Engineering a feedback inhibition-insensitive plant dihydrodipicolinate synthase to increase lysine content in Camelina sativa seeds

Camelina sativa (camelina) is emerging as an alternative oilseed crop due to its short growing cycle, low input requirements, adaptability to less favorable growing environments and a seed oil profile suitable for biofuel and industrial applications. Camelina meal and oil are also registered for use in animal and fish feeds; however, like meals derived from most cereals and oilseeds, it is deficient in certain essential amino acids, such as lysine. In higher plants, the reaction catalyzed by dihydrodipicolinate synthase (DHDPS) is the first committed step in the biosynthesis of lysine and is subject to regulation by lysine through feedback inhibition. Here, we report enhancement of lysine content in C. sativa seed via expression of a feedback inhibition-insensitive form of DHDPS from Corynebacterium glutamicums (CgDHDPS). Two genes encoding C. sativa DHDPS were identified and the endogenous enzyme is partially insensitive to lysine inhibition. Site-directed mutagenesis was used to examine the impact of alterations, alone and in combination, present in lysine-desensitized DHDPS isoforms from Arabidopsis thaliana DHDPS (W53R), Nicotiana tabacum (N80I) and Zea mays (E84K) on C. sativa DHDPS lysine sensitivity. When introduced alone, each of the alterations decreased sensitivity to lysine; however, enzyme specific activity was also affected. There was evidence of molecular or structural interplay between residues within the C. sativa DHDPS allosteric site as coupling of the W53R mutation with the N80V mutation decreased lysine sensitivity of the latter, but not to the level with the W53R mutation alone. Furthermore, the activity and lysine sensitivity of the triple mutant (W53R/N80V/E84T) was similar to the W53R mutation alone or the C. glutamicum DHDPS. The most active and most lysine-insensitive C. sativa DHDPS variant (W53R) was not inhibited by free lysine up to 1 mM, comparable to the C. glutamicums enzyme. Seed lysine content increased 13.6 -22.6% in CgDHDPS transgenic lines and 7.6–13.2% in the mCsDHDPS lines. The high lysine-accumulating lines from this work may be used to produce superior quality animal feed with improved essential amino acid profile.

The indices of sorghum seed quality in dependence on ecological and geographical origin

The current paper presents the study results of protein percentage, content of lysine and starch in the collection samples of grain sorghum of various ecological and geographical origin. It was determined that the seed protein percentage in the grain sorghum samples varied from 9.0 to 13.5%. The highest values were identified in the samples from Russia (KiM, Krymbel, Krusta, Ros’), Ukraine (Pioner 412/Milovskoe 6, No. 13-13, Pioner 878/Genicheskoe brown 129) and China (KX 8, No. 26-14) with 13.0-13.5%. The lysine content in protein of the studied samples was 2.5-4.3%, while the value of the standard variety Zernogradskoe 88 was 3.17% with the standard deviation of 0.24%. The maximum content (3.8-4.3%) was identified in the samples Early Hegari (Peru), Line ExF3 IS12606 (Australia), DN-35f (Ukraine), CS-175 (Philippines), H.S.-21 (Romania), Sorghum k-9553 (Korea). The starch content in seed of the collection samples varied within 70.2-77.5%. The highest intragroup values were showed by the samples from Peru (73.9%), Australia (73.8%), France (73.0%), Senegal (73.4%).

Effects of glutamine and glutamate on nursery piglets fed diets with different digestible lysine content

The objective of the study was to evaluate the effects of glutamine and glutamate (Gln/Glu) on the growth performance and immune response of nursery pigs fed different digestible lysine content. Two hundred and sixteen piglets, weaned at 21 days old, were assigned to a randomized block design according to their initial body weight (BW), in a 2 × 2 factorial arrangement with two levels of lysine (control-lys and low-lys) and two levels of Gln/Glu (0 and 12 g kg-1), with nine replicates. At 26 d, piglets consuming the low-lys diet not supplemented with Gln/Glu presented a higher (P < 0.01) incidence of diarrhea than the other treatments. From 21 to 32 d of age, the piglets fed the control-lys diets performed better than those fed low-lys diets (P < 0.01). From 21 to 42 d of age, there was a correlation (P < 0.01) between lysine level and Gln/Glu supplementation for average daily feed intake (ADFI) and feed conversion (FC). Gln/Glu supplementation improved (P < 0.05) the ADFI of pigs fed the low-lys diets, resulting in a higher (P < 0.01) average daily weight gain (ADG) and BW; however, worse (P < 0.05) FC. Piglets consuming control-lys diets had higher (P < 0.05) serum urea nitrogen concentration (SUN) and IgG than low-lys piglets. In addition, Gln/Glu supplementation correlated with higher (P < 0.01) SUN. Dietary supplementation of glutamine and glutamate improved the growth performance of weaned piglets from 21 to 42 days of age, regardless of the diets’ lysine levels. In addition, reducing lysine levels 10% below the requirement negatively affects the growth performance and the immune response of nursery piglets.

Proteome and Nutritional Shifts Observed in Hordein Double-Mutant Barley Lines

Lysine is the most limiting essential amino acid in cereals, and efforts have been made over the decades to improve the nutritional quality of these grains by limiting storage protein accumulation and increasing lysine content, while maintaining desired agronomic traits. The single lys3 mutation in barley has been shown to significantly increase lysine content but also reduces grain size. Herein, the regulatory effect of the lys3 mutation that controls storage protein accumulation as well as a plethora of critically important processes in cereal seeds was investigated in double mutant barley lines. This was enabled through the generation of three hordein double-mutants by inter-crossing three single hordein mutants, that had all been backcrossed three times to the malting barley cultivar Sloop. Proteome abundance measurements were integrated with their phenotype measurements; proteins were mapped to chromosomal locations and to their corresponding functional classes. These models enabled the prediction of previously unknown points of crosstalk that connect the impact of lys3 mutations to other signalling pathways. In combination, these results provide an improved understanding of how the mutation at the lys3 locus remodels cellular functions and impact phenotype that can be used in selective breeding to generate favourable agronomic traits.

