阅读说明：本技术 一种高效利用淀粉生产l-亮氨酸的方法 (Method for producing L-leucine by efficiently utilizing starch ) 是由 徐建中 史可 王颖妤 章洁颖 郝宇晨 *** 于 2019-10-25 设计创作，主要内容包括：本发明公开了一种高效利用淀粉生产L-亮氨酸的方法,属于生物催化技术领域。本发明通过对棒杆菌属的L-亮氨酸产生菌进行温度诱导的适应性进化处理,提高菌株对高温的耐受性。随后,在此突变菌株基础上异源表达来源于芽孢杆菌属微生物(如B.amyloliquefaciens)中α-淀粉酶编码基因,在棒杆菌属的L-亮氨酸产生菌中构建了可高效利用淀粉的代谢途径,从而改善了最适菌体生长温度与α-淀粉酶最适作用温度不一致的的缺点,获得了高效利用淀粉生产L-亮氨酸的重组谷氨酸棒杆菌。(The invention discloses a method for producing L-leucine by efficiently utilizing starch, belonging to the technical field of biocatalysis. The invention improves the high-temperature resistance of the strain by carrying out temperature-induced adaptive evolution treatment on the L-leucine producing strain of the corynebacterium. Then, on the basis of the mutant strain, an alpha-amylase coding gene derived from a bacillus microorganism (such as B. amyloliquefaciens) is expressed in a heterologous manner, and a metabolic pathway capable of efficiently utilizing starch is constructed in an L-leucine producing strain of corynebacterium, so that the defect that the optimal growth temperature of the strain is inconsistent with the optimal action temperature of the alpha-amylase is overcome, and the recombinant corynebacterium glutamicum for efficiently utilizing the starch to produce the L-leucine is obtained.)

1. A method for producing L-leucine by efficiently utilizing starch is characterized by comprising the following steps:

(1) inoculating starting Corynebacterium into LBG liquid medium, culturing at optimum temperature, and measuring specific growth rate χ of starting Corynebacterium at optimum growth temperature₀I.e. mu x₀；

(2) Inoculating the starting corynebacterium grown to the middle and later logarithmic growth stage in the step (1) into a fresh LBG liquid medium, culturing at 34-36 ℃, and measuring the specific growth rate mu x of the strain at 34-36 DEG C₁(ii) a Further, the above-mentioned strain grown to the middle and late logarithmic growth stage was inoculated into a fresh LBG liquid medium and cultured at 34-36 ℃ and the specific growth rate. mu. times.χ.of the strain at 34-36 ℃ was measured₂(ii) a The above steps are repeated until the strain has a specific growth rate mu ≈ chi at 34-36 DEG C₀Obtaining mutant strains which normally grow at the temperature of 34-36 ℃;

(3) inducing the mutant strain which normally grows at the temperature of 39-41 ℃ by using the mutant strain which normally grows at the temperature of 34-36 ℃ obtained in the step (2) as an original strain by adopting the same method as the step (2);

(4) inducing the mutant strain which normally grows at 44-46 ℃ by using the mutant strain which normally grows at 39-41 ℃ and is obtained in the step (3) as an original strain by adopting the same method as the step (2);

(5) taking the mutant strain which is obtained in the step (4) and normally grows at 44-46 ℃ as a host bacterium, and heterologously expressing an alpha-amylase gene to obtain a recombinant bacterium capable of expressing the alpha-amylase;

(6) and (3) taking starch as a unique carbon source, and fermenting and producing the L-leucine by using the recombinant bacteria obtained in the step (5).

2. The method of claim 1, wherein the corynebacterium is corynebacterium glutamicum.

3. The method as claimed in claim 1, wherein in step (1), the cultivation is carried out at a temperature of 28-32 ℃ and a rotation speed of 100-150 r/min.

4. The method as claimed in claim 1, wherein in step (2), the cultivation is performed at a rotation speed of 100-150 r/min.

5. The method according to claim 1, wherein in step (5), the alpha-amylase gene is derived from Bacillus amyloliquefaciens, Bacillus subtilis, or Bacillus licheniformis.

6. The method according to claim 1, wherein in the step (6), the fermentation comprises the following specific steps: inoculating the recombinant bacterium single colony into an LBG culture medium to be cultured to a logarithmic phase, and transferring the seed culture solution to an improved fermentation culture medium to carry out fermentation production of L-leucine.

