CN117264865B - Recombinant rhodococcus erythropolis and application thereof in compound synthesis - Google Patents

Recombinant rhodococcus erythropolis and application thereof in compound synthesis Download PDF

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CN117264865B
CN117264865B CN202311553921.6A CN202311553921A CN117264865B CN 117264865 B CN117264865 B CN 117264865B CN 202311553921 A CN202311553921 A CN 202311553921A CN 117264865 B CN117264865 B CN 117264865B
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rhodococcus erythropolis
recombinant
gene
synthesis
carotenoid
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CN117264865A (en
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于慧敏
梁有向
郑煜堃
陈博
王苗苗
李楠楠
赵明
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Beijing Yanwei Technology Co ltd
Tsinghua University
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Tsinghua University
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Abstract

The present application relates to recombinant rhodococcus erythropolis wherein the carotenoid synthesis gene is reduced or knocked out and its use in compound synthesis; the carotenoid synthesis genes with reduced expression or knockdown include genes encoding GGPP synthasecrtE‑1Gene encoding phytoene dehydrogenasecrtIGene encoding carotene synthesis-related membrane proteincrtMAnd genes encoding phytoene synthasecrtBAt least one of (2). The recombinant rhodococcus erythropolis does not affect the growth and enzyme production of rhodococcus erythropolis, and reduces pigment content.

Description

Recombinant rhodococcus erythropolis and application thereof in compound synthesis
Technical Field
The application relates to the technical fields of genetic engineering and synthetic biology, in particular to recombinant rhodococcus erythropolis and application thereof in compound synthesis.
Background
Rhodococcus erythropolis is a gram-positive bacterium and has important applications in biocatalysis, biodegradation, biomass utilization and the like. At present, rhodococcus containing important industrial enzymes such as nitrile hydratase, nitrilase, amidase, transaminase, epoxide hydrolase and the like is widely used as a biocatalyst in the industrial production of important compounds such as amide, carboxylic acid, chiral medicine intermediates and the like. However, when preparing a target chemical with high concentration, the problems of overhigh concentration of a substrate or a product, exothermic reaction and the like easily cause rhodococcus cell rupture, release pigment, deepen the color of a product solution, influence the quality of the product and increase the post-treatment cost of the product. Therefore, the reduction of the pigment content in the rhodococcus cells is significant for improving the product quality of the rhodococcus cell catalytic products and reducing the production cost.
Carotenoids are a class of natural pigments composed of isoprenoid polymers, which are widely found in animals, plants, fungi, algae and bacteria, and are more commonly tetraterpenoids such as C40-skeleton lycopene, beta-carotene, zeaxanthin, astaxanthin, capsanthin, and the like. Most rhodococci synthesize relatively large amounts of natural carotenoids, so the colony color appears orange or red. The pigments released by rhodococcus in biocatalytic reactions are mainly carotenoids, but to date, there have been no reports on weakening the synthesis of rhodococcus pigments by genetic engineering.
Although the main pathways for carotenoid biosynthesis are largely clear, the key genes for carotenoid synthesis in most rhodococcus have not been clear, and particularly the pathways related to carotenoid synthesis in rhodococcus erythropolis have not been reported so far. Rhodococcus usually contains redundant gene sequences, sometimes there are multiple different genes encoding proteins with the same function, but their activities often differ greatly, and it is difficult to predict which gene plays a major role, which further increases the difficulty of identifying key genes.
Disclosure of Invention
Accordingly, there is a need in the present application for a recombinant rhodococcus erythropolis having reduced pigment synthesis, and its use in the synthesis of amides, carboxylic acids, and the like.
The specific technical scheme is as follows:
a recombinant rhodococcus erythropolis in which the carotenoid synthesis gene is reduced in expression or knocked out; the carotenoid synthesis genes with reduced expression or knockdown include genes encoding GGPP synthasecrtE-1Gene encoding phytoene dehydrogenasecrtIGene encoding carotene synthesis-related membrane proteincrtMAnd genes encoding phytoene synthasecrtBAt least one of (2).
