Engineered cyanobacteria with enhanced lipid production

Abstract

A recombinant strain of F. diplosiphon was made by transforming wild type F. diplosiphon with a pGEM-7Zf (+) plasmid containing sterol desaturase gene (SD) via electroporation. The recombinant strain was designated B481-SD and overexpressed the sterol desaturase gene to result in enhanced lipid production. Selection made on NaCl enabled growth of the transformant to thrive up to 50 g L.sup.1 NaCl. This strain was designated B481-SDH.

Claims

1. A recombinant Fremyella diplosiphon cyanobacterium comprising at least one plasmid, said at least one plasmid including at least one polynucleotide having the sequence of SEQ ID NO: 1 which encodes a sterol desaturase polypeptide.

2. The recombinant Fremyella diplosiphon cyanobacterium according to claim 1, having a higher salt tolerance as compared to wild-type Fremyella diplosiphon cyanobacterium.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a representation of the identification of the sterol desaturase gene.

(2) FIG. 2 is a representation of the plasmid pGEM-7Zf.

(3) FIG. 3 shows electrophoresis of pGEM-7Zf-SD plasmid after triple digestion.

(4) FIG. 4 shows the relative quantification of sterol desaturase transcript levels of the wild type (WT) and transformed strain (B481-SD).

(5) FIG. 5 shows wild type B481 and transformant B481-SD grown on BG11 media containing 80 mg L.sup.1 ampicillin.

(6) FIG. 6 shows the growth of Fremyella diplosiphon wild type (WT) and transformant (B481-SD) in BG11/HEPES medium over a ten day-period.

(7) FIG. 7 shows the effect of sterol desaturase gene overexpression on growth rates of Fremyella diplosiphon wild type (WT) and transformant (B481-SD).

(8) FIG. 8 shows the comparison of total lipid content in wild type (WT) and transformant (B481-SD) Fremyella diplosiphon.

(9) FIG. 9 shows fatty acid methyl ester (FAME) composition of wild type (WT) and transformant (B481-SD) Fremyella diplosiphon total lipids subjected to direct transesterification.

(10) FIG. 10 shows fatty acid methyl ester (FAME) abundance in transesterified extractable lipids of wild type (WT) and transformant (B481-SD) Fremyella diplosiphon determined using GCGC-TOFMS.

(11) FIG. 11 shows the results of a growth experiment comparing wild type and transformant strains in media amended with various concentrations of sodium chloride.

(12) FIG. 12 shows relative quantification (RQ) of sterol desaturase transcript levels in Fremyella diplosiphon wild type (WT) and halotolerant transformant (B481-SDH).

DETAILED DESCRIPTION OF THE INVENTION

(13) 1. Methods and Materials

(14) 1.1. Strain and Culture Conditions

(15) F. diplosiphon strain B481 was used as the wild type for transformation. Cultures were grown in liquid BG-11 medium containing 20 mM HEPES (hereafter referred to as BG-11/HEPES) at 170 rpm and 28 C. for seven days under continuous white light adjusted to 30 mol m.sup.2s.sup.1 using the model LI-190SA quantum sensor (Li-Cor, USA). Escherichia coli FB5a competent cells (Thermo Fisher Scientific, USA) were grown at 37 C. in Luria-Bertani (LB) broth or agar plates containing 80 mg L.sup.1 ampicillin as the selective antibiotic.

(16) 1.2. Extraction of Complementary DNA

(17) Total RNA was extracted from F. diplosiphon B481 strain at an optical density of 0.6 at 750 nm (OD.sub.750) using Tri Reagent (Molecular Research Center, Inc., USA) according to the manufacture protocol. The quantity and quality of extracted RNA were verified using electrophoresis on agarose gels stained with GelRed (Phenix Research, USA) and A260/280 spectrophotometric ratio. Complementary DNA (cDNA) was reverse transcribed using high capacity RNA to cDNA (Life Technologies, USA).

(18) 1.3. PCR Screening and Sequencing of F. diplosiphon Sterol Desaturase Gene

(19) Homologs of the lipid production sterol desaturase gene were identified in F. diplosiphon. PCR amplification was performed with 50 ng cDNA using a C1000 Touch Thermocycler (Bio-Rad, USA). Primers designed to amplify the genes contained a HindIII restriction site added to the 5 ends and BamHI to the 3 ends. PCR products electrophoresed on a 1.2% agarose gel (FIG. 1) were excised at the expected size ranges and cDNA bands extracted using the Gel Recovery kit (Zymo Research, USA). Amplified fragments were sequenced using ABI 3130 XL Genetic Analyzer (Life Technologies, USA) and chromatograms were analyzed using Finch TV Version 1.4.0 (Geospiza Inc., USA). Basic Local Alignment Sequence Tool (BLAST) and BLASTx analysis were performed to confirm the homology of the genes. Sequences were compared to the NCBI nucleotide collection database to determine the percentage of identity relative to homologous genes in other species and submitted to GenBank.

