Emerging biotechnologies for production of itaconic acid and its applications as a platform chemical

Recently, itaconic acid (IA), an unsaturated C5-dicarboxylic acid, has attracted much attention as a biobased building block chemical. It is produced industrially (>80 g L^?1) from glucose by fermentation with Aspergillus terreus. The titer is low compared with citric acid production (>200 g L^?1). This review summarizes the latest progress on enhancing the yield and productivity of IA production. IA biosynthesis involves the decarboxylation of the TCA cycle intermediate cis-aconitate through the action of cis-aconitate decarboxylase (CAD) enzyme encoded by the CadA gene in A. terreus. A number of recombinant microorganisms have been developed in an effort to overproduce it. IA is used as a monomer for production of superabsorbent polymer, resins, plastics, paints, and synthetic fibers. Its applications as a platform chemical are highlighted. It has a strong potential to replace petroleum-based methylacrylic acid in industry which will create a huge market for IA.     

Overexpression of <Emphasis Type="Italic">ppc</Emphasis> or deletion of <Emphasis Type="Italic">mdh</Emphasis> for improving production of γ-aminobutyric acid in recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

l-Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced l-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, l-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3?±?3.4, 24.8?±?0.7, and 25.5?±?3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7?±?0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0?±?0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.     

The diversity of rhizobia nodulating the <Emphasis Type="Italic">Medicago</Emphasis>, <Emphasis Type="Italic">Melilotus</Emphasis> and <Emphasis Type="Italic">Trigonella</Emphasis> inoculation group in Egypt is marked by the dominance of two genetic types

Twenty four rhizobial strains were isolated from root nodules of Melilotus, Medicago and Trigonella plants growing wild in soils throughout Egypt. The nearly complete 16S rRNA gene sequence from each strain showed that 12 strains (50 %) were closely related to the Ensifer meliloti LMG6133^T type strain with identity values higher than 99.0 %, that 9 (37.5 %) strains were more than 99 % identical to the E. medicae WSM419^T type strain, and that 3 (12.5 %) strains showed 100 % identity with the type strain of N. huautlense S02^T. Accordingly, the diversity of rhizobial strains nodulating wild Melilotus, Medicago and Trigonella species in Egypt is marked by predominance of two genetic types, E. meliloti and E. medicae, although the frequency of isolation was slightly higher in E. meliloti. Sequencing of the symbiotic nodC gene from selected Medicago and Melilotus strains revealed that they were all similar to those of the E. meliloti LMG6133^T and E. medicae WSM419^T type strains, respectively. Similarly, nodC sequences of strains identified as members of the genus Neorhizobium were more than 99 % identical to that of N. galegae symbiovar officinalis HAMBI 114.     

Long-term adaptation of <Emphasis Type="Italic">Escherichia coli</Emphasis> to methanogenic co-culture enhanced succinate production from crude glycerol

Escherichia coli can hardly grow anaerobically on glycerol without exogenous electron acceptor. The formate-consuming methanogen Methanobacterium formicicum plays a role as a living electron acceptor in glycerol fermentation of E. coli. Wild-type and mutant E. coli strains were screened for succinate production using glycerol in a co-culture with M. formicicum. Subsequently, E. coli was adapted to glycerol fermentation over 39 rounds (273 days) by successive co-culture with M. formicicum. The adapted E. coli (19.9 mM) produced twice as much succinate as non-adapted E. coli (9.7 mM) and 62% more methane. This study demonstrated improved succinate production from waste glycerol using an adapted wild-type strain of E. coli with wild-type M. formicicum, which is more useful than genetically modified strains. Crude glycerol, an economical feedstock, was used for the cultivation. Furthermore, the increase in methane production by M. formicicum during co-culture with adapted E. coli illustrated the possibility of energy-saving effects for the fermentation process.     

