CN106191136B - A method of improving 1,3-PD biosynthesis - Google Patents

A method of improving 1,3-PD biosynthesis Download PDF

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CN106191136B
CN106191136B CN201610538284.9A CN201610538284A CN106191136B CN 106191136 B CN106191136 B CN 106191136B CN 201610538284 A CN201610538284 A CN 201610538284A CN 106191136 B CN106191136 B CN 106191136B
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pck
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butanediol
klebsiella pneumoniae
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宫衡
张永强
傅水林
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East China University of Science and Technology
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Abstract

The invention discloses a kind of gene pck knockout by Klebsiella pneumoniae Klebsiella pneumoniae, the methods of more efficient producing 1,3-propylene glycol by transforming glycerol.The advantages of method of the invention, is: drastically reducing the synthesis of by-product 2,3-butanediol after pck gene knockout, improves the production level that Klebsiella pneumoniae (Klebsiella pneumoniae) glycerine converting generates 1,3-PD.

Description

A method of improving 1,3-PD biosynthesis
Technical field
The invention belongs to technical field of bioengineering, specifically, it may be assumed that knock out in Klebsiella pneumoniae and encode phosphoric acid The gene of enol pyruvic acid carboxylation kinases synthesis, to improve the production level of 1,3-PD fermentation.
Background technique
1,3-PD (1,3-porpanediol, referred to as 1,3-PD) is a kind of important industrial chemicals, most heavy The purposes wanted is as monomer synthesizing new poly-vinegar --- poly terephthalic acid propylene glycol vinegar (PTT).Research shows that a kind of property of PTT Polyester material that can be especially excellent, thus the industrial value of 1,3-PD increasingly cause the attention of various countries.It is present research shows that: In 1,3-PD production method biological synthesis process because of mild condition, easy to operate, by-product is few, environmentally protective etc., closed than chemistry Cheng Fageng has the advantage of industrial application.In particular with biodiesel industrial expansion, cheap biodiesel industry is utilized By-product glycerin biosynthesis 1,3-PD becomes a research hotspot in nowadays industrial biotechnology field.
There are some microorganisms that can convert glycerol into 1,3-PD in nature.Wherein Klebsiella pneumoniae (K Lebsielapneumoniae) because biotransformation efficiency is high, biotransformation is easy to operate, research application is most.Cray primary Family name pneumobacillus generally passes through disproportionation approach using glycerol metabolism: i.e. glycerol, which is oxidized, generates intermediate product phosphoric acid enol form propanone Sour (phosphoenolpyruvate, abbreviation PEP), while glycerol is also reduced and generates product 1,3-PD.Intermediate product PEP meeting Further metabolism forms a series of by-product such as inside thallus, lactic acid, 2,3-butanediol and acetic acid, succinic acid etc..Especially It is 2,3-butanediol, production quantity is most in by-product, and due to its boiling point and product 1,3-PD is close, thus to subsequent The refining spearation process of 1,3-PD causes very big obstacle.
The invention discloses a kind of reduction by-product 2,3-butanediols, improve product 1, the method for 3-PD biosynthesis.It should Method significantly reduces the yield of by-product 2,3-butanediol, while also promoting by knocking out the gene pck in klebsiella Into the growth of thallus, product 1, the yield of 3-PD are improved.
PEP forms C4 compound oxaloacetic acid (oxaloacetate, abbreviation OAA) by carboxylation in biology, the way Diameter can supplement the C4 compound of reduction in tricarboxylic acid cycle (TCA circulation).But the related approach is to citric acid pneumonia bar The biological significance concern of bacterium is less.Pck negative gene responsible editor's code " PEP carboxylase kinases (abbreviation PCK) ", It is responsible for the ATP that the energy that carboxylation PEP forms OAA, while recycling energy-rich phosphate bond in PEP synthesizes 1 molecule in organism.Organism Interior there are also another enzymes, i.e. " phosphoric acid enol pyruvic acid carboxylase (abbreviation PPC) ", are encoded by gene ppc, although PPC Energy carboxylation PEP forms OAA, but the reaction of PPC catalysis does not generate ATP.It is a discovery of the invention that