Genetic identification of acetyl-CoA synthetases involved in acetate activation in Haloferax mediterranei

Abstract

Acetate/acetyl-CoA interconversion is an interesting metabolic node, primarily catalyzed by a set of various enzymes in prokaryotes. Haloferax mediterranei is a promising haloarchaeaon, capable of utilizing acetate as a sole carbon source for biosynthesis of high value-added products. Here, we have reported the key enzymes that catalyzed acetate activation in H. mediterranei. Based on bioinformatic and transcript analysis, thirteen possible candidate genes were screened. Simultaneous deletion of eleven genes led to a mutant strain (named as Δ11) that failed to grow on acetate. Gene complementation in Δ11 revealed six AMP-ACS (encoded by HFX_0870, HFX_1242, HFX_1451, HFX_6342, HFX_5131, and HFX_1643) and one ADP-ACS (encoded by HFX_0998) to be functional in acetate activation. Furthermore, heterologous expression of ADP-ACS genes from Haloarcula hispanica and Haloferax volcanii catalyzed acetate activation in Δ11. Subsequently, it was observed that, deletion of the six AMP-ACS genes in H. mediterranei ceased the cell growth of the resulting mutant (Δ6AMP-ACS) on acetate. An in vivo function of ADP-ACS in acetate activation could be excluded since ADP-ACS was downregulated on acetate. However, plasmid-based overexpression of ADP-ACS enabled Δ6AMP-ACS to grow on acetate, even better than the parent strain. Thus, it can be inferred that native ADP-ACS with low expression level was unable to mediate cell growth of Δ6AMP-ACS on acetate. This is the first genetic evidence exhibiting that overexpression of haloarchaeal ADP-ACS catalyzed acetate activation in vivo. Collectively, this is a comprehensive study of acetate activation in H. mediterranei, and the current findings would surely enrich the understanding of acetate metabolism in archaea.

Importance: Owing to the high demand and supply challenge of glucose, acetate might be considered a potential alternative carbon source for microbial growth and fermentation. Haloferax mediterranei is capable of utilizing acetate as a carbon source for growth and subsequent value-added product synthesis. Thus, it is essential to identify the genes responsible for acetate utilization in H. mediterranei. As per available literature, haloarchaeal ADP-forming acetyl-CoA synthetase (APD-ACS) catalyzes the reversible conversion of acetate to acetyl-CoA in vitro. However, in vivo, acetate activation and acetate formation are catalyzed by AMP-forming acetyl-CoA synthetase (AMP-ACS) and ADP-ACS, respectively. In this study, we have identified six AMP-ACS enzymes that catalyzed acetate activation in H. mediterranei. Deletion of these six genes abolished the growth of the resulting mutant (Δ6AMP-ACS) in acetate medium. The natively expressed ADP-ACS was unable to mediate its acetate activation in vivo. Interestingly, an artificial system based on plasmid overexpression of ADP-ACS in Δ6AMP-ACS restored its growth on acetate. This finding suggested that native ADP-ACS was unable to catalyze acetate activation in H. mediterranei due to its low expression level. Together, our study explored the acetate activation in H. mediterranei, and the obtained results would enrich the knowledge of acetate metabolism in archaea. Furthermore, the information offered in this study would benefit the improvement of acetate utilization in haloarchaea for value-added product synthesis.

Keywords: ADP-acetyl-CoA synthetase; AMP-acetyl-CoA synthetase; acetate activation; haloarchaea.

Fig 1

Upregulation of AMP-ACS genes and downregulation of ADP-ACS genes in H. mediterranei on acetate compared to glucose. qRT-PCR analysis for fold change in mRNA expression levels of the six putative AMP-ACS encoding genes, namely, HFX_0870, HFX_1242, HFX_1643, HFX_2150, HFX_5129, and HFX_5131 (represented by the gray column) and one putative ADP-ACS-encoding gene, namely, HFX_0998 (represented by white column) of H. mediterranei. DF50ΔEPS cultured to the exponential growth phase on 0.12 M acetate or 0.04 M glucose was used for qRT-PCR analysis. ns represents non-significant (p- value > 0.05), ** represents p-value < 0.01, and *** represents p-value < 0.005. Data are expressed as mean ± SD, n = 3.

Fig 2

Effect of single acs gene knockout on cell growth of H. mediterranei on 0.12 M acetate. (A) Growth analysis of Δ1242, Δ1643, Δ2150, Δ5129, and Δ5131. (B) Growth analysis of Δ0870. For A and B, the fermentation time lasted for 5 days, and they were cultured in two different batches. Δ1242 (red line), Δ1643 (blue line), Δ2150 (green line), Δ5129 (pink line), Δ5131 (yellow line), and Δ0870 (orange line) represent the single mutant of six putative AMP-ACS genes, namely, HFX_1242, HFX_1643, HFX_2150, HFX_5129, HFX_5131, and HFX_0870. (C) Growth analysis of Δ0998 (mutant of the putative ADP-ACS encoding gene, HFX_0998) (dark blue line). DF50ΔEPS (black line) represents the positive control. Data are expressed as mean ± SD, n = 3.