In one embodiment, the carotenoid synthesis gene with reduced expression or knockdown comprises a gene encoding GGPP synthasecrtE-1And/or genes encoding phytoene dehydrogenasecrtI。
The recombinant rhodococcus erythropolis is applied to compound synthesis.
In one embodiment, the compound includes one or more of an amide compound, a carboxylic acid compound, a chiral epoxide compound, and a chiral amine compound.
Compared with the traditional technology, the application has the following beneficial effects:
the application analyzes and identifies the key genes synthesized with the carotenoid of the rhodococcus erythropolis, constructs different recombinant rhodococcus erythropolis with weakened carotenoid synthesis by inactivating the key genes synthesized with the carotenoid, and screens the recombinant rhodococcus erythropolis which does not influence the growth and enzyme production of the rhodococcus erythropolis and reduces the pigment content. Further the method is used for synthesizing compounds such as amides, carboxylic acids, chiral epoxides, chiral amines and the like, and the quality of the compounds is improved.
Drawings
FIG. 1 shows carotenoid synthesis gene distribution of Rhodococcus erythropolis;
FIG. 2 is a graph showing the effect of inhibiting various carotenoid synthesis genes on carotenoid content;
FIG. 3 is a graph showing the effect of inhibiting the growth of rhodococcus by different carotenoid synthesis genes;
FIG. 4 is a graph showing the effect of inhibiting different carotenoid synthesis genes on rhodococcus nitrile hydratase activity;
FIG. 5 shows the color comparison of recombinant bacteria TH-crtE1 and TH-control;
FIG. 6 shows a color comparison of acrylamide hydration liquid synthesized by recombinant bacteria TH-crtE1 and TH-control.
Detailed Description
In order to make the above objects, features and advantages of the present application more comprehensible, embodiments accompanied with the present application are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is, however, susceptible of embodiment in many other forms than those described herein and similar modifications can be made by those skilled in the art without departing from the spirit of the application, and therefore the application is not to be limited to the specific embodiments disclosed below.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
To solve the problem that the pigment release of rhodococcus erythropolis affects the red during the reaction processThe present application first analyzes key genes for red rhodococcus carotene synthesis, including genes encoding GGPP synthetase, by bioinformaticscrtE-1AndcrtE-2gene encoding phytoene dehydrogenasecrtIGene encoding carotene synthesis-related membrane proteincrtMGene encoding phytoene synthasecrtBCoding lycopene beta-cyclasecrtY. Different recombinant rhodococcus erythropolis with weakened pigment synthesis are constructed through gene inactivation (inhibition or knockout), and strains which do not influence the growth and enzyme production of the rhodococcus erythropolis and reduce the pigment content are obtained through screening. The method is used for synthesizing compounds such as amides, carboxylic acids, chiral epoxides, chiral amines and the like, and improves the quality of the compounds.
An embodiment of the present application provides a recombinant rhodococcus erythropolis having reduced pigment synthesis, wherein the carotenoid synthesis gene in the recombinant rhodococcus erythropolis is inhibited or knocked out by expression. Wherein the carotenoid synthesis gene whose expression is suppressed or knocked out includes a gene encoding GGPP synthasecrtE-1Gene encoding phytoene dehydrogenasecrtIGene encoding carotene synthesis-related membrane proteincrtMGene encoding phytoene synthasecrtBAt least one of (2).
In a specific example, the carotenoid synthesis gene whose expression is inhibited or knocked out includes a gene encoding GGPP synthasecrtE-1And/or genes encoding phytoene dehydrogenasecrtI. Make the following stepscrtE1Or (b)crtIThe inactivation of the gene can reduce the yield of carotenoid to different degrees, and has no negative effect on the growth and enzyme production of bacteria. Wherein,crtE1inactivation of the gene reduces carotenoid content by more than 95%, biomass (OD 460 The value) is improved by 11%, the enzyme activity of nitrile hydratase fermentation is improved by 6%, the fermentation liquor of the strain is used for synthesizing high-concentration acrylamide, the chromaticity of the obtained hydration liquor is obviously reduced, and the product quality is improved. And (3) withcrtE1In contrast to the inactivation of the gene,crtE1andcrtIthe genes are inactivated at the same time, so that the enzyme activity of the nitrile hydratase is improved by 21%, and the carotenoid content is reduced by 57%.
