Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Microbial synthesis of the plant natural product precursor p-coumaric acid with Corynebacterium glutamicum.

BACKGROUND: Phenylpropanoids such as p-coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids, flavonoids, and lignans, which are of pharmacological interest due to their health-promoting properties. Although extraction from plant material or chemical synthesis is possible, microbial synthesis of p-coumaric acid from glucose has the advantage of being less expensive and more resource efficient. In this study, Corynebacterium glutamicum was engineered for the production of the plant polyphenol precursor p-coumaric acid from glucose. RESULTS: Heterologous expression of the tyrosine ammonia-lyase encoding gene from Flavobacterium johnsoniae enabled the conversion of endogenously provided tyrosine to p-coumaric acid. Product consumption was avoided by abolishing essential reactions of the phenylpropanoid degradation pathway. Accumulation of anthranilate as a major byproduct was eliminated by reducing the activity of anthranilate synthase through targeted mutagenesis to avoid tryptophan auxotrophy. Subsequently, the carbon flux into the shikimate pathway was increased, phenylalanine biosynthesis was reduced, and phosphoenolpyruvate availability was improved to boost p-coumaric acid accumulation. A maximum titer of 661 mg/L p-coumaric acid (4 mM) in defined mineral medium was reached. Finally, the production strain was utilized in co-cultivations with a C. glutamicum strain previously engineered for the conversion of p-coumaric acid into the polyphenol resveratrol. These co-cultivations enabled the synthesis of 31.2 mg/L (0.14 mM) resveratrol from glucose without any p-coumaric acid supplementation. CONCLUSIONS: The utilization of a heterologous tyrosine ammonia-lyase in combination with optimization of the shikimate pathway enabled the efficient production of p-coumaric acid with C. glutamicum. Reducing the carbon flux into the phenylalanine and tryptophan branches was the key to success along with the introduction of feedback-resistant enzyme variants.

From Aquaculture to Aquaculture: Production of the Fish Feed Additive Astaxanthin by Corynebacterium glutamicum Using Aquaculture Sidestream.

Circular economy holds great potential to minimize the use of finite resources, and reduce waste formation by the creation of closed-loop systems. This also pertains to the utilization of sidestreams in large-scale biotechnological processes. A flexible feedstock concept has been established for the industrially relevant Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin. In this study, we aimed to use a preprocessed aquaculture sidestream for production of carotenoids, including the fish feed ingredient astaxanthin by C. glutamicum. The addition of a preprocessed aquaculture sidestream to the culture medium did not inhibit growth, obviated the need for addition of several components of the mineral salt's medium, and notably enhanced production of astaxanthin by an engineered C. glutamicum producer strain. Improved astaxanthin production was scaled to 2 L bioreactor fermentations. This strategy to improve astaxanthin production was shown to be transferable to production of several native and non-native carotenoids. Thus, this study provides a proof-of-principle for improving carotenoid production by C. glutamicum upon supplementation of a preprocessed aquaculture sidestream. Moreover, in the case of astaxanthin production it may be a potential component of a circular economy in aquaculture.

High-efficiency production of the antimicrobial peptide pediocin PA-1 in metabolically engineered Corynebacterium glutamicum using a microaerobic process at acidic pH and elevated levels of bivalent calcium ions.

BACKGROUND: Pediocin PA-1 is a bacteriocin of recognized value with applications in food bio-preservation and the medical sector for the prevention of infection. To date, industrial manufacturing of pediocin PA-1 is limited by high cost and low-performance. The recent establishment of the biotechnological workhorse Corynebacterium glutamicum as recombinant host for pediocin PA-1 synthesis displays a promising starting point towards more efficient production. RESULTS: Here, we optimized the fermentative production process. Following successful simplification of the production medium, we carefully investigated the impact of dissolved oxygen, pH value, and the presence of bivalent calcium ions on pediocin production. It turned out that the formation of the peptide was strongly supported by an acidic pH of 5.7 and microaerobic conditions at a dissolved oxygen level of 2.5%. Furthermore, elevated levels of CaCl2 boosted production. The IPTG-inducible producer C. glutamicum CR099 pXMJ19 Ptac pedACDCg provided 66 mg L-1 of pediocin PA-1 in a two-phase batch process using the optimized set-up. In addition, the novel constitutive strain Ptuf pedACDCg allowed successful production without the need for IPTG. CONCLUSIONS: The achieved pediocin titer surpasses previous efforts in various microbes up to almost seven-fold, providing a valuable step to further explore and develop this important bacteriocin. In addition to its high biosynthetic performance C. glutamicum proved to be highly robust under the demanding producing conditions, suggesting its further use as host for bacteriocin production.

