CN112226399B

CN112226399B - Klebsiella (Klebsiella. spp) strain, engineering bacteria and application

Info

Publication number: CN112226399B
Application number: CN202011435733.XA
Authority: CN
Inventors: 郝健
Original assignee: Shanghai Advanced Research Institute of CAS
Current assignee: Shanghai Advanced Research Institute of CAS
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-02-26
Anticipated expiration: 2040-12-11
Also published as: CN112226399A

Abstract

The invention discloses a Klebsiella sp strain, engineering bacteria and application. The strain is named as Klebsiella pneumoniae SARI K01 and is preserved in China center for type culture Collection (Wuhan university, Wuhan), the preservation date is 10 months and 28 days in 2020, and the preservation number is CCTCC M2020648. When the strain is subjected to liquid culture, a great part of strains naturally aggregate to form a flocculent structure; after the fermentation liquor is stood still, the flocculent structures of the thalli are quickly settled. The strain and the engineering bacteria constructed by taking the strain as the starting strain can be used for producing chemicals such as 1, 3-propanediol, 2, 3-butanediol, acetoin, 2-ketogluconic acid, gluconic acid, xylonic acid, 2-ketoisovaleric acid, isobutanol, 2, 3-dihydroxyisovaleric acid and the like through fermentation. The strain or the engineering strain thereof provided by the invention has the advantages of easy separation of thalli from fermentation liquor, fast growth of thalli, high product concentration, high product production rate, high substrate conversion rate and the like, and has good application value and economic benefit.

Description

Klebsiella (Klebsiella. spp) strain, engineering bacteria and application

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a Klebsiella sp strain, and engineering bacteria and application thereof.

Background

Klebsiella bacterium (Klebsiella pneumoniae)Klebsiella. spp) The Klebsiella bacteria belong to the Enterobacteriaceae family, are widely distributed in nature and naturally exist in the environments of soil, water, plant rhizosphere, animal mucosa and the like. The Klebsiella has an important role in the biochemical and geochemical processes in the environment and is a major member of the micro-ecological community.

The Klebsiella has wide application in agriculture. The Klebsiella is a kind of rhizosphere symbiotic bacteria of plants, and can fix nitrogen and promote the growth of the plants. Meanwhile, the fertilizer also has the capability of dissolving phosphorus in soil and has obvious promotion effect on the growth of symbiotic plants. The Klebsiella can antagonize tobacco black shank bacteria and prevent and treat tobacco black shank. The Klebsiella may be used as biological pesticide for killing fall webworm.

The Klebsiella has wide application in environmental treatment. The Klebsiella can degrade thiocyanate which pollutes the environment. The Klebsiella can reduce hexavalent chromium ions in the environment and treat environmental pollution. The thallus can absorb bivalent cadmium in water body to eliminate pollution. The Klebsiella is used as a microbial inoculum for degrading pyridine in soil and phenol in sewage. The Klebsiella can decolorize azo dyes and is applied to treatment of printing and dyeing wastewater and polluted soil. The Klebsiella can be used for producing biological flocculant for sewage treatment.

The genetic resources of Klebsiella have wide application. The Klebsiella can be used for producing tannase and beta-galactosidase. The laccase gene of the Klebsiella can be exogenously expressed, and the obtained laccase is used for decoloring azo dyes and anthraquinone dyes. The lysine decarboxylase of the Klebsiella is exogenously expressed, and the obtained lysine decarboxylase polypeptide can be used for producing the pentanediamine.

Use of Klebsiella in food products. The Klebsiella can be used as bacteria for food fermentation to improve food quality, the bacteria has the capability of synthesizing VB12, and the bacterial strain is added in the process of preparing blue-streaked cheese, so that the content of VB12, folic acid and iron in the cheese is increased.

Application of Klebsiella in chemicals. The Klebsiella can be used as a production bacterium for producing chemicals such as 2, 3-butanediol, acetoin, 2-ketogluconic acid, gluconic acid, xylonic acid, 2-ketogluconic acid, 2, 3-dihydroxyisovaleric acid, isobutanol, 1, 3-propanediol and the like. Has the characteristics of fast growth, high raw material conversion rate and the like.

