have conducted experiments to produce xylitol from xylose by lowering the incubation temperature to 40 ° C, which is 5 ° C lower than 45 ° C. In this experiment, Kluyveromyces marxianus 36907 and Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) showed higher concentrations of xylitol and productivity than those of 45 ° C. At 40 ℃ a result of the 144 hours fermentation, Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus) 36907-FMEL1 (xylitol production was 25 g / L of KCTC18459P), Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus) xylitol production of 36907 was 14 g / L.

Meanwhile, in the present invention, an experiment for optimizing the stirring speed was performed in order to improve productivity of xylitol at high temperature. The productivity of xylitol was greatly improved at 40 ° C and 400 rpm.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example 1: 7 things Cluey Bromyces Marcianus  ( Kluyveromyces  marxianus ) Strain Xylitol  Superior production capacity K. marxianus  Strain selection]

(1) Culture of the strain

In this experiment, seven strains of Kluyveromyces marxianus , which are currently used for the production of xylitol, Were used.

A total of seven Kluyveromyces marxianus ) In order to culture the strains (124224, 46537, 200963, 36907, 26548, CBS 1555, and CBS 607), each strain was inoculated into YP medium (10 g / L yeast extract, 20 g / L Bacto peptone) at 200 rpm and 30 < 0 > C.

The cultured cells were then washed twice with sterile water and used in the following.

 (2) Xylitol  Superior production capacity Cluey Bromyces Marcianus  ( Kluyveromyces marxianus ) Selection of strains

The Kluyveromyces marxianus cultivated in the above- To confirm the ability of the strain to produce xylitol, each strain was cultured in YP medium supplemented with 80 g / L xylose at 200 rpm at 30 ° C for 4 days. After 4 days, the xylitol production capacity of each strain was measured and shown in Table 1 below.

Xylitol measurement was carried out by high performance liquid chromatography (HPLC) using a Rezex Roa-oRGANIC aCID H + column (Phenomenex Inc., Torrance, Calif.) Column and a '0.005 N H2SO4' 1200 Series, Agilent Technologies, Santa Clara, Calif.), And metabolites such as xylose and ethanol were measured and analyzed in the same manner.

Strain name Consumed xylose  (g / L) xylitol (g / L) xylitol yield
(g / g) Ethanol
(g / L) 124224 66.86 6.68 0.01 0.69 46537 75.46 0 0 1.91 200963 39.05 2.82 0.07 2.43 36907 27.85 13.27 0.48 1.03 26548 25.32 9.79 0.39 0.68 CBS 1555 72.73 0 0 0 CBS 607 59.11 0 0 0.72

As a result of measurement, Kluyveromyces marxianus ) 46 537 strain and Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus ) The CBS 1555 strain showed high xylose consumption of 75.46 g / L and 72.73 g / L, respectively, while xylitol was not produced at all and the ethanol production was 0 g / L and 1.91 g / L respectively.

On the other hand, Kluyveromyces marxianus ) In the case of strain 36907, the consumption of xylose was 27.85 g / L, the yield of xylitol was 13.27 g / L, the yield of xylitol was 0.48 g / g, and the yield of ethanol was found to be as low as 1.03 g / L .

On the other hand, Kluyveromyces marxianus ) For the strain 26548, 9.79 g / L xylitol was produced and the remaining Kluyveromyces marcescans marxianus ) 124224, 200963, and CBS 607 strains failed to produce xylitol or production was insufficient.

Taken together, the seven Kluyveromyces marxianus ( Kluyveromyces marxianus ) In the majority of the strains, the consumption of xylose was high, but it was found to be insufficient to produce xylitol from xylose by producing very little xylitol and also ethanol.

However, Kluyveromyces marxianus ) 36907 strains showed the highest xylitol yield and xylitol yield, confirming that it is a suitable strain for producing xylitol from xylose.

Thus, finally, Kluyveromyces marxianus ( Kluyveromyces marxianus ) 36907 strains were selected and used in Example 2 below.