Chromosomal editing of Corynebacterium glutamicum ATCC 13032 to produce gamma-aminobutyric acid.

Corynebacterium glutamicum has been used as a sustainable microbial producer for various bioproducts using cheap biomass resources. In this study, a high GABA-producing C. glutamicum strain was constructed by chromosomal editing. Lactobacillus brevis-derived gadB2 was introduced into the chromosome of C. glutamicum ATCC 13032 to produce gamma-aminobutyric acid and simultaneously blocked the biosynthesis of lactate and acetate. GABA transport and degradation in C. glutamicum were also blocked to improve GABA production. As precursor of GABA, l-glutamic acid synthesis in C. glutamicum was enhanced by introducing E. coli gdhA encoding glutamic dehydrogenase, and the copy numbers of gdhA and gadB2 were also optimized for higher GABA production. The final C. glutamicum strain CGY705 could produce 33.17 g/L GABA from glucose in a 2.4-L bioreactor after 78 h fed-batch fermentation. Since all deletion and expression of genes were performed using chromosomal editing, fermentation of the GABA-producing strains constructed in this study does not need supplementation of any antibiotics and inducers. The strategy used in this study has potential value in the development of GABA-producing bacteria.

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline.

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.

Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum.

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L-1 ) from d-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol d-xylose, which corresponds to 38% of the maximum theoretical yield. Under growth-decoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when d-glucose and d-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4%. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

Production of trans-cinnamic acid by whole-cell bioconversion from L-phenylalanine in engineered Corynebacterium glutamicum.

BACKGROUND: trans-cinnamic acid (t-CA) is a phenylpropanoid with a broad spectrum of biological activities including antioxidant and antibacterial activities, and it also has high potential in food and cosmetic applications. Although significant progress has been made in the production of t-CA using microorganisms, its relatively low product titers still need to be improved. In this study, we engineered Corynebacterium glutamicum as a whole-cell catalyst for the bioconversion of L-phenylalanine (L-Phe) into t-CA and developed a repeated bioconversion process. RESULTS: An expression module based on a phenylalanine ammonia lyase-encoding gene from Streptomyces maritimus (SmPAL), which mediates the conversion of L-Phe into t-CA, was constructed in C. glutamicum. Using the strong promoter PH36 and ribosome binding site (RBS) (in front of gene 10 of the T7 phage), and a high-copy number plasmid, SmPAL could be expressed to levels as high as 39.1% of the total proteins in C. glutamicum. Next, to improve t-CA production at an industrial scale, reaction conditions including temperature and pH were optimized; t-CA production reached up to 6.7 mM/h in a bioreactor under optimal conditions (50 °C and pH 8.5, using NaOH as base solution). Finally, a recycling system was developed by coupling membrane filtration with the bioreactor, and the engineered C. glutamicum successfully produced 13.7 mM of t-CA (24.3 g) from 18.2 mM of L-Phe (36 g) and thus with a yield of 75% (0.75 mol/mol) through repetitive supplementation. CONCLUSIONS: We developed a highly efficient bioconversion process using C. glutamicum as a biocatalyst and a micromembrane-based cell recycling system. To the best of our knowledge, this is the first report on t-CA production in C. glutamicum, and this robust platform will contribute to the development of an industrially relevant platform for the production of t-CA using microorganisms.

(Optochemical) Control of Synthetic Microbial Coculture Interactions on a Microcolony Level.

Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions.

A Set of Active Promoters with Different Activity Profiles for Superexpressing Rhodococcus Strain.