In a specific example, the knockout includes knocking out part or all of the carotenoid synthesis gene.
In a specific example, a gene encoding GGPP synthasecrtE-1The nucleotide sequence of (a) is shown as SEQ ID NO. 1, and/or a gene encoding phytoene dehydrogenasecrtIThe nucleotide sequence of (2) is shown as SEQ ID NO. 3, and/or the gene for encoding carotene synthesis related membrane proteincrtMThe nucleotide sequence of (a) is shown as SEQ ID NO. 4, and/or a gene encoding phytoene synthasecrtBThe nucleotide sequence of (2) is shown as SEQ ID NO. 5.
In a specific example, a genetically stable recombinant strain is constructed by knocking out a carotenoid synthesis gene, and the method for knocking out the carotenoid synthesis gene includes a homologous recombination double crossover method.
In a specific example, the method for constructing the recombinant rhodococcus erythropolis by using the homologous recombination double exchange method comprises the following steps of a-b:
and a step a, connecting an upstream homologous sequence and a downstream homologous sequence of the carotenoid synthesis gene into a suicide plasmid vector to obtain the recombinant suicide plasmid vector.
Primers are designed according to the upstream and downstream sequences of the carotenoid synthesis gene to amplify the upstream and downstream homologous sequences of the carotenoid synthesis gene, respectively.
And b, introducing the recombinant suicide plasmid vector into rhodococcus, and screening to obtain recombinant rhodococcus.
In a specific example, the method of screening comprises a first round of screening with an antibiotic-containing plate to obtain a first recombinant bacterium, and a second round of screening with a sucrose-containing plate to obtain a recombinant rhodococcus erythropolis.
Alternatively, the suicide plasmid vector comprises pK18mobsacB.
In a specific example, the method of reducing carotenoid synthesis gene expression comprises one or more of CRISPRi technology, antisense RNA technology, RNA interference, and promoter attenuation methods.
The CRISPRi technology inhibits transcription of target DNA through targeted combination of dCAS9-sgRNA complex and the target DNA to inhibit expression of the target gene. The dCas9-sgRNA complex can inhibit transcription of target DNA in two ways: (1) Transcription initiation is inhibited by preventing RNA polymerase from binding to the DNA promoter. (2) inhibition of transcriptional elongation by steric hindrance.
In a specific example, small molecule RNAs used in RNA interference techniques include miRNA, siRNA, dsRNA and shRNA.
In a specific example, a method of constructing a recombinant rhodococcus erythropolis using CRISPRi technology includes introducing a vector containing a dCas9 gene and a vector containing sgrnas targeting carotenoid synthesis genes into rhodococcus erythropolis to construct a recombinant rhodococcus erythropolis.
The two domains HNH and RuvC of the CRISPR/Cas9 protein with endonuclease activity are subjected to H840A and D10A site-directed mutagenesis respectively, so that dCAS9 protein losing endonuclease activity is obtained.
In a specific example, a gene encoding GGPP synthetase is targetedcrtE-1The recognition sequence of sgrnas of (a): GGCGTCCTCCTGGTCGTGTG (nucleotide sequence at positions 5-24 of SEQ ID NO: 11);
targeting genes encoding phytoene dehydrogenasecrtIThe recognition sequence of sgrnas of (a): CGACGGTCGCCTCGCGTTCG (nucleotide sequence 5-24 of SEQ ID NO: 15);
gene encoding carotene synthesis related membrane proteincrtMThe recognition sequence of sgrnas of (a): TCCGGGGGACGCTGCTGTTC (nucleotide sequence 5-24 in SEQ ID NO: 17);
gene encoding phytoene synthasecrtBThe recognition sequence of sgrnas of (a): ATAGTACGTCCGGCCGTGCG (nucleotide sequence at positions 5-24 of SEQ ID NO: 19).