same in Klebsiella pneumoniae When there is two kinds of enzymes, two kinds of enzymes all work during PEP carboxylation, but knock out pck gene disruption PCK enzyme activity after, show Landing reduces the generation of by-product 2,3-butanediol, improves growth and the product 1 of thallus, the synthesis of 3-PD.Have at present Specific effect of the said gene in Klebsiella research in detail not yet is closed, the related pck gene pairs that knocks out reduces by 2,3- fourth Glycol, the production quantifier elimination for improving 1,3-PD are not reported even more.By retrieving State Intellectual Property Office (www.sipo.gov.cn), World Intellectual Property Organization (www.wipo.int), EUROPEAN PATENT OFFICE (www.espacenet.com) and U.S.Patent & Trademark Office (www.uspto.gov) is also without finding that publication identical with this patent protection request or authorization are special Benefit.
Summary of the invention
Present inventor has found under study for action, knocks out the gene pck of coding PCK, can reduce by-product 2,3- fourth two The synthesis of alcohol improves the yield of 1,3-PD.So it is an object of that present invention to provide the method for 1,3-PD of more efficient production a kind of, That is: the gene pck in Klebsiella pneumoniae is knocked out, efficiently produces 1,3-PD.Compared with prior art, it utilizes When present invention production 1,3-PD, less by-product 2,3-butanediol is generated, the growth of thallus is promoted, improves 1,3- The concentration of PD.
The present invention is achieved by the following technical solutions: the gene pck in Klebsiella is knocked out, efficiently produces 1, 3-PD.The present invention includes that seed culture and ferment tank glycerine converting generate 1,3-PD.
According to the present invention, the pck negative gene responsible editor code PEP carboxylase kinases (EC 4.1.1.49). Enzymatic phosphoenolpyruvate (PEP) carboxylation forms oxaloacetic acid (OAA), is formed simultaneously the ATP of 1 molecule.
According to the present invention, the bacterial strain for generating 1,3-PD for glycerine converting is Klebsiella pneumoniae (K.pneumoniae).Compared to the method that existing glycerine converting produces 1,3-PD, the advantages of method of the invention, is: by-product Object 2,3-butanediol is few, and thalli growth speed is fast, production concentration is high.
Specific embodiment
Below in conjunction with specific embodiment, invention is further described in detail.It should be understood that following embodiment is only used for The bright present invention, not for limiting the scope of the invention.
In example, the formula of slant medium is as follows:
K2HPO43H2O 7g/L, (NH4)2SO41g/L, KH2PO42g/L, MgCl27H2O 0.1g/L, yeast extract 7g/L, Each 0.3mL of microelement adjusts pH 7.0, agar 2g/L.
The formula of seed culture medium is as follows:
K2HPO43H2O 7g/L, (NH4)2SO41g/L, KH2PO42g/L, MgCl27H2O 0.1g/L, yeast extract 7g/L, NaCl adjusting osmotic pressure is added after adjusting pH 7.0 in each 0.3mL of microelement.
The formula of fermentation tank culture medium is as follows:
KCl 0.75g/L, NaH2PO41.38g/L, (NH4)2SO45.35g/L Na2SO40.28g/L, MgSO4 6H2O0.26g/L, citric acid 0.42g/L, yeast powder 2g/L, each 0.3mL of microelement adjust pH 7.0.
The formula of microelement is as follows:
ZnCl234.2g/L FeCl36H2O 2.7g/L, MnCl24H2O 10g/L, CuCl22H2O 0.85g/L, CoCl22H2O 23.8g/L, H3BO30.31g/L, Na2MoO4 0.25g/L
In embodiment, the method for measuring dry cell weight in fermentation liquid is as follows:
It takes 1.0mL fermentation liquid to dilute 10 times of 7 ~, is control with deionized water, is read on 721 spectrophotometers in 620nm Take OD.The bacterium solution 10mL for taking different bacterium dense (i.e. different 620nm light absorption values), through thalline were collected by centrifugation, and is washed with deionized Two times washings dry the thallus after being collected by centrifugation again in 80 DEG C of baking ovens to constant weight.Weigh thallus make dry cell weight with OD620Standard curve, and return out relational expression.The dry weight of later thallus is according to the OD of the bacterium solution of measurement620Value is by standard curve Regression relation is calculated.The measurement of 1,3-PD and 2,3-butanediol uses gas chromatography in fermentation liquid.Produce 1,3-PD Bacterial strain use Klebsiella pneumoniae CCTCC M 2014574, hereinafter referred to as M 2014574.
Embodiment 1 knocks out pck reduction by-product 2, and the formation of 3-BD promotes thalli growth and 1, the synthesis of 3-PD
