CN105755028A - 4-Hydroxyphenylglycolic acid producing engineering bacteria and construction method thereof - Google Patents
4-Hydroxyphenylglycolic acid producing engineering bacteria and construction method thereof Download PDFInfo
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Abstract
The present invention discloses 4-hydroxyphenylglycolic acid producing engineering bacteria and a construction method thereof, the construction method is as follows: artificial synthesis of 4-hydroxyphenylglycolic acid synthase gene hmaS; synthesis of trc promoter; fusion of the trc promoter with the hmaS to obtain fragment trc-hmas; synthesis of expression vector backbone SyBE-002866, and enzyme-cut link up of the fragment trc-hmas and SyBE-002866 to obtain recombinant vector SyBE-002868; knockout of original chassis escherichia coli aspC gene to obtain optimized chassis strain BKT7; and conversion of the SyBE-002868 into BKT7, and monoclonal screening to obtain positive transformant SyBE-002872. 4-hydroxyphenylglycolic acid can be produced from glucose or glucose and xylose as a carbon source by use of engineering E. coli by fermentation without adding of any precursor, the problem of the source of 4-hydroxyphenylglycolic acid can be solved, and cost is reduced.
Description
Technical field
Biomedicine technical field belonging to the present invention, relates to the microbial production of a kind of 4-hydroxymandelic acid.
Background technology
4-hydroxymandelic acid (4-Hydroxymandelicacid), molecular formula is C8H8O4, molecular weight is 168.14, and pale red crystalline solid is soluble in water.4-parahydroxymandelic acid is widely used in the production of medicine, pesticide and spice, pharmaceutically as preparing antihypertensive atenolol, is also the intermediate of synthesis broad-spectrum antibiotic medicine amoxicillin, hydroxyl cefalexin etc..The staple product in many fine chemistry industries, new material can also be synthesized, such as D-pHPG, 4-Hydroxyphenyl hydantoin, p-hydroxyphenylaceticacid etc..
The synthesis of current parahydroxymandelic acid mainly has following three kinds of methods.
(1) Chloral method.First, phenol reacts with Chloral, generates α-trichloromethyl p-hydroxyphenylethanol, and then, the trichloromethyl hydrolysis of α-trichloromethyl p-hydroxyphenylethanol generates carboxyl, obtains L-parahydroxymandelic acid.This synthetic route response time was up to tens hours, and yield is relatively low.(2) hydroxy benzaldehyde method.First, hydroxy benzaldehyde and sodium sulfite addition, then react with Cyanogran., obtain p-hydroxybenzylcyanide, then, p-hydroxybenzylcyanide hydrolysis, obtain L-parahydroxymandelic acid.The same response time length of this route and yield are not high, and raw material Cyanogran. toxicity is relatively big, cause poor stability and are likely to produce Heavy environmental pollution.(3) phenol acetaldehyde acid system.In alkaline aqueous solution, with glyoxalic acid and phenol for raw material condensation, obtain adjacent hydroxymandelic acid and parahydroxymandelic acid, obtain L-parahydroxymandelic acid again through separation.These method raw material sources are extensive comparatively speaking, and technique is simple, and cost low-yield is high, is the main method of industrialized production.
But, above-mentioned organic synthesis raw material mostlys come from Fossil fuel, and raw material and production technology environmental pollution are seriously, do not meet the requirement of environmental protection sustainable development.In recent years, along with the continuous progress of synthesising biological technology, engineered microbes production of chemicals is used to be increasingly becoming the method received much concern.But so far but without any bibliographical information utilizing works Microbe synthesis 4-hydroxymandelic acid.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that produce the engineering bacteria of 4-hydroxymandelic acid.
Second purpose of the present invention is to provide the construction method of the engineering bacteria producing 4-hydroxymandelic acid.
3rd purpose of the present invention is to provide produces the method that the engineering bacterium fermentation of 4-hydroxymandelic acid prepares 4-hydroxymandelic acid.
