CN113373164A - Ribosome site sequence of over-expressed pyruvate dehydrogenase complex and application thereof - Google Patents

Ribosome site sequence of over-expressed pyruvate dehydrogenase complex and application thereof Download PDF

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CN113373164A
CN113373164A CN202110609494.3A CN202110609494A CN113373164A CN 113373164 A CN113373164 A CN 113373164A CN 202110609494 A CN202110609494 A CN 202110609494A CN 113373164 A CN113373164 A CN 113373164A
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宫衡
李胜明
傅水林
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East China University of Science and Technology
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Abstract

The invention discloses a ribosome binding site sequence capable of overexpressing a pyruvate dehydrogenase complex, and the pyruvate content in Redberg pneumonia bacillus with pyruvate accumulation and growth inhibition can be effectively reduced by carrying out overexpression on the pyruvate dehydrogenase complex by using the sequence, so that the growth of thalli is improved.

Description

Ribosome site sequence of over-expressed pyruvate dehydrogenase complex and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a section of ribosome binding site sequence capable of overexpressing a ketoacid dehydrogenase complex and application thereof.
Background
Pyruvate (pyruvate) is an important intermediate metabolite in prokaryotic metabolism, and is the final product of glycolysis, and can be converted into acetyl coenzyme A, so that the pyruvate enters into the citric acid cycle to provide energy for growth and metabolism of bacteria, and can also be converted into lactic acid to provide energy for anaerobic growth. The pyruvate content is maintained at a low level during cell growth. And in the pyruvate metabolism defective strain, a large amount of pyruvate is accumulated, and the pyruvate is used as organic acid, and the large amount of accumulation can cause irreversible damage to the thallus, thereby influencing the growth and metabolism of the thallus.
Based on the above problems, applicants designed a Ribosome Binding Site (RBS) sequence that is capable of overexpressing Pyruvate dehydrogenase complex (PDHC) to overexpress PDHC. The RBS sequence can over-express PDHC derived from Klebsiella pneumoniae, so that the content of pyruvic acid in pyruvic acid metabolism defective Klebsiella pneumoniae is increased, and the thallus growth of pyruvic acid metabolism defective strains is improved.
Disclosure of Invention
Aiming at the problems of pyruvate accumulation and growth inhibition, the invention aims to design an RBS sequence capable of overexpressing PDHC and an application of the overexpressed PDHC.
The RBS sequence is SEQ ID NO 1.
The PDHC coding gene is the genes aceE, aceF and lpdA of Klebsiella pneumoniae.
The application of the over-expression PDHC means that the PDHC coding gene of RBS is expressed and optimized through a plasmid vector, the content of pyruvic acid in pyruvic acid metabolism defective Klebsiella pneumoniae is reduced, and the growth of thalli is improved.
The construction method of the plasmid vector containing the RBS sequence overexpression PDHC is characterized by comprising the following process steps:
1) the gene fragments aceE, aceF and lpdA with the RBS sequence and the enzyme cutting site are obtained by taking a Klebsiella pneumoniae genome as a template and upstream and downstream primers SEQ ID NO 2 and SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5, and SEQ ID NO 6 and SEQ ID NO 7 respectively containing the enzyme cutting site and the RBS sequence.
2) Carrying out enzyme digestion on the gene aceE fragment by using SpeI, carrying out enzyme digestion on the gene aceF fragment by using Xba I, then connecting the genes at two ends together by using T4 ligase to obtain an aceE-aceF fragment, and carrying out amplification on the aceE-aceF fragment by using primers SEQ ID NO 2 and SEQ ID NO 5; then carrying out enzyme digestion on the amplified aceE-aceF fragment by using SpeI, carrying out enzyme digestion on the gene lpdA fragment by using Xba I, connecting the two genes together by using T4 ligase to obtain a PDHC coding gene fragment aceE-aceF-lpdA of the optimized RBS, and then carrying out amplification on the aceE-aceF-lpdA fragment by using primers SEQ ID NO 2 and SEQ ID NO 7.
3) Carrying out double enzyme digestion on PDHC coding gene segments of the optimized RBS by using enzymes Xba I and Hind III, carrying out double enzyme digestion on a plasmid pETNar by using enzymes Spe I and Hind III, connecting by using T4, transforming into escherichia coli DH5 alpha after connection, carrying out amplification, and then extracting the plasmid to obtain a recombinant plasmid pETNarHRBSPDHC for over-expression of PDHC.