[ Example  2: Cluey Bromyces Marcianus  ( Kluyveromyces marxianus ) 36907 < / RTI >

The Kluyveromyces marxianus , prepared above, 36907 strain was cultured in YP medium supplemented with 20 g / L of glucose for 12 hours, followed by centrifugation to obtain a cultured strain. First, 1.0 M of EMS was treated for 10 minutes, 20 minutes, 30 minutes, and 40 minutes, respectively, to determine the optimum time for inducing mutation of the obtained strain, and Kluyveromyces marxianus The cell death rate of strain 36907 was measured.

As a result of the measurement, it was confirmed that when treated with 1.0 M of EMS for 30 minutes, the cells exhibited an average cell death rate of 99.90 to 99.99%, and under the above conditions, random mutagenesis was induced to produce several Kluyveromyces marcans marxianus ) 36907 mutant strains were prepared.

Then, each strain was plated on a YP agar medium supplemented with 20 g / L of xylose, and a total of 580 Kluyveromyces marcans marxianus ) 36907 mutants were selected and isolated.

The 580 Kluyveromyces marxianus isolated from the above- In order to select the strains with the highest xylitol production capacity among the 36907 mutants, the production ability of xylitol of each strain was examined.

580 Kluyveromyces marxianus , isolated from the above, The 36907 mutant strain was cultured in a cell incubator for 4 days at a temperature of 30 DEG C in 1.5 mL of YP medium (24-well plate) supplemented with 80 g / L of xylose, and after 4 days, xylitol production And the strain with high xylitol production yield was selected.

As a result of screening, it was confirmed that 10 cloveromyces marcans ( Kluyveromyces marxianus ) 36907 mutants were selected and each strain was inoculated in a 250 ml flask with 50 ml of YP medium supplemented with 80 g / L of xylose to give an initial absorbance (OD ₆₀₀ ) of 1.0, , And the yield of xylitol production was measured.

As a result of the measurement, strains which produced the most xylitol among the 10 strains were selected, and they were classified into Kluyveromyces marcescans marxianus ) 36907-FMEL1 (KCTC18459P) and used in the following examples.

[ Example  3: Cluey Bromyces Marcianus  ( Kluyveromyces marxianus ) 36907 and Cluey Bromyces Marcianus  ( Kluyveromyces marxianus Comparison of xylitol production ability of strain 36907-FMEL1 (KCTC18459P)]

In this experiment, the above-mentioned Kluyveromyces marxianus ( Kluyveromyces marxianus ) 36907 and Kluyveromyces marxianus ( Cluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) strains.

The two strains were fermented in YP medium supplemented with 80 g / L of xylose at a temperature of 30 ° C and 100 rpm for 144 hours to measure and compare the production of xylitol.

As a result of measurement, Kluyveromyces The strain 36907-FMEL1 (KCTC18459P) consumed 78 g / L of xylose and produced 53 g / L of xylitol and showed productivity of 0.67 g / g of xylitol and 0.36 g / L ㆍ h of xylitol .

This is the parent strain Kluyveromyces marxianus ) Xylitol concentration was 120%, production yield was 39%, and xylitol production was doubled as compared to 36907 strain (Fig. 1). Figure 1 shows the results of a random mutagenesis in which Kluyveromyces marjans ( Kluyveromyces marxianus ) 36907 strain was fermented in a medium containing xylose to confirm the production of xylitol. (○: OD, ◆: xylitol, ■: xylose, △: ethanol)

Figure 1B shows the results of a random mutagenesis of Kluyveromyces < RTI ID = 0.0 > ( Kluyveromyces < / RTI > marxianus ) 36907-FMEL1 (KCTC18459P) strain was fermented in a medium containing xylose to confirm the production of xylitol. (○: OD, ◆: xylitol, ■: xylose, △: ethanol)

Interestingly, Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus) 36907-FMEL1 ( KCTC18459P) strains were stopped in the production of ethanol 1.7 g / L, an enhanced xylitol production ability of the strain was maintained even after more than 10 passages.

These results indicate that selected Kluyveromyces marxianus ( Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) is a suitable strain for mass production of xylitol.