7. The method of claim 6, wherein the modified fermentation medium is: soluble starch 40-50g/L, (NH)₄)₂SO₄ 10-20g/L，CH₃COONH₄ 10-20g/L，KH₂PO₄ 2-4g/L，MgSO₄·7H₂O 0.5-1.5g/L，MnSO₄·4H₂0.05-0.15g/L of O, 2-4g/L of sodium citrate, 2-5g/L of urea, 0.5-1.5g/L of L-methionine, 0.5-1.5g/L of L-glutamic acid, 0.05-0.15g/L of L-isoleucine, 1-2g/L of betaine hydrochloride, 0.0003-0.0008g/L of biotin, 0.0005-0.0010g/L of thiamine, CaCO₃ 30-50g/L。

8. The method of claim 6, wherein the culture conditions of the seed culture stage are: the culture temperature is 44-46 ℃, the rotation speed is 100-150r/min, and the culture time is 10-14 h.

9. The method of claim 6, wherein the inoculum size of the seed broth transferred to the fermentation medium is 5-15%.

10. The method according to claim 6, wherein the culture conditions of the fermentation stage are: the culture temperature is 44-46 ℃, the rotation speed is 100-150r/min, and the culture time is 60-80 h.

Technical Field

The invention relates to a method for producing L-leucine by efficiently utilizing starch, belonging to the technical field of biocatalysis.

Background

L-leucine is one of eight essential amino acids required for humans and animals, and is collectively called branched-chain amino acid because of having a methyl side chain branching structure together with L-valine and L-isoleucine. L-leucine has various physiological functions and is widely applied to food industry, feed industry, pharmaceutical industry and other industries. Meanwhile, the dosage of the L-leucine in aspects of amino acid intravenous infusion and the like is increasing day by day, is one of indispensable raw materials applied to clinical amino acid composite infusion, and plays a positive role in maintaining the nutritional requirements of critically ill patients and rescuing the lives of the patients. Corynebacterium glutamicum is the major industrial strain for the current microbial fermentation production of L-leucine. However, due to the long synthetic pathway and strict feedback control mechanism of L-leucine, the yield and transformation rate of the production strain are still low, and the yield cannot meet the increasing market demand at present. Therefore, research and development of a high-yield strain for efficiently synthesizing L-leucine by using a cheap substrate as a raw material are urgently needed.

At present, the method for breeding L-leucine high-yield strains at home and abroad mainly comprises the traditional physical-chemical mutagenesis, metabolic engineering regulation, cell recombination technology, genetic engineering technology and the like. The main ideas for breeding the L-leucine high-yield strain are as follows: the synergistic feedback repression effect of three branched chain amino acids on 3 common enzymes such as acetohydroxy acid synthetase (AHAS), acetohydroxy acid isomeroreductase (AHAIR), dihydroxy acid dehydratase (DHAD) in a biosynthesis pathway is relieved; secondly, the feedback inhibition effect of L-valine on AHAS is relieved; relieving the feedback inhibition and repression of alpha-isopropyl malate synthase (IPMS) by L-leucine, such as breeding mutants of resistance markers of structural analogues of L-leucine and L-valine; fourthly, the normal metabolism of the thalli is changed, so that a large amount of metabolites such as amino acid are accumulated, for example, mutant strains of resistance markers of certain drugs (such as sulfaguanidine) or antibiotics (such as rifampicin) are bred. Although the above methods have achieved a certain success, all carbon sources currently used for the fermentative production of L-leucine use glucose. Since the productivity and conversion rate of the strains currently used for the industrial production of L-leucine are still low, the growth cost is always high. How to adopt a cheap substrate as the only or main raw material to produce L-leucine becomes a problem to be solved urgently by amino acid production enterprises and amino acid producing bacteria breeding experts at home and abroad.

It is well known that microorganisms of the genus Corynebacterium utilize only a few sugars, such as glucose, sucrose, fructose and maltose, which are preferred carbon sources for microorganisms of the genus Corynebacterium, as carbon sources for bacterial growth and synthesis of certain metabolites. For most microorganisms of the genus Corynebacterium, they cannot utilize polysaccharides such as starch and cellulose because there is no enzyme decomposing and utilizing these polysaccharides in their cells. At present, although it has been reported that the synthesis of amino acids (e.g., L-lysine) from starch as a sole carbon source can be achieved by heterologous expression of α -amylase derived from microorganisms of the genus Streptomyces or Streptococcus in Corynebacterium glutamicum, the yield cannot be compared with that from glucose as a main carbon source, and industrial application cannot be achieved.

