CN106701811A - Binary expression vector for converting gpd1 gene agrobacterium of Chlamydomonas reinhardtii and construction method of binary expression vector - Google Patents
Binary expression vector for converting gpd1 gene agrobacterium of Chlamydomonas reinhardtii and construction method of binary expression vector Download PDFInfo
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Abstract
The invention discloses a binary expression vector containing a Saccharomyces cerevisiae gpd1 gene. A construction method of the binary expression vector comprises the steps of linking a gene extracted from Saccharomyces cerevisiae with a bleomycin gene Ble to obtain a linkage product, and linking the linkage product to multiple cloning sites of a plasmid pRI101-AN, so as to construct pRI-Ble-GPD1. According to the binary expression vector and the construction method, the manpower and material resources for constructing cell wall deficient algae strains can be saved, certain inspiration is provided for a transgenic method in which other cell wall deficient microalgaes cannot be constructed due to the lack of a database, and some thoughts are provided for the research of high-yield-oil transgenic microalgaes.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of for converting Chlamydomonas reinhardtiigpd1 gene agriculture bar
The structure of bacterium binary expression vector.
Background technology
Recently as the exhaustion that closes on of conventional fossil fuel, environmental problem also becomes increasingly conspicuous in addition, causes bioenergy
More and more widely paid close attention to, the production of the microalgae bio-fuel with microalgae oil-producing as representative is particularly subject to scientists from all over the world
Attention.Therefore using modern biotechnology reinforcing microalgae lipid metabolic pathways, the oil content for increasing microalgae, increase oil-rich microalgae
The accumulation of biomass turns into focus at present improving the competitiveness of microalgae oil-producing.Wherein Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) it is pattern algae in microalgae research, it is the preferable acceptor algae strain for studying lipid metabolism gene.Chlamydomonas reinhardtii
In oil synthesis approach, triglycerides（TAG）It is the main component of grease, the number of TAG contents is depending on it in metabolic pathway
The accumulation of precursor substance glycerol-3-phosphate (G-3-P), key enzyme glycerol-3-phosphate dehydrogenase（GPDH）Regulate and control the forward direction of G-3-P
How much synthesis, directly affect TAG contents.Vigeolas etc. recombinantly expresses saccharomycete glycerine 3- in Brassica napus cv.
Phosphate acyltransferase gene（gpd1）Gene, causing the oil content of seed increases by 40%, it was confirmed thatgpd1 gene is closed for TAG
Into key effect.Exist in the prior art and use external sourcegpd1 gene improve Chlamydomonas reinhardtii TAG contents technology, but its be
In Cell wall deficiency Chlamydomonas reinhardtii strain external source is reached using bead mill method translation tablegpd1 gene, Cell wall deficiency algae strain needs
By artificial reforming, waste time and energy.
The content of the invention
The present invention needs the shortcoming of Cell wall deficiency algae strain for prior art, by building for converting Chlamydomonas reinhardtii
'sgpd1 gene Agrobacterium binary expression vector, using Agrobacterium-mediated transformation wild type Chlamydomonas reinhardtii, to improve Rhein clothing
The oil content of algae.
Implementation process of the present invention is as follows：
One kind contains saccharomyces cerevisiaegpdThe binary expression vector of 1 gene, the construction method of the carrier is comprised the following steps：
（1）gpd1 gene is DNA molecular, the genome from saccharomyces cerevisiae, by embedding shown in SEQ ID No.3 in sequence table
Set PCR is obtained, and in the addition of gene two endsEcoRI andNdeI restriction enzyme sites, its primer sequence is SEQ ID No.1 in sequence table
With the DNA molecular shown in SEQ ID No.2；
（2）Reaction solution and carrier pDBle after PCR is expanded with NdeI andEcoRI double digestions, and the digestion products that will be obtained
Use T4DNA ligase is connected, and converts bacillus coli DH 5 alpha, and picking monoclonal Amplification Culture extracts the checking of plasmid double digestion, will
Positive recombinant plasmid is named as recombinant plasmid pDBle-GPD1；
（3）PDBle-GPD1 and pRI101-AN plasmids are usedBamHI andKpnI double digestions, reclaim connection, heat-shock transformed
The competent cell of DH5 α, Amp（100mg/L）The LB plate screening cultures of resistance, positive recombinant plasmid is named as pRI101-
GPD1。
The binary expression vector is pRI101-GPD1.