Technical scheme is summarized as follows:
Produce the construction method of the engineering bacteria of 4-hydroxymandelic acid, comprise the steps:
(1) synthetic 4-hvdroxymandelate acid synthase gene hmaS, described 4-hvdroxymandelate acid synthase gene hmaS is such as shown in SEQIDNo.27；
(2) full chemosynthesis trc promoter or gap promoter, described trc promoter is such as shown in SEQIDNo.28；Described gap promoter is such as shown in SEQIDNo.29；
(3) described trc promoter is merged by Overlap extension PCR with 4-hvdroxymandelate acid synthase gene hmaS, obtain fragment trc-hmas；Or described gap promoter is merged by Overlap extension PCR with 4-hvdroxymandelate acid synthase gene hmaS, obtain fragment gap-hmas；
(4) full chemosynthesis is with the expression vector skeleton SyBE-002866 shown in SEQIDNo.30, or full chemosynthesis is with the expression vector skeleton SyBE-002867 shown in SEQIDNo.31；
(5) by fragment trc-hmas and expression vector skeleton SyBE-002866 by enzyme action connected mode, recombinant vector SyBE-002868 is obtained；Or by fragment gap-hmas and expression vector skeleton SyBE-002867 by enzyme action connected mode, obtain recombinant vector SyBE-002869；Or by fragment trc-hmas and expression vector skeleton SyBE-002867 by enzyme action connected mode, obtain recombinant vector SyBE-002870；By fragment gap-hmas and expression vector skeleton SyBE-002866 by enzyme action connected mode, obtain recombinant vector SyBE-002871；
(6) utilize λ-red homologous recombination technique, knock out original chassis escherichia coli aspC gene, obtain the chassis bacterial strain BKT7 optimized；
(7) recombinant vector SyBE-002868 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002872；Or recombinant vector SyBE-002869 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002873；Or recombinant vector SyBE-002870 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002874；Or recombinant vector SyBE-002871 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002875.
The engineering bacteria producing 4-hydroxymandelic acid that said method builds.
Produce the method that the engineering bacterium fermentation of 4-hydroxymandelic acid prepares 4-hydroxymandelic acid, comprise the steps: engineering bacteria SyBE-002872, SyBE-002873, SyBE-002874 or SyBE-002875 batch fermentation or the fed-batch fermentation in the synthetic medium containing L-Aspartic acid respectively by producing 4-hydroxymandelic acid, obtain 4-hydroxymandelic acid.
Synthetic medium containing L-Aspartic acid is: 5.0g/L glucose, 2.9g/LNaCl, 2.7g/LNH4Cl、0.3g/LK2HPO4、0.2g/LMgSO4、0.05g/LCaCl2With 3.0g/LL-aspartic acid；Surplus is water, and 10MNaOH solution is adjusted to pH=7.0.
Synthetic medium containing L-Aspartic acid is: 2.5g/L glucose, 2.5g/L xylose, 2.9g/LNaCl, 2.7g/LNH4Cl、0.3g/LK2HPO4、0.2g/LMgSO4、0.05g/LCaCl2With 3.0g/LL-aspartic acid；Surplus is water, and 10MNaOH solution is adjusted to pH=7.0.
The step of fed-batch fermentation is: when in fermentation liquid, concentration of glucose is lower than 0.5g/L, and stream adds glucose, final concentration of 5.0g/L, feed supplement 2 times.
The step of fed-batch fermentation is: when in fermentation liquid, concentration of glucose is lower than 0.5g/L, with glucose and xylose etc. mass concentration mixed liquor carry out stream and add, the final concentration of 2.5g/L of glucose after feed supplement, feed supplement 2 times.
Advantages of the present invention:
4-hydroxymandelic acid is important chemical intermediate, is widely used in the production of medicine, pesticide and spice and new material.The present invention uses engineering colon bacillus, with glucose or glucose and xylose for carbon source, does not need to add any precursor, carries out fermenting and producing 4-hydroxymandelic acid.The present invention can solve the source problem that comes of 4-hydroxymandelic acid, reduces production cost to greatest extent simultaneously, is conducive to industrialized production.