Impact of lysine supplementation on growth and development of Hermetia illucens larvae

Hermetia illucens larvae are able to convert a broad range of organic substrates into animal protein and are believed to have the potential to help solving the supply problem for animal protein in the future. Many farmed animals such as poultry or pigs need an adequate amount of essential amino acids like lysine for optimal growth and performance. In most diets for farmed animals, essential amino acids are supplemented as feed additive to avoid excessive protein intake while ensuring a sufficient supply of essential amino acids. There is no information on the requirements of lysine for the growth of H. illucens larvae. Furthermore, it remains open whether lysine may have an extra effect on growth and development in the cultivation of such larvae. The aim of the study was to evaluate, if an addition of lysine to their feed substrate will affect these parameters. Therefore, in the present study up to 3.0% crystalline lysine was added to a basic substrate and survival, growth, and development of larvae were recorded. Furthermore, larval nutritional values like crude fat, crude protein, and lysine content were analysed. The addition of lysine did not improve growth, development or nutritional values of H. illucens larvae. In contrast, the addition of lysine led to reduced larval size, survival and prepupal rates, especially when high amounts of lysine were provided. The results of this study indicate that a lysine content of 0.3% in the substrate is sufficient for optimal larval growth. Using H. illucens larvae for animal protein production could, therefore, be accomplished with less lysine compared to many other farmed animals.

Bayesian Network Analysis of Lysine Biosynthesis Pathway in Rice

Lysine is the first limiting essential amino acid in rice because it is present in the lowest quantity compared to all the other amino acids. Amino acids are the building block of proteins and play an essential role in maintaining the human body’s healthy functioning. Rice is a staple food for more than half of the global population; thus, increasing the lysine content in rice will help improve global health. In this paper, we studied the lysine biosynthesis pathway in rice (Oryza sativa) to identify the regulators of the lysine reporter gene LYSA (LOC_Os02g24354). Genetically intervening at the regulators has the potential to increase the overall lysine content in rice. We modeled the lysine biosynthesis pathway in rice seedlings under normal and saline (NaCl) stress conditions using Bayesian networks. We estimated the model parameters using experimental data and identified the gene DAPF(LOC_Os12g37960) as a positive regulator of the lysine reporter gene LYSA under both normal and saline stress conditions. Based on this analysis, we conclude that the gene DAPF is a potent candidate for genetic intervention. Upregulating DAPF using methods such as CRISPR-Cas9 gene editing strategy has the potential to upregulate the lysine reporter gene LYSA and increase the overall lysine content in rice.

Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum

Cyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a biopolymer that could be used in various fields, for example, as a potential precursor for the synthesis of polyaspartic acid or for the production of CGP-derived dipeptides. To extend the applications of this polymer, it is therefore of interest to synthesize CGP with different compositions. A recent re-evaluation of the CGP synthesis in C. glutamicum has shown that C. glutamicum is a potentially interesting microorganism for CGP synthesis with a high content of alternative amino acids. This study shows that the amount of alternative amino acids can be increased by using mutants of C. glutamicum with altered amino acid biosynthesis. With the DM1729 mutant, the lysine content in the polymer could be increased up to 33.5 mol%. Furthermore, an ornithine content of up to 12.6 mol% was achieved with ORN2(Pgdh4). How much water-soluble or insoluble CGP is synthesized is strongly related to the used cyanophycin synthetase. CphADh synthesizes soluble CGP exclusively. However, soluble CGP could also be isolated from cells expressing CphA6308Δ1 or CphA6308Δ1_C595S in addition to insoluble CGP in all examined strains. The point mutation in CphA6308Δ1_C595S partially resulted in a higher lysine content. In addition, the CGP content could be increased to 36% of the cell dry weight under optimizing growth conditions in C. glutamicum ATCC13032. All known alternative major amino acids for CGP synthesis (lysine, ornithine, citrulline, and glutamic acid) could be incorporated into CGP in C. glutamicum.

Bayesian Networks Analysis of Lysine Biosynthesis Pathway in Rice

Lysine is the first limiting essential amino acid in rice because it is present in the lowest quantity compared to all the other amino acids. Amino acids are the building block of proteins and play an essential role in maintaining the human body&rsquo;s healthy functioning. Rice is a staple food for large proportion of the global population, thus increasing the lysine content in rice will improve its nutritional value. In this paper, we studied the lysine biosynthesis pathway in rice (Oryza Sativa) to identify the regulators of the lysine reporter gene LYSA (LOC_Os02g24354). Genetically intervening at the regulators has the potential to increase the overall lysine content in rice. We modeled the lysine biosynthesis pathway in rice seedlings under normal and saline (NaCl) stress conditions using Bayesian networks. We estimated the model parameters using experimental data and identified the gene DAPF(LOC_Os12g37960) as a positive regulator of the lysine reporter gene LYSA under both normal and saline stress conditions. Based on this analysis, we conclude that the gene DAPF is a potent candidate for genetic intervention. Upregulating DAPF using methods such as CRISPR-Cas9 has the potential to upregulate the lysine reporter gene LYSA and increase the overall lysine content in rice.