Rhodococcus bacteria are a promising platform for biodegradation, biocatalysis, and biosynthesis, but the use of rhodococci is hampered by the insufficient number of both platform strains for expression and promoters that are functional and thoroughly studied in these strains. To expand the list of such strains and promoters, we studied the expression capability of the Rhodococcus rhodochrous M33 strain, and the functioning of a set of recombinant promoters in it. We showed that the strain supports superexpression of the target enzyme (nitrile hydratase) using alternative inexpensive feedings-acetate and urea-without growth factor supplementation, thus being a suitable expression platform. The promoter set included Ptuf (elongation factor Tu) and Psod (superoxide dismutase) from Corynebacterium glutamicum ATCC13032, Pcpi (isocitrate lyase) from Rhodococcus erythropolis PR4, and Pnh (nitrile hydratase) from R. rhodochrous M8. Activity levels, regulation possibilities, and growth-phase-dependent activity profiles of these promoters were studied in derivatives of the M33 strain. The activities of the promoters were significantly different (Pcpi < Psod ≪ Ptuf < Pnh), covering 103-fold range, and the most active Pnh and Ptuf produced up to a 30-50% portion of target protein in soluble intracellular proteins. On the basis of the mRNA quantification and amount of target protein, the production level of Pnh was positioned close to the theoretical upper limit of expression in a bacterial cell. A selection method for the laboratory evolution of such active promoters directly in Rhodococcus was also proposed. Concerning regulation, Ptuf could not be regulated (2-fold change), while others were tunable (6-fold for Psod, 79-fold for Pnh, and 44-fold for Pcpi). The promoters possessed four different activity profiles, including three with peak of activity at different growth phases and one with constant activity throughout the growth phases. Ptuf and Pcpi did not change their activity profile under different growth conditions, whereas the Psod and Pnh profiles changed depending on the growth media. The results allow flexible construction of Rhodococcus strains using the studied promoters, and demonstrate a valuable approach for complex characterization of promoters intended for biotechnological strain construction.

Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars.

Efficient production of glutathione with multi-pathway engineering in Corynebacterium glutamicum.

Glutathione is a bioactive tripeptide composed of glycine, L-cysteine, and L-glutamate, and has been widely used in pharmaceutical, food, and healthy products. The current metabolic studies of glutathione were mainly focused on the native producing strains with precursor amino acid supplementation. In the present work, Corynebacterium glutamicum, a workhorse for industrial production of a series of amino acids, was engineered to produce glutathione. First, the introduction of glutathione synthetase gene gshF from Streptococcus agalactiae fulfilled the ability of glutathione production in C. glutamicum and revealed that L-cysteine was the limiting factor. Then, considering the inherent capability of L-glutamate synthesis and the availability of external addition of low-cost glycine, L-cysteine biosynthesis was enhanced using a varieties of pathway engineering methods, such as disrupting the degradation pathways of L-cysteine and L-serine, and removing the repressor responsible for sulfur metabolism. Finally, the simultaneously introduction of gshF and enhancement of cysteine formation enabled C. glutamicum strain to produce glutathione greatly. Without external addition of L-cysteine and L-glutamate, 756 mg/L glutathione was produced. This is first time to demonstrate the potential of the glutathione non-producing strain C. glutamicum for glutathione production and provide a novel strategy to construct glutathione-producing strains.

Development of a novel, robust and cost-efficient process for valorizing dairy waste exemplified by ethanol production.