Fig 3

H. mediterranei Δ11 lost its ability to grow on acetate. (A) Growth analysis of Δ7 on 0.12 M acetate. Δ7 (violet line) is the multiple acs mutant of DF50ΔEPS with the six putative AMP-ACS genes (HFX_0870, HFX_1242, HFX_1643, HFX_2150, HFX_5129, and HFX_5131) and one ADP-ACS gene (HFX_0998) knocked out. (B) Volcano plot of differentially expressed genes of Δ7 in 0.12 M acetate compared to 0.04 M glucose. Pink circles represent upregulated genes, green circles represent downregulated genes, and blue circles represent genes with no change in expression. (C) Changes in the expression level of potential AMP-ACS candidate genes in Δ7, represented as log₂(fold change) of gene expression in 0.12 M acetate compared to 0.04 M glucose. The candidate genes are annotated as following. HFX_1451, medium-chain-fatty-acid-CoA synthetase; HFX_4020, long-chain acyl-CoA synthetase; HFX_1837, medium-chain acyl-CoA synthetase; HFX_1860, CoA transferase; HFX_5190, putative acyl-CoA transferase; HFX_6342, medium-chain acyl-CoA synthetase. ns represents non-significant (p-value > 0.05), ** represents p-value < 0.01, and *** represents p-value < 0.005. (D) Growth analysis of Δ11 (gray line) on 0.12 M acetate. Δ11 represents the mutant of eleven genes, which includes ten possible AMP-ACS genes (HFX_0870, HFX_1242, HFX_1451, HFX_1643, HFX_1837, HFX_2150, HFX_4020, HFX_5129, HFX_5131, and HFX_6342) and one ADP-ACS gene (HFX_0998). DF50ΔEPS (black line) represents the positive control (A and D). Data are expressed as mean ± SD, n = 3 (A, C, and D).

Fig 4

Complementation of ADP-ACS in Δ11 restored acetate activation. (A) Growth analysis of Δ11 complementation strains on 0.12 M acetate. Red, blue, green, orange, and purple lines indicate growth of Δ11 complemented with HFX_0998, HFX_0870, HFX_6342, HFX_1451, and HFX_1242, respectively. (B) Growth analysis of Δ11 with heterologous expression of ADP-ACS genes (HVO_1000 and HAH_1525) from H. volcanii and H. hispanica on 0.12 M acetate. Blue and pink lines represent the expression of HVO_1000 and HAH_1525 in Δ11, respectively. Δ11 (pWL502) and DF50ΔEPS (pWL502) represent negative (gray line) and positive control (black line), respectively. Data are expressed as mean ± SD, n = 3.

Fig 5

HFX_5131 and HFX_1643 involved in acetate activation at low concentration of acetate. (A) Volcano plot of differentially expressed proteins of DF50ΔEPS in 0.12 M acetate compared to 0.04 M glucose. Pink circles represent upregulated proteins, green circles represent downregulated proteins, and gray circles represent proteins with no change in expression. (B) Changes in the expression level of the eleven possible acetate activation enzymes, represented as log₂(fold change) of expression in 0.12 M acetate compared to 0.04 M glucose. Pink columns represent upregulation of HFX_0870, HFX_1242, HFX_1451, HFX_1643, HFX_2150, HFX_5129, HFX_5131, and HFX_6342 in acetate. Green columns represent downregulation of HFX_1837, HFX_4020, and HFX_0998 in acetate. ns represents nonsignificant (p-value > 0.05), ** represents p-value < 0.01, and *** represents p-value < 0.005. (C) Growth analysis of the Δ11 complementation strain on 0.04 M acetate. Violet and blue lines represent growth of Δ11 complemented with HFX_5131 and HFX_1643, respectively. Δ11(pWL502) (gray line) and DF50ΔEPS(pWL502) (black line) represent negative and positive controls, respectively. Data are expressed as mean ± SD, n = 3 (B, C).

Fig 6

Plasmid-based overexpression of the ADP-ACS gene restored the growth of Δ6AMP-ACS on acetate. Growth of functional AMP-ACS mutants on 0.12 M (A) and 0.04 M (B) acetate. Δ1AMP-ACS represents DF50ΔEPSΔ6342 (pink line), Δ2AMP-ACS represents Δ1AMP-ACSΔ1451(blue line), Δ3AMP-ACS represents Δ2AMP-ACSΔ1242 (green line), Δ4AMP-ACS represents Δ3AMP-ACSΔ0870 (yellow line), Δ5AMP-ACS represents Δ4AMP-ACSΔ5131 (violet line), and Δ6AMP-ACS represents Δ5AMP-ACSΔ1643 (red). Δ11 (gray line) and DF50ΔEPS (black line) represent negative and positive controls, respectively. (C) Growth of Δ6AMP-ACS on 0.12 M acetate after HFX_0998 overexpression using the pWL502 plasmid, with the expression driven by its native promoter (orange line) or a strong promoter P_phaR (dark blue line). Δ11(pWL502) (gray line) and DF50ΔEPS(pWL502) (black line) represent negative and positive controls, respectively. Data are expressed as mean ± SD, n = 3.