CN115851803A - Construction method and application of photosynthetic bacteria for high yield of vanillyl alcohol - Google Patents
Construction method and application of photosynthetic bacteria for high yield of vanillyl alcohol Download PDFInfo
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- CN115851803A CN115851803A CN202211286235.2A CN202211286235A CN115851803A CN 115851803 A CN115851803 A CN 115851803A CN 202211286235 A CN202211286235 A CN 202211286235A CN 115851803 A CN115851803 A CN 115851803A
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- vanillyl alcohol
- rhodopseudomonas palustris
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- ZENOXNGFMSCLLL-UHFFFAOYSA-N vanillyl alcohol Chemical compound COC1=CC(CO)=CC=C1O ZENOXNGFMSCLLL-UHFFFAOYSA-N 0.000 title claims abstract description 105
- 241000894006 Bacteria Species 0.000 title claims abstract description 20
- 238000010276 construction Methods 0.000 title claims abstract description 19
- 230000000243 photosynthetic effect Effects 0.000 title claims abstract description 17
- 241000190950 Rhodopseudomonas palustris Species 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 22
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims abstract description 19
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims abstract description 19
- 235000001785 ferulic acid Nutrition 0.000 claims abstract description 19
- 229940114124 ferulic acid Drugs 0.000 claims abstract description 19
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims abstract description 19
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims abstract description 19
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Abstract
The invention relates to a construction method and application of photosynthetic bacteria for high yield of vanillyl alcohol, which construct an anaerobic regulated strong promoter expression system to efficiently express endogenous feruloyl coenzyme A synthetase CouB, enoyl coenzyme A hydratase/aldolase CouA and exogenous alcohol dehydrogenase ADH2 in rhodopseudomonas palustris to construct a vanillyl alcohol biosynthesis path. The recombinant photosynthetic bacteria take lignin monomer ferulic acid as a substrate and sodium thiosulfate as an electron donor, and vanillyl alcohol is efficiently produced through photocatalysis; no additional organic carbon source is needed, and the method has wide industrial application prospect.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a construction method and application of photosynthetic bacteria for high yield of vanillyl alcohol.
Background
Vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is a widely used flavoring agent, a natural aromatic compound, present in a variety of plants, such as gastrodia elata and vanilla. Vanillyl alcohol has various pharmacological activities such as antioxidation, antiangiogenesis, anti-inflammation, anti-pain, anti-asthma, anti-convulsion and the like, and shows that the vanillyl alcohol has potential therapeutic effects as a pharmaceutical ingredient. Currently, vanillyl alcohol is mainly extracted from various plants; however, these extraction methods still have the following disadvantages: limited supply of raw materials, harsh reaction conditions and lower yields. On the other hand, the chemical synthesis of vanillyl alcohol is mainly obtained by catalyzing vanillin, however, is limited by high catalyst costs (such as Pd/C, pt/C, and Au/C) and a high-value, unstable substrate, vanillin.
Therefore, the high-added-value vanillyl alcohol is produced by utilizing renewable cheap raw materials through an efficient biosynthesis mode, the sustainable development under the new economic situation is met, and the industrial development of the vanillyl alcohol is promoted. To date, studies on the biosynthesis of vanillyl alcohol have focused on the synthesis of the direct precursor vanillin, but the yield of vanillin has been limited by longer synthetic pathways, low catalytic efficiency of the enzyme, and unstable products. Recently, the p-hydroxybenzoic acid hydroxylase PobA, the carboxylic acid reductase CAR, the caffeic acid O-methyltransferase COMT were expressed heterologously in E.coli on the basis of the p-hydroxybenzoic acid pathway, and 240.69mg/L of vanillyl alcohol was obtained by fermentation with glycerol and glucose in the presence of endogenous alcohol dehydrogenase. In addition, an escherichia coli co-culture system is designed by constructing and optimizing an escherichia coli 3, 4-dihydroxybenzyl alcohol path and an escherichia coli methylation vanillyl alcohol generation path, and the vanillyl alcohol yield reaches 328.9mg/L under the optimal inoculation proportion; the aroE gene is further knocked out to enhance the synthetic capacity of the upstream strain, so that the yield is improved to 559.4mg/L, and the fed-batch fermentation yield reaches 3.89g/L. However, there are few reports on the biosynthesis of vanillyl alcohol, and more research and exploration is required to drive the production of vanillyl alcohol.