Accompanying drawing explanation
Fig. 1 is 4-hydroxymandelic acid synthetic route.
Fig. 2 is the building process of plasmid SyBE-002868, SyBE-002869, SyBE-002870, SyBE-002871 of the present invention.
Fig. 3 is the HPLC collection of illustrative plates of 4-hvdroxymandelate acid product checking, and wherein a is 4-hydroxymandelic acid standard substance figure, b is engineered strain SyBE-002872 fermentation broth sample.
Fig. 4 is engineered strain SyBE-002872, SyBE-002873, SyBE-002874, SyBE-002875 fermentation testing result Yield mapping built.
Detailed description of the invention
Original chassis colon bacillus (Escherichiacoli) is the bacterial strain of high yield tyrosine, name is called SyBE-002447, now in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, register on the books and be numbered CGMCCNo.7962 in preservation center.The preservation time is on July 22nd, 2013, and address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode 100101.
Below in conjunction with specific embodiment, the present invention is further illustrated.
The biosynthesis pathway of 4-hydroxymandelic acid of present invention design belongs to shikimic acid pathway, and detailed route of synthesis is as follows: with glucose or xylose or other biological matter for carbon source, synthesize Single-chip microcomputer through shikimic acid pathway.Single-chip microcomputer synthesizes 4-hydroxymandelic acid under the catalytic action of parahydroxymandelic acid synzyme (HmaS), and biosynthesis pathway schematic diagram is as shown in Figure 1.
The structure of embodiment 1hmaS gene overexpression recombinant vector
Preferred Amycolatopsis orientalis parahydroxymandelic acid synzyme, its aminoacid sequence is such as shown in SEQ ID No .32.Use the online codon optimized software of JCAT (http://www.jcat.de), with escherichia coli, the Preference of codon is optimized, design total length hmaS gene order, such as sequence table SEQ IDNo.27.
Utilize online oligonucleotide design software BuildingBlockDesign (http://www.genedesign.org), hmas full-length gene is split into about 60bp oligonucleotide primers 26, such as sequence table SEQ IDNo.1-26, adjacent two primers have 20bp homologous sequence.In the technical program, the synthesis of chemosynthesis company is entrusted in all related genes and primer synthesis.Undertaken 26 primers being synthetically derived total length hmaS gene with completing PCR assembling with overlap extension without template PCR.PCR system without template is: primer mixture 2 μ L, pfu enzyme 0.2 μ L, ultra-pure water 11.8 μ L, 5 × pfu enzyme buffer4 μ L, 2.5mMdNTP2 μ L.The program of PCR without template is: (1) 5 circulation: 94 DEG C, 30s；69℃,30s；72℃,15s；(2) 5 circulations: 94 DEG C, 30s；65℃,30s；72℃,15s；(3) 20 circulations: 94 DEG C, 30s；61℃,30s；72℃,15s；(4)72℃,3min；(5)4℃,+∞.Completing PCR system is: 2 μ L are without solution, 0.5 μ L50pMSEQIDNo.1,0.5 μ L50pMSEQIDNo.26,0.2 μ Lpfu enzyme, 11.8 μ L ultra-pure waters, 4 μ L5 × pfu enzyme buffer, 2 μ L2.5mMdNTP after template PCR primer 10 times dilution.Completing PCR program is: (1) 94 DEG C, 3min；(2) 25 circulations: 94 DEG C, 30s；55℃,30s；72℃,30s；(3)72℃,3min；(4)4℃,+∞.
Complete synthesis trc promoter and gap promoter, trc promoter sequence is such as shown in sequence table SEQ IDNo.28, and gap promoter sequence is such as shown in sequence table SEQ IDNo.29.Full chemosynthesis expression vector skeleton SyBE-002866, its sequence is such as shown in sequence table SEQ IDNo.30；Full chemosynthesis expression vector skeleton SyBE-002867, its sequence is such as shown in sequence table SEQ IDNo.31.