An embodiment of the application also provides application of the recombinant rhodococcus erythropolis in compound synthesis. In one specific example, the compound includes one or more of an amide compound, a carboxylic acid compound, a chiral epoxide compound, and a chiral amine compound.
In a specific example, the recombinant rhodococcus erythropolis includes an exogenous plasmid carrying a gene encoding an exogenous enzyme. Exogenous plasmid carrying exogenous enzyme coding gene is introduced into recombinant rhodococcus erythropolis to express exogenous enzyme, and the exogenous enzyme is used for catalyzing substrate to synthesize target compound, so that the quality and yield of the compound are improved.
In one specific example, the exogenous enzyme includes one or more of a nitrilase, a nitrile hydratase, a transaminase, and/or an epoxide hydrolase.
Specifically, carboxylic acid compounds can be synthesized by utilizing nitrilase to catalyze, chiral epoxide can be synthesized by utilizing epoxide hydrolase to catalyze, chiral amine compounds can be synthesized by utilizing aminotransferase to catalyze, and amide compounds can be synthesized by utilizing nitrile hydratase to catalyze.
In one specific example, the amide-based compound includes acrylamide and/or nicotinamide.
In a specific example, the method of acrylamide or nicotinamide synthesis comprises the steps of:
expressing nitrile hydratase by utilizing the genome of the recombinant rhodococcus erythropolis and/or expressing nitrile hydratase by introducing exogenous plasmid carrying nitrile hydratase encoding gene into the recombinant rhodococcus erythropolis;
the nitrile hydratase is used for catalyzing the hydration reaction of acrylonitrile or 3-cyanopyridine to synthesize acrylamide or nicotinamide.
In one specific example, the temperature of the hydration reaction is 15 ℃ to 45 ℃. Optionally, the temperature of the hydration reaction is 18-25 ℃.
In a specific example, the method for synthesizing acrylamide or nicotinamide comprises the steps of (1) to (3):
and (1) preparing the seed solution of the recombinant rhodococcus erythropolis.
In a specific example, the recombinant rhodococcus erythropolis may also carry an exogenous plasmid encoding a gene for a nitrile hydratase.
In a specific example, the seed culture medium used for preparing the seed solution of the recombinant rhodococcus erythropolis comprises glucose 10 g/L-50 g/L, yeast extract 1 g/L-4 g/L, and proteinPeptone 1 g/L-10 g/L KH 2 PO 4 0.2 g/L -3 g/L，K 2 HPO 4 0.2 g/L -3 g/L，MgSO 4 ·7H 2 O0.2 g/L-3 g/L and monosodium glutamate 1 g/L, and pH value is 7.0-7.5.
And (2) preparing the fermentation liquor of the recombinant rhodococcus erythropolis by using the seed liquor.
In a specific example, the fermentation medium for preparing the fermentation broth of the recombinant rhodococcus erythropolis comprises glucose 10 g/L-50 g/L, urea 5 g/L-20 g/L, yeast extract 1 g/L-4 g/L, peptone 1 g/L-10 g/L, KH 2 PO 4 0.2 g/L -3 g/L，K 2 HPO 4 0.2 g/L -3 g/L，MgSO 4 ·7H 2 O0.2 g/L-3 g/L, monosodium glutamate 1 g/L and CoCl 2 0.03 mM-0.5 mM, pH 7.0-7.5.
And (3) carrying out hydration reaction on the fermentation liquor and acrylonitrile or 3-cyanopyridine to synthesize acrylamide or nicotinamide.
Embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are preferably referred to in the guidelines given in the present application, may be according to the experimental manual or conventional conditions in the art, may be according to the conditions suggested by the manufacturer, or may be referred to experimental methods known in the art.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
EXAMPLE 1 annotation of carotenoid synthesis-related genes of Rhodococcus rhodochrous
Rhodococcus erythropolis (CN 101663389A) is cultured by an LB culture medium, then bacterial cells are collected, three generations of genome sequencing is carried out by Jin Weizhi biotechnology company, and RAST server (http:// RAST. Nmpdr. Org /) is used for analyzing and annotating the genome, and KEGG and GO databases are compared to determine the carotenoid production metabolic pathway. Major phaseThe genes involved include 2 genes encoding GGPP (geranylgeranyl pyrophosphate, geranylgeranyl pyrophosphat) synthetases (designated respectivelycrtE-1 and crtE-2The DNA sequences are shown as SEQ ID NO. 1 and SEQ ID NO.2, respectively), 1 gene encoding phytoene dehydrogenase (designated ascrtIThe DNA sequence is shown as SEQ ID NO: 3), 1 gene (named ascrtMThe DNA sequence is shown as SEQ ID NO. 4), 1 gene encoding phytoene synthase (namedcrtBThe DNA sequence is shown as SEQ ID NO. 5), 1 code lycopene beta-cyclase (namedcrtYThe DNA sequence is shown as SEQ ID NO: 6).
The partial synthesis pathway of beta-carotene and the distribution of the above genes are shown in FIG. 1,crtE-1、crtI、crtM、 crtBthe 4 genes are adjacent and are in the same gene cluster; whilecrtE-2、crtYAre far away from other genes and are independent.
The analysis of the gene annotation also confirms the redundancy of the rhodococcus gene. There are two genes encoding GGPP synthetases, and in particular which one plays a major role in carotenoid synthesis, yet to be analyzed.
Example 2 inhibition of carotenoid synthesis-related genes of rhodococcus erythropolis Using CRISPRi technology
In the embodiment, a CRISPRi system is constructed on the basis of a rhodococcus double-plasmid CRISPR gene knockout system (CN 110499274A) for inhibiting the carotene genes of the rhodococcus.
First, pNV-Pa2-dCAS9 was constructed as follows: pNV-Pa2-Cas9 (CN 110499274A) was digested with XbaI and KpnI to give plasmid backbone; using pNV-Pa2-Cas9 as a template, and using primers D10A-F and H840A-R, and amplifying the primers H840A-F and dCAS9-R to obtain two gene fragments; gibson ligation of the gene fragment and plasmid backbone resulted in pNV-Pa2-dCAs9, i.e., two mutations D10A and H840A were introduced into the Cas9 protein to make it lose nuclease activity, but retain DNA binding capacity.
Targeting was designed using the http:// www.rgenome.net/cas-designer/websitecrtE-1、crtE-2、crtI、crtEM、crtB5 genes of sgRNA, synthesizing primers, and constructing plasmid of pBNVCm-sgRNA series, concretely comprising the following steps: plasmid pBNVCm-sgRNA is digested with BbsI to obtain a plasmid backbone; the primers crtE-1-F and crtE-1-R, crtE-2-F and crtE-2-R, crtI-F and crtI-R, crtM-F and crtM-R, crtB-F and crtB-R, crtY-F and crtY-R are denatured at 95 ℃ respectively, and gradually cooled to 25 ℃ at 0.1 ℃/s, and the two primers are paired with each other to form dsDNA with sticky ends; the dsDNA was ligated to the plasmid backbone using T4 DNA ligase to give plasmids pBNVCm-crtE1-sgRNA, pBNVCm-crtE2-sgRNA, pBNVCm-crtI-sgRNA, pBNVCm-crtM-sgRNA, pBNVCm-crtB-sgRNA, pBNVCm-crtY-sgRNA targeting different genes.
Plasmids such as pBNVCm-crtE1-sgRNA, pBNVCm-crtE2-sgRNA, pBNVCm-crtI-sgRNA, pBNVCm-crtM-sgRNA, pBNVCm-crtB-sgRNA, pBNVCm-crtY-sgRNA and the like are respectively and simultaneously introduced into rhodococcus erythropolis with pNV-Pa2-dCAs9, so that recombinant strains which inhibit different carotene synthesis genes are obtained and are named as TH-crtE1, TH-crtE2, TH-crtI, TH-crtM, TH-crtB and TH-crtY respectively. The plasmids pBNVCm-sgRNA and pNV-Pa2-dCAs9 which do not target any genes are simultaneously introduced into rhodococcus erythropolis, and then a control strain TH-control is obtained.
The sequences of the primers used in this example are shown in Table 1, wherein the underlined parts are the recognition sequences of the designed sgRNAs.
