Engineering a glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O<Subscript>2</Subscript> deprivation

Objective

To explore the glycerol utilization pathway in Corynebacterium glutamicum for succinate production under O₂ deprivation.

Result

Overexpression of a glycerol facilitator, glycerol dehydrogenase and dihydroxyacetone kinase from Escherichia coli K-12 in C. glutamicum led to recombinant strains NC-3G diverting glycerol utilization towards succinate production under O₂ deprivation. Under these conditions, strain NC-3G efficiently consumed glycerol and produced succinate without growth. The recombinant C. glutamicum utilizing glycerol as the sole carbon source showed higher intracellular NADH/NAD⁺ ratio compare with utilizing glucose. The mass conversion of succinate increased from 0.64 to 0.95. Using an anaerobic fed-batch fermentation process, the final strain produced 38.4 g succinate/l with an average yield of 1.02 g/g.

Conclusions

The metabolically-engineered strains showed an efficient succinate production using glycerol as sole carbon source under O₂ deprivation.

     

Computational modeling and <Emphasis Type="Italic">in silico</Emphasis> analysis of differential regulation of <Emphasis Type="Italic">myo</Emphasis>-inositol catabolic enzymes in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Background  

Inositol is a key cellular metabolite for many organisms. Cryptococcus neoformans is an opportunistic pathogen which primarily infects the central nervous system, a region of high inositol concentration, of immunocompromised individuals. Through the use of myo-inositol oxygenase C. neoformans can catabolize inositol as a sole carbon source to support growth and viability.     

D-lactic acid production by a genetically engineered strain <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Based on its ability to produce lactic acid from glucose in mineral salt medium under anaerobic conditions, genetic modifications on Corynebacterium glutamicum Res 167 were carried out with the aim of producing optical pure D-lactic acid, involving the knockout of L-lactate dehydrogenase gene from C. glutamicum and the heterologous expression of D-lactate dehydrogenase gene from Lactobacillus bulgaricus into C. glutamicum. D-lactic acid production of the genetically engineered strain C. glutamicum Res 167Δldh/ldhA was 17.92 g/l (optical purity higher than 99.9%) after 16 h fermentation, which was 32.25% higher than the lactic acid production of the parental strain.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.     

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.

     

Isolation of the <Emphasis Type="Italic">phe</Emphasis>-operon from <Emphasis Type="Italic">G. stearothermophilus</Emphasis> comprising the phenol degradative <Emphasis Type="Italic">meta</Emphasis>-pathway genes and a novel transcriptional regulator

Background  

Geobacillus stearothermophilus is able to utilize phenol as a sole carbon source. A DNA fragment encoding a phenol hydroxylase catalyzing the first step in the meta-pathway has been isolated previously. Based on these findings a PCR-based DNA walk was performed initially to isolate a catechol 2,3-dioxygenase for biosensoric applications but was continued to elucidate the organisation of the genes encoding the proteins for the metabolization of phenol.     

Characterization of the biotin uptake system encoded by the biotin-inducible <Emphasis Type="Italic">bioYMN</Emphasis> operon of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Background  

The amino acid-producing Gram-positive Corynebacterium glutamicum is auxotrophic for biotin although biotin ring assembly starting from the precursor pimeloyl-CoA is still functional. It possesses AccBC, the α-subunit of the acyl-carboxylases involved in fatty acid and mycolic acid synthesis, and pyruvate carboxylase as the only biotin-containing proteins. Comparative genome analyses suggested that the putative transport system BioYMN encoded by cg2147, cg2148 and cg2149 might be involved in biotin uptake by C. glutamicum.     

Increasing l‐isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two‐component export system BrnFE

Aims

To increase the l ‐isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two‐component export system BrnFE.

Methods and Results

The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW‐8 individually or in combination. The constructed plasmids were transformed into an l ‐isoleucine‐producing strain C. glutamicum JHI3‐156, and the l ‐isoleucine production in these different strains was analysed and compared. More l ‐isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l ‐isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l ‐isoleucine production in JHI3‐156/pDXW‐8‐lrp‐brnFE increased 63% in flask cultivation, and the specific yield of l ‐isoleucine increased 72% in fed‐batch fermentation.

Conclusions

Both Lrp and BrnFE are important to enhance the l ‐isoleucine production in C. glutamicum.

Significance and Impact of the Study

The results provide useful information to enhance l ‐isoleucine or other branched‐chain amino acid production in C. glutamicum.     