Generally, during the liquid culture of bacteria, the bacterial cells are in a free suspension state. Cells can be flocculated by adding a flocculating agent into the culture solution, so that the aim of separating the cells from the culture solution is fulfilled. Some microorganisms have the characteristic of self-flocculation, which is the spontaneous aggregation of cells to form aggregates in liquid culture. The microorganism with self-flocculation characteristic is easy to separate from the culture solution, thereby reducing the separation and extraction cost of the product.

Disclosure of Invention

The invention aims to provide a Klebsiella bacterium (Klebsiella pneumoniae)Klebsiella. spp) Bacterial strain, engineering bacteria constructed by the same and application thereof.

The technical scheme adopted by the invention for realizing the purpose is as follows:

klebsiella bacterium (Klebsiella pneumoniae)Klebsiella. spp) Strain, name of the strainIs Klebsiella pneumoniae SARI K01, is preserved in China center for type culture Collection, is the Wuhan university in Wuhan, has a preservation date of 10 months and 28 days in 2020, and has a preservation number of CCTCC M2020648.

The invention also provides an engineering bacterium constructed by the Klebsiella SARI K01, which is constructed by at least one of the following modes,

inactivating butanediol dehydrogenase of the Klebsiella SARI K01 to construct engineering bacteria;

inactivating the gluconic acid dehydrogenase of the Klebsiella SARI K01 to construct engineering bacteria;

inactivating acetolactate decarboxylase of the Klebsiella SARI K01 to construct engineering bacteria;

simultaneously inactivating acetolactate decarboxylase and 2, 3-dihydroxyisovalerate dehydratase of the Klebsiella SARI K01 to construct engineering bacteria;

simultaneously inactivating acetolactate decarboxylase and lactate dehydrogenase of the Klebsiella SARI K01 to construct engineering bacteria;

inactivating the lactate dehydrogenase of the Klebsiella SARI K01 to construct engineering bacteria;

inactivating the alcohol dehydrogenase of the Klebsiella SARI K01 to construct engineering bacteria;

simultaneously inactivating the lactate dehydrogenase and the alcohol dehydrogenase of the Klebsiella SARI K01 to construct engineering bacteria;

inactivating the pyruvate dehydrogenase compound of the Klebsiella SARI K01 to construct engineering bacteria;

inactivating pyruvate decarboxylase of the Klebsiella SARI K01 to construct engineering bacteria;

simultaneously inactivating butanediol dehydrogenase and pyruvate dehydrogenase complexes of the Klebsiella SARI K01;

simultaneously inactivating acetolactate decarboxylase, lactate dehydrogenase and 2, 3-dihydroxyisovalerate dehydratase of the Klebsiella SARI K01.

The invention also provides application of the Klebsiella SARI K01 and the engineering bacteria constructed by the Klebsiella SARI K01 or the engineering bacteria constructed by utilizing the genetic resources of the Klebsiella in producing at least one chemical of 1, 3-propylene glycol, 2, 3-butanediol, acetoin, 2-ketogluconic acid, gluconic acid, xylonic acid, 2-ketoisovaleric acid, isobutanol or 2, 3-dihydroxyisovaleric acid.

As a preferred embodiment, the chemical is produced by culturing the Klebsiella SARI K01 or engineered bacteria by liquid fermentation. The Klebsiella SARI K01 or the engineering bacteria can form floccules through cell aggregation in liquid culture.

As a preferred embodiment, 1, 3-propanediol is produced by liquid fermentation culture of the Klebsiella SARI K01 or engineered bacteria, wherein the culture medium uses glycerol as the main carbon source.

As a preferred embodiment, 2, 3-butanediol, acetoin or 2-ketogluconic acid is produced by liquid fermentation culture of the Klebsiella SARI K01 or engineering bacteria, wherein the culture medium takes glucose as a main carbon source.

