Co-overexpression of native phospholipid-biosynthetic genes plsX and plsC enhances lipid production in Synechocystis sp. PCC 6803

Abstract

The overexpression of native plsX and plsC genes involving in fatty acid/phospholipid synthesis first timely-reported the significantly enhanced lipid contents in Synechocystis sp. PCC 6803. Growth rate, intracellular pigment contents including chlorophyll a and carotenoids, and oxygen evolution rate of all overexpressing (OX) strains were normally similar as wild type. For fatty acid compositions, saturated fatty acid, in particular palmitic acid (16:0) was dominantly increased in OX strains whereas slight increases of unsaturated fatty acids were observed, specifically linoleic acid (18:2) and alpha-linolenic acid (18:3). The plsC/plsX-overexpressing (OX + XC) strain produced high lipid content of about 24.3%w/dcw under normal condition and was further enhanced up to 39.1%w/dcw by acetate induction. This OX + XC engineered strain was capable of decreasing phaA transcript level which related to poly-3-hydroxybutyrate (PHB) synthesis under acetate treatment. Moreover, the expression level of gene transcripts revealed that the plsX- and plsC/plsX-overexpression strains had also increased accA transcript amounts which involved in the irreversible carboxylation of acetyl-CoA to malonyl-CoA. Altogether, these overexpressing strains significantly augmented higher lipid contents when compared to wild type by partly overcoming the limitation of lipid production.

Figure 1

Overview of metabolic pathways representing the conversion of acetyl-CoA to membrane lipid biosynthesis under growth condition in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (modified from and). Abbreviations AAS: putative acyl-ACP synthetase, accBCDA: Acetyl-CoA carboxylase gene subunits BCDA, ACP: Acyl Carrier Protein, CBB: Calvin-Benson-Bassham, G3P: Glyceraldehyde-3-phasphate, LipA: putative lipase, PhaA: beta-ketothiolase, PHB: poly-3-hydroxybutyrate, PlsX: fatty acid/phospholipid synthesis protein or putative phosphate acyltransferase, PlsY: putative acylglycerol-P acyltransferase (no data available in Cyanobase), PlsC: putative 1-acyl-glycerol-P acyltransferase, PtdOH: phosphatidic acid and TCA: Tricarboxylic acid.

Figure 2

Physical map representing psbA2 locus in Synechocystis sp. PCC 6803 wild type (WT), with the inserted plsX, plsC and plsC/plsX genes in different engineered strains, hereinafter OX + X, OX + C and OX + XC, respectively. The specific primers (Table 2) were used to recombine each gene into Synechocystis genome. The WT control cells contained an inserted Cm^r gene cassette in their genome. The size of each gene fragment was shown correspondingly under the map.

Figure 3

Confirmation of insertion and complete segregation using PCRs with genomic DNA as the template from WT, WTc and the OX strains, including OX + X (A), OX + C (B) and OX + XC (C), corresponding to the physical map in Fig. 2. Lane M: GeneRuler^TM DNA ladder (Fermentas). The different primer pairs was used to amplify each gene fragment as indicated in Table 3 including different 10 pairs of primers. The cropped gels (in C) were taken from the same gel cutting out the repeated bands of transformants as shown in Supplementary information.

Figure 4

Relative transcript levels of plsC, plsX and accA performed by RT-PCR in WT, WTc and OX strains grown under normal growth condition (A). The 16 s RNA was used as reference control. The ratios of relative band intensity of gene/16 s were shown in mean ± S.D. (n = 3) (B). The cropped gels of OX + X were taken from the different gels as shown in Supplementary information.

Figure 5

The optical density (OD) at 730 nm (A) of 16 day-cell culture, chlorophyll a (Chl a) and carotenoid (Car) contents (B) and oxygen evolution rate (C) of each strain grown at mid-log phase. Data represent mean ± S.D. (n = 3). Statistical significance between those levels of WT and OX strains was represented at p < 0.05.

Figure 6

The contents of total lipid and unsaturated lipid (A) and the fatty acid compositions measured by GC instrument (B) of WT, WTc, and OX strains. Data represent mean ± S.D. (n = 3). Statistical significance between those levels of WT and OX strains was represented at p < 0.05.

Figure 7

Effect of acetate supplementation on contents of total lipid (A) and unsaturated lipid (B) in WT, WTc, and OX strains at time indicated. Data represent mean ± S.D. (n = 3).

Figure 8

Relative transcript levels of plsC, plsX, accA, aas, lipA and phaA performed by RT-PCR in WT and OX + XC strain when cells supplemented with acetate at day 4 of treatment (A). The 16s RNA was used as reference control. The ratios of relative band intensity of gene/16s were shown in (B). The cropped gels of WT and OX + XC were taken from the different gels as shown in Supplementary information.