By 2014574 bacterial strain of M in LB culture medium (0.5% yeast extract, 1% tryptone, 1%NaCl, pH 7.0) In 37 DEG C of overnight incubations, extract genome.Cray using 2014574 genome of M extracted as template, according to NCBI login On the genome sequence (LOCUS:NC_009648) of Bai Shi pneumobacillus MGH 78578 pck gene (locus_tag: KPN_03773) design primer, glue recycles purpose band and is sequenced PCR after reaction, carries out base to purpose band after sequencing Because of analysis, the similarity with pck gene on MGH 78578 is 100%.With homologous recombination method, 2014574 genome of M is knocked out Pck gene (1623bp), the recombinant bacterium of acquisition is 2014574 △ pck of M.
By 2014574 △ pck of M 2014574 and M be inoculated in the triangular flask of 250ml respectively 37 DEG C detest it is aerobic with it is micro- good Oxygen culture 12 hours, culture medium was that seed culture medium adds 40g/L glycerol.Triangular flask is sealed with 8 layers of gauze when culture, and shaking table turns It states as 50rpm.
After culture 12 hours, cell concentration, 2,3-butanediol and 1 in measurement fermentation liquid, the concentration of 3-PD, as a result As shown in table 1.From table 1 it follows that significantly reducing the yield of by-product 2,3-butanediol after knocking out pck, but promote The growth of thallus and 1, the decline of the yield of 3-PD.
Table 1: the result of pck bacterial strain shaking flask culture is knocked out
Embodiment 2 knocks out ppc gene pairs thalli growth, and 2,3-butanediol and 1, the synthesis of 3-PD is almost without shadow It rings
By 2014574 bacterial strain of M in LB culture medium (0.5% yeast extract, 1% tryptone, 1%NaCl, pH 7.0) In 37 DEG C of overnight incubations, extract genome.Cray using 2014574 genome of M extracted as template, according to NCBI login On the genome sequence (LOCUS:NC_009648) of Bai Shi pneumobacillus MGH 78578 ppc gene (locus_tag: KPN_04245) design primer, glue recycles purpose band and is sequenced PCR after reaction, carries out base to purpose band after sequencing Because of analysis, the similarity with ppc gene on MGH78578 is 100%.With homologous recombination method, 2014574 genome of M is knocked out Pck gene (2676bp), the recombinant bacterium of acquisition is 2014574 △ ppc of M.
By 2014574 △ ppc of M 2014574 and M be inoculated in the triangular flask of 250ml respectively 37 DEG C detest it is aerobic with it is micro- good Oxygen culture 12 hours, culture medium was that seed culture medium adds 40g/L glycerol.Triangular flask is sealed with 8 layers of gauze when culture, and shaking table turns It states as 50rpm.
After culture 12 hours, cell concentration, 2,3-butanediol and 1 in measurement fermentation liquid, the concentration of 3-PD, as a result As shown in table 2.From Table 2, it can be seen that knocking out thalli growth and 1 after ppc, the synthesis of 3-PD and 2,3-butanediol does not almost have Have an impact.Thus while the enzyme of gene ppc coding, i.e. acid enol type pyruvate carboxylase (EC 4.1.1.38) can also be catalyzed PEP shape At OAA, but present invention discover that: only knock out the gene pck of coding enol pyruvic acid carboxylation kinases (EC 4.1.1.49) Can play reduces the production of by-product 2,3-butanediol, promotes product 1, the effect of 3-PD synthesis.
Table 2: the result of ppc bacterial strain shaking flask culture is knocked out
Embodiment 3 significantly reduces the generation of by-product 2,3-butanediol simultaneously after knocking out pck gene on 5L reactor Promote product 1, the synthesis of 3-PD
5L reactor fermenting experiment is as follows: by the shaking flask of bacterial strain (2014574 △ pck of M, M 2014574) access 250ml (liquid amount 50ml) is carried out seed culture 20 hours, rear to access in 5L fermentor (zymotic fluid liquid amount 2L), according to as follows Process regulation fermentation process.
Initial glycerol concentration 60g/L, 35 DEG C of fermentation temperature；Ventilatory capacity 1.0vvm；Speed of agitator 20rpm；In fermentation process In by be added NaOH solution control pH value be 5.5~7.5.Fermentation each period by filling into various concentration glycerite Glycerol concentration is controlled in 10~60g/L, fermentation terminates for 30 hours.
Shown in fermentation results table 3.From table 3 it is observed that on 5L reactor, compared with starting strain M 2014574, The yield for knocking out the 2014574 △ pck by-product 2,3-butanediol of bacterial strain M of pck gene significantly reduces, while product 1,3- The yield of PD significantly improves.
3: two strain fermentation result of table