In the structure of trc promoter regulation expression vector, first with complete synthesis trc promoter for template, trcF and trcR is that upper and lower primer PCR obtains trc promoter fragment；With complete synthesis gene hmaS for template, hmaSF12 and hmaSR12 is upper and lower primer PCR, obtains there is the hmaS genetic fragment of homologous sequence with trc fragment.Again with trc promoter fragment and hmaS genetic fragment for template, obtain promoter and gene fusion fragment trc-hmas for upper and lower primer by Overlap extension PCR with trcF and hmasR12.Merge fragment trc-hmas and utilize BamHI and HindIII enzyme action, be connected in the expression vector skeleton SyBE-002866 of identical linearization for enzyme restriction, obtain SyBE-002868.Primer trcF, trcR, hmasF12 and hmasR12 sequence are respectively as shown in sequence table SEQ IDNO.33, SEQIDNO.34, SEQIDNO.35 and SEQIDNO.36.
With complete synthesis trc promoter for template, trcF14 and trcR is that upper and lower primer PCR obtains trc promoter fragment；With complete synthesis gene hmaS for template, hmaSF12 and hmaSR13 is upper and lower primer PCR, obtains there is the hmaS genetic fragment of homologous sequence with trc fragment.Again with trc promoter fragment and hmaS genetic fragment for template, obtain promoter and gene fusion fragment trc-hmas for upper and lower primer by Overlap extension PCR with trcF14 and hmasR13.Merge fragment trc-hmas and utilize HindIII and KpnI enzyme action, be connected in the expression vector skeleton SyBE-002867 of identical linearization for enzyme restriction, obtain SyBE-002870.Primer trcF14 sequence is respectively as shown in sequence table SEQ IDNO.40.
In the structure of gap promoter regulation expression vector, first with complete synthesis gap promoter for template, gapF and gapR is that upper and lower primer PCR obtains gap promoter fragment；With complete synthesis gene hmaS for masterplate, hmaSF12 and hmaSR13 is upper and lower primer PCR, obtains there is the hmaS genetic fragment of homologous sequence with gap fragment.Again with gap promoter fragment and hmaS genetic fragment for template, obtain promoter and gene fusion fragment gap-hmas for upper and lower primer by Overlap extension PCR with gapF and hmasR3.Merge fragment gap-hmas and utilize HindIII and KpnI enzyme action, be connected in the expression vector skeleton SyBE-002867 of identical linearization for enzyme restriction, obtain SyBE-002869.Primer gapF, gapR and hmaSR13 sequence are respectively as shown in sequence table SEQ IDNO.37, SEQIDNO.38, SEQIDNO.39.
With complete synthesis gap promoter for template, gapF15 and gapR is that upper and lower primer PCR obtains gap promoter fragment；With complete synthesis gene hmaS for masterplate, hmaSF12 and hmaSR12 is upper and lower primer PCR, obtains there is the hmaS genetic fragment of homologous sequence with gap fragment.Again with gap promoter fragment and hmaS genetic fragment for template, obtain promoter and gene fusion fragment gap-hmas for upper and lower primer by Overlap extension PCR with gapF15 and hmasR12.Merge fragment gap-hmas and utilize BamHI and HindIII enzyme action, be connected in the expression vector skeleton SyBE-002866 of identical linearization for enzyme restriction, obtain SyBE-002871.Primer gapF15 sequence is respectively as shown in sequence table SEQ IDNO.41.
It it is the building process of recombinant expression carrier shown in Fig. 2.
Obtaining trc promoter fragment, gap promoter fragment, hmaS genetic fragment, gap-hmas fragment and trc-hmas fragment by PCR, General reactions system is template 1 μ L, each 0.5 μ L of upper and lower primer, Fastpfu enzyme 2.5 μ L, 5 × reaction buffer 10 μ L, dNTP5 μ L, ultra-pure water 30.5ul.95 DEG C of 30s of PCR response procedures, 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 15s or 60s, 72 DEG C of 5min, wherein the 2nd step circulates 30 times to the 5th step, 4th step adopts 15s for trc and gap promoter fragment, adopts 1min for hmas genetic fragment, gap-hmas fragment and trc-hmas fragment.Promoter and genetic fragment use after both passing through kits.After enzyme action connection, transformed competence colibacillus cell E.coliDH5 α, Fastpfu enzyme, dNTP and competent cell are all purchased from Beijing Quan Shi King Company, bacterium colony PCR screening positive clone upgrading grain, order-checking.