BACKGROUND: Delactosed whey permeate (DWP) is a side stream of whey processing, which often is discarded as waste, despite of its high residual content of lactose, typically 10-20%. Microbial fermentation is one of the most promising approaches for valorizing nutrient rich industrial waste streams, including those generated by the dairies. Here we present a novel microbial platform specifically designed to generate useful compounds from dairy waste. As a starting point we use Corynebacterium glutamicum, an important workhorse used for production of amino acids and other important compounds, which we have rewired and complemented with genes needed for lactose utilization. To demonstrate the potential of this novel platform we produce ethanol from lactose in DWP. RESULTS: First, we introduced the lacSZ operon from Streptococcus thermophilus, encoding a lactose transporter and a ß-galactosidase, and achieved slow growth on lactose. The strain could metabolize the glucose moiety of lactose, and galactose accumulated in the medium. After complementing with the Leloir pathway (galMKTE) from Lactococcus lactis, co-metabolization of galactose and glucose was accomplished. To further improve the growth and increase the sugar utilization rate, the strain underwent adaptive evolution in lactose minimal medium for 100 generations. The outcome was strain JS95 that grew fast in lactose mineral medium. Nevertheless, JS95 still grew poorly in DWP. The growth and final biomass accumulation were greatly stimulated after supplementation with NH4+, Mn2+, Fe2+ and trace minerals. In only 24 h of cultivation, a high cell density (OD600 of 56.8 ± 1.3) was attained. To demonstrate the usefulness of the platform, we introduced a plasmid expressing pyruvate decarboxylase and alcohol dehydrogenase, and managed to channel the metabolic flux towards ethanol. Under oxygen-deprived conditions, non-growing suspended cells could convert 100 g/L lactose into 46.1 ± 1.4 g/L ethanol in DWP, a yield of 88% of the theoretical. The resting cells could be re-used at least three times, and the ethanol productivities obtained were 0.96 g/L/h, 2.2 g/L/h, and 1.6 g/L/h, respectively. CONCLUSIONS: An efficient process for producing ethanol from DWP, based on C. glutamicum, was demonstrated. The results obtained clearly show a great potential for this newly developed platform for producing value-added chemicals from dairy waste.

Structural basis for substrate specificity of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum.

l-lysine is an essential amino acid that is widely used as a food supplement for humans and animals. meso-Diaminopimelic acid decarboxylase (DAPDC) catalyzes the final step in the de novol-lysine biosynthetic pathway by converting meso-diaminopimelic acid (meso-DAP) into l-lysine by decarboxylation reaction. To elucidate its molecular mechanisms, we determined the crystal structure of DAPDC from Corynebacterium glutamicum (CgDAPDC). The PLP cofactor is bound at the center of the barrel domain and forms a Schiff base with the catalytic Lys75 residue. We also determined the CgDAPDC structure in complex with both pyridoxal 5'-phosphate (PLP) and the l-lysine product and revealed that the protein has an optimal substrate binding pocket to accommodate meso-DAP as a substrate. Structural comparison of CgDAPDC with other amino acid decarboxylases with different substrate specificities revealed that the position of the α15 helix in CgDAPDC and the residues located on the helix are crucial for determining the substrate specificities of the amino acid decarboxylases.

Metabolic Design of Corynebacterium glutamicum for Production of l-Cysteine with Consideration of Sulfur-Supplemented Animal Feed.

l-Cysteine is a valuable sulfur-containing amino acid widely used as a nutrition supplement in industrial food production, agriculture, and animal feed. However, this amino acid is mostly produced by acid hydrolysis and extraction from human or animal hairs. In this study, we constructed recombinant Corynebacterium glutamicum strains that overexpress combinatorial genes for l-cysteine production. The aims of this work were to investigate the effect of the combined overexpression of serine acetyltransferase (CysE), O-acetylserine sulfhydrylase (CysK), and the transcriptional regulator CysR on l-cysteine production. The CysR-overexpressing strain accumulated approximately 2.7-fold more intracellular sulfide than the control strain (empty pMT-tac vector). Moreover, in the resulting CysEKR recombinant strain, combinatorial overexpression of genes involved in l-cysteine production successfully enhanced its production by approximately 3.0-fold relative to that in the control strain. This study demonstrates a biotechnological model for the production of animal feed supplements such as l-cysteine using metabolically engineered C. glutamicum.

Comparative analysis of the expression level of recombinant ginsenoside-transforming ß-glucosidase in GRAS hosts and mass production of the ginsenoside Rh2-Mix.

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A ß-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium glutamicum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E. coli pGEX 4T-1 expression system. The highly expressed BglPm_C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm_C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E. coli expression system.

Optimization of large-ring cyclodextrin production from starch by amylomaltase from Corynebacterium glutamicum and effect of organic solvent on product size.