TABLE 1 primers and sequences used in example 1
Example 3 determination of OD of recombinant rhodococcus erythropolis 460 Values and carotenoid content and nitrile hydratase Activity
Recombinant bacteria TH-crtE1, TH-crtE2, TH-crtI, TH-crtM, TH-crtB, TH-crtY and TH-control were inoculated into seed medium (glucose 10-50 g/L, yeast extract 1-4g/L, peptone 1-10g/L, KH) to which 25. Mu.g/mL kanamycin and 5. Mu.g/mL chloramphenicol were added, respectively 2 PO 4 0.2-3 g/L，K 2 HPO 4 0.2-3 g/L，MgSO 4 ·7H 2 O0.2-3 g/L, monosodium glutamate 1 gPer liter, pH 7.5), culturing at 25-37deg.C and 100-200 rpm for 48 hr, transferring to fermentation medium (glucose 10-50 g/L, urea 5-20 g/L, yeast extract 1-4g/L, peptone 1-10g/L, KH) at 10% ratio 2 PO 4 0.2-3 g/L，K 2 HPO 4 0.2-3 g/L，MgSO 4 ·7H 2 O0.2-3 g/L, monosodium glutamate 1 g/L, coCl 2 0.03-0.5mM, pH 7.5), fermenting at 28deg.C and 200rpm for 48 hr to obtain bacterial liquid, and measuring OD by using spectrophotometer 460 And the nitrile hydratase activity (method reference CN107177581 a) and carotenoid content were determined. The carotenoid determination method specifically comprises the following steps: 1mL of the fermentation broth was centrifuged at 10000 Xg for 3 minutes, and then the cells were collected, suspended with distilled water, centrifuged again, resuspended with 1mL of acetone, and then heated at 55℃for 15 minutes with light cut off, and centrifuged at 10000 Xg for 10 minutes, and the absorbance of the supernatant at 475nm was measured using acetone as a blank reference. The carotenoid yield (mg/L) was calculated according to the following formula: c= 3.994a 454 。
OD after 48 hours of fermentation of several different recombinant bacteria 460 The nitrile hydratase enzyme activity and carotenoid production are shown in FIGS. 2, 3 and 4, respectively.
Inhibition ofcrtMOD of Rhodococcus 460 Reduced by 33%, reduced by 49% in enzyme activity,crtMthe encoding membrane protein gene, and the reduction of the expression level thereof may affect the growth of rhodococcus. While suppressingcrtE1、crtE2、crtIAndcrtBthe gene has little influence on the growth and enzyme production of the rhodococcus; wherein inhibition is inhibitedcrtE1Has effect in promoting growth and enzyme production, and can make OD 460 The enzyme activity of the nitrile hydratase is improved by 11 percent and 6 percent.
Inhibition of crtE2 and crtY had no significant effect on carotenoid production, while inhibition was inhibitedcrtE1、crtI、crtMAndcrtBthe carotenoid production can be reduced to various degrees, wherein inhibition is achievedcrtE1The effect is most remarkable, and the yield of carotenoid is reduced by 97.5%. It was found that in rhodococcus erythropolis, two genes encoding GGPP synthase were presentcrtE1AndcrtE2in,crtE1plays a major role in the synthesis of carotenoids.
From the aspects of cell growth, enzyme production, carotenoid reduction and the like, TH-crtE1 is the optimal recombinant bacterium. By comparing TH-control with TH-crtE1, it can be seen that the control bacteria are orange in color, while TH-crtE1 appears white, as shown in FIG. 5. This was also the first reported white "rhodococcus".
EXAMPLE 4 recombinant Rhodococcus rhodochrous catalyzed hydration of acrylonitrile to acrylamide
400mL of cell suspensions of recombinant bacteria TH-control and TH-crtE1 (bacterial concentration 1.5 gdcw/L) were taken and placed in a 1L three-necked flask, and hydration reaction was performed under ice bath conditions. And (3) dropwise adding acrylonitrile while stirring, controlling the reaction temperature to be 18-25 ℃, stopping dropwise adding the acrylonitrile when the concentration of the acrylamide reaches 50%, and continuously reacting for 1 hour to completely consume the acrylonitrile. The reaction mixture was centrifuged at 10000 Xg for 20 minutes to separate the cells, thereby obtaining an acrylamide solution as a product.