Claims (3)

1. a kind of method for improving microorganism biological synthesis 1,3-PD, it is characterised in that: 1,3-PD is produced bacterium Gene pck in strain is knocked out, and is reduced the generation of by-product 2,3-butanediol, is promoted the production of product 1,3-PD.
2. the method according to claim 1, wherein the pck gene specifically: pck negative gene responsible editor's code The PEP carboxylase kinases that EC number is EC4.1.1.49.
3. the method according to claim 1, wherein the bacterial strain for glycerine converting to generate 1,3-PD is Klebsiella pneumoniae (Klebsielapneum oniae).
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Publication number Priority date Publication date Assignee Title
CN107099558B (en) * 2017-04-25 2021-06-11 华东理工大学 Novel method for simultaneously biosynthesizing 1, 3-propylene glycol and acetoin
CN108866114B (en) * 2018-07-26 2022-12-30 华东理工大学 Novel method for efficiently synthesizing polyol
CN111850055A (en) * 2020-08-09 2020-10-30 华东理工大学 Method for reducing by-products in 1, 3-propylene glycol production process

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104388476A (en) * 2014-12-01 2015-03-04 华东理工大学 Efficient and high-conversion-rate method for producing 1,3-propanediol
CN105154476A (en) * 2015-09-25 2015-12-16 华东理工大学 Method for efficiently producing 1, 3-propylene glycol by reducing by-product acetic acid

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104388476A (en) * 2014-12-01 2015-03-04 华东理工大学 Efficient and high-conversion-rate method for producing 1,3-propanediol
CN105154476A (en) * 2015-09-25 2015-12-16 华东理工大学 Method for efficiently producing 1, 3-propylene glycol by reducing by-product acetic acid

Non-Patent Citations (1)

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Title
Deleting pck improves growth and suppresses by-product formation during 1,3-propanediol fermentation by Klebsiella pneumoniae;Yongqiang Zhang等;《Journal of Applied Microbiology》;20170621;第123卷(第3期);678-687 *

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