Embodiment 2 λ-red methods of homologous recombination knocks out chassis strain gene aspC
The chassis strain construction process detailed step that gene aspC knocks out is as follows:
1, pKD46 is imported strain SyBE-002447 (CGMCCNo.7962), it is thus achieved that [SyBE-002447/pKD46], by the strain [SyBE-002447/pKD46] of activation, be inoculated in 10mlLB fluid medium, 30 DEG C, 200rpm, it is cultured to OD600For 0.4-0.6.Add final concentration of 10mML-arabinose, continue to cultivate 3h.At 4 DEG C, the centrifugal 8min of 4000rpm, collects cell.Abandon supernatant, 10% glycerol washed cell of addition ice pre-cooling 2 times, prepare into electricity and turn competent cell.Primer aspCF and aspCR sequence are respectively as sequence table SEQ IDNO.42, SEQIDNO.43, PCR preparation have chlorampenicol resistant fragment.Take resistance fragments 2 μ L, join 100 μ L electricity and turn in competent cell, rotate mixing gently.Feeding the mixture in the ice pre-cooling electric shock cup of 2mm, 2.5KV shocks by electricity 4-6ms.It is subsequently adding 1mLLB culture medium, the flat board of the corresponding resistance of coating, 42 DEG C of incubated overnight after 37 DEG C of recovery 3h.
2, the correct single bacterium colony of picking PCR checking, at 42 DEG C, chlorampenicol resistant flat board goes down to posterity 3 times, amicillin resistance flat board checking pKD46 disappearance, cultivates for 37 DEG C and preserves, it is thus achieved that knocks out the bacterial strain of aspC.
3, utilizing the recombinant bacterium obtained in step 2, prepare competent cell, same method electricity converts plasmid pCP20,30 DEG C of incubated overnight.Picking list bacterium colony, non-resistant plate streaking, 42 DEG C go down to posterity 3 times, the bacterial strain of screening chloromycetin and amicillin resistance disappearance, cultivate for 37 DEG C and preserve, the optimization chassis bacterial strain BKT7 of the traceless knockout aspC that succeeds.
Embodiment 3 recombinant vector converts and optimizes chassis bacterial strain and fermentation detection
The bacterial strain BKT7 obtained in embodiment 2 is utilized to prepare competent cell, in conversion embodiment 1, the SyBE-002868 of structure, SyBE-002869, SyBE-002870 and SyBE-002871 plasmid, obtain recombinant bacterial strain SyBE-002872, SyBE-002873, SyBE-002874 and SyBE-002875.The positive transformant of choosing colony PCR checking is to incubated overnight in 5mlLB culture medium.Then the bacterium solution of incubated overnight being transferred enters in the 250ml shaking flask containing 50ml synthetic medium, initial OD600It is about 0.1,37 DEG C, 220rpm cultivation and fermentation.
Fermentation basal medium consists of: the glucose of 5.0g/L, 2.9g/LNaCl, 2.7g/LNH4Cl、0.3g/LK2HPO4、0.2g/LMgSO4、0.05g/LCaCl2, regulate pH 7.0 with 10MNaOH solution.Culture medium is sterilizing 20min at 0.1Mpa pressure 121 DEG C.
Different time points during the fermentation, takes fermentation liquid 1ml, and then centrifugal 3 minutes of 12000r/min, take supernatant, with carrying out HPLC detection after the filtering with microporous membrane of 0.22 μm.Chromatographic condition is as follows: C18 (4.6 × 250mm) chromatographic column；Mobile phase is 10% methanol-90%50mMNaH2PO4 solution-0.01% phosphoric acid；Flow velocity 1mL/min；Sample size 20 μ L；Column temperature room temperature；UV-detector, detects wavelength 218nm.
