阅读说明：本技术 一种降低1,3-丙二醇生产过程中副产物的方法 (Method for reducing by-products in 1, 3-propylene glycol production process ) 是由 宫衡 魏仁全 董纾帆 傅水林 于 2020-08-09 设计创作，主要内容包括：本发明公开了一种降低1,3－丙二醇生产过程中副产物的方法,也即,将克雷伯氏肺炎杆菌(Klebsiella pneumoniae)中σ因子RpoS的编码基因敲除,从而降低了1,3－丙二醇生产过程中副产物的形成。相较于其他方法,本发明的优点是：不但彻底阻断了副产物2,3－丁二醇的形成,而且也显著降低了副产物有机酸的形成。因而,采用本发明的方法,极大地降低了1,3－丙二醇发酵过程中副产物的产生。(The invention discloses a method for reducing byproducts in the production process of 1, 3-propylene glycol, namely, knocking out the coding gene of a sigma factor Rpos in Klebsiella pneumoniae (Klebsiella pneumoniae), thereby reducing the formation of byproducts in the production process of 1, 3-propylene glycol. Compared with other methods, the method has the advantages that: not only completely blocks the formation of the byproduct 2, 3-butanediol, but also remarkably reduces the formation of the byproduct organic acid. Thus, the method of the invention greatly reduces the production of byproducts in the fermentation process of the 1, 3-propanediol.)

1. A method for reducing byproducts in the production process of 1, 3-propylene glycol is characterized in that a gene rpoS in Klebsiella pneumoniae is knocked out, so that the formation of byproducts in the production process of 1, 3-propylene glycol is reduced.

2. The method according to claim 1, characterized in that the gene rpoS is responsible for encoding the sigma subunit Rpos of the RNA polymerase.

3. The method of claim 1, wherein the byproducts are 2, 3-butanediol and organic acids.

4. The method according to claim 3, characterized in that the organic acids are in particular: acetic acid, alpha-ketoglutaric acid, succinic acid, and 3-hydroxypropionic acid.

5. The method according to claim 1, wherein the strain for producing 1, 3-propanediol is Klebsiella pneumoniae (Klebsiela pneumoniae).

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a biological engineering method, which comprises the following steps: by knocking out the coding gene of the sigma factor Rpos in the Klebsiella pneumoniae, the formation of byproducts in the production process of the 1, 3-propylene glycol is reduced.

Background

1, 3-propylene glycol is an important chemical raw material and has wide application. For example, 1, 3-propanediol can be used as a monomer to synthesize a novel poly-ester, namely, poly-propylene terephthalate (PTT), which is a polyester material with particularly excellent performance. Microorganisms that produce 1, 3-propanediol in nature broadly include: klebsiella pneumoniae (Klebsiela pneumoniae), Citrobacter freundi (Citrobacter freundi), Clostridium butyricum (Clostridium butyricum), and the like, among which Klebsiella pneumoniae produces the best.

It has been found that k. pneumoconiae produces 1, 3-propanediol and produces a number of by-products, the most predominant of which is 2, 3-butanediol, in addition to organic acids. The boiling point of the 2, 3-butanediol is close to that of the 1, 3-propanediol, and the cost of refining the 1, 3-propanediol by subsequent rectification is greatly increased due to the existence of the 2, 3-butanediol; in addition, organic acids are also the main impurities affecting the subsequent 1, 3-propanediol separation process. Therefore, the presence of these by-products not only increases the consumption of raw materials, but also causes difficulties in the subsequent separation and purification of the product.

Although various researches on knocking out a biosynthetic metabolic pathway of a specific byproduct exist at present, the effect of reducing the byproduct is not ideal. For example, knocking out the biosynthesis pathway of 2, 3-butanediol of k.pneumoniae can block the production of 2, 3-butanediol, but can severely inhibit the growth of cells. For another example, studies have shown that most organic acids (acetate, α -ketoglutarate, succinate, and 3-hydroxypropionate, etc.) are difficult to reduce their secretion by knocking out known biosynthetic pathways, mainly because of the more intricate metabolic pathways for organic acid biosynthesis. Therefore, new measures for effectively controlling the formation of byproducts in the fermentation process of 1, 3-propanediol are urgently needed.