As a preferred embodiment, the Klebsiella SARI K01 or the engineered bacterium is cultured by liquid fermentation to produce xylonic acid, wherein the culture medium uses xylose as a main carbon source.

As a preferred embodiment, 2-ketoisovalerate and/or isobutanol are produced by culturing the engineered bacteria by liquid fermentation; the engineering bacteria are acetolactate decarboxylase of inactivated Klebsiella SARI K01, or acetolactate decarboxylase and lactate dehydrogenase are simultaneously inactivated.

In a preferred embodiment, the engineering bacteria are cultured by liquid fermentation to produce the gluconic acid, wherein the culture medium takes the glucose as a main carbon source, and the engineering bacteria are the gluconic acid dehydrogenase of the inactivated Klebsiella SARI K01.

As a preferred embodiment, 2, 3-dihydroxyisovalerate is produced by culturing the engineered bacteria, which are acetolactate decarboxylase, lactate dehydrogenase and 2, 3-dihydroxyisovalerate dehydratase simultaneously inactivating Klebsiella SARI K01, by liquid fermentation.

Compared with the prior art, the invention has the beneficial effects that:

1, the Klebsiella SARI K01 of the invention is obtained by screening from soil, during the process of liquid culture using the bacterial strain or the engineering bacteria constructed by the genetic resources of the bacterial strain as the production bacterial strain, a part of the thalli can form aggregates, the aggregates are rapidly settled after the fermentation liquid is stood, meanwhile, the bacterial strain grows faster, the capability of resisting adverse environment is enhanced, and the product concentration, the product production strength and the raw material conversion rate are higher.

2, the Klebsiella SARI K01 and the engineering bacteria constructed by taking the Klebsiella as the starting strain can be used for producing chemicals such as 1, 3-propanediol, 2, 3-butanediol, acetoin, 2-ketogluconic acid, gluconic acid, xylonic acid, 2-ketoisovaleric acid, isobutanol, 2, 3-dihydroxyisovaleric acid and the like by fermentation with higher substrate conversion rate or production intensity.

Detailed Description

The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified.

Example 1

Acquisition of Klebsiella SARI K01.

1g of grass-negative surface soil of No. 99 green belt of Haichi city, Haichidae is taken.

The soil was inoculated into 50ml of liquid LB medium and shake-cultured overnight at 37 ℃.

Taking culture, diluting with distilled water 10⁶-10⁷And spreading the suspension on a solid LB culture medium, and culturing at 37 ℃ overnight.

And selecting single colonies with different shapes growing on the solid culture medium, and respectively inoculating the single colonies to other solid culture media to obtain pure culture microorganisms.

One of the pure cultured microorganisms obtained has ampicillin resistance, is classified as Klebsiella through genome sequencing and is named as Klebsiella SARI K01. The strain is preserved in China center for type culture Collection with the preservation number: CCTCC M2020648.

Example 2

Construction of engineering strain from Klebsiella SARI K01

The gene of the coding specific enzyme on the chromosome of the Klebsiella SARI K01 is inactivated by a gene recombination method to obtain a corresponding engineering strain. The lactate dehydrogenase was inactivated to obtain K01. delta. ldhA. Inactivation of alcohol dehydrogenase to obtain K01 delta adhE. Both lactate dehydrogenase and alcohol dehydrogenase were inactivated to obtain K01 Δ ldhA Δ adhE. Inactivation of pyruvate dehydrogenase complex yielded K01 Δ aco. Inactivation of pyruvate decarboxylase yielded K01. delta. pflB. Butanediol dehydrogenase was inactivated to obtain K01. delta. budC. Inactivation of both butanediol dehydrogenase and pyruvate dehydrogenase complexes yielded K01. delta. budC. DELTA. aco. Acetolactate decarboxylase was inactivated to obtain K01. delta. budA. Both acetolactate decarboxylase and lactate dehydrogenase were inactivated to obtain K01. delta. budA. delta. ldhA. The acetolactate decarboxylase, lactate dehydrogenase and 2, 3-dihydroxyisovalerate dehydratase were all inactivated to obtain K01 Δ budA Δ ldhA Δ ilvD. The gluconate dehydrogenase is inactivated to obtain K01 delta gad. The above specific construction method is a conventional technical means in the field and is not described in detail.