The color of the acrylamide solution is recorded by photographing, as shown in fig. 6, the carotenoid content of the control bacterium TH-control is higher, and a large amount of pigment is released in the hydration reaction process, so that the color of the product solution is darker; in comparison, the color of the product solution synthesized by the recombinant bacteria TH-crtE1 is lighter, and the product quality is obviously improved.
Example 5 knockout of the carotenoid synthesis-related Gene of Rhodococcus rhodochrous
The CRISPRi technology is adopted to inhibit carotenoid synthesis related genes, and exogenous plasmids are required to be introduced, so that on one hand, the physiological burden of rhodococcus is increased; on the other hand, plasmids have the problem of being lost due to unstable passage. For industrial production, it is necessary to knock out carotenoid synthesis related genes and construct strains with stable performance. Based on the results of example 3, in the carotenoid synthesis-related gene,crtE-2、crtYhas no effect on reducing pigment contentcrtMThe inhibition of (2) affects the growth of rhodococcus erythropolis, so the gene knockout of this example is aimed atcrtE-1AndcrtI。
knocking out by adopting homologous recombination double exchange methodcrtE-1AndcrtIspecifically, the following is described.
Construction of plasmid pK18mobsacB-crt for suicide: commercialized massThe granule pK18mobsacB is composed ofHindIIIXbaI, enzyme cutting to obtain a framework; using red rhodococcus as template, using crt-up-F, crt-up-R to amplify upstream homology arm, using primer crt-down-F, crt-down-R to amplify downstream homology arm; the three were joined by Gibson to give pK18mobsacB-crt.
The pK18mobsacB-crt is subjected to electric excitation transformation to red rhodococcus, and after resuscitating culture, the red rhodococcus is coated on LB plates containing 25 mug/mL kanamycin, and integrated into the genome by single exchange; single-exchanged colonies were further plated on LB plates of 100 g/L sucrose, grown for 3 days at 28℃to give colonies, and distinguished by colony PCR and colony colorcrtE-1AndcrtIwhether the gene was successfully knocked out, and the successfully knocked out colony was named as TH-Deltacrt. And inhibitcrtE1The recombinant strain TH-crtE1 of the gene is similar, and the thallus of the TH-delta crt is white, so that the synthesis path of carotenoid can be judged to be blocked.
The primers used in this example are shown in Table 2.
TABLE 2 primers and sequences used in example 5
Example 6 determination of growth of recombinant TH-Deltacrt and nitrile hydratase Activity
Inoculating recombinant bacteria TH-Deltacrt, TH-crtE1 into seed culture medium (glucose 10-50 g/L, yeast extract 1-4g/L, peptone 1-10g/L, KH) containing no antibiotics, kanamycin 25 μg/mL and chloramphenicol 5 μg/mL, respectively 2 PO 4 0.2-3 g/L，K 2 HPO 4 0.2-3 g/L，MgSO 4 ·7H 2 O0.2-3 g/L, monosodium glutamate 1-g/L, pH 7.5), 25-37 ℃ and 100-200 rpm for 48 hours, transferring to fermentation medium (glucose 10-50 g/L, urea 5-20 g/L, yeast extract 1-4g/L, peptone 1-10g/L, KH) according to 10% proportion 2 PO 4 0.2-3 g/L，K 2 HPO 4 0.2-3 g/L，MgSO 4 ·7H 2 O0.2-3 g/L, monosodium glutamate 1 g/L, coCl 2 0.03-0.5mM, pH 7.5), fermenting at 28deg.C and 200rpm for 48 hr to obtain bacterial liquid, and measuring OD by using spectrophotometer 460 And the nitrile hydratase activity (method reference CN 107177581A) and carotenoid production were measured.