It is above-mentioned to contain saccharomyces cerevisiaegpdThe genetic transforming method of the binary expression vector of 1 gene, comprises the following steps：
（1）Agrobacterium LBA4404 competence is prepared, recombinant plasmid pRI101-GPD1 is then transformed into root by freeze-thaw method
Cancer Agrobacterium, is containedgpdThe agrobacterium tumefaciens lba4404 of 1 gene；
（2）The positive Agrobacterium LBA4404 for obtaining is infected into Chlamydomonas reinhardtii FACHB479 to be turnedgpd1 gene Chlamydomonas reinhardtii；
（3）Extract Chlamydomonas reinhardtii genomic DNA and RNA and positive transgenic plant is identified by PCR and RT-PCR；
（4）Selection Chlamydomonas reinhardtii transformant, fat content mirror is carried out using the Nile red decoration method of improvement through multi-function microplate reader
It is fixed, and grease staining conditions are observed under laser confocal microscope.
Above-mentioned binary expression vector is carrying external sourcegpd1 gene has the Chlamydomonas reinhardtii base of cell membrane into wild type
Because of the application in group, infecting wild type using the Agrobacterium LBA4404 4 for carrying binary expression vector has the Rhein clothing of cell membrane
Algae FACHB-479, during genes of interest to be transferred to the genome of Chlamydomonas reinhardtii FACHB-479 in the presence of acetosyringone, and
Verified with PCR, RT-PCR and Nile red decoration method.
The present invention using the wild type Chlamydomonas reinhardtii FACHB479 with cell membrane as recipient cell, successfully construct for
Conversion Chlamydomonas reinhardtiigpd1 gene Agrobacterium binary expression vector.This can not only save and build the strain of Cell wall deficiency algae
Manpower and materials, and can pair other microalgaes that cannot build Cell wall deficiency because of no database transgenic method
With certain inspiration, for future, the research of oil-producing Transgenic Microalgae high provides some thinkings.
Brief description of the drawings
Fig. 1 is binary vector con- struction figure；
Fig. 2 is that gpd1 genes are nested PCR analysis charts, A first times PCR, B second PCR, M 2kb Marker;
Fig. 3 is pMD-GPD1 qualification figures, A PCR；BNde I and EcoR I double digestions:M 5kb Marker, 1 blank,
2 pMD-GPD1；
Fig. 4 is the gpd1 sequencing comparison charts of plasmid pMD-GPD1；
Fig. 5 is plasmid pDBle-GPD1 double digestion proof diagrams, M 15kb Marker, 1-2 pDBle-GPD1；
Fig. 6 is plasmid pRI101-GPD1 double digestion proof diagrams, M 15kb Marker, 1-2 pRI-Ble-GPD1；
Fig. 7 is the gpd1 sequencing comparison charts of plasmid pRI101-GPD1；
Fig. 8 is the culture figure for converting algae strain on Ble resistances TAP, 1：Wild type FACHB-479,2：FACHB-GPD1,3：It is unloaded
Body PRI101-AN conversion algaes FACHB-479；
Fig. 9 is the molecular biological analysis figure of gpd1 genes in different algae strains；A：PCR, B：RT-PCR M: 5kb Marker,
1：Negative control（Without template）, 2：Positive control, 3：Wild type FACHB-479,4-12：Conversion algae strain；
Figure 10 is frustule laser co-focusing figure；
Figure 11 is different algae strain fat content measure figures.