Example 3

Production of 1, 3-propanediol by using Klebsiella SARI K01 and its engineering bacteria

Klebsiella SARI K01, and control strains Klebsiella pneumoniae TUAC01 (accession number CGMCC 1.6366), K01. delta. ldhA, K01. delta. adhE, K01. delta. ldhA. delta. adhE, K01. delta. aco, and K01. delta. pflB were inoculated into 250ml conical flasks containing 50ml of a medium containing glycerol as a main carbon source, and cultured at 37 ℃ at 120 rpm.

The culture medium comprises the following components: (NH)₄)₂SO₄ 4 g/l，K₂HPO₄ 3H₂O 0.69 g/l，KH₂PO₄ 0.25 g/l，MgSO₄0.2 g/l，CaCO₃5g/l, yeast extract 1.5 g/l, glycerol 30 g/l, trace elements 1ml/l, 1l trace elements contain: MnSO₄4H₂O 100 mg，ZnCl₂ 70 mg，Na₂MoO₄2H₂O 35 mg，H₃BO₃ 60 mg，CoCl₂ 6H₂O 200 mg，CuSO₄5H₂O 29.28 mg，NiCl₂ 6H₂O 25 mg，37% HCl 0.9 ml。

The observation in the culture process shows that no flocculent aggregate is found in the culture solution of CGMCC1.6366A large number of flocculent aggregates appeared in the culture solutions of SARI K01 and K01. delta. ldhA, K01. delta. adhE, K01. delta. ldhA. delta. adhE, K01. delta. aco, K01. delta. pflB. After one day of culture, 30ml of each strain fermentation broth was poured into a measuring cylinder and left to stand, and most of the cells in SARI K01 and K01. delta. ldhA, K01. delta. adhE, K01. delta. ldhA. delta. adhE, K01. delta. aco, K01. delta. pflB fermentation broths after 2 minutes were left alone together with unconsumed CaCO₃Settle to a volume of 15 ml. Most of thallus of CGMCC1.6366 sample together with unconsumed CaCO₃No obvious sedimentation. After 10 minutes, most of the cells in the SARI K01 and K01. delta. ldhA, K01. delta. adhE, K01. delta. ldhA. delta. adhE, K01. delta. aco, K01. delta. pflB fermentation broths together with unconsumed CaCO₃Settle to a volume of 5 ml. Most of thallus of CGMCC1.6366 sample together with unconsumed CaCO₃No obvious sedimentation. After standing for 1 hour, most of the cells of the fermentation broth SARI K01, K01. delta. ldhA, K01. delta. adhE, K01. delta. ldhA. delta. adhE, K01. delta. aco and K01. delta. pflB were found to contain unconsumed CaCO₃Settle to a volume of 3 ml. Most thalli of CGMCC1.6366 fermentation liquor and unconsumed CaCO₃Settle to a volume of 10 ml. The standing time is continuously prolonged, and thallus of each strain together with unconsumed CaCO₃The volume of the sediment layer is not changed obviously. The results show that thalli in SARI K01 and the engineering strain fermentation liquor modified by SARI K01 as the original strain are easy to settle, and the thalli settling layer is more compact.

And (3) performing liquid chromatography analysis on the fermentation liquor sample, separating the components of the fermentation liquor by using an HPX-87H chromatographic column, and detecting by using a parallax detector. The mobile phase is 0.05mol/L dilute sulfuric acid solution, and the flow rate is 0.8 ml/min. The analysis results are shown in table 1.

