Significance of Arg3, Arg54, and Tyr58 of l-aspartate α-decarboxylase from Corynebacterium glutamicum in the process of self-cleavage

Purpose of work

We have elucidated the significance of three key amino acid residues of l-aspartate α-decarboxylase that act remotely from its cleavage site for its functional self-cleavage as well as for its catalytic activity. These results provide useful fundamental information for engineering l-aspartate α-decarboxylase. l-Aspartate α-decarboxylase (ADC) from Corynebacterium glutamicum, and encoded by panD, was cloned and expressed in Escherichia coli and then purified. Three amino acid residues were found to be related to ADC self-cleavage. Mutating R3 to either A, Q, N, L, D, or E produced only the unprocessed pro-enzyme. Although mutating R54 and Y58 into A or K and A or T, respectively, partly influenced ADC self-cleavage, the specific activity of each of the four ßmutants decreased to 3.5, 4, 2.4, and 2.6 U mg^?1, respectively, compared with a specific activity of 690 U mg^?1 for the wild-type enzyme. Thus, R3 triggers ADC self-cleavage and completes the modification of the active site with assistance by R54 and Y58. These results will help to engineer ADC for improved industrial applications.     

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of β-alanine

β-Alanine is an important β-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of β-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of β-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of β-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient β-alanine biosynthesis was used to construct a β-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative β-alanine exporter was newly identified, and its overexpression further improved the β-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of β-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.     

Metabolic engineering to improve 1,5-diaminopentane production from cellobiose using β-glucosidase-secreting Corynebacterium glutamicum

Microbial production of 1,5-diaminopentane (DAP) from renewable feedstock is a promising and sustainable approach for the production of polyamides. In this study, we constructed a β-glucosidase (BGL)-secreting Corynebacterium glutamicum and successfully used this strain to produce DAP from cellobiose and glucose. First, C. glutamicum was metabolically engineered to produce l -lysine (a direct precursor of DAP), followed by the coexpression of l -lysine decarboxylase and BGL derived from Escherichia coli and Thermobifida fusca YX (Tfu0937), respectively. This new engineered C. glutamicum strain produced 27 g/L of DAP from cellobiose in CGXII minimal medium using fed-batch cultivation. The yield of DAP was 0.43 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose), which is the highest yield reported to date. These results demonstrate the feasibility of DAP production from cellobiose or cellooligosaccharides using an engineered C. glutamicum strain.     

Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation

Mechanism-based inactivation of l -aspartate-α-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of β-alanine from L -aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanD^{I46V/I88M/K104S/I126*}) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l -aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L β-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.     

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s⁻¹), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s⁻¹), and high tolerance to product inhibition (K_i for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.     

Expression of Bacterial l-aspartate-α-decarboxylase in Tobacco Increases β-Alanine and Pantothenate Levels and Improves Thermotolerance

l-Aspartate-α-decarboxylase catalyzes the decarboxylation of l-aspartate to generate β-alanine and carbon dioxide. This is an unusual pyruvoyl-dependent enzyme unique to prokaryotes that undergoes limited self-processing. The Escherichia coli panD gene encoding l-aspartate-α-decarboxylase was expressed under a constitutive promoter in transgenic tobacco. Transgene expression was verified by assays based on RNA blots, immunoblots and enzyme activity in vitro. The panD lines had increased levels of leaf β-alanine (1.2- to 4-fold), pantothenate (3.2- to 4.1-fold) and total free amino acids (up to 3.7-fold) compared to wild-type and vector controls. Growth of homozygous lines expressing E. coli l-aspartate-α-decarboxylase was less affected than that of the control lines when the plants were stressed for 1 week at 35 °C. When transferred from 35 to 30 °C for 3 weeks, the PanD transgenic lines recovered significantly (P ≤ 0.001) better than the controls: PanD lines had on an average 54% and 84% greater fresh and dry weights respectively, compared to the controls. Homozygous lines expressing E. coli l-aspartate-α-decarboxylase had significantly greater thermotolerance (P ≤ 0.05) during germination. At 42 °C, 95% of two T3 PanD transgenic line seeds germinated after 12 days compared to 73% for the wild-type seeds. Our results indicated that E. colil-aspartate-α-decarboxylase was correctly processed and active in the transgenic eukaryotic host and its expression resulted in increased thermotolerance in tobacco. This is Florida Agricultural Experiment Station journal series number R-10355. W.M.F. was supported by the Egypt Development Training fellowship and by the UF College of Agriculture and Life Sciences assistantship.     

Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

Enhanced production of neoagarobiose from agar with Corynebacterium glutamicum producing exo-type and endo-type β-agarases

Neoagarobiose (NA2) derived from agar marine biomass is a rare reagent that acts as an anti-melanogenesis reagent and moisturizer. Here, for the economical manufacturing of NA2, we developed the co-secretory production system of endo-type β-agarases (DagA) and exo-type β-agarases (EXB3) in Corynebacterium glutamicum. For this purpose, we first developed a secretory system of DagA via Tat pathway. To improve the secretion efficiency, we coexpressed two Tat pathway components (TatA and TatC), and to improve the purity of secreted DagA in the culture supernatant, two endogenous protein genes (Cg2052 and Cg1514) were removed. Using the engineered strain (C. glutamicum SP002), we confirmed that DagA as high as 1.53 g l^-1 was successfully produced in the culture media with high purity (72.7% in the supernatant protein fraction). Next, we constructed the expression system (pHCP-CgR-DagA-EXB3) for the simultaneous secretion of EXB3 via Sec-pathway together with DagA, and it was clearly confirmed that DagA and EXB3 were successfully secreted as high as 54% and 24.5%, respectively. Finally, using culture medium containing DagA and EXB3, we successfully demonstrated the conversion of high-concentration agar (40 g l^-1) into NA2 via a two-stage hydrolysis process.     