Disclosure of Invention

In order to solve the problems, the invention provides a method for producing L-leucine by efficiently utilizing starch, which improves the high-temperature resistance of a strain by carrying out temperature-induced adaptive evolution treatment on an L-leucine producing strain of corynebacterium. Then, the alpha-amylase coding gene derived from the bacillus microorganism is heterologously expressed on the basis of the mutant strain, and a metabolic pathway capable of efficiently utilizing starch is constructed in an L-leucine producing strain of the corynebacterium, so that the defect that the optimal thallus growth temperature is inconsistent with the optimal action temperature of the alpha-amylase is overcome, and the recombinant corynebacterium glutamicum for efficiently utilizing the starch to produce the L-leucine is obtained.

The first purpose of the invention is to provide a method for producing L-leucine by using starch with high efficiency, which comprises the following steps:

(1) inoculating starting Corynebacterium into LBG liquid medium, culturing at optimum temperature, and measuring specific growth rate χ of starting Corynebacterium at optimum growth temperature₀I.e. mu x₀；

(2) Inoculating the starting corynebacterium grown to the middle and later logarithmic growth stage in the step (1) into a fresh LBG liquid medium, culturing at 34-36 ℃, and measuring the specific growth rate mu x of the strain at 34-36 DEG C₁(ii) a Further, the above-mentioned strain grown to the middle and late logarithmic growth stage was inoculated into a fresh LBG liquid medium and cultured at 34-36 ℃ and the specific growth rate. mu. times.χ.of the strain at 34-36 ℃ was measured₂(ii) a The above steps are repeated until the strain has a specific growth rate mu ≈ chi at 34-36 DEG C₀Obtaining mutant strains which normally grow at the temperature of 34-36 ℃;

(3) inducing the mutant strain which normally grows at the temperature of 39-41 ℃ by using the mutant strain which normally grows at the temperature of 34-36 ℃ obtained in the step (2) as an original strain by adopting the same method as the step (2);

(4) inducing the mutant strain which normally grows at 44-46 ℃ by using the mutant strain which normally grows at 39-41 ℃ and is obtained in the step (3) as an original strain by adopting the same method as the step (2);

(5) taking the mutant strain which is obtained in the step (4) and normally grows at 44-46 ℃ as a host bacterium, and heterologously expressing an alpha-amylase gene to obtain a recombinant bacterium capable of expressing the alpha-amylase;

(6) and (3) taking starch as a unique carbon source, and fermenting and producing the L-leucine by using the recombinant bacteria obtained in the step (5).

Further, the corynebacterium is corynebacterium glutamicum.

Further, in the step (1), the cultivation is carried out at a rotation speed of 100-150r/min at a temperature of 28-32 ℃.

Further, in step (2), the cultivation is performed at a rotation speed of 100-150 r/min.

Further, in the step (5), the alpha-amylase gene is derived from bacillus amyloliquefaciens, bacillus subtilis or bacillus licheniformis. Preferably from b.

Further, in the step (6), the fermentation specifically comprises the following steps: inoculating the recombinant bacterium single colony into an LBG culture medium to be cultured to a logarithmic phase, and transferring the seed culture solution to an improved fermentation culture medium to carry out fermentation production of L-leucine.

Further, the improved fermentation medium is as follows: soluble starch 40-50g/L, (NH)₄)₂SO₄10-20g/L，CH₃COONH₄ 10-20g/L，KH₂PO₄ 2-4g/L，MgSO₄·7H₂O 0.5-1.5g/L，MnSO₄·4H₂0.05-0.15g/L of O, 2-4g/L of sodium citrate, 2-5g/L of urea, 0.5-1.5g/L of L-methionine, 0.5-1.5g/L of L-glutamic acid, 0.05-0.15g/L of L-isoleucine, 1-2g/L of betaine hydrochloride, 0.0003-0.0008g/L of biotin, 0.0005-0.0010g/L of thiamine, CaCO₃30-50g/L。

Further, the culture conditions in the seed culture stage are: the culture temperature is 44-46 ℃, the rotation speed is 100-150r/min, and the culture time is 10-14 h.

Further, the inoculum size of the seed culture solution transferred to the fermentation medium is 5-15%.

Further, the culture conditions of the fermentation stage are: the culture temperature is 44-46 ℃, the rotation speed is 100-150r/min, and the culture time is 60-80 h.

