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© 2023 Elsevier B.V.Microbial production of aromatic compounds is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for L-tyrosine (L-Tyr) overproduction was attempted by pushing the carbon flux more towards L-Tyr. Translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes revealed that reduced expression of pheA was the major contributor to increased L-Tyr titer while codon exchange in trpE was effective to a lower extent. Overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, and of dapC (cg1253), which is predicted to encode prephenate aminotransferase, were futile to increase L-Tyr titer. Similarly, deletion of the qsuABD gene cluster had also not enhanced titer. As for increasing precursor supply, deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK) were not effective, but with utilization of xylose, enabled by overexpression of xylose isomerase (xylA) and xylulokinase (xylB), titer improved. Highest L-Tyr titer using the construct was 3.1 g/L on glucose and 3.6 g/L on a 1:3 (w/v) mixture of glucose and xylose. This result displays the potential of the constructed strain to produce L-Tyr from lignocellulosic renewable carbon sources.

Graphical Abstract

The physiological role of ubiquitous rhomboid proteases, membrane-integral proteins that cleave their substrates inside the lipid bilayer, is still ill-defined in many prokaryotes. The two rhomboid genes cg0049 and cg2767 of Corynebacterium glutamicum were mutated and it was the aim of this study to investigate consequences in respect to growth phenotype, stress resistance, transcriptome, proteome, and lipidome composition. Albeit increased amount of Cg2767 upon heat stress, its absence did not change the growth behavior of C. glutamicum during exponential and stationary phase. Quantitative shotgun mass spectrometry was used to compare the rhomboid mutant with wild type strain and revealed that proteins covering diverse cellular functions were differentially abundant with more proteins affected in the stationary than in the exponential growth phase. An observation common to both growth phases was a decrease in ribosomal subunits and RNA polymerase, differences in iron uptake proteins, and abundance changes in lipid and mycolic acid biosynthesis enzymes that suggested a functional link of rhomboids to cell envelope lipid biosynthesis. The latter was substantiated by shotgun lipidomics in the stationary growth phase, where in a strain-dependent manner phosphatidylglycerol, phosphatidic acid, diacylglycerol and phosphatidylinositol increased irrespective of cultivation temperature.

Carotenoids: Carotenoid and Apocarotenoid Biosynthesis, Metabolic Engineering and Synthetic Biology, Volume 671, the latest release in the Methods of Enzymology series highlights new advances in the field with chapters on Metabolomics-based analysis of carotenoids and related metabolites in various species via quantitative trait loci and genome wide association mapping approaches, Using bacteria for functional analysis of genes encoding carotenoid biosynthetic enzymes, Rice Callus as a High Throughput Platform for Synthetic Biology and Metabolic Engineering of Carotenoids, Transient expression in Nicotiana benthamiana: A simple platform to investigate genes encoding carotenoid biosynthesis enzymes from diverse algal lineages, and much more. Additional chapters in this new release cover Protein-protein interaction techniques to investigate post-translational regulation of carotenogenesis, The isolation of sub-chromoplast structures from tomato and capsicum fruit, Carrot protoplasts as a suitable method for protein subcellular localization, High throughput production and characterization of carotenoid enzymes for structural and functional studies, Production and structural characterization of the cytochrome P450 enzymes in carotene ring hydroxylation, and more.

Additional file 2. Codon optimised sequences of TNA genes optimized for expression in C. glutamicum used in this study.

Additional file 1. Fig. S1: Indole production of C1* (pGold-EctnaA-CgaroP) in bioreactor cultivation. Fig. S2. Exploration of sequence identity between TNA sequences grouped in 14 clusters. Fig. S3. Acrylamide gel of the soluble protein fraction of C1* expressing different TNA genes. Fig. S4. Partitioning of indole (A) and Trp (B) between water and the respective solvent.

Additional file 1: Figure S1. (A) DNA region of the plasmid pEC-XT99A-gabTDP134L with the 21 bp sequence deleted in ALE strain GluA T7 (boxed in red). (B) Determination of the minimum inhibitory concentration for tetracycline for strain GluA T0 with the orginal plasmid pEC-XT99A-gabTDP134L and GluA RG1 carrying this plasmid with the 21 bp deletion. Cells were grown in Duetz cultivation system using BHIS supplemented with increasing concentrations of tetracycline (0–80 µg µL−1). The ΔOD600 values were determined after 48 hours and are shown as means with standard deviations (n = 3 cultivations). Figure S2. Dependence of the specific acitvity of GOGAT on the glutamine concentration determined for GRLys1ΔgltB overexpressing the native version of gltBD (A) or the mutated gltBE686QD (B). Crude extracts of GRLys1ΔgltB (pEKEx3-gltBD) and GRLys1ΔgltB (pEKEx3-gltBE686QD) were assayed for specific activity of the glutamate synthase (GOGAT) in the presence of increasing glutamine concentrations (0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM, 5 mM, 10 mM). Values represent means and standard deviations of triplicate measurements. Figure S3. Glutarate production by GluA T7 in chloride-free medium. Cells were grown in Duetz cultivation system using either standard CGXII minimal medium or CGXII minimal medium without chloride supplemented with 40 g L−1 glucose, induced with 1 mM IPTG, and harvested after 48 h. Values and error bars represent means and standard deviations of glutarate (green) and 5AVA concentrations (red) in the culture supernatants (n = 3 cultivations).

