CN112877230A

CN112877230A - Yeast with improved vitamin D3 yield

Info

Publication number: CN112877230A
Application number: CN202110267085.XA
Authority: CN
Inventors: 刘龙; 陈坚; 吕雪芹; 堵国成; 李江华; 刘延峰; 曲丽莎
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-03-11
Filing date: 2021-03-11
Publication date: 2021-06-01

Abstract

The invention relates to a yeast with improved vitamin D3 yield, which utilizes a dCas9 system to dynamically regulate ERG6 gene, and the introduction of the system can divide saccharomyces cerevisiae cells into a prophase cell growth stage and a anaphase product accumulation stage. The dCas9 system does not play a role in inhibiting the ERG6 gene at the early stage, the cells realize normal growth and propagation, and when OD enters a higher level at the later stage, the dCas9 system plays a role in dynamic regulation, inhibits the normal transcription of the ERG6 gene, blocks a 7-DHC competitive pathway, and realizes that metabolic flow flows to a target product to a greater extent. Meanwhile, the normal growth and propagation of cells are ensured, and the fermentation yield reaches 123 mg/L.

Description

Yeast with improved vitamin D3 yield

Technical Field

The invention belongs to the technical field of metabolic engineering, and particularly relates to yeast with improved vitamin D3 yield.

Background

Vitamin D (VD), known as "sun Vitamin", is a fat-soluble Vitamin necessary to maintain the health of the body. Ergocalciferol (ergolciferol, VD2) and Cholecalciferol (Cholecalciferol, VD3) are the two predominant forms of presence in the VD series of compounds. Human skin tissue contains a large amount of 7-dehydrocholesterol (7-DHC), and after being irradiated by ultraviolet light, the 7-DHC is converted into VD3, which is the main source for obtaining VD by human bodies. VD is an important nutrient required for normal growth and development of bones. When the VD is absent in human body, rickets, osteomalacia and other diseases can be caused. Vitamin D can promote calcium and phosphorus absorption, act on cranial nerves, prevent schizophrenia, self-imposed diseases, cognitive disorder, neurodegeneration and other diseases, and can effectively treat and prevent diabetes by properly supplementing VD. In addition, more and more experiments prove that VD also has good physiological effect in the aspects of treating immune system diseases, cancers, cardiovascular diseases and the like.

In recent years, the production of target products by microbial fermentation has the characteristics of environmental protection and sustainability, the vitamin D3 microbial fermentation method mainly focuses on the aspects of screening and molecular modification of strains, the strain screening is time-consuming and labor-consuming, large-scale screening is needed, even if the strain for producing vitamin D3 is screened, the initial product concentration is extremely low, and the later-stage modification and industrial production are not facilitated. The molecular modification mainly focuses on the elimination of a branch path and the supply of precursor substances, but toxic intermediate products are easily accumulated, and the growth and the production of the strain are not facilitated.

The existing technology for producing 7-DHC mainly focuses on enhancing the cytoplasmic mevalonate pathway, knocking out ERG5 and ERG6 in the ergosterol synthetic pathway, realizing the accumulation of 7-DHC in Saccharomyces cerevisiae and further realizing the accumulation of VD 3. Ergosterol is an important constituent of the cell membrane of s.cerevisiae and determines the fluidity and permeability of structural membranes. The knockout of ERG6 gene leads to the synthesis of ergosterol to be hindered, thereby generating the problems of slow cell growth, increased free NADH/NAD + ratio, intracellular redox imbalance, glycerol and ethanol accumulation, lipid metabolism imbalance and the like.

In view of the above, the present inventors have actively studied and innovated to create a method for increasing the yield of vitamin D3, which is more industrially useful.