4) The obtained recombinant plasmid is transferred into a strain with pyruvate metabolism defect, pyruvate accumulation and growth inhibition for culture, so that the content of pyruvate in the thallus can be reduced, and the cell growth can be improved.
Drawings
FIG. 1 is a gel electrophoresis image of the amplification of genes aceE, aceF and lpdA;
FIG. 2 is a pETNar plasmid map;
FIG. 3 is a diagram of pETNar plasmid double-restriction enzyme gel electrophoresis;
FIG. 4 is an amplification map of a fragment of the PDHC-encoding gene aceE-aceF-lpdA (PDHC) of optimized RBS;
FIG. 5 is a map of the plasmid pETNarHRBSPDHC.
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 seed culture medium is as follows:
K2HPO4 3H2O 7g/L，(NH4)2SO4 1g/L，KH2PO4 2g/L，MgCl2 7H20.1g/L of O, 7g/L of yeast extract and 0.3mL of each trace element, and the pH value is adjusted to 7.0.
The formula of the fermentation medium is as follows:
KC l 0.75g/L，NaH2PO4 1.38g/L，(NH4)2SO4 5.35g/L，Na2SO4 0.28g/L，MgSO4 6H20.26g/L of O, 0.42g/L of citric acid, 2g/L of yeast powder, 0.3mL of each trace element and 40g/L of glycerol, and the pH value is adjusted to 7.0.
The formula of the trace elements is as follows:
ZnCl2 34.2g/L，FeCl3 6H2O 2.7g/L，MnCl2 4H2O 10g/L，CuCl2 2H2O 0.85g/L，CoCl2 2H2O 23.8g/L，H3BO3 0.31g/L，Na2MoO4 0.25g/L。
in the examples, the formulation of LB medium is as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of NaCl, and 1.5 percent of agar powder is additionally added into a solid culture medium.
In the examples, the method of measuring the dry weight of the cells in the fermentation broth was as follows:
1.0mL of fermentation broth is diluted 7 times, deionized water is used as a blank control, and the OD is read by setting the wavelength of a spectrophotometer at 620 nm. Taking 5mL of bacterial liquid with different bacterial concentrations, centrifuging at 12000rpm to collect thalli, washing for more than three times by using deionized water, placing the thalli after centrifugation and collection again on dried and weighed filter paper, and drying at 80 ℃. Weighing thallus to obtain thallus dry weight and OD620The standard curve of (2). The dry weight of the cells thereafter was measured by using the OD of the cells620The value of (d) is calculated by a standard curve relation.
In the example implementation, the kits are purchased from Tiangen corporation.
Example 1 construction of pyruvate metabolism-deficient Strain
The pflA (LOCUS: 1045721..1046461, completion) and pflB (LOCUS: 1046653..1048935, completion) gene sequences of Klebsiella pneumoniae MGH 78578 (LOCUS: NC _009648.1) registered according to NCBI were aligned with the genome sequencing result of Klebsiella pneumoniae CCTCC M2014574 (M2014574 for short), and the sequence similarity was 100%. Knocking out pflA and pflB genes in an M2014574 genome by using a lambda-Red homologous recombination method to obtain a recombinant bacterium M2014574 delta PFL, and performing fermentation culture.
Seed culture
Strains M2014574 and M2014574. delta. PFL were inoculated in an inoculum size of 1% into a test tube containing 5mL of liquid LB medium, incubated at 37 ℃ for 12 hours at 200rpm, and then inoculated in an inoculum size of 1% into a 250mL shake flask containing 50mL of liquid seed medium, and anaerobically cultured for 10 hours.
Shake flask fermentation
Inoculating the cultured seeds into 250mL shake flasks according to the inoculation amount of 1%, inoculating 3 shake flasks to each strain to perform a parallel test, wherein the liquid loading of the shake flasks is 50mL, the bottle openings are blocked by airtight rubber stoppers, and the seeds are cultured for 24 hours at 37 ℃ and 220 rpm. Detection of pyruvic acid by HPLC and by determination of fermentation broth OD620To determine the cell concentration.
As can be seen, compared with the starting strain M2014574, the pyruvate metabolism defective strain M2014574. delta. PFL has the advantages that the pyruvate content is greatly accumulated and is increased to 1.8g/L from 0.05g/L, so that the growth is obviously inhibited, and only 55 percent of the starting strain M2014574 is obtained.
TABLE 1 Shake flask fermentation results
Figure BDA0003095425540000051
Example 2 construction of pETNar plasmid
Since the T7 promoter carried by the pET28a (+) plasmid failed to function in Klebsiella pneumoniae, we modified the promoter of this plasmid. Firstly synthesizing a sequence SEQ ID NO 8 to obtain a nar promoter fragment with Bgl II and BamH I enzyme cutting sites, then carrying out double enzyme cutting on the nar promoter fragment and pET28a (+) plasmid by using Bgl II and BamH I, connecting by using T4 ligase, and finally transferring into escherichia coli DH5 alpha for amplification to obtain pETNar plasmid.
