The adc1 knockout with proC overexpression in Synechocystis sp. PCC 6803 induces a diversion of acetyl-CoA to produce more polyhydroxybutyrate

Abstract

Background: Lack of nutrients, in particular nitrogen and phosphorus, has been known in the field to sense glutamate production via 2-oxoglutarate and subsequently accelerate carbon storage, including glycogen and polyhydroxybutyrate (PHB), in cyanobacteria, but a few studies have focused on arginine catabolism. In this study, we first time demonstrated that gene manipulation on proC and adc1, related to proline and polyamine syntheses in arginine catabolism, had a significant impact on enhanced PHB production during late growth phase and nutrient-modified conditions. We constructed Synechocystis sp. PCC 6803 with an overexpressing proC gene, encoding Δ¹pyrroline-5-carboxylate reductase in proline production, and adc1 disruption resulted in lower polyamine synthesis.

Results: Three engineered Synechocystis sp. PCC 6803 strains, including a ProC-overexpressing strain (OXP), adc1 mutant, and an OXP strain lacking the adc1 gene (OXP/Δadc1), certainly increased the PHB accumulation under nitrogen and phosphorus deficiency. The possible advantages of single proC overexpression include improved PHB and glycogen storage in late phase of growth and long-term stress situations. However, on day 7 of treatment, the synergistic impact created by OXP/Δadc1 increased PHB synthesis by approximately 48.9% of dry cell weight, resulting in a shorter response to nutrient stress than the OXP strain. Notably, changes in proline and glutamate contents in engineered strains, in particular OXP and OXP/Δadc1, not only partially balanced the intracellular C/N metabolism but also helped cells acclimate under nitrogen (N) and phosphorus (P) stress with higher chlorophyll a content in comparison with wild-type control.

Conclusions: In Synechocystis sp. PCC 6803, overexpression of proC resulted in a striking signal to PHB and glycogen accumulation after prolonged nutrient deprivation. When combined with the adc1 disruption, there was a notable increase in PHB production, particularly in situations where there was a strong C supply and a lack of N and P.

Keywords: Glutamate; Glycogen; PHB; Proline; Synechocystis sp. PCC 6803.

Fig. 1

Overview of the polyamine-proline-glutamate pathways connected to the tricarboxylic acid (TCA) cycle and related biosynthetic pathways of polyhydroxybutyrate (PHB), and glycogen in cyanobacterium Synechocystis sp. PCC 6803. Abbreviations of genes are: acc, a multisubunit acetyl-CoA carboxylase; ach, acetyl-CoA hydrolase; ackA, acetate kinase; acs, acetyl-CoA synthase; adc, arginine decarboxylase; argD, N-acetylornithine aminotransferase; gad, glutamate decarboxylase; gdhA, glutamate dehydrogenase; glgC, ADP-glucose pyrophosphorylase; glgX, glycogen debranching enzyme; gltA, citrate synthase; plsX, fatty acid/phospholipid synthesis protein; phaA, β-ketothiolase; phaB, acetoacetyl-CoA reductase; phaC and phaE, the heterodimeric PHB synthase; proA, gamma-glutamyl phosphate reductase; proC, Δ¹pyrroline-5-carboxylate reductase; pta, phosphotransacetylase; putA, proline oxidase; speB1, arginase; speB2, agmatinase. Abbreviations of intermediates are: FASII, fatty acid synthesis type II; GABA, gamma-aminobutyric acid; GOGAT, glutamate synthase; G6P, glucose-6-phosphate; GS, glutamine synthetase; 3-PGA, 3-phosphoglycerate; PHB, polyhydroxybutyrate