Disclosure of Invention
The present invention is directed to solving, at least in part, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a method for constructing photosynthetic bacteria with high vanillyl alcohol yield. The engineering bacteria constructed by the method can enable ferulic acid to produce vanillyl alcohol with the conversion efficiency of more than 99%, and the catalytic process is simple and environment-friendly, and has good economic value and industrial application prospect.
To this end, in a first aspect of the present invention, the present invention provides a method for constructing a photosynthetic bacterium that produces vanillyl alcohol at a high yield, comprising the steps of:
constructing strong promoter expression vectors pBRPt334O and pGenPt334O for anaerobic regulation;
constructing a plasmid pBRT334O-CouBA for expressing the feruloyl-CoA synthetase CouB and the enoyl-CoA hydratase/aldolase CouA and a plasmid pGenT334O-ADH2 for expressing the alcohol dehydrogenase ADH2;
knocking out aldehyde dehydrogenase in the rhodopseudomonas palustris, and transferring the combination of the plasmid pBRT334O-CouBA and the plasmid pGenT334O-ADH2 into the rhodopseudomonas palustris to obtain the photosynthetic bacteria with high vanillyl alcohol yield.
According to the embodiment of the invention, the rhodopseudomonas palustris constructed by the method knocks down aldehyde dehydrogenase, and three enzymes are expressed based on a strong promoter for anaerobic regulation: feruloyl-CoA synthetase (CouB) and enoyl-CoA hydratase/aldolase (CouA) from rhodopseudomonas palustris r. By using photosynthetic bacteria simultaneously containing the three enzymes, lignin-derived ferulic acid is converted into vanillyl alcohol through simple thallus collection and enzyme cascade catalysis, and according to the embodiment of the invention, the experimental result shows that the yield of the vanillyl alcohol can be up to about 100% by using a 30mM ferulic acid substrate; therefore, the lignin waste can be used for producing the vanillyl alcohol, the waste is changed into valuable, and the method has good economic value and industrial application prospect.
Optionally, the expression vector pBRPt334O is constructed with a nucleotide sequence of SEQ ID NO:25 of the promoter P T334-6 As template, SEQ ID NO: 1. SEQ ID NO:2 is used as a primer, and the strong promoter P for anaerobic regulation is obtained by PCR amplification T334O Connecting the expression vector pBRPt334O with a vector pBRT to obtain an expression vector pBRPt334O with kanamycin resistance; the construction of the expression vector pGenPt334O takes pZJD29a plasmid as a template and SEQ ID NO: 4. SEQ ID NO:5 is used as a primer, a gentamicin resistance gene is obtained by PCR amplification, and an expression vector pGenPt334O with gentamicin resistance is obtained by antibiotic screening marker replacement.
Optionally, the plasmid pBRT334O-CouBA is constructed by using the rhodopseudomonas palustris whole genome as a template, and by using SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO: 8. the amino acid sequence of SEQ ID NO:9 as primer, PCR amplified to obtain CouB gene and CouA gene, cloning the two genes to expression vector pBRPt334O to obtain plasmid pBRT334O-CouBA.
Optionally, the plasmid pGenT334O-ADH2 is constructed by taking a saccharomyces cerevisiae genome as a template and SEQ ID NO: 10. the amino acid sequence of SEQ ID NO:11 is a primer, and the ADH2 gene is obtained through PCR amplification, and then the gene is cloned to an expression vector pGenPt334O, so as to obtain a plasmid pGenT334O-ADH2.
Alternatively, aldehyde dehydrogenases RPA1206, RPA1687 and RPA1725 in rhodopseudomonas palustris are knocked out.
Further, the genome of rhodopseudomonas palustris is taken as a template, and the genome of the rhodopseudomonas palustris is expressed by SEQ ID NO: 12. the amino acid sequence of SEQ ID NO:13 and SEQ ID NO: 14. SEQ ID NO:15 is used as a primer, and overlapping extension PCR is carried out to obtain the sequences of the upstream and downstream homology arms of aldehyde dehydrogenase RPA 1206; as set forth in SEQ ID NO: 16. SEQ ID NO:17 and SEQ ID NO: 18. SEQ ID NO:19 is used as a primer to carry out overlap extension PCR to obtain the upstream and downstream homologous arm sequences of aldehyde dehydrogenase RPA 1687; as set forth in SEQ ID NO: 20. SEQ ID NO:21 and SEQ ID NO: 22. SEQ ID NO:23 as primers, obtaining the sequences of the upstream and downstream homology arms of the aldehyde dehydrogenase RPA1725, respectively inserting the sequences of the upstream and downstream homology arms into a suicide plasmid pZJD29c to obtain pZJ-delta 1206, pZJ-delta 1687 and pZJ-delta 1725, and then introducing the plasmids pZJ-delta 1206, pZJ-delta 1687 and pZJ-delta 1725 into rhodopseudomonas palustris by combined transfer.