Example 3 the situation where the expression of PDHC using the RBS sequence of the original PDHC did not improve pyruvate accumulation and growth inhibition
The gene sequences of aceE (LOCUS: 137677..140340), aceF (LOCUS: 140355..142253) and lpdA (LOCUS: 142460..143884) in the genomic sequence of Klebsiella pneumoniae MGH 78578 (LOCUS: NC-009648.1) registered according to NCBI were aligned with the genomic sequencing result of Klebsiella pneumoniae CCTCC M2014574, and the sequence similarity was 100%.
Firstly, the genome of Klebsiella pneumoniae CCTCC M2014574 is taken as a template, the whole sequence of three gene segments coding PDHC is amplified by primers SEQ ID NO 9 and SEQ ID NO 10 containing enzyme cutting sites to obtain a YSPHDC segment, then the obtained YSPHDC segment is subjected to double enzyme cutting by Xba I and Hind III, meanwhile, the pETNar plasmid obtained in the embodiment 2 is subjected to double enzyme cutting by SpeI and Hind III, then the plasmid is connected by T4 ligase, and then the plasmid is transformed into escherichia coli DH5 alpha for amplification, and then the plasmid is extracted to obtain the recombinant plasmid pETNarPDHC expressing PDHC by using the original RBS site. Then, the recombinant plasmid pETNarPDHC is transferred into the pyruvate metabolism deficiency strain M2014574 delta PFL obtained in the example 1 by an electrotransformation method to obtain a strain M2014574 delta PFL: : and YS. Strain M2014574 Δ PFL and strain M2014574 Δ PFL were fermented as in example 1: : and YS is subjected to fermentation culture. Detection of pyruvic acid by HPLC and by determination of fermentation broth OD620The cell concentration was determined, and the results are shown in Table 2.
It can be seen that: after the expression of PDHC by using the original RBS sequence of the coding PDHC gene, the content of pyruvic acid in the thallus is not obviously reduced, and the growth of the thallus is not obviously increased.
TABLE 2 Shake flask fermentation results
Figure BDA0003095425540000061
Example 4 improvement of pyruvate accumulation and growth inhibition by PDHC overexpression Using the optimized RBS sequences of the invention
Firstly, taking the genome of Klebsiella pneumoniae CCTCC M2014574 as a template, and respectively obtaining gene fragments aceE, aceF and lpdA with an enzyme cutting site and an RBS sequence by using upstream and downstream primers SEQ ID NO 2 and SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5, and SEQ ID NO 6 and SEQ ID NO 7 containing the enzyme cutting site and the RBS sequence.
Then carrying out enzyme digestion on the gene aceE fragment by using SpeI, carrying out enzyme digestion on the gene aceF fragment by using Xba I, connecting the genes at two ends together by using T4 ligase to obtain an aceE-aceF fragment, and amplifying the aceE-aceF fragment by using primers SEQ ID NO 2 and SEQ ID NO 5; then carrying out enzyme digestion on the amplified aceE-aceF fragment by using SpeI, carrying out enzyme digestion on the gene lpdA fragment by using Xba I, then connecting the two genes together by using T4 ligase to obtain a PDHC coding gene fragment aceE-aceF-lpdA of the optimized RBS, and then carrying out amplification on the aceE-aceF-lpdA fragment by using primers SEQ ID NO 2 and SEQ ID NO 7.
Then, the PDHC coding gene segment of the optimized RBS is subjected to double enzyme digestion by using enzymes Xba I and Hind III, meanwhile, pETNar plasmid obtained in the embodiment 2 is subjected to double enzyme digestion by using enzymes Spe I and Hind III, the pETNar plasmid is connected by using T4, the connected pETNar plasmid is transformed into escherichia coli DH5 alpha for amplification, and then the plasmid is extracted, so that the recombinant plasmid pETNarHRBSPDHC of the over-expressed PDHC can be obtained.
The obtained recombinant plasmid pETNarHRBSPDHC was then transferred into the pyruvate metabolism deficient strain M2014574 Δ PFL obtained in example 1 to obtain strain M2014574 Δ PFL: : HRBS. Finally, strain M2014574 Δ PFL and strain M2014574 Δ PFL were fermented as in example 1: : HRBS (high resolution binding library) for shake flask fermentation, detection of pyruvic acid by HPLC (high Performance liquid chromatography) and determination of OD (optical density) of fermentation liquor620The cell concentration was determined, and the results are shown in Table 3.
It can be seen that: after PDHC overexpression is carried out by using the designed RBS sequence, the content of pyruvic acid is obviously reduced, and the growth of thalli is obviously improved.
TABLE 3 Shake flask fermentation results
Figure BDA0003095425540000071
Figure BDA0003095425540000081
Figure BDA0003095425540000091
Figure BDA0003095425540000101
SEQUENCE LISTING
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Claims (3)