OD of two recombinant bacteria 460 And the nitrile hydratase activity is shown in Table 3, compared with TH-crtE1, the growth of recombinant strain TH-Deltacrt with the carotenoid synthesis gene knocked out is almost unchanged, the carotenoid yield is further reduced by 57%, and the nitrile hydratase activity is improved by 21%. The reason for this is that recombinant TH-Deltacrt does not contain exogenous plasmid, and more cell resources can be used for expression of nitrile hydratase.
TABLE 3 OD of TH-crtE1 and TH- Δcrt 460 Comparison of nitrile hydratase Activity
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. The scope of the patent is, therefore, indicated by the appended claims, and the description may be used to interpret the contents of the claims.

Claims (9)

1. A recombinant rhodococcus erythropolis, wherein a carotenoid synthesis gene in said recombinant rhodococcus erythropolis is reduced in expression or knocked out; the carotenoid synthesis gene with reduced expression or knockdown is selected from genes encoding GGPP synthetasecrtE-1Gene encoding phytoene dehydrogenasecrtIAnd genes encoding phytoene synthasecrtBOne of them; or (b)
The carotenoid synthesis gene with reduced expression or knockdown is a gene encoding GGPP synthetasecrtE-1And genes encoding phytoene dehydrogenasecrtI；
Wherein the gene encoding GGPP synthasecrtE-1The nucleotide sequence of (2) is shown as SEQ ID NO. 1;
wherein the original strain of the recombinant rhodococcus erythropolis is rhodococcus erythropolis TH (Rhodococcus ruber TH) or a mutant strain thereof, the rhodococcus erythropolis TH is preserved in the China general microbiological culture collection center (CGMCC) of China general microbiological culture collection center (China general microbiological culture collection center) with the accession number of CGMCC No.2380;
the mutant strain is rhodococcus erythropolis TH3 (amdA-) preserved in China general microbiological culture Collection center with a strain preservation registration number of CGMCC No.2381.
2. The recombinant rhodococcus erythropolis of claim 1, wherein said gene encoding phytoene dehydrogenasecrtIThe nucleotide sequence of the gene is shown as SEQ ID NO. 3, and the gene for encoding phytoene synthetasecrtBThe nucleotide sequence of (2) is shown as SEQ ID NO. 5.
3. The recombinant rhodococcus erythropolis of claim 1 or 2, wherein the method of knocking out carotenoid synthesis genes comprises a homologous recombination double crossover method.
4. The recombinant rhodococcus erythropolis of claim 3, wherein the method for constructing the recombinant rhodococcus erythropolis using the homologous recombination double crossover method comprises the steps of:
connecting an upstream homologous sequence and a downstream homologous sequence of a carotenoid synthesis gene into a suicide plasmid vector to obtain a recombinant suicide plasmid vector;
and (3) introducing the recombinant suicide plasmid vector into rhodococcus, and screening to obtain recombinant rhodococcus.
5. The recombinant rhodococcus erythropolis of claim 1 or 2, wherein the method of reducing carotenoid synthesis gene expression comprises one or more of CRISPRi technology, antisense RNA technology, RNA interference technology and promoter attenuation method.
6. The recombinant rhodococcus erythropolis of claim 5, wherein the method for constructing the recombinant rhodococcus erythropolis using the CRISPRi technique comprises introducing a vector containing dCAS9 gene and a vector containing sgRNA targeting carotenoid synthesis gene into rhodococcus erythropolis to construct the recombinant rhodococcus erythropolis.
7. The use of the recombinant rhodococcus erythropolis of any one of claims 1 to 6 in the synthesis of a compound, wherein the compound is an amide compound;
the amide compound comprises acrylamide.
8. The use according to claim 7, wherein the method of acrylamide synthesis comprises the steps of: expressing a nitrile hydratase by using the genome of the recombinant rhodococcus erythropolis and/or expressing a nitrile hydratase by introducing an exogenous plasmid carrying a gene encoding a nitrile hydratase into the recombinant rhodococcus erythropolis;
and catalyzing acrylonitrile hydration reaction by using the nitrile hydratase to synthesize acrylamide.
9. The use according to claim 8, wherein the hydration reaction temperature is 15 ℃ to 45 ℃.
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