In a second aspect of the present invention, the present invention provides a photosynthetic bacterium that produces vanillyl alcohol at a high yield, obtained by the above construction method.
In a third aspect of the invention, the invention proposes a method for the photocatalytic biosynthesis of vanillyl alcohol, comprising the following steps:
activating and expanding photosynthetic bacteria as recited in claim 7, inoculating to MedS culture medium for 24 hr, culturing, inoculating to MedC culture medium for aerobic growth at ratio of 1;
catalyzing ferulic acid to react into vanillyl alcohol at 30 ℃ by adopting a phosphate buffer reaction system with pH =7, and detecting the vanillyl alcohol yield by liquid chromatography.
According to the method for producing vanillyl alcohol, which is disclosed by the embodiment of the invention, vanillyl alcohol with a molar yield of 100% is obtained after catalyzing for 48 hours by using 30mM ferulic acid as a substrate; the highest reported yield of 4.63g/L is obtained by utilizing the recombinant rhodopseudomonas palustris to biologically convert the lignin derivative ferulic acid to synthesize vanillyl alcohol, the catalytic process is simple and environment-friendly, no carbohydrate is required to be added additionally, and the method has a wide industrial application prospect.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows three paths for enzymatically synthesizing vanillyl alcohol by CouB, couA and ADH2 according to the embodiment of the present invention;
FIG. 2 is a strong promoter expression system for anaerobic regulation according to an embodiment of the present invention;
FIG. 3 is an agarose gel electrophoresis image of PCR-verified Rhodopseudomonas palustris aldehyde dehydrogenase knockdown according to an embodiment of the invention;
FIG. 4 is a graph of different recombinant Rhodopseudomonas palustris strains RVA1, RVA2, RVA3 for vanillyl alcohol production with 5mM ferulic acid as substrate and corresponding HPLC validation according to an embodiment of the invention;
FIG. 5 is a graph showing the yields of vanillyl alcohol produced by recombinant Rhodopseudomonas palustris strain RVA3 under dark and light conditions at various concentrations of ferulic acid according to an embodiment of the invention;
FIG. 6 is a graph of the time course of production of vanillyl alcohol by the recombinant Rhodopseudomonas palustris strain RVA3 by fed batch at a substrate concentration of 30mM according to an embodiment of the invention.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps recited herein do not preclude the presence of additional method steps before or after the recited combining step or that additional method steps can be inserted between the explicitly recited steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market; the related experiments are all routine experimental methods if not specifically stated.
Sources of materials used: rhodopseudomonas palustris CGA009 and Escherichia coli S17-1 are commercially available, wherein Escherichia coli S17-1 is used for all vector constructions and conjugal transfer with Rhodopseudomonas palustris in the present invention. Rhodopseudomonas palustris expression vectors pBRPt334O and pGenPt334O are derived from vector pBdRSf (ACS Synthetic Biology 2021,10, 1545-1552), the plasmid skeleton of the vector is pBBR1MCS-2 derived from Novagen, the suicide plasmid pZJD29c (Appl Environ Microbiol 2005,71, 3014-24) for gene editing is obtained from Fevzi Dalal laboratory gift of university of pennsylvania, and the Phusion high fidelity DNA polymerase and restriction endonuclease are purchased from Xiamen Lulong biotechnology development Limited. Plasmid extraction kits, DNA purification kits, gel recovery kits, and bacterial genomic DNA extraction kits were purchased from shanghai bioengineering, ltd.
The LB medium consisted of: 10 g.L -1 Peptone, 5 g. L -1 Yeast powder, 5 g.L -1 NaCl, the balance double distilled water, 0.1Mpa pressure 121 deg.C sterilization for 20min.