Specific embodiment
1.gpdThe construction method of 1 gene Chlamydomonas reinhardtii binary expression vector pRI101-GPD1
According togpd1 gene order（NCBI GenBank accession number is NC_001136.10）, increase restriction enzyme site in upstream and downstream,
Oligo7 software Design primers are used, sequence is sequence 1 and sequence 2 in sequence table, is synthesized by company.RNA isolation kit extracts wine brewing ferment
Female DNA, is expanded using nested PCR processgpd1.PCR system is 20 μ L, containing each 0.5 μm of ol/L of upstream and downstream primer final concentration,
μ L, the Pyrobest DNA Polymerase 1U of dNTPs final concentration of 125 μm of ol/L, 10 × Pyrobest Buffer 2.Expand
Increasing program is 94 DEG C of 10min, 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 2min, carries out 30 circulations, 72 DEG C of 10min.1% fine jade
Sepharose is separated by electrophoresis PCR primer, reclaimsgpd1 gene, electrophoresis result shows the DNA fragmentation for amplifying 1175bp（Fig. 2）.gpd13 ' ends plus base A are connected to carrier pMD18-T, and heat shock is transferred to DH5 α competent cells, is applied to and is attached with 100mg/L
The LB flat boards of Amp, 37 DEG C are turned out single bacterium colony, then Liquid Culture is overnight.gpdThe PCR amplifications of 1 fragment（Fig. 3）And Nde
I and EcoR I double digestion results（Fig. 3）The expected electrophoretic band of 1175bp, company's sequence verification can be obtained（Fig. 4）Restructuring
In plasmid pMD-GPD1gpd1 sequence and saccharomyces cerevisiaegpd1 gene（NCBI GenBank accession number NC_001136.10）
100% coincide..
WithNdeI andEcoRI enzymes 37 DEG C of 4h difference digested plasmid pMD-GPD1 and pDBle, recovery product connection, heat
Conversion DH5 α competent cells are swashed, in Amp（100mg/L）The LB flat board cultures of resistance.Positive single bacterium colony digestion is accredited as restructuring
Plasmid pDBle-GPD1.Empirical tests, plasmid pDBle-GPD1 passes throughNdeI andEcoRI double digestions can obtain the pre- of 1175bp
Phase band（Fig. 5）.
PDBle-GPD1 is carried out with pRI101-AN plasmidsBamHI andKpn37 DEG C of 2h digestions of I, reclaim connection, heat shock
The competent cell of conversion DH5 α, Amp（100mg/L）The LB plate screening cultures of resistance, positive single bacterium colony digestion identification name
It is pRI101-GPD1, the plasmid pRI101-GPD1 for successfully constructing is usedBamHI andKpnI carries out double digestion obtains 4930bp bars
Band（Fig. 6）.
2.pRI101-GPD1 converts Agrobacterium
The competent cell that binary vector pRI101-GPD1 freeze-thaw methods are transformed into LBA4404 is taken, in Amp（100mg/L）Resistance
YEB plate screenings, positive colony bacterium sequence verification carriergpd1 sequence is correct（Fig. 7）.Simultaneously with empty carrier pRI 101-AN phases
The competent cell of LBA4404 is converted with condition, culture obtains empty carrier conversion Agrobacterium.
Above-mentioned freeze-thaw method method for transformation is as follows：
Take 0.1 μ g recombinant plasmids pRI101-GPD1 to be added in the LBA4404 competent cells of 100 μ l, ice puts 60-
90min；Liquid nitrogen flash freezer 5min, rapid to take out, 28 DEG C of water-bath heat shock 5min；Add 500 μ l YEB fluid nutrient mediums, 28 DEG C,
100 rpm cultivate 2-3h；Bacterium solution is uniformly coated on and contains Str (100mg/l）YEB with Amp (100mg/L) consolidates
Screened on body culture medium.Picking positive colony is sent into the sequencing of row company, the sequence in objective gene sequence and Genebank
Row (GenBank accession number is NC_001136.10) 100% is identical, and insertion point is correct (Fig. 7), and explanation containsgpd1 gene
Recombinant plasmid pRI101-GPD1 is successfully transferred in Agrobacterium LBA4404.
3. the genetic transformation of Chlamydomonas reinhardtii
By FACHB479 algae solutions in adding on 100 μm of TAP flat boards of ol/l acetosyringones, the algae of preculture 5d falls to receiving 200 μ
The even spread of LBA4404 suspension of the l containing carrier pRI101-GPD1 is contaminated, in 25 ± 2 DEG C, 1500-2000lx, brightness 1:1
After condition co-cultures 48h, frustule is harvested.Synchronously carry out the Agrobacterium-mediated Transformation culture containing empty carrier of FACHB479.