Being the HPLC collection of illustrative plates of product checking in SyBE-002872 fermentation broth sample as shown in Figure 3, wherein a is standard substance 4-hydroxymandelic acids, and b is engineered strain fermentation broth sample.
Add 3.0g/LL-aspartic acid (synthetic medium containing L-Aspartic acid) and yield can be significantly improved.
Using SyBE-002873 bacterial strain, when culture medium is without L-Aspartic acid, 4-hydroxymandelic acid fermentation yield is 60.6mg/L；Add 1.0g/L, 3.0g/LL-aspartic acid and carry out fermentation 48 hours, 4-hvdroxymandelate acid yield respectively 272.8mg/L and 701.8mg/L.
Synthetic medium containing L-Aspartic acid is: 5.0g/L glucose, 2.9g/LNaCl, 2.7g/LNH4Cl、0.3g/LK2HPO4、0.2g/LMgSO4、0.05g/LCaCl2With 3.0g/LL-aspartic acid；Surplus is water, and 10MNaOH solution is adjusted to pH=7.0.
Fig. 4 is the engineered strain built, in the culture medium containing 3.0g/LL-aspartic acid during fermentation 48h, 4-hydroxymandelic acid Yield mapping, SyBE-002872, SyBE-002873, SyBE-002874 and SyBE-002875 yield respectively 861.7mg/L, 706.2mg/L, 648.3mg/L and 566.9mg/L.
Embodiment 4 glucose fed-batch fermentation
In the fermentation basal medium of embodiment 3, add 3.0g/LL-aspartic acid, for fed-batch fermentation basal medium (synthetic medium containing L-Aspartic acid).For bacterial strain SyBE-002872, carry out stream and add glucose fed-batch fermentation.When concentration of glucose is lower than 0.5g/L in fermentation liquid, add the final concentration of 5.0g/L after glucose, continuous feeding 2 times.During fed-batch fermentation 120h, L-parahydroxymandelic acid yield reaches 2.3g/L respectively.SyBE-002873, SyBE-002874 and SyBE-002875 yield respectively 1.9,1.7,1.5g/L.
Embodiment 5 glucose and xylose muscovado batch fermentation
For bacterial strain SyBE-002872, carry out muscovado batch fermentation.Muscovado fermentation basal medium is glucose 2.5g/L, xylose 2.5g/L, 2.9g/LNaCl, 2.7g/LNH4Cl、0.3g/LK2HPO4、0.2g/LMgSO4、0.05g/LCaCl2, the L-Aspartic acid of 3.0g/L；Surplus is water, and 10MNaOH solution is adjusted to pH=7.0.L-parahydroxymandelic acid measures as described in Example 3.During fermentation 36h, the L-parahydroxymandelic acid yield of SyBE-002872 reaches 1.2g/L.SyBE-002873, SyBE-002874 and SyBE-002875 yield respectively 1.1,0.9,0.8g/L.
Embodiment 6 glucose and xylose muscovado fed-batch fermentation
For bacterial strain SyBE-002872, carry out muscovado fed-batch fermentation.Muscovado fermentation basal medium composition is identical with embodiment 5.When concentration of glucose is lower than 0.5g/L in fermentation liquid, adds glucose and xylose (mass ratio is 1:1) muscovado, add the final concentration of 2.5g/L of rear glucose, feed supplement 2 times.During fermentation 120h, L-parahydroxymandelic acid yield reaches 1.9g/L.
SyBE-002873, SyBE-002874 and SyBE-002875 yield respectively 1.6,1.5,1.3g/L.
Above the present invention has been done exemplary description; should be noted that; when without departing from the core of the present invention, any simple deformation, amendment or other those skilled in the art can not spend the equivalent replacement of creative work to each fall within protection scope of the present invention.