The MedS culture medium comprises the following components: succinic acid 2 g.L -1 ，(NH 4 ) 2 SO 4 0.5g·L -1 0.1 g.L of L-glutamic acid -1 L-aspartic acid 0.04 g.L -1 ，NaCl 1g·L -1 ，Solution C 20mL·L -1 Adjusting pH to 7.0, adding double distilled water, and sterilizing at 121 deg.C under 0.1Mpa for 20min. Adding 1 mL/L of compound vitamin after sterilization -1 And 20 mL. L of phosphate buffer solution -1 (K 2 HPO 4 174.18g·L -1 ，KH 2 PO 4 136.1g·L -1 pH 7.0). Wherein the Solution C component comprises: nitrilotriacetic acid 10 g.L -1 ，MgSO 4 14.4g·L -1 ，CaCl 2 ·2H 2 O 3.335g·L -1 ，FeSO 4 ·7H 2 O 0.099g·L -1 ，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 0.0093g·L -1 Trace elements 50 mL. L -1 (the composition comprises ZnSO4 & 7H2O 10.95g & L -1 ，EDTA 2.5g·L -1 ，FeSO4·7H2O 5.0g·L -1 ，H3BO3 0.114g·L -1 ，MnSO4·H2O 1.54g·L -1 ，CuSO4·5H2O 0.392g·L -1 ，Co(NO3)2·6H2O 0.25g·L -1 pH 7.0); compoundingThe vitamin comprises the following components: thiamine hydrochloride 5 mg.L -1 Nicotinic acid 10 mg.L -1 Biotin 0.1 mg. L -1 。
The MedC medium is improved MedS medium, wherein the succinic acid is 2 g.L -1 Replacement with 10mM NaHCO 3 and 1mM Na 2 S 2 O 3 The other components are unchanged.
Phosphate buffer (200 mM), KP buffer, was composed of: k 2 HPO 4 ·3H 2 O 42.90g/L,KH 2 PO 4 1.63g/L,pH 7.0。
The biosynthesis pathway of hydroxytyrosol in the examples of the present invention is shown in FIG. 1:
taking ferulic acid as a substrate, generating feruloyl coenzyme A under the action of feruloyl coenzyme A synthetase CouB of rhodopseudomonas palustris, generating vanillin under the action of enoyl coenzyme A hydratase/aldolase, and finally synthesizing vanillyl alcohol under the action of yeast alcohol dehydrogenase ADH2; meanwhile, endogenous aldehyde dehydrogenase ALDHs are knocked out, and the generation of vanillic acid by vanillin is limited.
Table 1: primers for PCR amplification
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1 construction of anaerobically regulated Strong promoter expression vectors
The nucleotide sequence is SEQ ID NO:24, digested with XhoI and SalI, and subsequently ligated into XhoI-digested vector pBdRsf, resulting in plasmid pBRT by gene sequencing.
The nucleotide sequence is SEQ ID NO:25 of the synthetic promoter P T334-6 As a template, withThe amino acid sequence of SEQ ID NO:1 and SEQ ID NO:3 is an upstream primer and a downstream primer (table 1), and the strong promoter P is obtained by PCR amplification T334-6 (ii) a With a synthetic promoter P T334-6 Sequence as template, SEQ ID NO:1 and SEQ ID NO:2 is an upstream primer and a downstream primer, wherein the downstream primer is SEQ ID NO:2, the strong promoter P for anaerobic regulation is obtained by PCR amplification and contains an oxygen regulatory protein binding site sequence from rhodobacter sphaeroides T334O . The promoter P T334-6 The fragment was double-digested with EcoRI and KpnI, followed by ligation into the EcoRI and KpnI double-digested vector pBRT, to obtain an expression vector pBRPt334-6 having kanamycin resistance. The promoter P T334O The fragment was double-digested with EcoRI and KpnI, followed by ligation into the EcoRI and KpnI double-digested vector pBRT to obtain an expression vector pBRPt334O having kanamycin resistance.
pZJD29a plasmid was used as a template, and the sequence shown in SEQ ID NO:4 and SEQ ID NO:5 are upstream and downstream primers, a gentamicin resistance gene is obtained through PCR amplification, and an expression vector pGenPt334O with gentamicin resistance is obtained through antibiotic selection marker replacement.
Taking a green fluorescent protein eGFP sequence (accession number: JQ 064507.1) as a reporter gene template, and taking the sequence shown in SEQ ID NO:26 and SEQ ID NO:27 is a sequence obtained by the PCR amplification of a primer; the eGFP fragment was double-digested with BamHI and XhoI, followed by ligation into BamHI and XhoI double-digested vectors pBRPt334-6 and pBRPt334O, respectively, to give plasmids pBRPt3346-eGFP and pBRPt334O-eGFP. The plasmid was transformed into E.coli S17-1, followed by conjugative transfer with Rhodopseudomonas palustris and into Rhodopseudomonas palustris. By observing the green fluorescent signal (see FIG. 2), the promoter P was found T334-6 There was no significant difference in eGFP expression under aerobic, microaerobic, anaerobic conditions. And the rhodopseudomonas palustris is found to be capable of recognizing the oxygen regulatory protein binding site from rhodobacter sphaeroides and is subjected to oxygen inhibition, namely, the oxygen regulatory protein binding DNA and RNA polymerase binding DNA form competition to inhibit gene transcription. eGFP in P in aerobic/microaerobic culture T334O Hardly expressed under control; and is efficiently expressed under anaerobic conditions and is linked with a promoter P T334-6 Activity is not shownThe difference is significant. Therefore, anaerobic regulated strong promoter expression vectors pBRPt334O and pGenPt334O were successfully constructed.