Culture can harvest positive transformants algae（Fig. 8）, single algae fall numbering after Ble（10ng/μL）The TAP liquid of resistance after
It is commissioned to train foster, the frustule of substantial amounts of different transformation plants can be collected into.And wild type FACHB-479 and empty carrier PRI101-
AN conversions algae FACHB-479 can not in the TAP culture mediums containing 10ng/ μ LBle on grow.
4. the positive identification of Chlamydomonas reinhardtii algae strain is converted
Take culture to mid log phase (cell concentration 3 × 105Individual/mL) conversion algae strain, using improvement CTAB methods extract
Frustule genomic DNA, PCR detectionsgpd1 integration.Trizol methods extract frustule RNA, with TaKaRa's
St Strand cDNA Synthesis Kit kit reverse transcriptions cDNA, the rtPCR detections of PrimeScriptTM II 1gpd1
Expression.Wherein PCR and rtPCR primers are usedgpd1-F,gpd1-R；In the reaction system of 20 μ L, comprising each 1 μ L of template,
Each 1 μ L of upstream and downstream primer（10μmol/L）, Premix Taq10 μ L；Response procedures are 94 DEG C of 10min, 94 DEG C of 30s, 59 DEG C
30s, 72 DEG C of 1min, 30 cyclic program amplifications, 72 DEG C of 10min.
The resistance algae strain for screening, the strain of display portion resistance algae it is amplifiable go out about 1175 bpgpd1 gene target band,
And wild type FFACHB-479 and empty carrier pRI 1001-AN conversion algae strains do not amplify purpose band.Extract the total of algae strain
RNA, reverse transcription PCR（RT-PCR）Detection（Fig. 9）To the strain of Partial Conversion algaegpdThe bp bands of 1 gene target 1175 clearly may be used
See, T4, T5, T6, T7, T10, T12 are defined as transgenic algae strain.
Above-mentioned CTAB methods are comprised the following steps that：
0.2g Chlamydomonas reinhardtiis are taken, liquid nitrogen is fully ground, plus every 10 during 650 2 × CTAB of μ l, 65 DEG C of water-bath 1h
Min shakes once；It is subsequently adding isometric chloroform：Isoamyl alcohol (24:1), room temperature places 30 min, 12 000 rpm centrifugations
10min, takes supernatant (with the pipette tips for subtracting, it is to avoid destruction genomic DNA), is repeated once；Supernatant adds 2 times of -20 DEG C of volumes
The absolute ethyl alcohol of precooling, precipitates more than 2h, and 12 000 rpm are centrifuged 10 min, and two are precipitated with 70% absolute ethanol washing
Secondary, ethanol is thoroughly blotted in centrifugation, it is to be dried after, add the ddH2O of 200 μ l, 4 DEG C overnight dissolve, and put -20 DEG C of preservations standby
With.
Above-mentioned Trizol methods are comprised the following steps that：
0.2g Chlamydomonas reinhardtiis are taken, liquid nitrogen is fully ground, be transferred to rapidly in the EP pipes of the 1.5ml of precooling, add 1ml
TRIzol reagents are mixed；15 ~ 30 DEG C of placement 5min, are kept completely separate nucleic acid-protein compound；4 DEG C, 10 000 rpm centrifugations
10min, takes supernatant；Plus 0.2ml chloroforms cover lid, 15s is acutely shaken, room temperature places 3min；4 DEG C, 10 000 rpm
10 min are centrifuged, sample divides three layers (bottom organic phase/interphase/upper strata colourless aqueous phase), and RNA is present in the Shui Xiang the superiors,
Transfer water is managed to new EP, adds 0.5ml isopropanol precipitatings, and 4 DEG C, 10 000 rpm are centrifuged 10 min, take supernatant；Plus
The ethanol of 1ml 75% (water treated using DEPC is prepared) washing precipitation, 4 DEG C, 7 500 rpm are centrifuged 10 min, abandon
Clearly；Room temperature is placed and dried, and is precipitated as RNA, plus 25 ~ 200 μ l DEPC water, and 55 ~ 60 DEG C are placed 10min dissolvings, put -80 DEG C
Save backup.