TABLE 1

As can be seen from Table 1, the SARI K01 has stronger ability to synthesize 1, 3-propanediol by using glycerol as a main carbon source compared with CGMCC1.6366, high conversion rate of raw materials, high product concentration and lower total synthesis amount of byproducts. The capability of the strain to synthesize 1, 3-propanediol is further improved after lactate dehydrogenase and ethanol dehydrogenase are inactivated, or simultaneously lactate dehydrogenase and ethanol dehydrogenase are inactivated, or pyruvate decarboxylase is inactivated. The conversion rate of 1, 3-propanediol synthesized by the strain K01 delta aco with the inactivated pyruvate dehydrogenase complex is reduced, but the rate of 1, 3-propanediol synthesized by the strain is improved by 12-hour culture detection.

The strain CGMCC1.6366 delta ldhA delta adhE constructed by K01 delta ldhA delta adhE and a reference strain CGMCC1.6366 is used for shaking the flask to culture seeds by using LB culture medium. 50ml of the seed culture solution cultured overnight was transferred to 5L fermentors each containing 3L of fermentation medium, and subjected to fermentation culture. After the glycerol in the fermentation liquid is consumed, the glycerol is fed into the fermentation liquid. After the fermentation of CGMCC1.6366 delta ldhA delta adhE is finished for 72 hours, the final concentration of 1, 3-propanediol is 75g/L, the conversion rate of the raw material glycerol is 0.40g/g, during the culture process, the thalli are not visually aggregated, and the non-light-absorbing substances in the culture solution are directly observed by using an optical microscope; the fermentation liquor is placed for 20 minutes without obvious layering phenomenon by visual observation. K01 DeltaldhA DeltaadhE was fermented for 48 hours, the final 1, 3-propanediol concentration was 110g/L, the raw material glycerol conversion rate was 0.48g/g, aggregates were visually observed during the culture, a large amount of irregular spherical substances having a diameter of 0.05-0.3mm were directly observed in the culture by an optical microscope, the cells settled quickly after the fermentation broth was settled, and a cell layer having a thickness of 2cm was formed at the bottom of the pot after the fermentation broth was settled for 20 minutes.

Therefore, K01 Δ ldhA Δ adhE has higher raw material conversion rate and product concentration and faster cell sedimentation in the fermentation liquid when used for synthesizing 1, 3-propanediol.

Example 4

Production of 2, 3-butanediol by using SARI K01 and engineering bacteria thereof

The CGMCC1.6366, SARI K01 and K01 delta ldhA delta adhE strains were inoculated into 250ml conical flasks, respectively, and 50ml of a medium containing glucose as a main carbon source was cultured at 37 ℃ at 200 rpm.

The culture medium comprises the following components: 50g/L glucose, 4 g/L corn steep liquor, (NH)₄)₂SO₄5g/L and 3 g/L of sodium acetate.

During the culture process, a large amount of flocculent aggregates appear in the culture solution of SARI K01 and K01 delta ldhA delta adhE, but no flocculent aggregates appear in the culture solution of CGMCC 1.6366. After one day of culture, 50ml of the fermentation broth was poured inThe cylinder was allowed to stand and after 2 minutes most of the cells of the SARI K01 and K01 Δ ldhA Δ adhE samples were found to be along with unconsumed CaCO₃Settle to a volume of 15 ml. Most of thallus of CGMCC1.6366 sample together with unconsumed CaCO₃Settle to a volume of 25 ml. After 5 minutes most of the cells of SARI K01 and K01 Δ ldhA Δ adhE samples were found to contain unconsumed CaCO₃Settle to a volume of 5 ml. Most of thallus of CGMCC1.6366 sample together with unconsumed CaCO₃Settle to a volume of 10 ml. The standing time was further prolonged, and the volume of the cells of each strain together with the deposited layer of unconsumed CaCO3 did not change significantly. The results show that the thalli in the fermentation liquor of the SARI K01 and the engineering strain are easy to settle.

Samples were taken at 12 hours and 24 hours during the culture and the fermentation samples were subjected to liquid chromatography analysis in the same manner as in example 3. The analysis results are shown in Table 2.