Disclosure of Invention

For solving vitamin D₃The invention provides a genetically engineered bacterium for improving vitamin D3 and a construction method and application thereof, the construction method takes Saccharomyces cerevisiae BY4742 as an initial strain, introduces exogenous gene DHCR24 gene, constructs 7-DHC synthetic pathway in yeast cells, and the fermentation yield of vitamin D3 reaches 0.7 mg/L. 8 key genes (ERG10, ERG13, ERG12, ERG20, tHMG1, IDI, ERG8 and ERG19) in the MVA pathway are overexpressed by using an inducible promoter GAL, and the fermentation yield reaches 57.6 mg/L. The inducible promoter GAL is used for over-expressing 4 genes (ERG1, ERG11, ERG7 and ERG24) of the posterior squalene pathway, and the fermentation yield reaches 73 mg/L. And then the dCas9 system is used for dynamically regulating ERG6 gene, and the fermentation yield reaches 123 mg/L.

The first object of the present invention is to provide a yeast with improved vitamin D3 production, which heterologously expresses 3 β -hydroxysteroid-D24 reductase DHCR24 gene in yeast host, and overexpresses acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, mevalonate kinase ERG12, farnesyl pyrophosphate synthase ERG20, 3-hydroxy-3-methylglutaryl-CoA reductase tmg 1, isopentenyl pyrophosphate isomerase IDI, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, squalene epoxidase ERG1, sterol 14 α -demethylase ERG11, lanosterol synthase ERG7 and C-14 sterol reductase ERG24 genes.

Furthermore, in the yeast, a dCas9 system is adopted to dynamically regulate sterol C-24 methyltransferase ERG6 gene expression, specifically, a dCas9 protein gene fragment using GAL1 as a promoter and a gRNA expression fragment are introduced into saccharomyces cerevisiae, and the gRNA expression fragment contains a 20nt site on an ERG6 gene promoter.

Furthermore, the nucleotide sequence of the 20nt site is shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.

Further, the dCas9 protein gene fragment and the gRNA expression fragment were introduced into 106a site of yeast.

Further, the yeast is saccharomyces cerevisiae, pichia pastoris or candida tropicalis.

Furthermore, the nucleotide sequence of the GAL1 promoter is shown in SEQ ID NO. 6.

Further, 3 β -hydroxysteroid-D24 reductase DHCR24 is numbered as ID:424661, acetoacetyl-CoA thiolase ERG10 is numbered as ID:856079, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 is numbered as ID:854913, mevalonate kinase ERG12 is numbered as ID:855248, farnesyl pyrophosphate synthase ERG20 is numbered as ID: 853272, 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, ID:42650, ID:855986 for isopentenyl pyrophosphate isomerase IDI, ID:855260 for phosphomevalonate kinase ERG8, ID:855779 for mevalonate pyrophosphate decarboxylase ERG19, ID:853086 for squalene epoxidase ERG1, ID:856398 for sterol 14 alpha-demethylase ERG11, ID:856470 for lanosterol synthase ERG7, and ID:855441 for C-14 sterol reductase ERG24 gene.

Further, sterol C-24 methyltransferase ERG6 is numbered ID: 855003.

The second purpose of the invention is to provide the application of the yeast in the fermentation production of vitamin D3.

Further, the yeast seed liquid is cultured in a YPD culture medium at the temperature of 28-32 ℃ and the rpm of 150-300.

By the scheme, the invention at least has the following advantages:

the invention utilizes a dCas9 system to dynamically regulate ERG6 gene, and the introduction of the system can divide the saccharomyces cerevisiae cell into a prophase cell growth stage and a later-stage product accumulation stage. The dCas9 system does not play a role in inhibiting the ERG6 gene at the early stage, the cells realize normal growth and propagation, and when OD enters a higher level at the later stage, the dCas9 system plays a role in dynamic regulation, inhibits the normal transcription of the ERG6 gene, blocks a 7-DHC competitive pathway, and realizes that metabolic flow flows to a target product to a greater extent. Meanwhile, the normal growth and propagation of cells are ensured, and the fermentation yield reaches 123 mg/L.

The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a preferred embodiment of the present invention and is described in detail below.