The invention has the following function principle: in the process of producing L-leucine by fermentation using starch as a sole carbon source, starch needs to be decomposed into fermentable sugars such as monosaccharides or disaccharides in order to be effectively utilized. Alpha-amylases play an important role in the enzymatic hydrolysis of starch. According to the difference of the optimal action temperature, the alpha-amylase can be divided into medium-temperature amylase and high-temperature amylase, wherein the optimal action temperature of the former is 50-70 ℃, and the optimal action temperature of the latter is more than 90 ℃. However, most of microorganisms belonging to the genus Corynebacterium have an optimum growth temperature of 25 to 37 ℃ and cannot efficiently synthesize L-leucine using starch because the optimum temperature for the enzyme does not match the optimum temperature for growth of the cells. The invention utilizes a temperature-induced adaptive evolution method to obtain a strain with the growth optimum temperature close to the optimum temperature of enzyme, constructs a starch metabolic pathway, and utilizes the starch metabolic pathway to ferment and produce L-leucine by taking starch as a unique carbon source.

The invention has the beneficial effects that:

the invention improves the high-temperature resistance of the strain by carrying out temperature-induced adaptive evolution treatment on the L-leucine producing strain of the corynebacterium. Then, on the basis of the mutant strain, an alpha-amylase coding gene derived from a bacillus microorganism (such as B. amyloliquefaciens) is expressed in a heterologous manner, and a metabolic pathway capable of efficiently utilizing starch is constructed in an L-leucine producing strain of corynebacterium, so that the defect that the optimal growth temperature of the strain is inconsistent with the optimal action temperature of the alpha-amylase is overcome, and the recombinant corynebacterium glutamicum for efficiently utilizing the starch to produce the L-leucine is obtained.

Drawings

FIG. 1 shows the synthesis of L-leucine and two other branched chain amino acid metabolic pathways from starch and their key enzyme genes;

FIG. 2 shows the growth rate and L-leucine production of starting strain XQ at different temperatures;

FIG. 3 is a graph showing the change in specific growth rate at each cycle temperature;

FIG. 4 shows the screening of strains that produce L-leucine optimally at 35 deg.C, 40 deg.C and 45 deg.C;

FIG. 5 shows the growth of recombinant strain L45-52/pEC-XK99E-amy at 45 ℃ and L-leucine.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Qualitative and quantitative analysis of products and monitoring of thallus growth conditions: the leucine product is monitored in real time and measured with an amino acid analyzer. And (3) measuring the concentration of the bacterial liquid: sucking sample bacteria liquid, diluting with distilled water by a certain time, taking distilled water as blank control, and measuring OD in 1cm optical path by using spectrophotometer_600nm。

The LBG medium is: 5g/L glucose, 10g/L peptone, 5g/L yeast extract and 10g/L NaCl.

Glucose fermentation medium: 80-120g/L glucose, 20-30g/L corn steep liquor, (NH)₄)₂SO₄ 10-20g/L，CH₃COONH₄ 10-20g/L，KH₂PO₄ 2-4g/L，MgSO₄·7H₂O 0.5-1.5g/L，MnSO₄·4H₂0.05-0.15g/L of O, 2-4g/L of sodium citrate, 2-5g/L of urea, 0.5-1.5g/L of L-methionine, 0.5-1.5g/L of L-glutamic acid, 0.05-0.15g/L of L-isoleucine, 1-2g/L of betaine hydrochloride, 0.0003-0.0008g/L of biotin, 0.0005-0.0010g/L of thiamine, CaCO₃ 30-50g/L。

Improving a fermentation medium: soluble starch 40-50g/L, (NH)₄)₂SO₄ 10-20g/L，CH₃COONH₄10-20g/L，KH₂PO₄ 2-4g/L，MgSO₄·7H₂O 0.5-1.5g/L，MnSO₄·4H₂0.05-0.15g/L of O, 2-4g/L of sodium citrate, 2-5g/L of urea, 0.5-1.5g/L of L-methionine, 0.5-1.5g/L of L-glutamic acid, 0.05-0.15g/L of L-isoleucine, 1-2g/L of betaine hydrochloride, 0.0003-0.0008g/L of biotin, 0.0005-0.0010g/L of thiamine, CaCO₃30-50g/L。

The application takes the L-leucine producing bacterium corynebacterium glutamicum with the optimal growth temperature of 30 ℃ as an example to illustrate the feasibility of the technical scheme.