(20) 1.4. Construction of Expression Plasmids

(21) Expression plasmids pGEM-7Zf-SD (FIG. 2) with native promoters were constructed to overexpress the sterol desaturase gene. Amplified gene product was double digested with HindIII, BamHI, and pGEM-7Zf (+) vector (Promega, USA) was triple digested with HindIII, BamHI and ScaI enzymes and bands extracted. Purified inserts were cloned and ligated into the vector with T4 DNA ligase (NEB, USA) at the corresponding restriction sites. Plasmids were transformed into E. coli FB5u competent cells (Thermo Fisher Scientific, USA) via heat shock at 42 C. for 20 s and incubated in super optimal broth with catabolite repression media at 37 C. for 1 h (Thermo Fisher Scientific, USA). Transformed competent cells were plated on LB agar plates containing 80 mg L.sup.1 ampicillin and incubated for 16 h at 37 C. Resistant colonies were selected and grown overnight in liquid LB medium containing ampicillin. Plasmids were extracted from transformed cells and checked for the presence of the insert by PCR and further confirmed by triple digestion and sequence analysis.

(22) 1.5. Electroporation-Mediated Transformation

(23) Expression plasmids harboring the sterol desaturase gene were transformed into wild type F. diplosiphon using electroporation as described by Kehoe and Grossman. Cultures were grown in BG-11/HEPES to an optical density at 750 nm (OD.sub.750) of 0.5 under continuous white light at 15 mol m.sup.2s.sup.1 followed by 72 hours in dark at 170 rpm. After centrifugation at 3450g for 10 min, the pellet was washed thrice with distilled water and the supernatant removed. Concentrated cells (40 l) were mixed with 6 g purified plasmid DNA on ice and electroporated using a GenePulser Xcell (Bio-Rad, USA) at 200 resistance, 1.0 kV, and 25 g capacitance. After incubation on ice for 20 min, cells were grown in 10 ml BG11/HEPES liquid cultures for 16 h and transferred to BG-11/HEPES solid plates supplemented with 80 mg L.sup.1 ampicillin for selection of electrotransformants. Stability of sterol desaturase in the transformants was confirmed by weekly subculture on 80 mg L.sup.1 ampicillin for over 28 generations.

(24) 1.6. PCR Analysis of Transformed Strains

(25) To verify the insertion of the sterol desaturase gene, we performed PCR with primers specific for this gene. Amplifications were performed in a 50 l, reaction volume containing 25 l of 2KOD Hot Start Master Mix (EMD Millipore, USA), 1.5 l each of 10 M forward and reverse primers, 50 ng template cDNA and nuclease free water. PCR amplifications were performed as mentioned above and products visualized on a 1.2% agarose gel with a GeneRuler 100 bp plus ladder.

(26) 1.7. RNA Extraction and cDNA Synthesis

(27) Total RNA in WT and B481-SD was extracted and cDNA synthesized according to the manufacture's protocol.

(28) 1.8. Quantification of F. Diplosiphon Gene Expression Levels Using RT-qPCR

(29) Reverse Transcription-quantitative PCR (RT-qPCR) was used to quantify gene overexpression in the transformant. Real-time amplifications performed using SYBR green master mix (Applied Biosystem, USA). A Thermal Cycler CFX96 Real Time machine (Bio-Rad, USA) was used to perform RT-qPCR. The reaction was performed in 20 l volume containing 10 l SYBR Master Mix, 10 ng cDNA template. Amplifications were performed under the following conditions: 95 C. for 20 s; 95 C. for 20 s; and 40 cycles of 95 C. for 30 s. Four replicates were maintained for each treatment type and relative quantification (RQ) data of the wild type and transformant was analyzed using the Ct method with CFX Manager 3.1 (Bio-Rad, USA).

(30) 1.9. Culture of Transformant in 80 mg L.sup.1 Ampicillin

(31) The transformant was cultured in liquid BG-11/HEPES media containing 80 mg L.sup.1 ampicillin over a ten day-period under conditions described above. WT grown in the absence of ampicillin served as control. Culture conditions were maintained as described above and growth (OD.sub.750) measured every 24 h using a Cynmar 1105 spectrophotometer (Cynmar, USA). B481-SD was grown in BG11/HEPES media containing 80 mg L.sup.1 ampicillin over 20 generations.