Drawings

FIG. 1 is a schematic diagram of the 7-DHC synthetic pathway;

FIG. 2 is a graph of the fermentation inhibition effect of a recombinant strain 96-well plate;

FIG. 3 is a graph of the fermentation inhibition effect of a recombinant strain 96-well plate;

FIG. 4 is a diagram of the effect of recombinant bacteria on shake flask fermentation inhibition.

Detailed Description

The related detection method comprises the following steps:

gas phase-mass spectrometry combined detection of vitamin D3: detection was performed using an Agilent Technologies 7890A instrument with a FID detector and a Chirasil-DEX-CB chromatography column (Agilent; 25m, 0.32mm, 0.25 μm). The injector temperature was 180 deg.C, the carrier gas was helium, the flow rate was 1mL/min, and the pressure was 5.8 psi. The program started at 70 ℃ and the temperature was raised to 150 ℃ at a rate of 20 ℃/min for 3 minutes and then to 190 ℃ at a rate of 2 ℃/min for 3 minutes.

Fermentation culture of engineering bacteria:

inoculating single colony of engineering Saccharomyces cerevisiae on solid YPD plate in 2mL YPD culture medium, culturing at 30 deg.c and 220rpm for 16-20 hr, inoculating 1% of the strain into 25mL YPD liquid culture medium in 250mL round bottom shake flask, and culturing at 30 deg.c and 220rpm for 108 hr.

Post-treatment of fermentation liquor:

the fermentation liquor is centrifuged for 5min at 5000r, the supernatant is discarded, and the thallus is collected and washed once by sterile water. The cells were disrupted by liquid nitrogen milling. The triturated material was added to 5ml of a 1.5mol/L KOH-methanol solution. Saponifying at 80 deg.C for 30 min. Adding n-hexane, extracting for 3 times (3 ml each time), and keeping the upper layer liquid. Drying with a vacuum centrifugal concentrator, and keeping the crystal. Standard weighing 1.5mg dissolved in 1ml of derivatizing agent, derivatizing at 37 deg.C for 2h, and derivatizing the sample with 200. mu.l of derivatizing agent. Blowing and drying by a nitrogen blowing instrument after the derivatization, and adding n-hexane into the residual crystal for redissolving.

Example 1: introduction of heterologous gene DHCR24 to construct exogenous pathway

(a) PGAL1-DHCR24-TCYC1 gene was synthesized by Jinwei Zhi Co., Ltd, and gene fragments 106a-U and 106a-D were obtained by amplification with primers.

(b) Subjecting the three fragments PGAL1-DHCR24-TCYC1, 106a-U, and 106a-D gene fragments obtained in step (a) to overlap extension PCR under the conditions: pre-denaturation at 98 ℃ for 5min, then denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ for 2min for 30 cycles in total, cutting gel and recovering fragments with correct sizes to obtain fusion gene fragments 106a-U-PGAL1-DHCR24-TCYC1-106a-D gene fragments.

(c) Taking pML104 plasmid as a template, adopting primers to amplify to obtain a fragment 106a, carrying out hot shock transfer to Escherichia coli JM109 to be competent, carrying out colony PCR verification, carrying out sequencing, inoculating a single colony with correct sequencing into 2mL LB culture medium, culturing for 16h, and extracting by using a plasmid extraction kit to obtain the plasmid pML104-106 a.

(d) Preparing original saccharomyces cerevisiae competence, transferring the constructed fusion gene segment 106a-U-PGAL1-DHCR24-TCYC1-106a-D and the plasmid pML104-106a into the competence together, and carrying out colony PCR verification by using primers after a single bacterium grows out on an SD-Ura screening solid plate.

(e) And (d) inoculating the single colony verified to be correct by the colony PCR in the step (d) into a YPD liquid culture medium for culturing for 16h, then scribing on a YPD solid plate containing 5-FOA, culturing for 3d at 30 ℃, then respectively transferring the grown single colony onto the YPD solid plate and an SD-Ura screening solid plate for comparison verification, wherein the single colony which normally grows on the YPD solid plate but cannot grow on the SD-Ura screening solid plate is the correct modified bacterium and is named as SCD 1.