Claims (7)
1. produce the construction method of the engineering bacteria of 4-hydroxymandelic acid, it is characterized in that comprising the steps:
(1) synthetic 4-hvdroxymandelate acid synthase gene hmaS, described 4-hvdroxymandelate acid synthase gene hmaS is such as shown in SEQIDNo.27；
(2) full chemosynthesis trc promoter or gap promoter, described trc promoter is such as shown in SEQIDNo.28；Described gap promoter is such as shown in SEQIDNo.29；
(3) described trc promoter is merged by Overlap extension PCR with 4-hvdroxymandelate acid synthase gene hmaS, obtain fragment trc-hmas；Or described gap promoter is merged by Overlap extension PCR with 4-hvdroxymandelate acid synthase gene hmaS, obtain fragment gap-hmas；
(4) full chemosynthesis is with the expression vector skeleton SyBE-002866 shown in SEQIDNo.30, or full chemosynthesis is with the expression vector skeleton SyBE-002867 shown in SEQIDNo.31；
(5) by fragment trc-hmas and expression vector skeleton SyBE-002866 by enzyme action connected mode, recombinant vector SyBE-002868 is obtained；Or by fragment gap-hmas and expression vector skeleton SyBE-002867 by enzyme action connected mode, obtain recombinant vector SyBE-002869；Or by fragment trc-hmas and expression vector skeleton SyBE-002867 by enzyme action connected mode, obtain recombinant vector SyBE-002870；By fragment gap-hmas and expression vector skeleton SyBE-002866 by enzyme action connected mode, obtain recombinant vector SyBE-002871；
(6) utilize λ-red homologous recombination technique, knock out original chassis escherichia coli aspC gene, obtain the chassis bacterial strain BKT7 optimized；
(7) recombinant vector SyBE-002868 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002872；Or recombinant vector SyBE-002869 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002873；Or recombinant vector SyBE-002870 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002874；Or recombinant vector SyBE-002871 is converted chassis bacterial strain BKT7, monoclonal is screened, obtains positive transformant SyBE-002875.
2. the engineering bacteria producing 4-hydroxymandelic acid that the method for claim 1 builds.
3. the method that the engineering bacterium fermentation producing 4-hydroxymandelic acid of claim 2 prepares 4-hydroxymandelic acid, it is characterized in that engineering bacteria SyBE-002872, SyBE-002873, SyBE-002874 or the SyBE-002875 batch fermentation or the fed-batch fermentation in the synthetic medium containing L-Aspartic acid respectively that comprise the steps: producing 4-hydroxymandelic acid, obtain 4-hydroxymandelic acid.
4. method according to claim 3, is characterized in that the described synthetic medium containing L-Aspartic acid is: 5.0g/L glucose, 2.9g/LNaCl, 2.7g/LNH4Cl、0.3g/LK2HPO4、0.2g/LMgSO4、0.05g/LCaCl2With 3.0g/LL-aspartic acid；Surplus is water, and 10MNaOH solution is adjusted to pH=7.0.
5. method according to claim 3, is characterized in that the described synthetic medium containing L-Aspartic acid is: 2.5g/L glucose, 2.5g/L xylose, 2.9g/LNaCl, 2.7g/LNH4Cl、0.3g/LK2HPO4、0.2g/LMgSO4、0.05g/LCaCl2With 3.0g/LL-aspartic acid；Surplus is water, and 10MNaOH solution is adjusted to pH=7.0.
6. method according to claim 3, is characterized in that the step of described fed-batch fermentation is: when concentration of glucose is lower than 0.5g/L in fermentation liquid, and stream adds glucose, final concentration of 5.0g/L, feed supplement 2 times.
7. method according to claim 3, it is characterized in that the step of described fed-batch fermentation is: when concentration of glucose is lower than 0.5g/L in fermentation liquid, with glucose and xylose etc. mass concentration mixed liquor carry out stream and add, the final concentration of 2.5g/L of glucose after feed supplement, feed supplement 2 times.
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| CN110982771A (en) * | 2019-12-26 | 2020-04-10 | 江南大学 | Method for synthesizing p-hydroxymandelic acid |
| CN114150009A (en) * | 2020-09-08 | 2022-03-08 | 厦门大学 | Construction of engineering bacteria for preparing ephedrine and preparation method of ephedrine |
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