(32) 1.10. Analysis of Transformant's Photosynthetic Efficiency

(33) Photosynthetic efficiency of the transformant was examined by extraction and quantification of chla and phycobiliproteins and compared to the wild type. For estimation of chla, absorption spectra of WT and transformant were measured at A.sub.665 while for phycobiliproteins quantified at A.sub.565, A.sub.620 and A.sub.650.

(34) 1.11. Lipid Production Analysis

(35) Total lipid contents in WT and B481-SD F. diplosiphon was determined using a chloroform:methanol extraction method based on Folch, et al. reported in Wahlen, et al. Dried samples (80-100 mg) were sonicated in 5 mL of chloroform:methanol (2:1 by volume) for 30 s and centrifuged at 6000 rpm, washed with 1 mL distilled water, and centrifuged at 2000 rpm to facilitate phase separation. Methanol and sulfuric acid partitioned with water in the upper phase, while lipids separated with chloroform in the lower phase. The organic phase was transferred into a new tube and extraction was repeated twice to collect residual lipids. The organic phase was transferred into pre-weighted flasks followed by drying in a rotary evaporator and weighed to determine lipid yield of each sample. Three biological replicates were maintained and the experiment repeated once. Significance among cumulative treatment means was determined using ANOVA and Tukey's honest significant differences post hoc test at 95% confidence intervals (P<0.05). The single factor, fixed-effect ANOVA model, Yij=+Si+ij, was used where Y is the total lipid yield content in strain i and biological replicate j. The represents overall total lipid content mean with adjustments from the effects of strain (S), and ij is the experimental error from genotype i and biological replicate j.

(36) 1.12 Gas Chromatography-Mass Spectrometry of Transesterified Product for FAME Analysis:

(37) We determined FAME composition of transesterified material using a Shimadzu GC17A/QP5050A GC-MS combination (Shimadzu Instruments, USA) at the Mass Spectrometry Facility at Johns Hopkins University (Baltimore, Md.). The GC17A was equipped with a low-polarity (5% phenyl-, 95% methyl-siloxane) capillary column (30 m length, 0.25 mm ID, 0.25 m film thickness, and 10 m length guard column). We identified peaks by comparing mass spectra to The Lipid Web Archive of FAME Mass Spectra. Three biological replicates of each sample were analyzed, and the experiment repeated once. In addition, we also calculated theoretical chemical and physical properties of the transesterified lipids from FAME composition (w %) using BiodieselAnalyzer software Version 2.2.

(38) 1.13 GCGC-TOFMS analysis of total transesterified lipids

(39) High-resolution GCGC-TOFMS from LECO (USA) was used to identify FAMEs from the WT and B481-SD strains. Total lipids were extracted, subjected to direct transesterification as explained above, dried under dry N.sub.2 (g), and reconstituted in 2 mL dichloromethane with cholestane (50 g mL.sup.1) spiked as an internal standard.

(40) 1.14 Growth of Transformant in Increasing NaCl Concentrations

(41) Growth of the transformant and wild type were evaluated in BG11/HEPES containing various concentrations of NaCl. B481-SD was cultured in 250 ml conical flasks containing 100 ml liquid BG-11/HEPES media at NaCl concentrations ranging from 10 to 50 g L.sup.1 over a ten day-period under conditions described above.

(42) 1.15. RNA Extraction and cDNA Synthesis

(43) Total RNA of the halotolerant strain (B481-SDH) was extracted and cDNA synthesized according to the manufacturer's protocol.

(44) 1.16. Quantification of B481-SDH Gene Expression Levels Using RT-qPCR

(45) SD gene overexpression in B481-SDH was quantified using RT-qPCR. Real-time amplifications were performed using SYBR green master mix (Applied Biosystem, USA) as mentioned above.

(46) 2. Results

(47) 2.1. Identification of Sterol Desaturase Homologues in Fremyella Diplosiphon

(48) Primer-specific amplification of F. diplosiphon revealed a single discrete band at the expected size of 1314 base pairs for the SD gene (FIG. 1). Purified products were successfully subjected to Sanger sequencing and NCBI BLAST analysis to confirm identity of the SD gene and its encoded proteins. Results of this study revealed an open reading frame of 1314 base pairs encoding 437 amino acids for the SD protein. The sequence alignment identified a 94% match to SD gene in F. diplosiphon, thus confirming the identity of the gene. The amino acid sequence of SD revealed 93%, 86%, 77%, and 77% identities to Nostoc carneum NIES-2107, Calothrix sp. NIES-2100, Nodularia sp. NIES-3585, and Fortiea contorta respectively. The SD sequence was deposited at NCBI Genbank with the accession number MH329183.