(f) The transformed strain SCD1 is subjected to shake flask fermentation and post-treatment, and the yield of 7-DHC in gas quality detection reaches 0.8 mg/L.

Example 2: engineering bacterium of reconstructed saccharomyces cerevisiae

(a) Gene fragment PGAL1-tHMG1-TADH1 is artificially synthesized, and gene 416D-U and gene 416D-D are obtained BY using a saccharomyces cerevisiae BY4742 genome as a template and adopting primer amplification.

(b) Subjecting the three fragments PGAL1-tHMG1-TADH1, 416D-U, and 416D-D gene fragments obtained in step (a) to overlap extension PCR under the conditions: pre-denaturation at 98 ℃ for 5min, followed by denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, 72 ℃ and extension for 2min for a total of 30 cycles, and gel cutting to recover the correct size fragment, resulting in the fusion gene fragment 416D-U-PGAL1-tHMG1-TADH1-416D-D gene fragment.

(c) Taking pML104 plasmid as a template, adopting primers to amplify to obtain a fragment 416d, carrying out hot shock transfer to escherichia coli JM109 to be competent, carrying out colony PCR verification, carrying out sequencing, inoculating a single colony with correct sequencing into 2mL LB culture medium, culturing for 16h, and extracting by using a plasmid extraction kit to obtain the plasmid pML104-416 d.

(d) SCD1 Saccharomyces cerevisiae competence is prepared, the constructed fusion gene segment 416D-U-PGAL1-tHMG1-TADH1-416D-D and the plasmid pML104-416D are together transferred into competence, and colony PCR verification is carried out by adopting primers after single bacteria grow on an SD-Ura screening solid plate.

(e) And (d) inoculating the single colony verified to be correct by the colony PCR in the step (d) into a YPD liquid culture medium for culturing for 16h, then scribing on a YPD solid plate containing 5-FOA, culturing for 3d at 30 ℃, then respectively transferring the grown single colony onto the YPD solid plate and an SD-Ura screening solid plate for comparison verification, wherein the single colony which normally grows on the YPD solid plate but cannot grow on the SD-Ura screening solid plate is the correct modified bacterium and is named as SCD 2.

(f) The overexpression genes of MVA and the posterior squalene pathway (ERG10, ERG13, ERG12, ERG20, IDI, ERG8, ERG19, ERG1, ERG11, ERG7 and ERG24) were sequentially introduced by the similar method to obtain a modified strain SCD 8.

Example 3: construction of plasmids containing fluorescent protein characterization

(a) A PY13 free plasmid (containing His label) is used as a plasmid vector of a gene, an original promoter gene sequence of an ERG6 gene is inserted, GFP fluorescent protein is inserted at the same time, and the 20nt site with the best inhibitory effect of dcas9 protein is reflected by detecting the fluorescence intensity of the GFP fluorescent protein. A fluorescent degradation tag is added behind the GFP fluorescent protein gene sequence to shorten the half-life of the fluorescent protein. The gene fragment PY13 was obtained by amplification with the primers. Artificially synthesized gene fragment P_ERG6-eGFP-T_CYC1The gene fragment P is obtained BY taking a saccharomyces cerevisiae BY4742 genome as a template and adopting primer amplification_ERG6And using plasmids containing eGFP as templates and adopting primers to amplify to obtain the gene eGFP. PCR conditions were as follows: pre-denaturation at 98 ℃ for 5min, then denaturation at 98 ℃ for 10s and annealing at 55 ℃ for 5s and 72 ℃ for 2min, 30 cycles in total, and recovery of the glue.

(b) The three fragments were ligated using one-step cloning enzyme and ligated for 1h at 50 ℃. The pcr product was introduced into E.coli competence, incubated on ice for 30min, heat-shocked at 42 ℃ for 90s, incubated with 200. mu.l of non-resistant LB medium at 37 ℃ for 1h, and plated on LB solid medium with A antibody. And (5) verifying bacterium p.

