Improving the Plasmid Stability by a Hok/Sok System for L-Homoserine Production in Escherichia Coli

Background: The production of bioactive compounds using microbial hosts is considered a safe, cost competitive and scalable approach. However, the efficient engineering of cell factories with well stability, such as for the production of L-aspartate family amino acids and derivatives, remains an outstanding challenge.Results: In the work, the toxin/antitoxin system and genome modification strategy were used to construct a stable Escherichia coli strain for L-homoserine production. The metabolic engineering strategies were focused on the enhancement of precursors for L-homoserine synthesis, reinforcement of the NADPH generation and efflux transporters using CRISPR-Cas9 system at the genome level. To improve the plasmid stability, two strategies were explored, including construction of the aspartate-auxotrophic and hok/sok systems. Constructing the auxotrophic complementation system to maintain plasmid stability was failed herein. The plasmid stability was improved by introducing the hok/sok system, resulting in 6.1 g/L (shake flask) and 44.4 g/L (5 L fermenter) L-homoserine production of the final engineered strain SHL19 without antibiotics addition. Moreover, the hok/sok system was also used to improve the plasmid stability for ectoine production, resulting in 36.7% and 46.5% higher titer of ectoine at shake flask and 5L fermenter without antibiotics addition, respectively. Conclusion: This work provides valuable strategies to improve plasmid stability for producing L-aspartate family amino acids and derivatives and eliminate environmental concerns associated with the application of antibiotics.

Genetic Code Evolution Reconstructed with Aligned Metrics

Sequence homology in pre-divergence tRNA species revealed cofactor/adaptors cognate for 16 amino acids derived from oxaloacetate, pyruvate, phosphoglycerate, or phosphoenolpyruvate were related. Synthesis path-distances of these amino acids correlated with phylogenetic depth, reflecting relative residue frequency in pre-divergence sequences. Both metrics were thus aligned in the four sub-families of the Aspartate family, and misaligned in the small Glutamate family; a functional difference was noted and seen to parallel synthetase duality. Amino acid synthetic order, based on path-distances, indicate NH4+ fixer amino acids, Asp1, Asn2, and homologues, Glu1, Gln2, formed the first code. Together with a termination signal, they acquired all four triplet 4-sets in the XAN column (X, 5’ coding site; N, any 3’-base). An invariant mid-A conformed with pre-code translation on a poly(A) template by a ratchet-equipped ribosome resulting in random, polyanionic polypeptides. Code expansion occurred in a compact (mutation minimizing) columnwise pattern, (XAN) ➔ XCN ➔ XGN ➔ XUN; with increasing mean path-distance, (1.5) ➔ 4 ➔ 5 ➔ 7 steps; amino acid side-chain hydrophobicity, (+6.6) ➔ −0.8 ➔ −1.5 ➔ −3.2 kcal/ mol; codon:anticodon H bond enthalpy (selection for bond-strength), (−12.5) ➔ −17.5 ➔ −15.5 ➔ −14.5 kcal/ mol; and precursorspecific 5’-base, A, oxaloacetate, G, pyruvate/oxaloacetate, U, phosphoglycerate/oxaloacetate, C, oxoglutarate, forming horizontal code domains. Codon bias evidence corroborated the XCN ➔ XGN step in expansion, and revealed row GNN coevolved with ANN, on correction for overprinting. Extended surfaceattachment (Fajan-Paneth principle) by pro-Fd[5] and bilayer partitioning by H+ ATPase proteolipid-h1 subunit implicated expansion phase proteins in driving increases in side-chain hydrophobicity during code expansion. 3’-Base recruitment in pre-assigned codon boxes added six long (9-to 14-step) path amino acid, bearing a basic, or cyclic, side-chain; 3 of 4 polar, post-expansion amino acids acquired polar cluster NAN codons and 2 of 3 non-polar (Ile7 included) acquired non-polar cluster NUN codons, yieldng a split-box pair homology of p = 5.4×10-3. All eight overprinted codon boxes (GAYR for Asp1, Glu1 included) exhibit weak codon:anticodon H-bond enthalpy, −14 kcal/mol or higher, in three of six distinct code enthalpy states.

Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 hours. We also engineered E. coli strain to produced 24 mg/L of prenol from 10 g/L of ethanol in 48 hours, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.HighlightsEngineered Escherichia coli strains to grow on ethanol as sole carbon sourceDemonstrated that ethanol was converted into acetyl-CoA (AcCoA) through two pathways (acetaldehyde-acetate-AcCoA and acetaldehyde-AcCoA)Converted ethanol into two acetyl-CoA derived products with low structural similarity (polyhydroxybutyrate and prenol)Discovered that supplementation of the aspartate family amino acids can substantially improve cell growth on ethanol

Comparative Metabolomics of Early Development of the Parasitic Plants Phelipanche aegyptiaca and Triphysaria versicolor

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. The connection of the haustorium to the host marks a shift in parasite metabolism from autotrophy to at least partial heterotrophy, depending on the level of parasite dependence. Species within the family Orobanchaceae span the spectrum of host nutrient dependency, yet the diversity of parasitic plant metabolism remains poorly understood, particularly during the key metabolic shift surrounding haustorial attachment. Comparative profiling of major metabolites in the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor before and after attachment to the hosts revealed several metabolic shifts implicating remodeling of energy and amino acid metabolism. After attachment, both parasites showed metabolite profiles that were different from their respective hosts. In P. aegyptiaca, prominent changes in metabolite profiles were also associated with transitioning between different tissue types before and after attachment, with aspartate levels increasing significantly after the attachment. Based on the results from 15N labeling experiments, asparagine and/or aspartate-rich proteins were enriched in host-derived nitrogen in T. versicolor. These results point to the importance of aspartate and/or asparagine in the early stages of attachment in these plant parasites and provide a rationale for targeting aspartate-family amino acid biosynthesis for disrupting the growth of parasitic weeds.

Revisiting the attempts to fortify methionine content in plant seeds

The sulfur-containing amino acid methionine belongs to the group of essential amino acids, meaning that humans and animals must consume it in their diets. However, plant seeds have low levels of methionine, limiting their nutritional potential. For this reason, efforts have been made over the years to increase methionine levels in seeds. Here, we summarize these efforts and focus particularly on those utilizing diverse genetic and molecular tools. Four main approaches are described: (i) expression of methionine-rich storage proteins in a seed-specific manner to incorporate more soluble methionine into the protein fraction; (ii) reduction of methionine-poor storage proteins inside the seeds to reinforce the accumulation of methionine-rich proteins; (iii) silencing methionine catabolic enzymes; and (iv) up-regulation of key biosynthetic enzymes participating in methionine synthesis. We focus on the biosynthetic genes that operate de novo in seeds and that belong to the sulfur assimilation and aspartate family pathways, as well as genes from the methionine-specific pathway. We also include those enzymes that operate in non-seed tissues that contribute to the accumulation of methionine in seeds, such as S-methylmethionine enzymes. Finally, we discuss the biotechnological potential of these manipulations to increase methionine content in plant seeds and their effect on seed germination.

Mechanistic insights into the allosteric regulation of Pseudomonas aeruginosa aspartate kinase

In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from Pseudomonas aeruginosa (PaAK), a typical α2β2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2β2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.