TABLE 2

As can be seen from table 2, the first 12 hour rates of the synthesis of 2, 3-butanediol and acetoin by SARI K01 and K01 Δ ldhA Δ adhE using glucose as the main carbon source were faster, but the final conversion was lower relative to CGMCC 1.6366.

Example 5

Production of acetoin by using SARI K01 and engineering bacteria thereof

Using CGMCC1.6366 as an initial strain, and inactivating butanediol dehydrogenase to construct an engineering bacterium CGMCC1.6366 delta budC; and (3) constructing the engineering bacteria CGMCC1.6366 delta budC delta aco by inactivating the butanediol dehydrogenase and pyruvate dehydrogenase compounds. Respectively inoculating K01 delta budC, K01 delta budC delta aco, CGMCC1.6366 delta budC and CGMCC1.6366 delta budC delta aco into a 250ml conical flask, wherein the flask is filled with 50ml LB culture medium, 200 turns and is cultured overnight at 37 ℃, and transferring the seed culture solution into a 5L fermentation tank, wherein the fermentation medium is filled with 3L for high aerobic fermentation culture.

The fermentation medium comprises the following components: 100 g/L glucose, 2 g/L yeast extract, 4 g/L corn steep liquor, (NH)₄)₂SO₄5g/L, sodium acetate 3 g/L KCl, 0.4 g/L，MgSO₄，0.1 g/L。

During the culture process, a large amount of flocculent aggregates appear in the culture solution of K01 delta budC and K01 delta budC delta aco, and flocculent aggregates do not appear in the culture solution of CGMCC1.6366 delta budC and CGMCC1.6366 delta budC delta aco. Both K01 Δ budC and K01 Δ budC Δ aco produced 28g/L acetoin at 14 hours. CGMCC1.6366 delta budC and CGMCC1.6366 delta aco produced 28g/L acetoin at 16 hours.

K01 delta budC and K01 delta budC delta aco have faster rates of acetoin synthesis compared to CGMCC1.6366 delta budC and CGMCC1.6366 delta aco which use glucose as a main carbon source.

Example 6

Production of 2-ketogluconic acid using SARI K01

Respectively inoculating SARI K01 and control strain Klebsiella pneumoniae TUAC01 (CGMCC 1.6366) into 250ml conical flask, culturing overnight at 37 ℃ in 50ml LB culture medium, transferring the seed culture solution into 5L fermentation tank containing 3L fermentation medium, and performing high aerobic fermentation culture.

The fermentation medium comprises the following components: 150 g/L glucose, 4 g/L corn steep liquor, 45 g/L MgSO, and 3 g/L sodium acetate.

The pH value in the fermentation process is controlled to be neutral in the first stage, and a large amount of flocculent aggregates appear in the SARI K01 culture solution and are not seen in the CGMCC1.6366 culture solution during observation in the culture process. After 3 hours of culture, the second stage was carried out, and the fermentation pH was adjusted to 5. SARI K01 converted all of the glucose to 2-ketogluconic acid in 24 hours, and CGMCC1.6366 converted all of the glucose to 2-ketogluconic acid in 28 hours.

The SARI K01 has a faster rate of synthesizing 2-ketogluconic acid compared with CGMCC1.6366 which utilizes glucose as a main carbon source.

Example 7

Production of gluconic acid by using SARI K01 engineering strain

The CGMCC1.6366 is used as an original strain, and the inactivated gluconate dehydrogenase is used for constructing the engineering bacterium CGMCC1.6366 delta gad. Respectively inoculating K01 delta gad and CGMCC1.6366 delta gad into a 250ml conical flask, wherein 50ml LB culture medium is filled in the conical flask, culturing overnight at the temperature of 37 ℃ after 200 turns, transferring the seed culture solution into a 5L fermentation tank, wherein 3L fermentation culture medium is filled in the fermentation tank, and performing high aerobic fermentation culture.

The fermentation medium comprises the following components: glucose 90 g/L, corn steep liquor 4 g/L, MgSO₄5g/L and 3 g/L of sodium acetate.