Example 2 construction of recombinant Rhodopseudomonas palustris
1. Construction of pBRT334O-CouBA plasmid
As set forth in SEQ ID NO:6 and SEQ ID NO:7 is an upstream primer and a downstream primer (table 1), and a CouB gene is amplified by PCR by taking a rhodopseudomonas palustris whole genome (accession number: NC-005296.1) as a template; as set forth in SEQ ID NO:8 and SEQ ID NO:9 is an upstream primer and a downstream primer, and a total genome of rhodopseudomonas palustris is used as a template to amplify the CouA gene; PCR amplification conditions included 1min at 98 ℃,30 cycles: 15s at 98 ℃,15 s at 68 ℃, 1min at 72 ℃ and 3min at 72 ℃. Digesting the CouB gene fragment after glue recovery with BamHI and HindIII at 37 ℃ for 2h, digesting the CouA gene fragment with Esp3I at 37 ℃ for 2h, purifying and recovering the digested fragment; connecting the enzyme-cut CouBA fragment with an expression vector pBRPt334O subjected to double enzyme cutting by BamHI and XhoI; 20 mu L of the connection system comprises 3 mu L of enzyme digestion vector pBRPt334O, 7 mu L of enzyme digestion segment CouA, 7.5 mu L of enzyme digestion segment CouB, 2 mu L of T4 ligase buffer solution and 0.5 mu L of T4 ligase, and the connection is carried out overnight at the temperature of 16 ℃; the ligation product was transferred into E.coli S17-1 competent cells, followed by ligation with primers SEQ ID NO:6 and SEQ ID NO: and 9, verifying and obtaining a positive clone colony, and extracting and obtaining a plasmid pBRT334O-CouBA.
2. Construction of pGenT334O-ADH2 plasmid
The genome of Saccharomyces cerevisiae (accession number: BK 006946.2) is used as a template, and the nucleotide sequence shown in SEQ ID NO:10 and SEQ ID NO:11 is the ADH2 gene segment amplified by the upstream primer and the downstream primer through PCR; PCR amplification conditions included 1min at 98 ℃ with 30 cycles: 15s at 98 ℃,15 s at 64 ℃, 45s at 72 ℃ and 2min at 72 ℃. The ADH2 gene fragment was double-digested with BamHI and XhoI, then ligated with the BamHI and XhoI double-digested expression vector pGenPt334O, ligated overnight at 16 ℃ and then transferred into E.coli S17-1 competent cells, followed by ligation with SEQ ID NO:10 and SEQ ID NO: and (3) verifying by using the 11 primer to obtain a positive clone colony, and extracting a plasmid pGenT334O-ADH2.
3. Construction of pZJ-Delta 1206 plasmid
Taking the genome of rhodopseudomonas palustris as a template, and taking the nucleotide sequence shown in SEQ ID NO:12 and SEQ ID NO:13 is a primer, and the upstream homologous arm sequence of the aldehyde dehydrogenase RPA1206 gene is amplified by PCR; as set forth in SEQ ID NO:14 and SEQ ID NO:15 is a primer, and the downstream homology arm sequence of the aldehyde dehydrogenase RPA1206 gene is amplified by PCR; taking a mixed fragment of an upstream homology arm and a downstream homology arm of the RPA1206 gene as a template, and taking the sequence shown in SEQ ID NO:12 and SEQ ID NO:15 is used as primer, and the upstream and downstream homologous arm segments of the RPA1206 gene are connected together by overlap extension PCR amplification. The RPA1206 homology arm fragment was digested with SacI and KpnI, followed by ligation into SacI and KpnI double-digested suicide plasmid pZJD29c, resulting in plasmid pZJ-. DELTA.1206.