The invention discloses a method, namely knocking out a coding gene rpoS of a global regulation sigma factor RpoS in K.pneumoniae, wherein the knocking out of the gene not only can thoroughly block the formation of a byproduct 2, 3-butanediol, but also can effectively reduce the formation of a byproduct organic acid. The same published or granted patents as the patent protection request have not been found by searching the national intellectual property office (www.sipo.gov.cn), the world property organization (www.wipo.int), the european patent office (www.espacenet.com), and the united states patent and trademark office (www.uspto.gov).

Disclosure of Invention

Sigma factors are subunits of bacterial RNA polymerase (RNAP), play a key role in transcription initiation, and are global regulatory factors. RpoS is one of sigma factors, generally considered to play a role in the stationary phase of bacterial growth, and is often regarded as a stress factor in k. However, in research, the inventors of the present application found that the knock-out of RpoS, a gene encoding RpoS in k.pneumoniae, greatly reduced the production of 1, 3-propanediol fermentation byproducts, as shown in: 1) the generation of the byproduct 2, 3-butanediol is completely blocked; 2) the formation of by-product organic acids is also significantly reduced. Compared with the prior art, the method can obviously reduce the formation of byproducts when the 1, 3-propylene glycol is produced.

The invention is realized by the following technical scheme: the rpoS gene in the K.pneumoniae is knocked out, so that the formation of byproducts of 1, 3-propylene glycol fermentation is reduced.

According to the invention, the gene rpoS is responsible for the sigma subunit rpoS of RNA polymerase;

according to the invention, the by-products are 2, 3-butanediol and organic acids;

according to the invention, the organic acid is specifically: acetic acid, alpha-ketoglutaric acid, succinic acid, and 3-hydroxypropionic acid.

According to the invention, the strain used for the production of 1, 3-propanediol is Klebsiella pneumoniae (K. pneumoconiae). Compared with the existing method for producing 1, 3-propanediol, the method has the advantages that the formation of organic acid as a byproduct can be remarkably reduced while the byproduct 2, 3-butanediol is completely removed.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are illustrative of the present invention only and are not intended to limit the scope of the present invention.

In the examples, the formulation of the slant medium is as follows:

K₂HPO₄3H₂O 7g/L，(NH₄)₂SO₄1g/L，KH₂PO₄2g/L，MgCl₂7H₂0.1g/L of O, 7g/L of yeast extract and 0.3mL of each trace element, and adjusting the pH value to 7.0 and the agar to 2 g/L.

The formula of the seed culture medium is as follows:

K₂HPO₄3H₂O 7g/L，(NH₄)₂SO₄1g/L，KH₂PO₄2g/L，MgCl₂7H₂0.1g/L of O, 7g/L of yeast extract and 0.3mL of each trace element are added after the pH is adjusted to 7.0NaCl regulates the osmotic pressure.

The formulation of the fermenter medium was as follows:

KC l 0.75g/L，NaH₂PO₄1.38g/L，(NH₄)₂SO₄5.35g/L，Na₂SO₄0.28 g/L，MgSO₄6H₂0.26g/L of O, 0.42g/L of citric acid, 2g/L of yeast powder and 0.3mL of each trace element, and the pH value is adjusted to 7.0.

The formula of the trace elements is as follows:

ZnCl₂34.2g/L，FeCl₃6H₂O 2.7g/L，MnCl₂4H₂O 10g/L，CuCl₂2H₂O 0.85g/L，CoCl₂2H₂O 23.8g/L，H₃BO₃0.31g/L，Na₂MoO₄0.25g/L