Fig. 2

Genomic maps and transcript levels of Synechocystis sp. PCC 6803 strains. The four constructed strains are Synechocystis sp. PCC 6803 wild-type control (WTc), Synechocystis sp. PCC 6803 lacking adc1 gene (Δadc1c), Synechocystis sp. PCC 6803 overexpressing proC gene (OXP), and Δadc1 mutant overexpressing proC gene (OXP/Δadc1). PCR analysis employing two pairs of specific primers (Supplementary information Table S1) was used to confirm the accurate integration and placement of each gene fragment into the Synechocystis genome. (A) The double homologous recombination of both Cm^R gene occurred between the conserved sequences of psbA2 gene in WTc and Δadc1c, and a proC:Cm^R fragment occurred between the conserved sequences of psbA2 gene in OXP and OXP/Δadc1 strains when compared to WT. (B) For PCR products using UP_psbA2-F and Dw_psbA2-R primers, (B.1) For OXP strain, Lane M: GeneRuler DNA ladder, Lanes OX1, OX2, and OX3: three clones no. 1–3 containing a 3.2 kb fragment of Up_psbA2-proC-Cm^R-Dw_psbA2, Lanes WT and WTc: negative controls of a 2.4 kb fragment in WT and a 2.2 kb fragment in WTc, respectively. (B.2) For OXP/Δadc1 strain, Lane M: GeneRuler DNA ladder, Lanes OX1 and OX2: two clones no. 1 and 2 containing a 3.2 kb fragment of Up_psbA2-proC-Cm^R-Dw_psbA2, Lanes Δadc1 and Δadc1c: negative controls of a 2.4 kb fragment in Δadc1 and a 2.2 kb fragment in Δadc1c, respectively. (C) For PCR products using ProC-F and Cm^R-R primers, (C.1) For OXP strain, Lane M: GeneRuler DNA ladder, Lanes OX1, OX2, and OX3: three clones no. 1–3 containing a 1.9 kb fragment of proC-Cm^R, Lanes WT and WTc: negative controls (no band) using WT and WTc as template, respectively. (C.2) For OXP/Δadc1 strain, Lane M: GeneRuler DNA ladder, Lanes Δadc1 and Δadc1c: negative controls (no band) using Δadc1 and Δadc1c as template, respectively, Lanes OX1 and OX2: two clones no. 1 and 2 containing a 1.9 kb fragment of proC-Cm^R. (D) Transcript levels of proC gene determined by RT-PCR using RT-ProC-F and RT-ProC-R primers (Additional file 1: Table S1) in WT, WTc, Δadc1, Δadc1c, and two overexpressing strains, including OXP and OXP/Δadc1. The 0.8% agarose gel electrophoresis of PCR products was performed from cells grown for 6 days in normal BG₁₁ medium. The 16s rRNA was used as reference. The cropped gels (in D) were taken from the original images of RT-PCR products on agarose gels as shown in Supplementary information Figure S1

Fig. 3

Growth curve (A), chlorophyll a content (B), carotenoid content (C), oxygen evolution rate (D), contents of polyamines (E), proline (F), glutamate (G), GABA (H), and PHB (I) of WTc, Δadc1c, OXP, and OXP/Δadc1 Synechocystis sp. PCC 6803 strains. In (A–C), and (I), cells grown in BG₁₁ medium for 16 days. In (D–H), cells were grown in normal BG₁₁ medium for 7 days, and harvested for metabolite contents. The error bars represent standard deviations of means (mean ± S.D., n = 3). In (D–I), the statistical difference of the results between those values of WTc and that engineered strain is indicated by an asterisk at *P < 0.05

Fig. 4

Growth curve (A–C), chlorophyll a content (D–F), and carotenoid content (G–I) of Synechocystis WTc, Δadc1c, OXP, and OXP/Δadc1 strains adapted in normal BG₁₁ medium, BG₁₁ medium with N and P deprivation (BG₁₁-N-P), and BG₁₁-N-P supplemented with 4%(w/v) acetate (BG₁₁-N-P + A) medium for 11 days. The error bars represent standard deviations of means (mean ± S.D., n = 3). The statistical difference of the results between those values of WTc and that engineered strain is indicated by an asterisk at *P < 0.05

Fig. 5

Contents of PHB (A–C) and glycogen (D–F) of Synechocystis WTc, Δadc1c, OXP, and OXP/Δadc1 strains adapted in normal BG₁₁ medium, BG₁₁ medium with N and P deprivation (BG₁₁-N-P), and BG₁₁-N-P supplemented with 4%(w/v) acetate (BG₁₁-N-P + A) medium for 11 days. The error bars represent standard deviations of means (mean ± S.D., n = 3). An asterisk (*P < 0.05) denotes the statistical difference in results between those WTc values and that engineered strain at each day

Fig. 6

Fold changes of obtained results of metabolite products in three engineered strains compared with those in Synechocystis WTc after adapting cells in BG₁₁, BG₁₁-N-P, and BG₁₁-N-P + A for 7 days. In each box, the number represents the fold change of that value of each engineered strain under each stress condition divided by that value of WTc. The statistical difference in the data between those values of WT and the engineered strain is represented by an asterisk at *P < 0.05

Fig. 7

The Nile red stained PHB granules (A), relative transcript levels (B), and their band intensity ratios of gene/16s (C) of genes involved in PHB synthesis, glycogen degradation, proline-glutamate conversion, and neighboring pathways in Synechocystis WTc, Δadc1c, OXP, and OXP/Δadc1 strains under BG₁₁-N-P + A condition at day 7 of treatment. The 16s rRNA was used as reference control

Fig. 8

Fold changes of obtained results of gene transcript levels and metabolite contents in three engineered strains compared with those in Synechocystis WTc after adapting cells in BG₁₁-N-P + A for 7 days. In each box and graph, the number and bar graph represents the fold change of that value of each engineered strain divided by that value of WTc, respectively