Identification of Differentially Expressed Genes and Pathways Involved in Growth and Development of Mesona chinensis Benth Under Red- and Blue-Light Conditions

Mesona chinensis Benth (MCB) is an important Chinese herbal medicine. The plant factories might be one of the ways to solve the shortage of MCB supply. In this study, the MCB seedlings were treated under the red (R) and blue (B) lights in the plant factory. Results showed that the red light promoted the growth and development of MCB in comparison with the blue light. Under the red-light condition, the biomass, plant height, and root characteristics were significantly higher than those under blue-light condition, while the soil and plant analyzer development (SPAD) under the red-light treatment was significantly lower than that under the blue-light treatment. Red light also significantly promoted the content of soluble sugar and pectin of MCB compared with blue light. Transcriptome analysis showed that a total of 4,165 differentially expressed genes (DEGs) were detected including 2,034 upregulated and 2,131 downregulated. Of these, 1,112 DEGs including 410 upregulated and 702 downregulated genes were associated with 111 pathways. Moreover, a total of 8,723 differentially expressed transcription factors (TFs) were identified in R vs. B, and these TFs were distributed in 56 gene families. Metabonomic results revealed that a total of 184 metabolites and 99 differentially expressed metabolites (DEMs) (42 upregulated and 57 downregulated) were identified in the red- and blue-light treatments. Integrative analysis of transcriptome and metabolome unveiled that a total of 24 pathways included 70 compounds (metabolites) and were associated with 28 unigenes. In particular, these pathways included starch and sucrose metabolism, phenylpropanoid biosynthesis, cysteine and methionine metabolism, glycolysis/gluconeogenesis, and pentose and glucuronate interconversions. The unigenes included asparagine synthetase (AS), thymidine kinase (TK), alpha, alpha-trehalose-phosphate synthase (TPS), phosphatase IMPL1 (IMPL1), dihydroflavonol 4-reductase (D4R), and 4-coumarate-CoA ligase-like 6 (4CL6), bifunctional aspartokinase-homoserine dehydrogenase 1 (thrA), and abscisic acid 8′-hydroxylase 2 isoform X1 (ABA8). It was indicated that these pathways and genes might play important roles in the growth and development of MCB. This study laid a foundation for the future research of MCB.

Enhancement of lysine biosynthesis confers high-temperature stress tolerance to Escherichia coli cells

Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds. Key points • Lysine supplementation improved the growth of E. coli cells at high temperature. • The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells. • The G474D substitution in ThrA reduced homoserine dehydrogenase activity. • E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.

Metabolomic Analysis of Biosynthesis Mechanism of ε-Polylysine Produced by Streptomyces diastatochromogenes

ε-Polylysine (ε-PL), a natural preservative with broad-spectrum antimicrobial activity, has been widely used as a green food additive, and it is now mainly produced by Streptomyces in industry. In the previous study, strain 6#-7 of high-yield ε-PL was obtained from the original strain TUST by mutagenesis. However, the biosynthesis mechanism of ε-PL in 6#-7 is still unclear. In this study, the metabolomic analyses of the biosynthesis mechanism of ε-PL in both strains are investigated. Results show that the difference in metabolisms between TUST and 6#-7 is significant. Based on the results of both metabolomic and enzymatic activities, a metabolic regulation mechanism of the high-yield strain is revealed. The transport and absorption capacity for glucose of 6#-7 is improved. The enzymatic activity benefits ε-PL synthesis, such as pyruvate kinase and aspartokinase, is strengthened. On the contrary, the activity of homoserine dehydrogenase in the branched-chain pathways is decreased. Meanwhile, the increase of trehalose, glutamic acid, etc. makes 6#-7 more resistant to ε-PL. Thus, the ability of the mutagenized strain 6#-7 to synthesize ε-PL is enhanced, and it can produce more ε-PLs compared with the original strain. For the first time, the metabolomic analysis of the biosynthesis mechanism of ε-PL in the high-yield strain 6#-7 is investigated, and a possible mechanism is then revealed. These findings provide a theoretical basis for further improving the production of ε-PL.

Characterization of a Novel Type Homoserine Dehydrogenase Only with High Oxidation Activity from Arthrobacter nicotinovorans

Homoserine dehydrogenase (HSD) is a key enzyme in the synthesis pathway of the aspartate family of amino acids. HSD can catalyze the reversible reaction of L-aspartate-β-semialdehyde (L-ASA) to L-homoserine (L-Hse). In direct contrast, growth characteristic studies of some bacterial such as Arthrobacter nicotinovorans showed that the bacterium could grow well in medium with L-homoserine as sole carbon, nitrogen and energy source, but the genes responsible for the degradation of L-Hse remain unknown. Based on the function and sequence analysis of HSD, one putative homoserine dehydrogenase from A.nicotinovorans was named AnHSD, which was different from those HSDs that from the aspartic acid metabolic pathway, might be responsible for the degradation of L-Hse. Surprisingly, the analysis showed that the purified AnHSD exhibited specific L-Hse oxidation activity without reducing activity. At pH 10.0 and 40 °C, The Km and Kcat of AnHSD was 6.30 ± 1.03 mM and 462.71 s-1, respectively. AnHSD was partiality for NAD+ cofactor, as well as insensitive to feedback inhibition of downstream amino acids of aspartic acid family. The physiological role of AnHSD in A.nicotinovorans is discussed. These findings provide a novel insight for a better understanding of an alternative genetic pathway for L-Hse catabolism which was dominated by the novel HSD.ImportanceL-homoserine is an important building block for the synthesis of L-threonine, L-methionine, L-lysine which from aspartic acid family amino acids. However, some bacteria can make use of L-homoserine as a sole carbon and nitrogen source. Although the microbial degradation of L-homoserine has been studied several times, the genes involved and the molecular mechanisms remain unclear. In this study, we show that AnHSD responsible for the catabolism of L-homoserine in strain Arthrobacter nicotinovorans, as a special homoserine dehydrogenase with high diversity exists in Arthrobacter, Microbacterium, Rhizobium. We report for the first time that this novel homoserine dehydrogenase is now proposed to play a crucial role in that L-homoserine can use as a sole carbon and nitrogen source. This study is aimed at elucidating the enzymatic properties and function features of homoserine dehydrogenase from Arthrobacter nicotinovorans. These findings provide new insight into the catabolism of L-homoserine in bacteria.