From zero to hero – Production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum

Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2 g L⁻¹ h⁻¹, and a final titer of 88 g L⁻¹. The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.     

Involvement of meso-α,∊-Diamino-pimelate D-Dehydrogenase in Lysine Biosynthesis in Corynebacterium glutamicum

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

The β-ketoadipate pathway, a primarily chromo-somally encoded catabolic route that plays a signifi-cant role in the degradation of aromatic compounds, is widely distributed in soil bacteria and fungi[1]. This pathway consists of two parallel branches of which the aromatic rings are cleaved by either protocatechuate 3,4-dioxygenase or catechol 1,2-dioxygenase. The two branches converge at β-ketoadipate enol-lactone in bacteria, and three additional steps complete the con-version of the latter…     

Mechanism of Concerted Inhibition of α2β2-type Hetero-oligomeric Aspartate Kinase from Corynebacterium glutamicum

Aspartate kinase (AK) is the first and committed enzyme of the biosynthetic pathway producing aspartate family amino acids, lysine, threonine, and methionine. AK from Corynebacterium glutamicum (CgAK), a bacterium used for industrial fermentation of amino acids, including glutamate and lysine, is inhibited by lysine and threonine in a concerted manner. To elucidate the mechanism of this unique regulation in CgAK, we determined the crystal structures in several forms: an inhibitory form complexed with both lysine and threonine, an active form complexed with only threonine, and a feedback inhibition-resistant mutant (S301F) complexed with both lysine and threonine. CgAK has a characteristic α₂β₂-type heterotetrameric structure made up of two α subunits and two β subunits. Comparison of the crystal structures between inhibitory and active forms revealed that binding inhibitors causes a conformational change to a closed inhibitory form, and the interaction between the catalytic domain in the α subunit and β subunit (regulatory subunit) is a key event for stabilizing the inhibitory form. This study shows not only the first crystal structures of α₂β₂-type AK but also the mechanism of concerted inhibition in CgAK.     

N-Acetylornithine-δ-aminotransferase-deficient and N-Acetylglutamokinase-deficient Arginine Auxotrophs of Corynebacterium glutamicum

An N-acetylglutamokinase-deficient arginine-requiring mutant, KY9390 and an N-acetylornithine aminotransferase-deficient arginine-requiring mutant, AA-7 were derived from a wild-type strain and an l-arginine-producing mutant of Corynebacterium glutamicum, respectively. KY 9390 accumulated 7.5 mg per ml of N-acetylglutamic acid and AA-7 accumulated 2 mg per ml of N-acetylglutamate-γ-semialdehyde, intermediates of arginine biosynthesis, in a culture medium.

The production of N-acetylglutamate-γ-semialdehyde by AA-7 was not affected by the concentration of l-arginine in the medium, whereas that of N-acetylglutamic acid by KY 9390 was inhibited by the addition of l-arginine in the medium.     

Corynebacterium glutamicum,a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro‐aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild‐type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L^?1) corresponded to the lower k_La value of 5 h^?1. Above 5 h^?1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L^?1. Taking into account this threshold, a fed‐batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild‐type strain was able to produce 93.6 g L^?1 of succinate, which is one of the highest concentration reported in the literature.     

Identification and expression of the 11β-steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industries. Particularly, the 11β-hydroxylation of steroids is a reaction of biotechnological importance currently carried out at industrial scale by the fungus Cochliobolus lunatus. In this work, we have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11β-hydroxylase activity in this fungus, which is the key step for the preparative synthesis of cortisol in industry. A recombinant Corynebacterium glutamicum strain harbouring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11β-hydroxylate several steroids as substrates. This is a new example to show that the industrial strain C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during isoleucine formation from α-hydroxybutyric acid

When Corynebacterium glutamicum ATCC 14310 (leu^-) was cultured with 200 mg/l leucine and 150 mM -hydroxybutyric acid the acetohydroxy acid synthase activity was increased to 0.17 U/mg as compared to 0.03 U/mg in the wildtype. This increase was a combined effect of the limiting amounts of leucine added, together with an apparent additional internal leucine/valine shortage resulting from accumulated -ketobutyric acid (5 mM) and the kinetic characteristics of the acetohydroxy acid synthase. The increase in the specific AHAS activity by the appropriate amino acid limitation resulted in an increased isoleucine yield of 71 mmol/l as compared to 27 mmol/l obtained under non-limiting conditions.Abbreviation AHAS Acetohydroxy acid synthase     

Transfection of Corynebacterium glutamicum with Temperate Phage ϕ CG1

Versatile plasmid vectors useful for gene cloning in Brevibacterium lactofermentum, a glutamic acid-producing bacterium, have been constructed. The trimethoprim (Tp)-resistant dihydrofolate reductase gene derived from chromosomal DNA of the Tp-resistant mutant of B. lactofermentum was introduced into pAM330, a cryptic plasmid in B. lactofermentum. The constructed cloning vector pAJ228 (7.6kb) exists in 10 to 20 copies in cells of B. lactofermentum and donated Tp-resistance, which is a useful selective marker of transformants. pAJ228 was further improved to a versatile plasmid vector pAJ224 having some profitable characteristics such as smaller size (3.7 kb), higher copy number (60 ~ 80 copies), and additional useful cloning sites (Bam HI, Pst I and Sal I) equipped with two different promoters arranged at both orientations for the expression of passenger DNA without promoter. These plasmids were stably retained in B. lactofermentum even in the absence of Tp over many generations. Thus, they have been found very powerful vectors for gene cloning in Brevibacterium and the related bacteria.