Additional file 1.: Fig. S1. Sequence alignment of cphA6308 and cphA6308Δ1_C595S. Fig. S2. Graphical representation of the results obtained with the different C. glutamicum strains for CGP synthesis.

Corynebacterium glutamicum strain development has adopted each metabolic engineering tool immediately upon its conception and has contributed to developing these tools further. In the last more than five decades, C. glutamicum strains have been selected and screened after undirected mutagenesis. This approach yielded industrially relevant strains, however, it was limited by the little insight that could be gained hampering transfer to related processes. Genetic engineering, the complete genome sequence and omics tools, systems biology and synthetic biology have closed this gap and provided a profound knowledge base to develop engineering strategies driven by metabolic insight. Quite recently, serendipitous untargeted approaches came into focus again since mutants selected classically e.g. using genetically encoded biosensors or by adaptive laboratory evolution could be understood when genome resequencing was combined with genetic and biochemical experiments. We will discuss metabolic engineering from the classical, genetic engineering, systems biology eras to the era of synthetic biology for C. glutamicum strain development and forecast the impact of the most recent methods such as CRISPR technology and adaptive laboratory evolution.

Background The Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C1 carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus. Results The Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed KM of 0.74 mM and Vmax of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts. Conclusions While it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License

Amino acid fermentation is one of the major pillars of industrial biotechnology. The multi-billion USD amino acid market is rising steadily and is diversifying. Metabolic engineering is no longer focused solely on strain development for the bulk amino acids L-glutamate and L-lysine that are produced at the million-ton scale, but targets specialty amino acids. These demands are met by the development and application of new metabolic engineering tools including CRISPR and biosensor technologies as well as production processes by enabling a flexible feedstock concept, co-production and co-cultivation schemes. Metabolic engineering advances are exemplified for specialty proteinogenic amino acids, cyclic amino acids, omega-amino acids, and amino acids functionalized by hydroxylation, halogenation and N-methylation.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of l-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of l-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L

Corynebacterium glutamicum is the workhorse of the production of proteinogenic amino acids used in food and feed biotechnology. After more than 50 years of safe amino acid production, C. glutamicum has recently also been engineered for the production of amino acid-derived compounds, which find various applications, e.g., as synthons for the chemical industry in several markets including the polymer market. The amino acid-derived compounds such as non-proteinogenic ω-amino acids, α,ω-diamines, and cyclic or hydroxylated amino acids have similar carbon backbones and functional groups as their amino acid precursors. Decarboxylation of amino acids may yield ω-amino acids such as β-alanine, γ-aminobutyrate, and δ-aminovalerate as well as α,ω-diamines such as putrescine and cadaverine. Since transamination is the final step in several amino acid biosynthesis pathways, 2-keto acids as immediate amino acid precursors are also amenable to production using recombinant C. glutamicum strains. Approaches for metabolic engineering of C. glutamicum for production of amino acid-derived compounds will be described, and where applicable, production from alternative carbon sources or use of genome streamline will be referred to. The excellent large-scale fermentation experience with C. glutamicum offers the possibility that these amino acid-derived speciality products may enter large-volume markets.

Background In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578–5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed. Results The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-taCv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily responsible for pyruvate catabolism during redox self-sufficient amination of alcohols using this whole cell biocatalyst. Conclusion The replacement of the transaminase TaVf by TaCv, which showed higher activity at 42°C, in the artificial operon ald-adh-ta improved amination of alcohols in whole cell biotransformation. The addition of L-alanine, which was consumed by E. coli via pyruvate catabolism, was required for 100% product formation possibly by providing maintenance energy. Metabolic engineering revealed that pyruvate catabolism occurred primarily via oxidative decarboxylation to acetate by PoxB under the chosen biotranformation conditions. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0189-x) contains supplementary material, which is available to authorized users.

An increase in the number of elderly people suffering from the symptoms of Parkinson's disease is leading to an expansion in the market size of 3,4-dihydroxyphenyl-L-alanine (L-DOPA), which is the most commonly used drug for the treatment of this disease. Need for better quality products through economically feasible and sustainable processes makes biotechnological approaches attractive. The current study is focused on heterologous expression of Ralstonia solanacearum tyrosinase in Corynebacterium glutamicum cells to produce L-DOPA during growth on glucose or glucose/xylose mixtures. Whole-cells pre-grown on glucose were further exploited for biotransformation of L-tyrosine to L-DOPA. To prevent L-DOPA oxidation, not only the most commonly used agent, ascorbic acid, but also for the first time, thymol was evaluated. The highest L-DOPA titer was 0.26 ± 0.02 g/L at the end of growth on a mixture of 1% xylose and 3% glucose in the presence of 200 μM thymol as the oxidation inhibitor. The ability to co-utilize glucose and xylose to reach this titer could make these cells ideal for L-DOPA production using hydrolyzed lignocellulosic biomass. When the pre-grown cells were further used for biotransformation, the highest L-DOPA yield was 0.61 ± 0.02 g/gDCW with 4 mM ascorbic acid. Since L-tyrosine biotransformation is primarily dependent on tyrosinase activity, yield in this route could be improved by optimizing reaction conditions. As the industrial workhorse for amino acid production, these C. glutamicum cells will clearly benefit from strain development efforts and bioprocess optimization towards sustainable and economically feasible L-DOPA production. KEY POINTS: • Fermentative l-DOPA production was achieved in C. glutamicum. • Tyrosinase produced by C. glutamicum cells successfully transformed l-Tyr. • Thymol proved to be a significant oxidation inhibitor for l-DOPA production.