[ Example  4: Cluey Bromyces Marcianus  ( Kluyveromyces marxianus ) 36907 and Cluey Bromyces Marcianus  ( Kluyveromyces marxianus ) Identification of heat resistance of strain 36907-FMEL1 (KCTC18459P)

In this experiment, the heat resistance of Kluyveromyces marxianus 36907 and Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) was examined.

The heat-resistant strain Kluyveromyces marxianus ) 36907 and Cluyveromyces marxanus ( Kluyveromyces marxianus ) The strain 36907-FMEL1 (KCTC18459P) was fermented at a high temperature of 45 ° C for 144 hours using YL medium supplemented with xylose.

As a result of the experiment, it was confirmed that both of the strains consumed 22 ~ 29 g / L of xylose and produced 10 ~ 12 g / L of xylitol (not shown)

Based on the above results, it was tried to further confirm the heat resistance of the two strains. The two strains were subjected to glucose fermentation test at 30 ° C and 45 ° C for 12 hours using YP medium supplemented with 80 g / L of glucose Respectively.

As a result of the experiment, Kluyveromyces marxianus ) 36907 strains and Kluyveromyces marcescans marxianus ) When 36907-FMEL1 (KCTC18459P) strain used glucose as a carbon source, it was confirmed that the strain exhibited excellent heat resistance. However, the glucose consumption rate and the ethanol production rate when cultured at 30 ° C were slightly or almost similar to those when cultured at 45 ° C.

As a result of the above experiment, it was confirmed that when the xylose was used as a carbon source, the two strains had very low ATP biosynthesis for maintaining heat resistance (Fig. 2). Therefore, it was judged that it is necessary to lower the incubation temperature in order to produce xylitol from xylose.

Figures 2 A and B are the results of fermentation with Kluyveromyces marxianus 36907 supplied at 30 ° C and 40 ° C, respectively, with glucose. (?: Glucose,?: OD,?: Ethanol)

2 C and D show results of fermentation of Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) by feeding glucose at 30 ° C and 40 ° C, respectively. (?: Glucose,?: OD,?: Ethanol)

[ Example 5: 40 ° C  Under the conditions, Cluey Bromyces Marcianus  ( Kluyveromyces marxianus ) 36907 and Cluey Bromyces Marcianus  ( Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) strain Xylitol  Production confirmation]

As a result of the above experiment, it was confirmed that the present invention, Kluyveromyces marxianus , 36907 strain and Kluyveromyces marxianus ( Kluyveromyces marxianus ) The 36907-FMEL1 (KCTC18459P) strain did not show significant heat resistance at 45 ° C culture with xylose as a carbon source. In this experiment, xylitol production of these strains was determined at 40 ° C, which is 5 ° C lower than 45 ° C Respectively.

The experiment was carried out in the same manner as in Example 4 except that the temperature condition was 40 ° C, and the fermentation was carried out in the YP medium supplemented with xylose for 144 hours.

As a result of the experiment, Kluyveromyces Xylitol production marxianus) 36907-FMEL1 (KCTC18459P) was 25 g / L, Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus ) The xylitol production of 36907 was 14 g / L. Unusually, Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) produced about twice the yield compared to 45 ° C.

In addition, Kluyveromyces marxianus ) 36907-FMEL1 (KCTC18459P) was deposited in Kluyveromyces marxianus ( Kluyveromyces marxianus ) Compared with 36907 (FIG. 3).

From the above results, it was confirmed that Kluyveromyces marxianus ( Kluyveromyces marxianus ) The xylitol production capacity of 36907-FMEL1 (KCTC18459P) was higher than that of Kluyveromyces marxianus ) Which is higher than that of 36907.