Example 4: construction of dCas9 System Gene fragment

(a) 4 20nt sites were randomly selected on the original promoter of the ERG6 gene, and as shown in the following ERG6 gene promoter sequence, the gRNA containing the 20nt site guided the dCas9 protein to a specific part of the promoter. Introducing a constructed gene segment into a 106a site of the saccharomyces cerevisiae, wherein the segment comprises a Leu label; the GAL1 promoter induces the transcribed dCas9 protein; randomly selected 20nt sites. A gene fragment Leu is amplified BY using a primer, a plasmid containing dCas9 protein is used as a template, a gene fragment dCas9 is amplified BY using the primer, a genome of saccharomyces cerevisiae BY4742 is used as the template, and a gene fragment 20nt is amplified BY using the primer.

(b) Introducing the dCas9 system into the yeast genome: by utilizing the cre-loxp system, the gene segments realize homologous recombination in saccharomyces cerevisiae and are integrated into a yeast genome. In the early stage of cell growth, glucose in the culture medium is used for cell propagation, and meanwhile, the GAL promoter is inhibited, and dCas9 protein does not play an inhibiting role; when glucose is depleted, the GAL promoter is activated, transcribes dCas9 protein, represses the ERG6 gene, and metabolic flux flows to the 7-DHC synthetic pathway. 5 yeast cells are prepared by chemical transformation, 4 SCD8 yeast cells are transferred into 4 different 20nt sites, ERG6 original promoter-GFP plasmid and dCas9 gene fragment respectively, besides, another 1 yeast competence is transferred into only the constructed ERG6 original promoter-GFP plasmid as a control. Plates were plated on Leu and His deficient medium for screening. And (5) verifying a colony pcr.

ERG6 gene promoter sequence (SEQ ID NO. 1):

example 5: fluorescence intensity detection

ERG6 gene regulated by dCas9 protein is fused with fluorescent protein GFP, and the expression strength of ERG6 gene is positively correlated with fluorescence strength. And (3) fermenting the constructed 5 strains by using a 96-pore plate shaker and a shaking flask, setting 3 parallel samples for each strain, using the strain converted into GFP plasmid as a control, and detecting the fluorescence intensity under a microplate reader, wherein the weaker the fluorescence intensity is, the better the inhibition effect is.

FIGS. 2 and 3 show two 96-well plate fermentations, in which the 2 and 4 sites have the weakest fluorescence intensity and the better inhibition effect. FIG. 4 shows the results of shake flask fermentation, and the 2 and 4 sites were found to be more effective in inhibiting.

The result of detecting the vitamin D3 by adopting gas phase-mass spectrometry shows that the yield of VD3 reaches 123 mg/L.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of south of the Yangtze river

<120> a yeast having an improved vitamin D3 production

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 502

<212> DNA

<213> (Artificial sequence)

<400> 1

tcttatgctg cctactatat tattatttta ttcttgtttt tttaaattat gtaaaactta 60

aatcgaaagt aaaacagata agggaaactt gaatgaaaat taacagctta taaattacaa 120

agacgaatgg aaagaaaaag agagcagcag aggataggag aaaacacccg aactatataa 180

aaagcaatga agaagttatc ggcaacagca agtattggag gtttctcacc aatcttcgca 240

taaaacgcat gcttcgtaag gcgataaaaa tgaagtgtac cattgagacg aagtggagca 300

gtttacagtg aataatacat tgtctgctcc tcacttactc tcgcttctcg aaaacttttt 360

tgtatttatc ggaaacggca gtaaacgacg ccaaaaattg aaaataagta cgggaacgag 420

gtaccatata cgaagatcgg ggctttgttc ggccctaaat tttgctgatc ttaccctgtt 480

gcatcttttc aaaaatagta ac 502

<210> 2

<211> 24

<212> DNA

<213> (Artificial sequence)