5. the grease identification of positive transgenic algae strain
Growth medium of taking the logarithm (cell concentration 3 × 105Individual/mL)gpd1 conversion algae strain 1mL, adds the storage of 800 μ L Nile reds
Liquid (0.1g/L Nile red/Methanol) is mixed（Final concentration 44mg/L）, centrifuge tube, 23 DEG C of lucifuge dyeing are wrapped with aluminium foil
15min, 3000rpm, 30s collect frustule, and unnecessary dyeing liquor is washed away with TAP fluid nutrient mediums.Excitation wavelength 485nm, hair
Under the conditions of ejected wave 590nm long, confocal laser scanning microscope record.The μ L of chlamydonomas reinhardtii cells 200 of above-mentioned dyeing are taken simultaneously
It is separately added into 96 orifice plates, multi-function microplate reader determines fluorescent value under the conditions of excitation wavelength 485nm, Detection wavelength 590nm
（a.u.）, analyze different algae strain grease situations.
Using the fat content of grease Nile red colouring method analysis transgenic algae FACHB-GPD.It is aobvious in laser co-focusing
In microscope fields, FACHB-GPD cells integrally take on a red color, and glassy yellow shows the grease of intracellular presence, and size and number show
It is higher than wild type algae FACHB to write（Figure 10）.Fat content, the Nile red dyeing-fluorescence of optimization are determined using multi-function microplate reader
Testing conditions are the Nile red methanol solution of final concentration 44mg/L, and 23 DEG C of lucifuges dye 15min, the transgenosis algae solution for obtaining it is glimmering
Light value is above the strain of wild type algae（Figure 11）, detection algae strain fat content is compare algae strain 1.3 ~ 1.52 times.
SEQUENCE LISTING
<110>Northwest University
<120>A kind of construction method for converting the gpd1 gene Agrobacterium binary expression vectors of Chlamydomonas reinhardtii
<130> 2016
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223>Primer
<400> 1
ggaattccat atgtctgctg ctgctgatag 30
<210> 2
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223>Primer
<400> 2
cggaattcct aatcttcatg tagatc 26
<210> 3
<211> 1176
<212> DNA
<213> Saccharomyces cerevisiae
<400> 3
atgtctgctg ctgctgatag attaaactta acttccggcc acttgaatgc tggtagaaag 60
agaagttcct cttctgtttc tttgaaggct gccgaaaagc ctttcaaggt tactgtgatt 120
ggatctggta actggggtac tactattgcc aaggtggttg ccgaaaattg taagggatac 180
ccagaagttt tcgctccaat agtacaaatg tgggtgttcg aagaagagat caatggtgaa 240
aaattgactg aaatcataaa tactagacat caaaacgtga aatacttgcc tggcatcact 300
ctacccgaca atttggttgc taatccagac ttgattgatt cagtcaagga tgtcgacatc 360
atcgttttca acattccaca tcaatttttg ccccgtatct gtagccaatt gaaaggtcat 420
gttgattcac acgtcagagc tatctcctgt ctaaagggtt ttgaagttgg tgctaaaggt 480
gtccaattgc tatcctctta catcactgag gaactaggta ttcaatgtgg tgctctatct 540
ggtgctaaca ttgccaccga agtcgctcaa gaacactggt ctgaaacaac agttgcttac 600
cacattccaa aggatttcag aggcgagggc aaggacgtcg accataaggt tctaaaggcc 660
ttgttccaca gaccttactt ccacgttagt gtcatcgaag atgttgctgg tatctccatc 720
tgtggtgctt tgaagaacgt tgttgcctta ggttgtggtt tcgtcgaagg tctaggctgg 780
ggtaacaacg cttctgctgc catccaaaga gtcggtttgg gtgagatcat cagattcggt 840
caaatgtttt tcccagaatc tagagaagaa acatactacc aagagtctgc tggtgttgct 900
gatttgatca ccacctgcgc tggtggtaga aacgtcaagg ttgctaggct aatggctact 960
tctggtaagg acgcctggga atgtgaaaag gagttgttga atggccaatc cgctcaaggt 1020
ttaattacct gcaaagaagt tcacgaatgg ttggaaacat gtggctctgt cgaagacttc 1080
ccattatttg aagccgtata ccaaatcgtt tacaacaact acccaatgaa gaacctgccg 1140
gacatgattg aagaattaga tctacatgaa gattag 1176
Claims (6)
1. one kind contains saccharomyces cerevisiaegpdThe binary expression vector of 1 gene, it is characterised in that the construction method bag of the carrier
Include following steps：
（1）gpd1 gene is DNA molecular, the genome from saccharomyces cerevisiae, by embedding shown in SEQ ID No.3 in sequence table
Set PCR is obtained, and in the addition of gene two endsEcoRI andNdeI restriction enzyme sites, its primer sequence is SEQ ID No.1 in sequence table
With the DNA molecular shown in SEQ ID No.2；
（2）Reaction solution and carrier pDBle after PCR is expanded with NdeI andEcoRI double digestions, and the digestion products that will be obtained
Use T4DNA ligase is connected, and converts bacillus coli DH 5 alpha, and picking monoclonal Amplification Culture extracts the checking of plasmid double digestion, will
Positive recombinant plasmid is named as recombinant plasmid pDBle-GPD1；
（3）PDBle-GPD1 and pRI101-AN plasmids are usedBamHI andKpnI double digestions, reclaim connection, heat-shock transformed
The competent cell of DH5 α, Amp（100mg/L）The LB plate screening cultures of resistance, positive recombinant plasmid is named as pRI101-
GPD1。
2. it is according to claim 1 to contain saccharomyces cerevisiaegpdThe binary expression vector of 1 gene, it is characterised in that described double
First expression vector is pRI101-GPD1.
