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.2022 Sep 24;15(1):99.
doi: 10.1186/s13068-022-02197-9.

Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803

Affiliations

Enhanced productivity of extracellular free fatty acids by gene disruptions of acyl-ACP synthetase and S-layer protein in Synechocystis sp. PCC 6803

Kamonchanock Eungrasamee et al. Biotechnol Biofuels Bioprod..

Abstract

Background: Based on known metabolic response to excess free fatty acid (FFA) products, cyanobacterium Synechocystis sp. PCC 6803 preferentially both recycles via FFA recycling process and secrets them into medium. Engineered cyanobacteria with well growth and highly secreted FFA capability are considered best resources for biofuel production and sustainable biotechnology. In this study, to achieve the higher FFA secretion goal, we successfully constructs Synechocystis sp. PCC 6803 mutants disrupting genes related to FFA recycling reaction (aas gene encoding acyl-acyl carrier protein synthetase), and surface layer protein (encoded by sll1951).

Results: Three Synechocystis sp. PCC 6803 engineered strains, including two single mutants lacking aas (KA) and sll1951 (KS), and one double mutant lacking both aas and sll1951 (KAS), significantly secreted FFAs higher than that of wild type (WT). Certain increase of secreted FFAs was noted when cells were exposed to nitrogen-deficient conditions, BG11-half N and BG11-N conditions, with the exception of strain KS. Under BG11-N condition at day 10, strain KAS strikingly secreted FFAs products up to 40%w/DCW or 238.1 mg/L, with trace amounts of PHB. Unexpectedly, strain KS, with S-layer disruption, appeared to have endured longer in BG11-N growth medium. This strain KS significantly acclimated to the BG11-N environment by accumulating a greater glycogen pool with lower FFA production, whereas strain KA favored higher PHB and intracellular lipid accumulations with moderate FFA secretion.

Conclusions: Mutations of both aas and sll1951 genes in Synechocystis sp. PCC 6803 significantly improved the productivity of secreted FFAs, especially under nitrogen deprivation.

Keywords: Acyl–acyl carrier protein synthetase; Free fatty acid secretion; Nitrogen deprivation; S-layer protein; Synechocystis sp. PCC 6803.