The pH value in the fermentation process is controlled to be neutral in the first stage, and a large amount of flocculent aggregates appear in the K01 delta gad culture solution and are not seen in the CGMCC1.6366 delta gad culture solution during observation in the culture process. After 3 hours of culture, the second stage was carried out, and the fermentation pH was adjusted to 5. K01 Δ gad converted all of the glucose to gluconic acid in 10 hours, and CGMCC1.6366 Δ gad converted all of the glucose to gluconic acid in 12 hours. After glucose is consumed, glucose is supplemented for fed-batch fermentation, the K01 delta gad strain finally synthesizes 450g/L gluconic acid, and the CGMCC1.6366 delta gad finally synthesizes 420 g/L gluconic acid.

Compared with CGMCC1.6366 delta gad, the K01 delta gad has a faster rate of synthesizing gluconic acid by using glucose as a main carbon source, and the final gluconic acid concentration is higher.

Example 8

Production of xylonic acid using SARI K01

The fermentation medium comprises the following components: 30 g/L of xylose, 4 g/L of corn steep liquor and MgSO₄5g/L and 3 g/L of sodium acetate.

The pH value in the fermentation process is controlled to be neutral in the first stage, and a large amount of flocculent aggregates appear in the SARI K01 culture solution and are not seen in the CGMCC1.6366 culture solution during observation in the culture process. After 4 hours of culture, the second stage was carried out, and the fermentation pH was adjusted to 5. SARI K01 converted xylose to xylonic acid completely in 8 hours, and CGMCC1.6366 converted xylose to xylose completely in 9 hours.

The SARI K01 has a faster rate of xylonic acid synthesis compared with CGMCC1.6366 which utilizes xylose as a main carbon source.

Example 9

Production of 2-ketoisovalerate and isobutanol by SARI K01 engineering strain

The CGMCC1.6366 is used as an initial strain, the inactivated acetolactate decarboxylase constructs engineering bacteria CGMCC1.6366 delta budA, and the inactivated acetolactate decarboxylase and lactate dehydrogenase construct engineering bacteria CGMCC1.6366 delta budA delta ldhA. K01 delta budA, K01 delta budA delta ldhA, CGMCC1.6366 delta budA and CGMCC1.6366 delta budA delta ldhA are respectively inoculated into a 250ml conical flask which is filled with 50ml LB culture medium, 200 turns is carried out, and culture is carried out overnight at 37 ℃, and the seed culture solution is transferred into a 5L fermentation tank which is filled with 3L fermentation culture medium for fermentation culture.

The fermentation medium comprises the following components: 100 g/L glucose, 5g/L yeast extract, 4 g/L corn steep liquor, (NH)₄)₂SO₄5g/L, sodium acetate 3 g/L KCl, 0.4 g/L, MgSO₄，0.1 g/L。

A large amount of flocculent aggregates appear in the culture solution of K01 delta budA and K01 delta budA delta ldhA, but no flocculent aggregates appear in the culture solution of CGMCC1.6366 delta budA and CGMCC1.6366 delta budA delta ldhA. Fermenting and culturing for 24 hours, detecting the components in the fermentation liquor by liquid chromatography, and showing in table 3.

TABLE 3

Compared with CGMCC1.6366 delta budA and CGMCC1.6366 delta budA delta ldhA, the substrate conversion rate of synthesizing 2-ketoisovalerate and isobutanol by K01 delta budA and K01 delta budA delta ldhA is lower.

Example 10

Production of 2, 3-dihydroxy isovaleric acid by SARI K01 engineering strain

The CGMCC1.6366 is used as an initial strain, and the engineering bacteria CGMCC1.6366 delta budA delta ldhA delta ilvD is constructed by inactivating acetolactate decarboxylase, lactate dehydrogenase and 2, 3-dihydroxy isovalerate dehydratase. K01. delta. budA. delta. ldhA. delta. ilvD and CGMCC 1.6366. delta. budA. delta. ldhA. delta. ilvD were inoculated into 250ml flasks containing 50ml of the fermentation medium, and fermentation culture was carried out at 37 ℃ at 200 rpm.