3. saccharomyces cerevisiae is contained described in claim 1gpdThe construction method of the binary expression vector of 1 gene, it is characterised in that bag
Include following steps：
（1）gpd1 gene is DNA molecular, the genome from saccharomyces cerevisiae, by embedding shown in SEQ ID No.3 in sequence table
Set PCR is obtained, and in the addition of gene two endsEcoRI andNdeI restriction enzyme sites, its primer sequence is SEQ ID No.1 in sequence table
With the DNA molecular shown in SEQ ID No.2；
（2）Reaction solution and carrier pDBle after PCR is expanded with NdeI andEcoRI double digestions, and the digestion products that will be obtained
Use T4DNA ligase is connected, and converts bacillus coli DH 5 alpha, and picking monoclonal Amplification Culture extracts the checking of plasmid double digestion, will
Positive recombinant plasmid is named as recombinant plasmid pDBle-GPD1；
（3）PDBle-GPD1 and pRI101-AN plasmids are usedBamHI andKpnI double digestions, reclaim connection, heat-shock transformed
The competent cell of DH5 α, Amp（100mg/L）The LB plate screening cultures of resistance, positive recombinant plasmid is named as pRI101-
GPD1。
4. saccharomyces cerevisiae is contained described in claim 1gpdThe genetic transforming method of the binary expression vector of 1 gene, its feature exists
In comprising the following steps：
（1）Agrobacterium LBA4404 competence is prepared, recombinant plasmid pRI101-GPD1 is then transformed into root by freeze-thaw method
Cancer Agrobacterium, is containedgpdThe agrobacterium tumefaciens lba4404 of 1 gene；
（2）The positive Agrobacterium LBA4404 for obtaining is infected into Chlamydomonas reinhardtii FACHB479 to be turnedgpd1 gene Chlamydomonas reinhardtii；
（3）Extract Chlamydomonas reinhardtii genomic DNA and RNA and positive transgenic plant is identified by PCR and RT-PCR；
（4）Selection Chlamydomonas reinhardtii transformant, fat content mirror is carried out using the Nile red decoration method of improvement through multi-function microplate reader
It is fixed, and grease staining conditions are observed under laser confocal microscope.
5. the binary expression vector described in claim 1 is carrying external sourcegpd1 gene has the Rhein of cell membrane into wild type
Application in chlamydomonas genome.
6. apply according to claim 5, it is characterised in that：Invaded using the Agrobacterium LBA4404 4 for carrying binary expression vector
Dye wild type has the Chlamydomonas reinhardtii FACHB-479 of cell membrane, and genes of interest is transferred into Rhein in the presence of acetosyringone
In the genome of chlamydomonas FACHB-479, and verified with PCR, RT-PCR and Nile red decoration method.
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Citations (3)
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| WO2015023989A1 (en) * | 2013-08-15 | 2015-02-19 | Lallemand Hungary Liquidity Management Llc | Methods for the improvement of product yield and production in a microorganism through glycerol recycling |
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