4. Construction of pZJ- Δ 1687 plasmid
Taking the genome of rhodopseudomonas palustris as a template, and taking the nucleotide sequence shown in SEQ ID NO:16 and SEQ ID NO:17 is a primer, and the upstream homologous arm sequence of the aldehyde dehydrogenase RPA1687 gene is amplified by PCR; as set forth in SEQ ID NO:18 and SEQ ID NO:19 is a primer, and the downstream homologous arm sequence of the aldehyde dehydrogenase RPA1687 gene is amplified by PCR; taking the mixed fragment of the upstream and downstream homology arms of the RPA1687 gene as a template, and taking the sequence shown in SEQ ID NO:16 and SEQ ID NO:19 is used as a primer, and the upstream and downstream homologous arm fragments of the RPA1687 gene which are connected together are obtained by overlap extension PCR amplification. The upstream and downstream homology arm fragments of RPA1687 were digested with XbaI and KpnI, and then ligated into XbaI and KpnI double digested suicide plasmid pZJD29c, resulting in plasmid pZJ-. DELTA.1687.
5. Construction of pZJ-Delta 1725 plasmid
Using the genome of rhodopseudomonas palustris as a template, and using the nucleotide sequence shown in SEQ ID NO:20 and SEQ ID NO:21 is a primer, and the upstream homologous arm sequence of the aldehyde dehydrogenase RPA1725 gene is amplified by PCR; the peptide represented by SEQ ID NO:22 and SEQ ID NO:23 is a primer, and the downstream homologous arm sequence of the aldehyde dehydrogenase RPA1725 gene is amplified by PCR; taking the upstream and downstream homology arm mixed fragment of the RPA1725 gene as a template, and taking the sequence shown in SEQ ID NO:20 and SEQ ID NO:23 as primers, and performing overlap extension PCR amplification to obtain the upstream and downstream homologous arm fragments of the RPA1687 gene which are connected together. The upstream and downstream homology arm fragments of RPA1725 were digested with XbaI and KpnI, and then ligated into XbaI and KpnI double-digested suicide plasmid pZJD29c, resulting in plasmid pZJ-. DELTA.1725.
6. Obtaining rhodopseudomonas palustris recombinant strain
The method for transforming the plasmid into the rhodopseudomonas palustris is combined transfer, firstly 10mL of MedS culture medium is used for culturing the rhodopseudomonas palustris 24h at 35 ℃, and 5mL of LB culture medium is used for culturing escherichia coli S17-1 containing the target plasmid overnight at 37 ℃. Each 1mL of the suspension was centrifuged (7000rpm, 3min) to collect the cells, which were washed once with 1mL of MedS medium, and then 100. Mu.L of MedS was used to resuspend E.coli S17-1 and Rhodopseudomonas palustris, respectively, after centrifugation to collect the cells. mu.L of S17-1 and 100. Mu.L of Rhodopseudomonas palustris were each aspirated, mixed and spread on a MedS plate to form a circular patch having a diameter of about 2 cm. After the circular spots were dried, the plates were inverted and incubated at 35 ℃ for 24h. Thereafter, the round plaque cells were scraped off with 1mL of MedS and a spreader bar, the bacteria were collected by centrifugation, washed once with 1mL of MedS medium, and finally the mixed bacteria were resuspended with 800. Mu.L of MedS. 100. Mu.L of the resuspension solution was pipetted and spread on correspondingly resistant MedS plates and cultured in an inverted state at 35 ℃ for 2-3 days until a single colony of red color (Rhodopseudomonas palustris) appeared.
The plasmids pZJ-. DELTA.1206, pZJ-. DELTA.1687 and pZJ-. DELTA.1725 obtained in 3,4 and 5 above were introduced into Rhodopseudomonas palustris CGA009 by conjugative transfer, and a single colony of Rhodopseudomonas palustris containing a pZJD29 c-derived plasmid was selected and inoculated with 1mL of MedS and cultured overnight at 35 ℃. The bacterial liquid of 2-10 μ L is taken the next day, spread on MedS plate containing 10% sucrose, and cultured upside down at 35 deg.C for 2-3 days. At this time, only the wild type photosynthetic bacteria and the mutant photosynthetic bacteria can grow due to the influence of the sacB gene in the plasmid. Selecting single colony, culturing, and performing PCR verification with the primer of the target gene to obtain RPA1206 single gene knockout strain YC1 and RPA1206, RPA1687, and RPA1725 three gene knockout strain YC2 (shown in FIG. 3).
The plasmids pBRT334O-CouBA and pGent334O-ADH2 obtained in the steps 1 and 2 are transferred by combination, introduced into rhodopseudomonas palustris CGA009, YC1 and YC2 strains, and screened in a culture medium containing two antibiotics, namely kanamycin and gentamycin, to respectively obtain rhodopseudomonas palustris recombinant strains RVA1, RVA2 and RVA3 for producing vanillyl alcohol.