© 2022. The Author(s).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Overview of the production of lipids and free fatty acid (FFA), and FFA secretion into the growth medium in the cyanobacteriumSynechocystis sp. PCC 6803 (modified from [9, 36]). Abbreviations of genes:accDACB, a multisubunit acetyl-CoA carboxylase gene;aas, acyl-ACP synthetase;glgA, glycogen synthase;glgC, ADP-glucose pyrophosphorylase;glgP, glycogen phosphorylase;glgX, glycogen isoamylase;glpD, glycerol-3-phosphate dehydrogenase;lipA, a lipolytic enzyme-encoding gene;phaA, β-ketothiolase;phaB, acetoacetyl-CoA reductase;phaEC, the heterodimeric PHB synthase;plsX andplsC, putative phosphate acyl-transferases;RuBisCO, the RuBisCO gene cluster includingrbcLSX, encoding RuBisCo large, small and chaperone subunits, respectively;sll1951, the surface (S) layer protein. Abbreviations of intermediates: DHAP, dihydroxyacetone phosphate; FASII, fatty acid synthesis type II; fatty acyl-ACP, fatty acyl–acyl carrier protein; FFAs, free fatty acids; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; Gro3P, glycerol-3-phosphate; 3PG, 3-phosphoglycerate; PHB, polyhydroxybutyrate; RuBP, ribulose-1,5-bisphosphate; TCA cycle, Tricarboxylic acid cycle
Fig. 2
Fig. 2
Genomic maps of the engineeredSynechocystis sp. PCC 6803 strains, KS and KAS.A Double homologous recombination occurred between the conserved sequences ofsll1951 orS-layer gene on the recombinant pJSKm plasmid containing an antibiotic kanamycin resistant cassette (Kmr) and genomic DNA of WT or KA host strain, generating KS or KAS strain, respectively. Confirmations of engineered strains were performed by PCR analysis using selected pairs of specific primers (shown in Table 2). For (B) KS strain; Lane M: GeneRuler DNA ladder (Fermentus); Lane 1: Negative control using WT as template (a–c), a Lanes 2–3: clone numbers 1 to 2 using Km_F and Km_R primers, b Lanes 2–3: clone numbers 1 to 2 using Sll1951_F and Sll1951_R primers, and c Lanes 2–4: clone numbers 1 to 3 using Sll1951_UF and Sll1951_R primers, and only positive clones (numbers 2 and 3) were selected for next experiments. For (C) KAS strain. Lane M: GeneRuler DNA ladder (Fermentus); Lane 1: Negative control using KA as template (a–c), a Lanes 2–5: clone numbers 1 to 4 using Km_F and Km_R primers, b Lanes 2–5: clone numbers 1 to 4 using Sll1951_UF and Km_SR primers, and c Lane 2: clone number 1 using Sll1951_UF and Sll1951_R as the primers, and only positive clone number 1 was selected for next experimentsq4
Fig. 3
Fig. 3
Growth curve (A), oxygen evolution rate (B), chlorophylla content (C), and carotenoid content (D) of WT, KS, KA, and KASSynechocystis sp. PCC 6803 strains cultured in BG11 medium for 14 days. In (A), (C), and (D), the error bars represent standard deviations of means (mean ± S.D.,n = 3). In (B), the oxygen evolution rate was measured using log phase-growing cells (5 days). Data represent mean ± S.D.,n = 3. Means with the same letter are not significantly different with the significance level atP < 0.05
Fig. 4
Fig. 4
Growth curve (A), chlorophylla content (B), carotenoid content (C), and images of cultured flasks of WT, KS, KA, and KASSynechocystis sp. PCC 6803 strains cultured in BG11 containing 8.8 mM NaNO3 medium (BG11-half N) during 15 days of cultivation. The error bars represent standard deviations of means (mean ± S.D.,n = 3)
Fig. 5
Fig. 5
Growth curve (A), chlorophylla content (B), carotenoid content (C), and images of cultured flasks of WT, KS, KA, and KASSynechocystis sp. PCC 6803 strains cultured in BG11 without the addition of NaNO3 medium (BG11-N) during 15 days of cultivation. The error bars represent standard deviations of means (mean ± S.D.,n = 3)
Fig. 6
Fig. 6
Contents of total intracellular lipids (A) and extracellular FFAs (B), and total contents of total intracellular lipids and extracellular FFAs (C) of WT, KS, KA, and KASSynechocystis sp. PCC 6803 strains growing in BG11-half N and BG11-N at 0, 5 and 10 days, respectively. The error bars represent standard deviations of means (mean ± S.D.,n = 3). Means with the same letter are not significantly different with the significance level atP < 0.05
Fig. 7
Fig. 7
Contents of polyhydroxybutyrate (PHB) (A) and glycogen (B) ofSynechocystis sp. PCC 6803 WT, KS, KA, KAS, and KAOL strains cultured under BG11-half N and BG11-N at day 10. The error bars represent standard deviations of means (mean ± S.D.,n = 3). Means with the same letter are not significantly different with the significance level atP < 0.05
Fig. 8
Fig. 8
Transcript levels of genes includingglgX, phaA, accA,aas, plsX, lipA and16S rRNA of WT, KS, KA, and KASSynechocystis sp. PCC 6803 strains under BG11 (A) and BG11-N (B) conditions. Cell culture at day 10 of treatment were harvested and analyzed. On the right hand side, the relative intensity ratios of each gene/16S rRNA were analyzed by GelQuant.NET program. Data represent mean ± S.D.,n = 3. Means with the same letter are not significantly different with the significance level atP < 0.05
Fig. 9
Fig. 9
Summary of obtained results, products and gene expression levels, in the engineered strains compared toSynechocystis sp. PCC 6803 WT cells after 10 days of growth in BG11-N. Each box's number represents the fold of that value divided by WT. When compared to WT, the green and pink colored boxes show lower and higher folds of that product, respectively. For yellow and blue boxes represent lower and higher folds of that transcript amount when compared to WT, respectively
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