FIG. 3A shows the results of confirming the production of xylitol by fermenting Kluyveromyces marxianus strain 36907 at 40 DEG C in a medium supplemented with xylose. (○: OD, ◆: xylitol, ■: xylose, Δ: ethanol)

FIG. 3B shows the results of confirming the production of xylitol by fermenting Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strain at 40 ° C in a medium supplemented with xylose. (○: OD, ◆: xylitol, ■: xylose, △: ethanol)

[ Example 6: Effect of xylitol on growth of Cluyveromyces Marcia Taunus (Kluyveromyces marxianus) optimize agitation speed of 36907-FLEL1 strain;

(One) Stirring speed  Check optimization conditions

Kluyveromyces marxianus , which was excavated in the above example, 36907-FMEL1 (KCTC18459P), the fermentation agitation speed was optimized to improve the productivity of xylitol.

Kluyveromyces The flask was cultured in YP medium supplemented with 80 g / L of xylose at a temperature of 300, 350, 400, 450 and 500 rpm at 30 ° C and 40 ° C for 4 days, And the production of xylitol of each strain was compared. The results of culturing are shown in Tables 3 to 4 and 4 to 5, respectively.

After culturing at 300, 350, 400, 450 and 500 rpm at 30 ° C rpm Optical Density
(A ₆₀₀ nm) Consumed
xylose (g / L) Xylitol (g / L) Yield (g / g) Productivity
(g / Lh) 300 12 49 23 0.46 0.23 350 37 68 35 0.44 0.29 400 31 77 41 0.53 0.57 450 55 78 26 0.33 0.44 500 55 71 9 0.12 0.14

After culturing at 300, 350, 400, 450 and 500 rpm at 40 ° C rpm Optical Density
 (A 600 nm) Consumed
xylose (g / L) Xylitol  (g / L) Yield (g / g) Productivity
(g / Lh) 300 5.62 42 24 0.57 0.25 350 10.40 60 36 0.59 0.49 400 18.60 68 43 0.64 0.90 450 36.00 77 31 0.42 0.46 500 36.04 75 17 0.20 0.36

As a result of the experiment, it was confirmed that Kluyveromyces marcans ( Kluyveromyces) cultured at 40 ° C and 400 rpm marxianus ) The production rate of xylitol of 36907-FMEL1 (KCTC18459P) strain was 43 g / L and the production rate was 0.90 (g / L · h), which was higher than other stirring speed conditions.

Therefore, it was confirmed that the optimization conditions for high production of xylitol at high temperature were 40 ° C and 400 rpm (FIGS. 4 and 5).

Figure 4 shows that Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strains were tested at 300 (A), 350 (B), 400 (C), 450 (D) and 500 , And the amount of xylitol produced was measured. (■: xylose, ◇: xylitol, ●: OD, △: ethanol)

FIG. 5 shows the results of a strain of Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strain at 300 (A), 350 (B), 400 (C), 450 (D) and 500 , And the amount of xylitol produced was measured. (■: xylose, ◇: xylitol, ●: OD, △: ethanol)

(2) Stirring speed  Comparison of productivity of xylitol using flask incubator and fermenter under optimized conditions

In this example, based on the conditions of the stirring speed optimization as confirmed in the above experiment, Kluyveromyces marxianus ) 36907-FLEL1 cultures and fermentation broth cultures.

Under the conditions of '40 ° C, 400 rpm' and '30 ° C, 400 rpm', Kluyveromyces marxianus ) The 36907-FMEL1 (KCTC18459P) strain was cultured in YP medium supplemented with 80 g / L xylose, and cultured for 144 hours in the flask culture and 72 hours and 60 hours in the fermentation culture, respectively.

As a result of the experiment, it was confirmed that the productivity of xylitol at the optimum condition (40 ° C, 400 rpm) of the fermenter was increased about 6 times as compared with that of the flask culture (FIG. 6). FIG. 6 shows the results of comparing the amounts of xylitol produced by fermentation of Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) strain at 30 and 40 ° C using a flask and a fermenter, respectively.

Korea Biotechnology Research Institute KCTC18459P 20160405

Claims (3)

Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) with improved xylitol production.
A method for producing xylitol, which comprises culturing Kluyveromyces marxianus 36907-FMEL1 (KCTC18459P) inoculated on a culture medium containing xylose.
3. The method of claim 2,
The above-
30 to 40 DEG C, and 350 to 450 rpm.

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