Example 3 Whole cell bioconversion of recombinant strains
The Rhodopseudomonas palustris recombinant strain prepared in example 2 (RVA 1, RVA,RVA2 or RVA 3) is inoculated in 10mL MedS culture solution, aerobic culture is carried out for 24h at 35 ℃, then the culture solution is inoculated into MedC culture solution according to the proportion of 1 600 After reaching 0.4-06 KPa, introducing 50KPa mixed gas (80% H) 2 /20％ CO 2 ) And (3) illuminating at 30 ℃ for 16-24 h under anaerobic induction protein expression, and centrifuging to collect cells.
Catalyzed by a 2mL reaction system (10 g/L dry cell weight, ferulic acid substrate, 10mM NaHCO) 3 ,1mM Na 2 S 2 O 3 ) Finally adding phosphate buffer for filling (200mM, pH = 7.0), and detecting the vanillyl alcohol yield by liquid chromatography after whole-cell catalysis for 1-24h at 30 ℃ under anaerobic illumination (4000 Lux).
Product quantitative analysis: detecting and analyzing the conversion solution by using a Shimadzu high performance liquid chromatograph, and detecting by using a photodiode array detector (the working wavelength is 210 nm); adopting Shimadzu C18 chromatographic column (4.6 × 250mm,5 μm), flow rate of 1mL/min, column temperature of 40 deg.C, and sample amount of 10 μ L; mobile phase: 90% ddH 2 O,0.1% TFA,10% acetonitrile.
As shown in FIG. 4, the RVA1 strain synthesized 2.59mM vanillyl alcohol (VA, 51.8% by weight) with the byproduct 2.41mM vanillic acid (VAc, 49.2% by weight) 10h catalyzed by 5mM Ferulic Acid (FA). When the aldehyde dehydrogenase RPA1206 is knocked out, the RVA1 strain improves the vanillyl alcohol yield to 2.98mM (59.6 percent); when the aldehyde dehydrogenases RPA1206, RPA1687 and RPA1725 are further knocked out, the RVA3 strain produces 5mM vanillyl alcohol (accounting for 100 percent), the molar yield reaches 100 percent, and the liquid chromatogram can also show that no side product vanillic acid is produced.
The results are shown in FIG. 5, the RVA3 strain is used as a whole cell to carry out light-dark contrast catalysis by using 10mM, 20mM and 30mM Ferulic Acid (FA) as a substrate, and the results show that the yield and the molar conversion rate under the light condition are far higher than those under the dark condition, and the light energy drives the regeneration of the cofactors ATP and NADH to accelerate the whole catalytic process.
As shown in FIG. 6, the RVA3 strain as a whole-cell photocatalyst with 30mM Ferulic Acid (FA) as a substrate, fed 10mM ferulic acid every 6h in a fed-batch mode, yielded 30.01mM Vanillyl Alcohol (VA) and a molar yield of-100%.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (8)
1. A method for constructing photosynthetic bacteria with high vanillyl alcohol yield is characterized by comprising the following steps:
constructing strong promoter expression vectors pBRPt334O and pGenPt334O for anaerobic regulation;
constructing a plasmid pBRT334O-CouBA for expressing the feruloyl-CoA synthetase CouB and the enoyl-CoA hydratase/aldolase CouA and a plasmid pGenT334O-ADH2 for expressing the alcohol dehydrogenase ADH2;
knocking out aldehyde dehydrogenase in the rhodopseudomonas palustris, and transferring the combination of the plasmid pBRT334O-CouBA and the plasmid pGenT334O-ADH2 into the rhodopseudomonas palustris to obtain the photosynthetic bacteria with high vanillyl alcohol yield.
2. The construction method according to claim 1, wherein the expression vector pBRPt334O is constructed by using a nucleotide sequence of SEQ ID NO:25 of the sequence listing T334-6 As template, SEQ ID NO: 1. SEQ ID NO:2 is a primer, and the anaerobic regulation and control is obtained by PCR amplificationStrong promoter P T334O Connecting the expression vector pBRPt334O with a vector pBRT to obtain an expression vector pBRPt334O with kanamycin resistance; the construction of the expression vector pGenPt334O takes pZJD29a plasmid as a template and SEQ ID NO: 4. SEQ ID NO:5 is used as a primer, a gentamicin resistance gene is obtained by PCR amplification, and an expression vector pGenPt334O with gentamicin resistance is obtained by antibiotic screening marker replacement.