The fermentation medium comprises the following components: 25 g/L glucose, 5g/L yeast extract, 4 g/L corn steep liquor, (NH)₄)₂SO₄5g/L, sodium acetate 3 g/L, KCl 0.4 g/L, MgSO₄0.1 g/L，FeSO₄0.02 g/L。

A large number of flocculent aggregates were observed in the K01 Δ budA Δ ldhA Δ ilvD culture solution during the culture, but no flocculent aggregates were observed in the CGMCC1.6366 Δ budA Δ ldhA Δ ilvD culture solution. K01 Δ budA Δ ldhA Δ ilvD produced 3.8g/L of 2, 3-dihydroxyisovalerate at 24 hours. CGMCC 1.6366. delta. budA. delta. ldhA. delta. ilvD produced 3.3 g/L of 2, 3-dihydroxyisovalerate at 24 hours.

The substrate conversion rate of K01 delta budA delta ldhA delta ilvD in the synthesis of 2, 3-dihydroxyisovalerate is high.

The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.

Claims

1. Klebsiella bacterium (Klebsiella pneumoniae)Klebsiella. spp) A strain characterized by: the strain is named as Klebsiella pneumoniae SARI K01 and is preserved in China center for type culture Collection (Wuhan university, Wuhan), with the preservation date of 10 and 28 days in 2020 and the preservation number of CCTCC M2020648.

2. The engineered bacterium constructed from the Klebsiella strain of claim 1, wherein: the engineering bacteria are constructed by at least one of the following modes,

3. Use of the klebsiella strain of claim 1 or the engineered bacterium of claim 2 or the engineered bacterium constructed using genetic resources of the klebsiella strain of claim 1 for producing at least one chemical of 1, 3-propanediol, 2, 3-butanediol, acetoin, 2-ketogluconate, gluconate, xylonate, 2-ketoisovalerate, isobutanol, or 2, 3-dihydroxyisovalerate.

4. Use according to claim 3, characterized in that: and culturing the Klebsiella SARI K01 or engineering bacteria by liquid fermentation to produce the chemical.

5. The use of claim 4, wherein: the Klebsiella SARI K01 or the engineering bacteria can form floccules through cell aggregation in liquid culture.

6. Use according to claim 3, characterized in that: and culturing the Klebsiella SARI K01 or engineering bacteria by liquid fermentation to produce 1, 3-propanediol, wherein the culture medium uses glycerol as a main carbon source.

7. Use according to claim 3, characterized in that: and culturing the Klebsiella SARI K01 or engineering bacteria through liquid fermentation to produce 2, 3-butanediol, acetoin or 2-ketogluconic acid, wherein the culture medium takes glucose as a main carbon source.

8. Use according to claim 3, characterized in that: and (3) culturing the Klebsiella SARI K01 or the engineering bacteria through liquid fermentation to produce xylonic acid, wherein the culture medium takes xylose as a main carbon source.

9. Use according to claim 3, characterized in that: culturing the engineering bacteria by liquid fermentation to produce 2-ketoisovalerate and/or isobutanol; the engineering bacteria are acetolactate decarboxylase of inactivated Klebsiella SARI K01, or acetolactate decarboxylase and lactate dehydrogenase are simultaneously inactivated.

10. Use according to claim 3, characterized in that: and culturing the engineering bacteria through liquid fermentation to produce gluconic acid, wherein the culture medium takes glucose as a main carbon source, and the engineering bacteria are the gluconic acid dehydrogenase of inactivated Klebsiella SARI K01.

11. Use according to claim 3, characterized in that: and culturing the engineering bacteria through liquid fermentation to produce the 2, 3-dihydroxyl isovalerate, wherein the engineering bacteria are acetolactate decarboxylase, lactate dehydrogenase and 2, 3-dihydroxyl isovalerate dehydratase which simultaneously inactivate the Klebsiella SARI K01.