(49) 2.2. Cloning and Transformation of Sterol Desaturase Gene in F. Diplosiphon

(50) Amplified products and pGEM-7Zf (+) vector were double and triple digested using HindIII, BamHI, and ScaI followed by ligation of purified inserts into the vector at the corresponding restriction sites (FIG. 3). pGEM-7Zf-SD plasmid containing the SD gene was constructed, cloned, and transformed into WT via electroporation. Gene insertion was confirmed by primers specific to the SD gene and Sanger sequencing. The resultant SD-expressing F. diplosiphon strain was designated B481-SD.

(51) 2.3. RT-qPCR Analysis Confirms Overexpression of Sterol Desaturase in B481-SD

(52) A significant increase in mRNA transcript level was detected in F. diplosiphon transformant. Relative quantity values revealed a 64-fold increase in expression of SD gene in B481-SD compared to WT (FIG. 4).

(53) 2.4 Validation of Transformant Growth and Photosynthetic Pigment Accumulation in Ampicillin Media

(54) No significant difference was observed in growth and growth rate of B481-SD in liquid BG-11/HEPES medium containing 80 mg L.sup.1 ampicillin (FIGS. 5-7). B481-SD was persistently grown in BG11/HEPES media containing 80 mg L.sup.1 ampicillin over 28 generations.

(55) 2.5 Determination of Total Lipid Content in Transformant Strain by Gravimetric Analysis

(56) Results of gravimetric analysis revealed a significant increase in B481-SD total lipid yield compared to the WT (FIG. 8). While a total lipid content of 17% cellular dry weight (CDW) was detected in the wild type, 23% CDW lipid yield was quantified in the transformant. Thus, a 27% increase in total lipid was observed in B481-SD relative to SVT.

(57) 2.6 Characterization of FAME in Wild Type and Transformant Strain by GC-MS

(58) We identified methyl palmitate, the methyl ester of hexadecanoic acid (C16:0) as the most abundant FAME, which accounted for 76.35% and 65.93% of total FAMEs produced from WT and B481-SD total lipids respectively (Table 1).

(59) Table 1 shows breakdown of saturated and unsaturated fatty acid methyl ester (FAME) proportions in wild type (WT) and transformant (B481-SD) Fremyella diplosiphon.

(60) TABLE-US-00001 TABLE 1 FAME Type (%) Ratio of FAME Strain Saturated Unsaturated Saturated/Unsaturated WT 80.99 19.01 4.26 B481-SD 76.62 23.38 3.27

(61) In addition to methyl palmitate, other FAMEs including methyl tetradeconate (C14:1), methyl hexadecenoate (C16:1), methyl octadecanoate (C18:0), methyl octadecenoa to (C18:1), and methyl octadecadienoate (C18:2) were identified (Table 2).

(62) Table 2 shows composition of fatty acid methyl ester in transesterified lipids of Fremyella diplosiphon wild type (WT) and transformant (B481-SD) strains.

(63) TABLE-US-00002 TABLE 2 WT B481-SD :0.sup.b :1 :2 SUM :0 :1 :2 SUM C14.sup.a 3.07 3.07 3.21 3.21 C16 72.39 11.66 84.05 65.67 7.67 73.34 C18 8.60 1.79 2.49 12.88 10.95 5.16 7.34 23.45 SUM 80.99 16.52 2.49 100 76.62 16.04 7.34 100 .sup.aColumn represents length of carbon chain. .sup.bRow represents degree of saturation (number of double bonds in chain).

(64) Results of the study revealed significant increases in methyl octadecenoate (C18:1), and methyl octadecadienoate (C18:2) levels from B481-SD transesterified lipids, while no significant differences were observed in other components obtained from F. diplosiphon transesterified lipids (FIG. 9).

(65) 2.7 Lipid Characterization in Transformant Strain by GCGC-TOFMS

(66) GCGC-TOFMS analysis revealed the presence of FAMEs with carbon number from 12-18, as well as alkanes from C11 to C34. However, FAME abundance in B481-SD (80.92% TL) was significantly higher than WT (77.92% TL) (FIG. 10). Our results revealed FAME compounds such as C12:0 C15:0, C18:3, and C18:4 identified by GCGC-TOFMS which were not detected in 1D GC-MS.

(67) 2.8. Transformed Strain Exhibit Enhanced Halotolerance

(68) F. diplosiphon B481-SD was capable of growth in liquid BG-11/HEPES medium at concentrations up to 50 g L.sup.1 NaCl (FIG. 11).