3. The construction method according to claim 1, wherein the plasmid pBRT334O-CouBA is constructed by using Rhodopseudomonas palustris genome as template and SEQ ID NO: 6. SEQ ID NO:7 and SEQ ID NO: 8. SEQ ID NO:9 as primer, PCR amplified to obtain CouB gene and CouA gene, cloning the two genes to expression vector pBRPt334O to obtain plasmid pBRT334O-CouBA.
4. The construction method according to claim 1, wherein the plasmid pGenT334O-ADH2 is constructed by using a Saccharomyces cerevisiae genome as a template and SEQ ID NO: 10. SEQ ID NO:11 as a primer, obtaining ADH2 gene by PCR amplification, and cloning the gene to an expression vector pGenPt334O to obtain plasmid pGenT334O-ADH2.
5. The method of claim 1, wherein the aldehyde dehydrogenases RPA1206, RPA1687 and RPA1725 in Rhodopseudomonas palustris are knocked out.
6. The method according to claim 5, wherein the genome of Rhodopseudomonas palustris is used as a template, and the genome of Rhodopseudomonas palustris is expressed by SEQ ID NO: 12. SEQ ID NO:13 and SEQ ID NO: 14. SEQ ID NO:15 as a primer, and performing overlap extension PCR to obtain the sequences of the upstream and downstream homologous arms of the aldehyde dehydrogenase RPA 1206; as set forth in SEQ ID NO: 16. SEQ ID NO:17 and SEQ ID NO: 18. SEQ ID NO:19 is used as a primer to carry out overlap extension PCR to obtain the upstream and downstream homologous arm sequences of aldehyde dehydrogenase RPA 1687; as set forth in SEQ ID NO: 20. SEQ ID NO:21 and SEQ ID NO: 22. SEQ ID NO:23 as the primer, performing overlap extension PCR to obtain the upstream and downstream homology arm sequences of the aldehyde dehydrogenase RPA1725, respectively inserting the upstream and downstream homology arm sequences into a suicide plasmid pZJD29c to obtain pZJ-delta 1206, pZJ-delta 1687 and pZJ-delta 1725, and then introducing the plasmids pZJ-delta 1206, pZJ-delta 1687 and pZJ-delta 1725 into rhodopseudomonas palustris by combined transfer.
7. A photosynthetic bacterium producing vanillyl alcohol at a high yield, which is constructed by the construction method according to any one of claims 2 to 6.
8. A method for the photocatalytic biosynthesis of vanillyl alcohol, comprising the steps of:
activating and expanding the photosynthetic bacteria of claim 7, inoculating in MedS culture medium, culturing for 24h, transferring the culture into MedC culture solution at a ratio of 1;
catalyzing ferulic acid to react to vanillyl alcohol at 30 ℃ by adopting a phosphate buffer reaction system with pH =7, and detecting the vanillyl alcohol yield by liquid chromatography.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN105000934A (en) * | 2015-07-22 | 2015-10-28 | 浙江工商大学 | Method for obtaining high biological activity lignan and mushroom by using edible mushroom to degrade flax seed shell |
| CN107075465A (en) * | 2014-09-29 | 2017-08-18 | 西姆莱斯股份公司 | The method that forulic acid is converted into vanillic aldehyde |
| US20210198679A1 (en) * | 2019-12-27 | 2021-07-01 | Tianjin University Of Science And Technology | Saccharomyces cerevisiae strain with high yield of ethyl butyrate and construction method and application of saccharomyces cerevisiae strain |
| CN113416664A (en) * | 2021-02-02 | 2021-09-21 | 天津科技大学 | Saccharomyces cerevisiae genetic engineering strain, construction method thereof and application thereof in high-yield ethyl acetate |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107075465A (en) * | 2014-09-29 | 2017-08-18 | 西姆莱斯股份公司 | The method that forulic acid is converted into vanillic aldehyde |
| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN105000934A (en) * | 2015-07-22 | 2015-10-28 | 浙江工商大学 | Method for obtaining high biological activity lignan and mushroom by using edible mushroom to degrade flax seed shell |
| US20210198679A1 (en) * | 2019-12-27 | 2021-07-01 | Tianjin University Of Science And Technology | Saccharomyces cerevisiae strain with high yield of ethyl butyrate and construction method and application of saccharomyces cerevisiae strain |
| CN113416664A (en) * | 2021-02-02 | 2021-09-21 | 天津科技大学 | Saccharomyces cerevisiae genetic engineering strain, construction method thereof and application thereof in high-yield ethyl acetate |
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