Molecular Evolution of Lysine Biosynthesis in Agaricomycetes

As an indispensable essential amino acid in the human body, lysine is extremely rich in edible mushrooms. The α-aminoadipic acid (AAA) pathway is regarded as the biosynthetic pathway of lysine in higher fungal species in Agaricomycetes. However, there is no deep understanding about the molecular evolutionary relationship between lysine biosynthesis and species in Agaricomycetes. Herein, we analyzed the molecular evolution of lysine biosynthesis in Agaricomycetes. The phylogenetic relationships of 93 species in 34 families and nine orders in Agaricomycetes were constructed with six sequences of LSU, SSU, ITS (5.8 S), RPB1, RPB2, and EF1-α datasets, and then the phylogeny of enzymes involved in the AAA pathway were analyzed, especially homocitrate synthase (HCS), α-aminoadipate reductase (AAR), and saccharopine dehydrogenase (SDH). We found that the evolution of the AAA pathway of lysine biosynthesis is consistent with the evolution of species at the order level in Agaricomycetes. The conservation of primary, secondary, predicted tertiary structures, and substrate-binding sites of the enzymes of HCS, AAR, and SDH further exhibited the evolutionary conservation of lysine biosynthesis in Agaricomycetes. Our results provide a better understanding of the evolutionary conservation of the AAA pathway of lysine biosynthesis in Agaricomycetes.

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.

High-Level Production of Lysine in the Yeast Saccharomyces cerevisiae by Rational Design of Homocitrate Synthase

Homocitrate synthase (HCS) catalyzes the aldol condensation of 2-oxoglutarate (2-OG) and acetyl coenzyme A (AcCoA) to form homocitrate, which is the first enzyme of the lysine biosynthetic pathway in the yeast Saccharomyces cerevisiae. The HCS activity is tightly regulated via feedback inhibition by the end product lysine. Here, we designed a feedback inhibition-insensitive HCS of S. cerevisiae (ScLys20) for high-level production of lysine in yeast cells. In silico docking of the substrate 2-OG and the inhibitor lysine to ScLys20 predicted that the substitution of serine to glutamate at position 385 would be more suitable for desensitization of the lysine feedback inhibition than the substitution from serine to phenylalanine in the already-known variant Ser385Phe. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine content in yeast cells expressing the Ser385Glu variant was 4.62-fold and 1.47-fold higher than that of cells expressing the wild-type HCS and Ser385Phe variant, respectively, due to the extreme desensitization to feedback inhibition. In this study, we obtained highly feedback inhibition-insensitive HCS using in silico docking and enzymatic analysis. Our results indicate that the rational engineering of HCS for feedback-inhibition desensitization by lysine and could be useful for constructing new yeast strains with higher lysine productivity. IMPORTANCE A traditional method for screening toxic analogue-resistant mutants has been established for the breeding of microbes that produce high levels of amino acids, including lysine. However, another efficient strategy is required to further improve their productivity. Homocitrate synthase (HCS) catalyzes the first step of lysine biosynthesis in the yeast Saccharomyces cerevisiae, and its activity is subject to feedback inhibition by lysine. Here, in silico design of a key enzyme that regulates the biosynthesis of lysine was utilized to increase the productivity of lysine. We designed HCS for the high level production of lysine in yeast cells by in silico docking simulation. The engineered HCS exhibited much less sensitivity to lysine and conferred higher production of lysine than the already-known variant obtained by traditional breeding. The combination of in silico design and experimental analysis of a key enzyme will contribute to advances in metabolic engineering for the construction of industrial microorganisms.

Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

How a Taxonomically-Ambiguous Cyanobiont and Vanadate Assist in the Phytoremediation of Cadmium by Azolla pinnata: Implications for CKDu

We employed scientific tools to investigate the ex situ phytoremediation of cadmium by Azolla pinnata. Azolla pinnata was capable of efficient sequestration of cadmium up to a concentration of 1 ppm, though with a visibly high “physiological cost”. The sequestration of cadmium (1 ppm) was strongly reduced after 24 hours, in Azolla plants pre-treated with the gram-negative antibiotic erythromycin (60 µg/l), suggesting that the cyanobacterial population was important for phytoremediation. Only the co-treatment of 1 ppm cadmium with 1 ppm vanadate, showed significantly higher phytoremediation (P<0.05) compared to the “cadmium+erythromycin” treatment. The phytoremediation of Cadmium by the Azolla-Nostoc symbiosis was significantly (p<0.05) improved by the addition of citrate at 10 ppm in the presence of 1 ppm vanadate, compared to the 1 ppm cadmium only treatment. We hypothesize that citrate acting either as “vanadophores” or working as a cofactor in the Homocitrate Synthase enzyme, facilitates remediation of cadmium. When phylogeny was inferred using Homocitrate Synthases, the cyanobiont was approximated to a taxonomical twilight zone between Nostoc and Anabaena, although showing more proximity to the Anabaena cluster. It is proposed here that the cyanobacterial contribution appears to be crucial for the ability of Azolla pinnata to efficiently remediate cadmium and a “helping hand” appears to be provided by a vanadate dependent mechanism, which is likely to be nitrogen fixation. The association between vanadate-assisted phytoremediation by Azolla pinnata and the heightened bioavailability of vanadium in CKDu endemic areas, could serve as a vital stepping stone in developing a biological solution to CKDu.