(69) Selection of the transformant was conducted at high salt content (40 g L.sup.1 NaCl). The resultant strain was named B481-SDH. RT-PCR shows B481-SDH transcript levels were 38-fold higher at 40 g L.sup.1 NaCl compared to WT (See FIG. 12).

(70) TABLE-US-00003 SEQ.ID.NO.1 LOCUS Seq1[Fremyella1314bpmRNAlinearBCT10-MAY-2018 DEFINITION diplosiphon]UTEXB481F.diplosiphonSteroldesaturasemRNA, completecds. ACCESSION Seq1[Fremyella VERSION KEYWORDS . SOURCE Tolypothrixsp.PCC7601(FremyelladiplosiphonCCAP1429/1) ORGANISM Tolypothrixsp.PCC7601 Bacteria;Cyanobacteria;Nostocales;Tolypothrichaceae; Tolypothrix. REFERENCE 1(bases1to1314) AUTHORS Sitther,V.,GharaieFathabad,S.,SigamaniArumanayagam,A.and Tabatabai,B. TITLE OverexpressionofsteroldesaturasegeneinFremyelladiplosiphon JOURNAL Unpublished REFERENCE 2(bases1to1314) AUTHORS Sitther,V.,GharaieFathabad,S.,SigamaniArumanayagam,A.and Tabatabai,B. TITLE DirectSubmission JOURNAL Submitted(09-MAY-2018)Biology,MorganStateUniversity,1700East ColdSpringLane,Baltimore,MD21251,UnitedStates COMMENT BankitComment:ALTEMAIL:sogha1@morgan.edu. BankitComment:TOTAL#OFSEQS:1. ##Assembly-Data-START## SequencingTechnology::Sangerdideoxysequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1...1314 /organism=Tolypothrixsp.PCC7601 /moltype=mRNA /strain-UTEXB481 /isolationsource=Freshwater /dbxref=taxon:1188 /note=[culturedbacterialsource] gene 1..1314 /gene=Steroldesaturase /note=Increaselipidcontent CDS 1...1314 /gene=Steroldesaturase /note=[intronlessgene];Increaselipidcontent;Lipid enhancement /codon_start=1 /transl_table=11 /product=Steroldesaturase 1 ttgacttttgacttcttcatagcgggactagcgctgataaaaaccgagtacagaaaaatt 61 acgatgaatgttttagcacaaagctgggctgagattgcggctcaattacagataaattgg 121 aatctggtaaatacctgcttgcagtttgctagttggggattagtctcgctgttgttggta 181 gagatagtgagagatagctatcatgctttgtgtcactatgtcccctcgcttggtaaatgg 241 cataataagcaccacatggcgtatcgccgcgatttatcggtagtttctttaaaaatttac 301 caagagtctcagttatacaatgatattgtcgagtcaacgctactggttgtagttttgact 361 gtgatggctttactgctacagcaatggggcttttggttgggagtagtctatgctttcacc 421 tttttatatggcgcgtcccggcgatattttctcggtaaaattgatacagattacactcac 481 ctccccgggccattagaaactattccctcggtttggtgggtaaatcgttcttaccactgg 541 cgacatcattttgatgatgttaacgcctattacagtggtgtgtttcctttagtagatacg 601 gtattgggaacaggtttatctcttaaaggtaaaaccattgctttaactggtgcttccggt 661 gctttagggcaagcattgactgctgaattgattaaaaataatgccaaggtagtagcctta 721 actaccaatcccgaaaaactacagcctcaagaaaagctaactgtaattgcttgggaattg 781 ggtaaggaagcagagttaaaagctgctttagagaaagttgatattttgattatcaatcac 841 ggtgtcaatgtctacgctaaccgcacctcagaagcaattgagtcttcttatgaggtgaat 901 actttttctacattgcggttgatggatatatttttggcaaccgttaccgggccgcaatcc 961 aaagcaactaaagaaatttgggttaacacttccgaagctgaagtatctccggctttaagt 1021 cctctttatgaactcagtaaacgcgctatcggagatattgttaccctcaagcgtttggat 1081 ggggattgtataattcgcaagttaattctggggccgtttaagagtcaacttaatccttat 1141 ggggtgatgtcagcgccgcaagtagcccgtgcaattttgtttttagcaaagcgggacttc 1201 cgcaatattattgtgtccatcaatcccctgacatatctgctgtttccgttgaaggaagtt 1261 agcacttggctttactaccgaatctttagtaaaaaggttcaatctttgaactaa //