1. A ribosome binding site sequence which can over-express pyruvate dehydrogenase complex and is shown as SEQ ID No. 1.
2. The sequence of claim 1, which can construct a plasmid vector for overexpression of a pyruvate dehydrogenase complex encoded by genes lpdA, aceE and aceF derived from Klebsiella pneumoniae, wherein the plasmid vector can reduce the pyruvate content in Klebsiella pneumoniae in which accumulation and growth of pyruvate are inhibited, thereby improving growth of bacteria.
3. The method of plasmid vector according to claim 2, characterized by the following process steps:
1) using Klebsiella pneumoniae genome as a template, and obtaining gene fragments aceE, aceF and lpdA with enzyme cutting sites by using upstream and downstream primers SEQ ID NO 2 and SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7;
2) carrying out enzyme digestion on the gene aceE fragment by using SpeI, carrying out enzyme digestion on the gene aceF fragment by using Xba I, then connecting the genes at two ends together by using T4 ligase to obtain an aceE-aceF fragment, and carrying out amplification on the aceE-aceF fragment by using primers SEQ ID NO 2 and SEQ ID NO 5; then carrying out enzyme digestion on the amplified aceE-aceF fragment by using SpeI, carrying out enzyme digestion on the gene lpdA fragment by using Xba I, connecting the two genes together by using T4 ligase to obtain a gene fragment aceE-aceF-lpdA, and then amplifying the aceE-aceF-lpdA fragment by using primers SEQ ID NO 2 and SEQ ID NO 7;
3) carrying out double enzyme digestion on the aceE-aceF-lpdA fragment by using Xba I and Hind III, carrying out double enzyme digestion on a plasmid pETNar by using Spe I and Hind III, connecting by using T4 chain, transforming into escherichia coli DH5 alpha after connection, carrying out amplification, and then extracting the plasmid to obtain a recombinant plasmid for over-expressing a pyruvate dehydrogenase compound;
4) the obtained recombinant plasmid is transferred into Klebsiella pneumoniae of which the accumulation and the growth of pyruvic acid are inhibited to be cultured, so that the content of the pyruvic acid in the thallus can be reduced, and the cell growth can be improved.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2010146281A (en) * 2010-11-13 2012-05-20 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленн BACTERIA OF THE GENUS Escherichia Possessing Enhanced Expression of the ACEEF-lpdA GENES OPERON AND METHOD FOR PRODUCING SUITIC ACID USING SUCH BACTERIA
US20150064758A1 (en) * 2013-08-29 2015-03-05 Samsung Electronics Co., Ltd. Microorganism comprising pyruvate dehydrogenase variant and method of producing c4-chemicals using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2010146281A (en) * 2010-11-13 2012-05-20 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленн BACTERIA OF THE GENUS Escherichia Possessing Enhanced Expression of the ACEEF-lpdA GENES OPERON AND METHOD FOR PRODUCING SUITIC ACID USING SUCH BACTERIA
US20150064758A1 (en) * 2013-08-29 2015-03-05 Samsung Electronics Co., Ltd. Microorganism comprising pyruvate dehydrogenase variant and method of producing c4-chemicals using the same

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DANFENG XU等: "Analysis of the Growth and Metabolites of a Pyruvate Dehydrogenase Complex- Deficient Klebsiella pneumoniae Mutant in a Glycerol-Based Medium", 《J MICROBIOL BIOTECHNOL》, vol. 30, no. 5, pages 753 - 761 *
MCCLELLAND, M.等: "MULTISPECIES: pyruvate dehydrogenase complex dihydrolipoyllysine-residue acetyltransferase [Klebsiella]", NCBOI, pages 1 - 8 *
MCCLELLAND, M等: "MULTISPECIES: pyruvate dehydrogenase (acetyl-transferring), homodimeric type [Enterobacteriaceae]", NCBI, pages 1 - 9 *
WATANABE, N.等: "MULTISPECIES: dihydrolipoyl dehydrogenase [Enterobacteriaceae]", NCBI, pages 1 - 7 *

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