<400> 2

gcctactata ttattatttt attc 24

<210> 3

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 3

atcgaaagta aaacagataa ggg 23

<210> 4

<211> 23

<212> DNA

<213> (Artificial sequence)

<400> 4

tcggcaacag caagtattgg agg 23

<210> 5

<211> 17

<212> DNA

<213> (Artificial sequence)

<400> 5

cctcacttac tctcgct 17

<210> 6

<211> 600

<212> DNA

<213> (Artificial sequence)

<400> 6

tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60

ttttatgaca tttgaataag aagtaataca aactgaaaat gttgaaagta ttagttaaag 120

tggttatgca gcttttccat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180

attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tatcatccta tggttgttaa 240

tttgattcgt taatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300

accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360

ctgcgtttca ggaacgcgac cggtgaagac gaggacgcac ggaggagagt cttccgtcgg 420

agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480

gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540

catttccaca acatataagt aagattagat atggatatgt atatggtggt aatgccatgt 600

Claims

1. A yeast with improved vitamin D3 yield is characterized in that 3 beta-hydroxysteroid-D24 reductase DHCR24 gene is heterologously expressed in a yeast host, and acetoacetyl-CoA thiolase ERG10, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13, mevalonate kinase ERG12, farnesyl pyrophosphate synthase ERG20, 3-hydroxy-3-methylglutaryl-CoA reductase tHMG1, isopentenyl pyrophosphate isomerase IDI, phosphomevalonate kinase ERG8, mevalonate pyrophosphate decarboxylase ERG19, squalene epoxidase ERG1, 14 alpha-demethylase ERG11, lanosterol synthase ERG7 and C-14 sterol reductase ERG24 gene are overexpressed.

2. The yeast according to claim 1, wherein the sterol C-24 methyltransferase ERG6 gene expression is dynamically regulated by a dCas9 system, specifically, a dCas9 protein gene fragment using GAL1 as a promoter and a gRNA expression fragment are introduced into saccharomyces cerevisiae, and the gRNA expression fragment contains 20nt site on the ERG6 gene promoter.

3. The yeast according to claim 2, wherein the nucleotide sequence of the 20nt locus is shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 5.

4. The yeast of claim 2, wherein the dCas9 protein gene fragment and the gRNA expression fragment are introduced into the yeast at position 106 a.

5. The yeast of claim 1, wherein the nucleotide sequence of the GAL1 promoter is shown in SEQ ID No. 6.

6. The yeast of claim 1, wherein the yeast is saccharomyces cerevisiae, pichia pastoris, or candida tropicalis.

7. The yeast of claim 1, wherein 3 β -hydroxysteroid-D24 reductase DHCR24 is numbered ID 424661, acetoacetyl-CoA thiolase ERG10 is numbered ID 856079, 3-hydroxy-3-methylglutaryl-CoA synthase ERG13 is numbered ID 854913, mevalonate kinase ERG12 is numbered 855248, farnesyl pyrophosphate synthase ERG20 is numbered ID: 853272, 3-hydroxy-3-methylglutaryl coenzyme A reductase tHMG1, ID:42650, ID:855986 for isopentenyl pyrophosphate isomerase IDI, ID:855260 for phosphomevalonate kinase ERG8, ID:855779 for mevalonate pyrophosphate decarboxylase ERG19, ID:853086 for squalene epoxidase ERG1, ID:856398 for sterol 14 alpha-demethylase ERG11, ID:856470 for lanosterol synthase ERG7, and ID:855441 for C-14 sterol reductase ERG24 gene.

8. The yeast of claim 2, wherein sterol C-24 methyltransferase ERG6 is numbered ID 855003.

9. Use of the yeast of any one of claims 1 to 8 for the fermentative production of vitamin D3.

10. The use according to claim 9, wherein the seed solution of yeast is cultured in YPD medium at 28-32 ℃ and 150-300 rpm.