Redirecting carbon flux through <Emphasis Type="Italic">pgi</Emphasis>-deficient and heterologous transhydrogenase toward efficient succinate production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is particularly known for its potentiality in succinate production. We engineered C. glutamicum for the production of succinate. To enhance C₃–C₄ carboxylation efficiency, chromosomal integration of the pyruvate carboxylase gene pyc resulted in strain NC-4. To increase intracellular NADH pools, the pntAB gene from Escherichia coli, encoding for transhydrogenase, was chromosomally integrated into NC-4, leading to strain NC-5. Furthermore, we deleted pgi gene in strain NC-5 to redirect carbon flux to the pentose phosphate pathway (PPP). To solve the drastic reduction of PTS-mediated glucose uptake, the ptsG gene from C. glutamicum, encoding for the glucose-specific transporter, was chromosomally integrated into pgi-deficient strain resulted in strain NC-6. In anaerobic batch fermentation, the production of succinate in pntAB-overexpressing strain NC-5 increased by 14% and a product yield of 1.22 mol/mol was obtained. In anaerobic fed-batch process, succinic acid concentration reached 856 mM by NC-6. The yields of succinate from glucose were 1.37 mol/mol accompanied by a very low level of by-products. Activating PPP and transhydrogenase in combination led to a succinate yield of 1.37 mol/mol, suggesting that they exhibited a synergistic effect for improving succinate yield.     

Optimization of C16 and C18 fatty alcohol production by an engineered strain of <Emphasis Type="Italic">Lipomyces starkeyi</Emphasis>

The oleaginous yeast Lipomyces starkeyi was engineered for the production of long-chain fatty alcohols by expressing a fatty acyl-CoA reductase, mFAR1, from Mus musculus. The optimal conditions for production of fatty alcohols by this strain were investigated. Increased carbon-to-nitrogen ratios led to efficient C16 and C18 fatty alcohol production from glucose, xylose and glycerol. Batch cultivation resulted in a titer of 1.7 g/L fatty alcohol from glucose which represents a yield of 28 mg of fatty alcohols per gram of glucose. This relatively high level of production with minimal genetic modification indicates that L. starkeyi may be an excellent host for the bioconversion of carbon-rich waste streams, particularly lignocellulosic waste, to C16 and C18 fatty alcohols.     

Genetic engineering of Clostridium thermocellum DSM1313 for enhanced ethanol production

Background

The twin problem of shortage in fossil fuel and increase in environmental pollution can be partly addressed by blending of ethanol with transport fuel. Increasing the ethanol production for this purpose without affecting the food security of the countries would require the use of cellulosic plant materials as substrate. Clostridium thermocellum is an anaerobic thermophilic bacterium with cellulolytic property and the ability to produce ethanol. But its application as biocatalyst for ethanol production is limited because pyruvate ferredoxin oxidoreductase, which diverts pyruvate to ethanol production pathway, has low affinity to the substrate. Therefore, the present study was undertaken to genetically modify C. thermocellum for enhancing its ethanol production capacity by transferring pyruvate carboxylase (pdc) and alcohol dehydrogenase (adh) genes of the homoethanol pathway from Zymomonas mobilis.

Results

The pdc and adh genes from Z. mobilis were cloned in pNW33N, and transformed to Clostridium thermocellum DSM 1313 by electroporation to generate recombinant CTH-pdc, CTH-adh and CTH-pdc-adh strains that carried heterologous pdc, adh, and both genes, respectively. The plasmids were stably maintained in the recombinant strains. Though both pdc and adh were functional in C. thermocellum, the presence of adh severely limited the growth of the recombinant strains, irrespective of the presence or absence of the pdc gene. The recombinant CTH-pdc strain showed two-fold increase in pyruvate carboxylase activity and ethanol production when compared with the wild type strain.

Conclusions

Pyruvate decarboxylase gene of the homoethanol pathway from Z mobilis was functional in recombinant C. thermocellum strain and enhanced its ability to produced ethanol. Strain improvement and bioprocess optimizations may further increase the ethanol production from this recombinant strain.

     

Antibiotic activity of bacterial endobionts of basidiomycete fruit bodies

Bacterial strains (93 isolates) capable of growth on full-strength nutrient media were isolated from 86 fungal fruit bodies collected in the Moscow region. Antimicrobial activity of the endobiont isolates against 12 bacterial and fungal test strains (including drug-resistant ones) was studied in submerged cultures. Most of the strains (84.9%) were found to produce antibiotic compounds with different antimicrobial properties, including antifungal activity in 18.3% of the strains. Morphological characteristics and analysis of the 16S rRNA gene sequences were used to determine the taxonomic position of 16 bacterial strains of the following 10 species: Bacillus subtilis, Ewingella americana, Pseudomonas sp., Stenotrophomonas maltophilia, as well as Achromobacter spanius, B. licheniformis, Hafnia paralvei, Micrococcus terreus, Nocardia coeliaca, and St. rhizophila, which have not been previously known to be endobionts of basidiomycete fruit bodies. Antimicrobial activity of A. spanius, E. americana, H. paralvei, M. terreus, N. coeliaca, and St. rhizophila has not been reported previously. Complex mechanisms of symbiotic relations between fungi and bacteria, including those associated with antibiotic formation, probably developed in the course of co-evolution.     

