Unusual Phosphoenolpyruvate (PEP) Synthetase-Like Protein Crucial to Enhancement of Polyhydroxyalkanoate Accumulation in Haloferax mediterranei Revealed by Dissection of PEP-Pyruvate Interconversion Mechanism

Abstract

Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis and is catalyzed by various sets of enzymes in different Archaea groups. In this study, we report the key enzymes that catalyze the anabolic and catabolic directions of the PEP/pyruvate interconversion in Haloferax mediterranei. The in silico analysis showed the presence of a potassium-dependent pyruvate kinase (PYKHm [HFX_0773]) and two phosphoenol pyruvate synthetase (PPS) candidates (PPSHm [HFX_0782] and a PPS homolog protein named PPS-like [HFX_2676]) in this strain. Expression of the pykHm gene and ppsHm was induced by glycerol and pyruvate, respectively; whereas the pps-like gene was not induced at all. Similarly, genetic analysis and enzyme activities of purified proteins showed that PYKHm catalyzed the conversion from PEP to pyruvate and that PPSHm catalyzed the reverse reaction, while PPS-like protein displayed no function in PEP/pyruvate interconversion. Interestingly, knockout of the pps-like gene led to a 70.46% increase in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production. The transcriptome sequencing (RNA-Seq) and quantitative reverse transcription-PCR (qRT-PCR) results showed that many genes responsible for PHBV monomer supply and for PHBV synthesis were upregulated in a pps-like gene deletion strain and thereby improved PHBV accumulation. Additionally, our phylogenetic evidence suggested that PPS-like protein diverged from PPS enzyme and evolved as a distinct protein with novel function in haloarchaea. Our findings attempt to fill the gaps in central metabolism of Archaea by providing comprehensive information about key enzymes involved in the haloarchaeal PEP/pyruvate interconversion, and we also report a high-yielding PHBV strain with great future potentials. IMPORTANCE Archaea, the third domain of life, have evolved diversified metabolic pathways to cope with their extreme habitats. Phosphoenol pyruvate (PEP)/pyruvate interconversion during carbohydrate metabolism is one such important metabolic process that is highly differentiated among Archaea. However, this process is still uncharacterized in the haloarchaeal group. Haloferax mediterranei is a well-studied haloarchaeon that has the ability to produce polyhydroxyalkanoates (PHAs) under unbalanced nutritional conditions. In this study, we identified the key enzymes involved in this interconversion and discussed their differences with their counterparts from other members of the Archaea and Bacteria domains. Notably, we found a novel protein, phosphoenolpyruvate synthetase-like (PPS-like), which exhibited high homology to PPS enzyme. However, PPS-like protein has evolved some distinct sequence features and functions, and strikingly the corresponding gene deletion helped to enhance poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) synthesis significantly. Overall, we have filled the gap in knowledge about PEP/pyruvate interconversion in haloarchaea and reported an efficient strategy for improving PHBV production in H. mediterranei.

Acetate Metabolism in Archaea: Characterization of an Acetate Transporter and of Enzymes Involved in Acetate Activation and Gluconeogenesis in Haloferax volcanii

“…solfataricus (Eyzaguirre et al, 1982;Cicicopol et al, 1999;Hutchins et al, 2001;Tjaden et al, 2006;Chen et al, 2019;Haferkamp et al, 2019). All archaeal PPSs show similar subunit sizes (75-90 kDa) that have also been reported for PPSs from bacteria and eukarya.…”

Rational engineering ofKluyveromyces marxianusto create a chassis for the production of aromatic products

“…Although a previous study suggested that they could not function in this manner in K. marxianus (Kim et al, 2014), we found that the choice of enzyme and strain engineering played a role in the extent to which PEP synthase overexpression improved AAA or shikimate production. As some PEPSes are exclusively used by their host organisms for either gluconeogenesis or PEP synthesis (Chen et al, 2019;Zhang and Anderson, 2013), a further investigation of more PEPSes with similar functions and kinetics may help us identify the best way to integrate this class of enzyme in yeast cell factories. In our efforts to increase E4P production, we overexpressed phosphoketolases that tap into K. marxianus' high fructose-6-phosphate levels and PPP flux (Bergman et al, 2019;Jung et al, 2016) to further boost available E4P.…”

“…We overexpressed five codon-optimized PEPSes: EcppsA, an archaeal PEPS and three fungal PEPSes (Table 3). The fungal PEPSes were chosen based on their sequence homology to EcppsA while the archaeal PEPS was chosen since it was found to solely catalyse PEP formation (Chen et al, 2019). We also chose to test a codon-optimised PPDK from Arabidopsis thaliana as it has a 10-fold higher K m for pyruvate than PEP as substrate, which could allow for a fastidious conversion of pyruvate to PEP (Chastain et al, 2011).…”

Dietary Supplementation With Creatine Pyruvate Alter Rumen Microbiota Protein Function in Heat-Stressed Beef Cattle

“…Our metaproteomics analyses showed that the administration of CrPyr signi cantly up-regulated the pyruvate metabolism and glycolysis/gluconeogenesis pathway. Phosphoenolpyruvate (PEP)/pyruvate interconversion is a major metabolic point in glycolysis and gluconeogenesis [29]. In the case of the pyruvate-to-PEP conversion, the reaction proceeds through diversi ed metabolic reactions.…”