Multiplex Design of the Metabolic Network for Production of L-Homoserine in Escherichia coli

ABSTRACT l-Homoserine, which is one of the few amino acids that is not produced on a large scale by microbial fermentation, plays a significant role in the synthesis of a series of valuable chemicals. In this study, systematic metabolic engineering was applied to target Escherichia coli W3110 for the production of l-homoserine. Initially, a basic l-homoserine producer was engineered through the strategies of overexpressing thrA (encoding homoserine dehydrogenase), removing the degradative and competitive pathways by knocking out metA (encoding homoserine O-succinyltransferase) and thrB (encoding homoserine kinase), reinforcing the transport system, and redirecting the carbon flux by deleting iclR (encoding the isocitrate lyase regulator). The resulting strain constructed by these strategies yielded 3.21 g/liter of l-homoserine in batch cultures. Moreover, based on CRISPR-Cas9/dCas9 (nuclease-dead Cas9)-mediated gene repression for 50 genes, the iterative genetic modifications of biosynthesis pathways improved the l-homoserine yield in a stepwise manner. The rational integration of glucose uptake and recovery of l-glutamate increased l-homoserine production to 7.25 g/liter in shake flask cultivation. Furthermore, the intracellular metabolic analysis further provided targets for strain modification by introducing the anaplerotic route afforded by pyruvate carboxylase to oxaloacetate formation, which resulted in accumulating 8.54 g/liter l-homoserine (0.33 g/g glucose, 62.4% of the maximum theoretical yield) in shake flask cultivation. Finally, a rationally designed strain gave 37.57 g/liter l-homoserine under fed-batch fermentation, with a yield of 0.31 g/g glucose. IMPORTANCE In this study, the bottlenecks that sequentially limit l-homoserine biosynthesis were identified and resolved, based on rational and efficient metabolic-engineering strategies, coupled with CRISPR interference (CRISPRi)-based systematic analysis. The metabolomics data largely expanded our understanding of metabolic effects and revealed relevant targets for further modification to achieve better performance. The systematic analysis strategy, as well as metabolomics analysis, can be used to rationally design cell factories for the production of highly valuable chemicals.

Proteomic Analysis of a hom-disrupted, Cephamycin C Overproducing Streptomyces clavuligerus

Background: Streptomyces clavuligerus is prolific producer of cephamycin C, a medically important antibiotic. In our former study, cephamycin C titer was 2-fold improved by disrupting homoserine dehydrogenase (hom) gene of aspartate pahway in Streptomyces clavuligerus NRRL3585. Objective: In this article, we aimed to provide a comprehensive understanding at the proteome level on potential complex metabolic changes as a consequence of hom disruption in Streptomyces clavuligerus AK39. Methods: A comparative proteomics study was carried out between the wild type and its hom disrupted AK39 strain by 2 Dimensional Electrophoresis-Matrix Assisted Laser Desorption and Ionization Time-Of-Flight Mass Spectrometry (2DE MALDI-TOF/MS) and Nanoscale Liquid Chromatography-Tandem Mass Spectrometry (nanoLC-MS/MS) analyses. Clusters of Orthologous Groups (COG) database was used to determine the functional categories of the proteins. The theoretical pI and Mw values of the proteins were calculated using Expasy pI/Mw tool. Results: “Hypothetical/Unknown” and “Secondary Metabolism” were the most prominent categories of the differentially expressed proteins. Upto 8.7-fold increased level of the positive regulator CcaR was a key finding since CcaR was shown to bind to cefF promoter thereby direcly controlling its expression. Consistently, CeaS2, the first enzyme of CA biosynthetic pathway, was 3.3-fold elevated. There were also many underrepresented proteins associated with the biosynthesis of several Non-Ribosomal Peptide Synthases (NRPSs), clavams, hybrid NRPS/Polyketide synthases (PKSs) and tunicamycin. The most conspicuously underrepresented protein of amino acid metabolism was 4-Hydroxyphenylpyruvate dioxygenase (HppD) acting in tyrosine catabolism. The levels of a Two Component System (TCS) response regulator containing a CheYlike receiver domain and an HTH DNA-binding domain as well as DNA-binding protein HU were elevated while a TetRfamily transcriptional regulator was underexpressed. Conclusion: The results obtained herein will aid in finding out new targets for further improvement of cephamycin C production in Streptomyces clavuligerus.