High yield production of four-carbon dicarboxylic acids by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61–0.67 mol (mole glucose)^?1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38 mM (5.41 g/L) and 50.34 mM (6.75 g/L) malate with a molar yield of 0.53 and 0.55 mol (mole succinate)^?1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133 mM (17.83 g/L) malate in 47 h, with a high yield of 1.3 mol (mole glucose)^?1 and productivity of 0.38 g L^?1 h^?1.     

Enhancement of 5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Objective

To construct a strain of Corynebacterium glutamicum capable of efficiently producing 5-aminolevulinic acid (5-ALA) via the C4 pathway by modification of serine and glycine pathway using glucose as sole carbon source.

Results

The recombinant C. glutamicum strain AP2 harboring a codon-optimized hemA gene from Rhodobacter sphaeroides was used as host strain for 5-ALA production. A plasmid harboring the serine operon, which contained serB, serC and the site-specific mutant serA ^Δ197 , was constructed and introduced into C. glutamicumAP2, leading to an increase of 70% in 5-ALA production. Further overexpression of the glyA gene increased production of 5-ALA by 150% over the control. 5-ALA production was thus significantly enhanced by engineering the glycine biosynthetic pathway. C.glutamicum AG3 produced 3.4 ± 0.2 g 5-ALA/l in shake-flask cultures in CGIIIM medium with the addition of 7.5 g glycine/l.

Conclusion

This is the first report of remodeling the serine and glycine biosynthetic pathway to improve the production of 5-ALA in C. glutamicum.

     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Objective

α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to construct recombinant Aspergillus niger strains with high α-galactosidase production.

Results

Two recombinant A. niger strains were constructed: AB and AGB. The recombinant AB strain contained the α-galactosidase aglB gene from A. niger with its native AglB signal peptide regulated by the glucoamylase promoter. In the AGB recombinant strain, the AglB signal peptide was replaced with the glucoamylase (GlaA) signal peptide. The extracellular maximum α-galactosidase activity of the AGB strain was 215.7 U/ml and that of the AB strain was 9.8 U/mL. The optimal conditions for α-galactosidase were pH 3.5 and 35 °C.

Conclusions

The GlaA signal peptide substantially increased the yield of secreted α-galactosidase in A. niger. This recombinant strain holds great potential for industrial applications.

     

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.     

Production of sapogenins (stigmasterol and hecogenin) from genetically transformed hairy root cultures of <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis> (Safed musli)

Chlorophytum borivilianum belonging to the family Liliaceae, is distributed in the pantropical regions of India and South Africa. The sapogenins (stigmasterol and hecogenin) of C. borivilianum are well known for their appetizing and aphrodisiac properties. The present study involves enhancing the sapogenin content in C. borivilianum by genetic transformations with Agrobacterium rhizogenes strains (MTCC 2364 and 532, PRT Gus). A maximum transformation frequency of 98% was obtained with Agrobacterium rhizogenes MTCC 2364 strain with rhizome explants after a co-cultivation period of 48 h. Two potential rhizoclones (2364a and 2364b) were selected for the production of stigmasterol and hecogenin. The maximum production of stigmasterol (83.952?±?0.01 mg/g) was seen in 2364b rhizoclone, whereas, the highest accumulation of hecogenin (81.52?±?0.02 mg/g) was observed in 2364a rhizoclone. The C. borivilianum hairy root cultures obtained in this study provide a continuous and sustainable production of stigmasterol and hecogenin on a commercial scale.     

Development of <Emphasis Type="Italic">mazF</Emphasis>-based markerless genome editing system and metabolic pathway engineering in <Emphasis Type="Italic">Candida tropicalis</Emphasis> for producing long-chain dicarboxylic acids

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the β-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH–cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.