AIMS: To increase yield of starch conversion to large-ring cyclodextrins (LR-CDs) by amylomaltase from Corynebacterium glutamicum (CgAM). METHODS AND RESULTS: In this work, LR-CDs produced from pea, tapioca, corn, potato, rice and glutinous-rice starch by the recombinant CgAM were analysed by High-Performance Anion-Exchange Chromatography Using Pulsed Amperometric Detection (HPAEC-PAD). Among these, pea starch gave the highest yield of LR-CDs. Pretreatment of pea starch with isoamylase prior to incubation with CgAM resulted in the increase in LR-CD products by 20%. Surprisingly, CgAM converted starch into LR-CDs within a wide pH range (pH 5·5-9·0). LR-CD syntheses at alkaline pH or at a long incubation time favoured low-degree of polymerization (DP) products (CD22-CD32). Addition of 5-15% dimethyl sulfoxide (DMSO) promoted the synthesis of medium-DP species (CD33-CD43) by 10-25%. CONCLUSIONS: Pretreatment of pea starch with isoamylase could enhance the yield of LR-CDs. The ratio of LR-CD products depends on pH, incubation time and addition of organic solvents such as DMSO. SIGNIFICANCE AND IMPACT OF THE STUDY: LR-CD yield can be increased by thorough optimization of starch types, starch concentrations, enzyme activities, pH and incubation times. This study is the first report of the effect of organic solvents on LR-CD production by amylomaltase.

Metabolic engineering of Corynebacterium glutamicum ATCC13032 to produce S-adenosyl-L-methionine.

As an important biological methyl group donor, S-adenosyl-L-methionine is used as nutritional supplement or drug for various diseases, but bacterial strains that can efficiently produce S-adenosyl-L-methionine are not available. In this study, Corynebacterium glutamicum strain HW104 which can accumulate S-adenosyl-L-methionine was constructed from C. glutamicum ATCC13032 by deleting four genes thrB, metB, mcbR and Ncgl2640, and six genes metK, vgb, lysC(m), hom(m), metX and metY were overexpressed in HW104 in different combinations, forming strains HW104/pJYW-4-metK-vgb, HW104/pJYW-4-SAM2C-vgb, HW104/pJYW-4-metK-vgb-metYX, and HW104/pJYW-4-metK-vgb-metYX-hom(m)-lysC(m). Fermentation experiments showed that HW104/pJYW-4-metK-vgb produced more S-adenosyl-L-methionine than other strains, and the yield achieved 196.7 mg/L (12.15 mg/g DCW) after 48h. The results demonstrate the potential application of C. glutamicum for production of S-adenosyl-L-methionine without addition of L-methionine.

Metabolic engineering of Escherichia coli for the production of 3-aminopropionic acid.

A novel metabolic pathway was designed for the production of 3-aminopropionic acid (3-AP), an important platform chemical for manufacturing acrylamide and acrylonitrile. Using a fumaric acid producing Escherichia coli strain as a host, the Corynebacterium glutamicum panD gene (encoding L-aspartate-α-decarboxylase) was overexpressed and the native promoter of the aspA gene was replaced with the strong trc promoter, which allowed aspartic acid production through the aspartase-catalyzed reaction. Additional overexpression of aspA and ppc genes, and supplementation of ammonium sulfate in the medium allowed production of 3.49 g/L 3-AP. The 3-AP titer was further increased to 3.94 g/L by optimizing the expression level of PPC using synthetic promoters and RBS sequences. Finally, native promoter of the acs gene was replaced with strong trc promoter to reduce acetic acid accumulation. Fed-batch culture of the final strain allowed production of 32.3 g/L 3-AP in 39 h.

Monitoring the enzyme expression in a respiratory chain of Corynebacterium glutamicum in a copper ion-supplemented culture medium.

Corynebacterium glutamicum has a branched respiratory chain: one of the branches is cytochrome bcc complex and cytochrome aa3-type cytochrome c oxidase, and the other is cytochrome bd-type menaquinol oxidase. The factors that influence the expression patterns of these respiratory enzymes remain unclear. To investigate the expressional control mechanism of the enzymes, we have previously constructed a promoter assay system utilizing enhanced green fluorescence protein. Here, we monitored respiratory enzymes' expression by using this system during growth in various culture media, with and without Cu(2+) ion supplementation. The promoter activities of cytochrome aa3 oxidase in the early stationary phase in the media supplemented with Cu(2+) ion at 40 or 400 µM were significantly increased 1.49-fold or 1.99-fold, respectively, as compared to the control. Moreover, the H(+)/O ratio, or the proton-pumping activity of the cells, increased about 1.6 times by the Cu(2+) supplementation. These facts indicate that copper ions can switch the branches.