Isolation,evaluation and use of two strong,carbon source‐inducible promoters from Corynebacterium glutamicum

Aims: To obtain strong, carbon source‐inducible promoters useful for industrial applications of Corynebacterium glutamicum. Methods and Results: DNA microarray and qRT‐PCR enabled identification of the promoters of cgR_2367 (malE1) and cgR_2459 (git1) as strong, maltose‐ and gluconate‐inducible promoters, respectively, in C. glutamicum. Promoter probe assays revealed that in the presence of the inducing sugars, PmalE1 and Pgit1, respectively, facilitated 3·4‐ and 4·2‐fold increased β‐galactosidase activities compared to the same activity induced by glucose. In addition, PmalE1 was not functional in Escherichia coli, in which Pgit1 function was repressible, which enabled the cloning of a hitherto ‘difficult‐to‐clone’ heterologous gene of a lignocellulolytic enzyme, whose secretion was consequently induced by the carbon sources. Conclusions: PmalE1 and Pgit1 are strong, carbon source‐inducible promoters of C. glutamicum whose characteristics in E. coli are integral to the secretion ability of C. glutamicum to secrete lignocellulolytic enzyme. Significance and Impact of the Study: Corynebacterium glutamicum, like its counterpart industrial workhorses E. coli and Bacillus subtilis, does exhibit strong, carbon source‐inducible promoters, and the functionality of two of which was demonstrated in this study. While this study may be most relevant in the ongoing efforts to establish technologies of the biorefinery, it should also be of interest to general microbiologists exploring the versatility of industrial micro‐organisms. In so doing, the study should impact future advances in industrial microbiology.     

High-level recombinant protein production with Corynebacterium glutamicum using acetate as carbon source

In recent years, biotechnological conversion of the alternative carbon source acetate has attracted much attention. So far, acetate has been mainly used for microbial production of bioproducts with bulk applications. In this study, we aimed to investigate the potential of acetate as carbon source for heterologous protein production using the acetate-utilizing platform organism Corynebacterium glutamicum. For this purpose, expression of model protein eYFP with the promoter systems T7lac and tac was characterized during growth of C. glutamicum on acetate as sole carbon source. The results indicated a 3.3-fold higher fluorescence level for acetate-based eYFP production with T7 expression strain MB001(DE3) pMKEx2-eyfp compared to MB001 pEKEx2-eyfp. Interestingly, comparative eyfp expression studies on acetate or glucose revealed an up to 83% higher biomass-specific production for T7 RNAP-dependent eYFP production using acetate as carbon source. Furthermore, high-level protein accumulation on acetate was demonstrated for the first time in a high cell density cultivation process with pH-coupled online feeding control, resulting in a final protein titer of 2.7 g/L and product yield of 4 g per 100 g cell dry weight. This study presents a first proof of concept for efficient microbial upgrading of potentially low-cost acetate into high-value bioproducts, such as recombinant proteins.     

Characterization of the mannitol catabolic operon of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum encodes a mannitol catabolic operon, which comprises three genes: the DeoR-type repressor coding gene mtlR (sucR), an MFS transporter gene (mtlT), and a mannitol 2-dehydrogenase gene (mtlD). The mtlR gene is located upstream of the mtlTD genes in the opposite orientation. In spite of this, wild-type C. glutamicum lacks the ability to utilize mannitol. This wild-type phenotype results from the genetic regulation of the genes coding for mannitol transport and catalytic proteins mediated by the autoregulated MtlR protein since mtlR mutants grow on mannitol as the sole carbon source. MtlR binds to sites near the mtlR (two sites) and mtlTD promoters (one site downstream of the promoter), with the consensus sequence 5′-TCTAACA-3′ being required for its binding. The newly discovered operon comprises the three basic functional elements required for mannitol utilization: regulation, transport, and metabolism to fructose, further processed to the common intermediate of glycolysis fructose-6-phosphate. When relieved from MtlR repression, C. glutamicum, which lacks a functional fructokinase, excretes the fructose derived from mannitol and imports it by the fructose-specific PTS. In order to use mannitol from seaweed biomass hydrolysates as a carbon source for the production of useful commodity chemicals and materials, an overexpression system using the tac promoter was developed. For congruence with the operon, we propose to rename sucR as the mtlR gene.     

Chemotaxis of <Emphasis Type="Italic">Burkholderia</Emphasis> sp. Strain SJ98 towards chloronitroaromatic compounds that it can metabolise

Background  

Burkholderia sp. strain SJ98 is known for its chemotaxis towards nitroaromatic compounds (NACs) that are either utilized as sole sources of carbon and energy or co-metabolized in the presence of alternative carbon sources. Here we test for the chemotaxis of this strain towards six chloro-nitroaromatic compounds (CNACs), namely 2-chloro-4-nitrophenol (2C4NP), 2-chloro-3-nitrophenol (2C3NP), 4-chloro-2-nitrophenol (4C2NP), 2-chloro-4-nitrobenzoate (2C4NB), 4-chloro-2-nitrobenzoate (4C2NB) and 5-chloro-2-nitrobenzoate (5C2NB), and examine its relationship to the degradation of such compounds.     

Direct production of cadaverine from soluble starch using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> coexpressing α-amylase and lysine decarboxylase

Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 α-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli–C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5’-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation of l-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.     

Excision dynamics of <Emphasis Type="Italic">Vibrio</Emphasis> pathogenicity island-2 from <Emphasis Type="Italic">Vibrio cholerae:</Emphasis> role of a recombination directionality factor VefA

Background  

Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758) that belongs to the tyrosine recombinase family. IntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate.     

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.     

Direct l-lysine production from cellobiose by Corynebacterium glutamicum displaying beta-glucosidase on its cell surface

We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum, and direct l-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of l-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced l-lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum.