Enhanced production of glutaric acid by biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption.

Product inhibition caused by organic acids is a serious issue in establishing economical biochemical production systems. Herein, for enhanced production of glutaric acid by overcoming product inhibition triggered by glutaric acid, a whole-cell bioconversion system equipped with biocatalyst recycling process and in situ product recovery by adsorption was developed successfully. From the whole-cell bioconversion reaction, we found that both dissociated and undissociated forms of glutaric acid acted as an inhibitor in the whole-cell bioconversion reaction, wherein bioconversion was hindered beyond 200 mM glutaric acid regardless of reaction pH. Therefore, as the promising solution for the inhibition issue by glutaric acid, the biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption was introduced in the whole-cell bioconversion. As a result, 592 mM glutaric acid was produced from 1000 mM 5-aminovaleric acid with 59.2% conversion. We believe that our system will be a promising candidate for economically producing organic acids with high titer.

Development of a glutaric acid production system equipped with stepwise feeding of monosodium glutamate by whole-cell bioconversion.

In the bioproduction of glutaric acid, an emerging bioplastic monomer, α-ketoglutaric acid (α-KG) is required as an amine acceptor for 4-aminobutyrate aminotransferase (GabT)-driven conversion of 5-aminovalerate (5-AVA) to glutarate semialdehyde. Herein, instead of using expensive α-KG, an indirect α-KG supply system was developed using a relatively cheap alternative, monosodium glutamate (MSG), for l-glutamate oxidase (Gox)-based whole-cell conversion. Using 200 mM 5-AVA and 30 mM MSG initially with Gox, 67.1 mM of glutaric acid was produced. By applying the stepwise feeding strategy of MSG, the glutaric acid production capability was increased to 159.1 mM glutaric acid with a conversion yield of 79.6%. In addition, a buffer-free one-pot reaction from l-lysine was also applied in a 5 L bioreactor to evaluate its industrial applicability, resulting in a conversion yield of 54.2%. The system developed herein might have great potential for the large-scale, economically feasible production of glutaric acid by whole-cell conversion.

Construction of an Artificial Biosynthetic Pathway for Zingerone Production in Escherichia coli Using Benzalacetone Synthase from Piper methysticum.

Zingerone (vanillylacetone; 4-hydroxy-3-methoxyphenylethyl methyl ketone) is a key component responsible for the pungency of ginger (Zingiber officinale). In this study, it was confirmed that a type III polyketide synthase (PKS) gene (pmpks) from Piper methysticum exhibits feruloyl-CoA-preferred benzalacetone synthase (BAS) activity. Based on these results, we constructed an artificial biosynthetic pathway for zingerone production from supplemented ferulic acid with 4-coumarate CoA ligase (4CL), PmPKS, and benzalacetone reductase (BAR). Furthermore, a de novo pathway for the production of zingerone was assembled using six heterologous genes, encoding tyrosine ammonia-lyase (optal), cinnamate-4-hydroxlase (sam5), caffeic acid O-methyltransferase (com), 4CL (4cl2nt), BAS (pmpks), and BAR (rzs1), in Escherichia coli. Using the engineered l-tyrosine-overproducing E. coli ΔCOS4 strain as a host, a maximum yield of 24.03 ± 2.53 mg/L zingerone was achieved by complete de novo synthesis.

Construction of an Artificial Biosynthetic Pathway for the Styrylpyrone Compound 11-Methoxy-Bisnoryangonin Produced in Engineered Escherichia coli.

A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.

Engineering of CYP153A33 With Enhanced Ratio of Hydroxylation to Overoxidation Activity in Whole-Cell Biotransformation of Medium-Chain 1-Alkanols.

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.

Engineered Escherichia coli strains as platforms for biological production of isoprene.

Volatile compounds can be produced by fermentation from genetically engineered microorganisms. Escherichia coli strains are mainly used for isoprene production owing to their higher titers; however, this has thus far been confined to only strains BL21, BL21 (DE3), Rosetta, and BW25113. Here, we tested four groups of E. coli strains for improved isoprene production, including K-12 (DH5α, BW25113, W3110, MG1655, XL1-Blue, and JM109), B [Rosetta (DE3), BL21, and BL21 (DE3)], Crooks C, and Waksman W strains. The isoprene productivity of BL21 and MG1655 was remarkably higher than that of the others in 5-L fermentation, and scale-up fermentation (300 L) of BL21 was successfully performed. This system shows potential for biobased production of fuel and volatile compounds in industrial applications.

Whole-cell biocatalysis using cytochrome P450 monooxygenases for biotransformation of sustainable bioresources (fatty acids, fatty alkanes, and aromatic amino acids).

Cytochrome P450s (CYPs) are heme-thiolated enzymes that catalyze the oxidation of CH bonds in a regio and stereoselective manner. Activation of the non-activated carbon atom can be further enhanced by multistep chemo-enzymatic reactions; moreover, several useful chemicals can be synthesized to provide alternative organic synthesis routes. Given their versatile functionality, CYPs show promise in a number of biotechnological fields. Recently, various CYPs, along with their sequences and functionalities, have been identified owing to rapid developments in sequencing technology and molecular biotechnology. In addition to these discoveries, attempts have been made to utilize CYPs to industrially produce biochemicals from available and sustainable bioresources such as oil, amino acids, carbohydrates, and lignin. Here, these accomplishments, particularly those involving the use of CYP enzymes as whole-cell biocatalysts for bioresource biotransformation, will be reviewed. Further, recently developed biotransformation pathways that result in gram-scale yields of fatty acids and fatty alkanes as well as aromatic amino acids, which depend on the hosts used for CYP expression, and the nature of the multistep reactions will be discussed. These pathways are similar regardless of whether the hosts are CYP-producing or non-CYP-producing; the limitations of these methods and the ways to overcome them are reviewed here.

Development of glutaric acid production consortium system with α-ketoglutaric acid regeneration by glutamate oxidase in Escherichia coli.

Glutaric acid is a C5 dicarboxylic acid that can be used as a building block for bioplastics. Although high concentrations of glutaric acid can be produced by fermentation or bioconversion, a large amount of α-ketoglutaric acid (α-KG) is necessary to accept the amine group from 5-aminovaleric acid. To decrease the demand for α-KG, we introduced l-glutamate oxidase (GOX) from Streptomyces mobaraensis in our previous system for cofactor regeneration in combination with a glutaric acid production system from 5-aminovaleric acid. To enhance glutaric acid production, critical factors were optimized such as the expression vector, pH, temperature, and cell ratio. As a result, the demand for α-KG was decreased by more than 6-fold under optimized conditions. Additionally, the effect of catalase was also demonstrated by blocking the degradation of α-KG to succinic acid because of the hydrogen peroxide. Finally, 468.5 mM glutaric acid was produced from 800 mM 5-aminovaleric acid using only 120 mM α-KG. Moreover, this system containing davBA, gabTD-nox, and gox can be applied to produce glutaric acid from L-lysine by reusing α-KG with GOX. This improved cofactor regeneration system has a potential to apply much larger production of glutaric acid.

Production of glutaric acid from 5-aminovaleric acid by robust whole-cell immobilized with polyvinyl alcohol and polyethylene glycol.

Glutaric acid is an attractive C5 dicarboxylic acid with wide applications in the biochemical industry. Glutaric acid can be produced by fermentation and bioconversion, and several of its biosynthesis pathways have been well characterized, especially the simple pathway involving glutaric acid from l-lysine using 5-aminovaleric acid. We previously reported the production of glutaric acid using 5-aminovaleric acid and α-ketoglutaric acid by a whole-cell reaction, resulting in a high conversion yield. In this study, we sought to enhance the stability and reusability of this whole-cell system for realizing the efficient production of glutaric acid under harsh reaction conditions. To this end, various matrices were screened to immobilize Escherichia coli whole-cell overexpressing 4-aminobutyrate aminotransferase (GabT), succinate semi-aldehyde dehydrogenase (GabD), and NAD(P)H oxidase (NOX). We ultimately selected a PVA-PEG gel (LentiKats®) for cell entrapment, and several factors of the reaction were optimized. The optimal temperature and pH were 35 °C and 8.5, respectively. Treatment with Tween 80 as a surfactant, as well as additional NOX, was found to be effective. Under the optimized conditions, an immobilized cell retained 55% of its initial activity even after the eighth cycle, achieving 995.2 mM accumulated glutaric acid, whereas free cell lost most of their activity after only two cycles. This optimized whole-cell system can be used in the large-scale production of glutaric acid.

Melamine-promoted formation of bright and stable DNA-silver nanoclusters and their antimicrobial properties.

A new method has been developed for the preparation of brightly fluorescent and stable DNA-silver nanoclusters (DNA-AgNCs). The approach takes advantage of specific interactions occurring between melamine and thymine residues in a DNA template. These interactions cause the formation of a melamine-DNA-AgNC complex (Mel-DNA-AgNCs), in which a change in the environment of the DNA template causes binding of additional Ag+ and an enhancement in the fluorescence efficiency and stability. The effects of the nature of the template DNA, DNA : Ag+ : NaBH4 ratio, pH and temperature were systematically assessed in order to maximize the melamine-promoted fluorescence enhancement. The results show that the Mel-DNA-AgNCs, generated under the optimal conditions, exhibit a ca. 3-fold larger fluorescence efficiency and long-term stability (70 d) in contrast to those of DNA-AgNCs in the absence of melamine. Importantly, the bright and stable Mel-DNA-AgNCs exhibit antimicrobial activities against Gram-positive and Gram-negative bacteria that are superior to those of DNA-AgNCs alone. To the best of our knowledge, this is the first report describing the synthesis of DNA-AgNCs that have improved fluorescence efficiencies and that function as effective antimicrobial agents.

Enhanced production of glutaric acid by NADH oxidase and GabD-reinforced bioconversion from l-lysine.

Glutaric acid is a promising alternative chemical to phthalate plasticizer since it can be produced by the bioconversion of lysine. Though, recent studies have enabled the high-yield production of its precursor, 5-aminovaleric acid (AMV), glutaric acid production via the AMV pathway has been limited by the need for cofactors. Introduction of NAD(P)H oxidase (Nox) with GabTD enzyme remarkably diminished the demand for oxidized nicotinamide adenine dinucleotide (NAD+ ). Supply of oxygen through vigorous shaking had a significant effect on the conversion of AMV with a reduced requirement of NAD + . A high conversion rate was achieved in Nox coupled GabTD reaction under optimized expression vector, terrific broth (TB), and pH 8.5 at high cell density. Supplementary expression of GabD resulted in the production of 353 ± 35 mM glutaric acid with 88.3 ± 8.7% conversion from 400 mM AMV. Moreover, the reaction with a higher concentration of AMV could produce 528 ± 21 mM glutaric acid with 66.0 ± 2.7% conversion. In addition, the co-biotransformation strategy of GabTD and DavBA whole cells could produce 282 mM glutaric acid with 70.8% conversion from lysine, compared to the 111 mM glutaric acid yield from the combined GabTD-DavBA system.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Production of glutaric acid from 5-aminovaleric acid using Escherichia coli whole cell bio-catalyst overexpressing GabTD from Bacillus subtilis.

Glutaric acid is one of the promising C5 platform compounds in the biochemical industry. It can be produced chemically, through the ring-opening of butyrolactone followed by hydrolysis. Alternatively, glutaric acid can be produced via lysine degradation pathways by microorganisms. In microorganisms, the overexpression of enzymes involved in this pathway from E. coli and C. glutamicum has resulted in high accumulation of 5-aminovaleric acid. However, the conversion from 5-aminovaleric acid to glutaric acid has resulted in a relatively low conversion yield for unknown reasons. In this study, as a solution to improve the production of glutaric acid, we introduced gabTD genes from B. subtilis to E. coli for a whole cell biocatalytic approach. This approach enabled us to determine the effect of co-factors on reaction and to achieve a high conversion yield from 5-aminovaleric acid at the optimized reaction condition. Optimization of whole cell reaction by different plasmids, pH, temperature, substrate concentration, and cofactor concentration achieved full conversion with 100 mM of 5-aminovaleric acid to glutaric acid. Nicotinamide adenine dinucleotide phosphate (NAD(P)+) and α-ketoglutaric acid were found to be critical factors in the enhancement of conversion in selected conditions. Whole cell reaction with a higher concentration of substrates gave 141 mM of glutaric acid from 300 mM 5-aminovaleric acid, 150 mM α-ketoglutaric acid, and 60 mM NAD+ at 30 °C, with a pH of 8.5 within 24 h (47.1% and 94.2% of conversion based on 5-aminovaleric acid and α-ketoglutaric acid, respectively). The whole cell biocatalyst was recycled 5 times with the addition of substrates; this enabled the accumulation of extra glutaric acid.

Enhanced Photodynamic Cancer Treatment by Mitochondria-Targeting and Brominated Near-Infrared Fluorophores.

A noninvasive and selective therapy, photodynamic therapy (PDT) is widely researched in clinical fields; however, the lower efficiency of PDT can induce unexpected side effects. Mitochondria are extensively researched as target sites to maximize PDT effects because they play crucial roles in metabolism and can be used as cancer markers due to their high transmembrane potential. Here, a mitochondria targeting photodynamic therapeutic agent (MitDt) is developed. This photosensitizer is synthesized from heptamethine cyanine dyes, which are conjugated or modified as follows. The heptamethine meso-position is conjugated with a triphenylphosphonium derivative for mitochondrial targeting, the N-alkyl side chain is modified for regulation of charge balance and solubility, and the indolenine groups are brominated to enhance reactive oxygen species generation (ROS) after laser irradiation. The synthesized MitDt increases the cancer uptake efficiency due to the lipo-cationic properties of the triphenylphosphonium, and the PDT effects of MitDt are amplified after laser irradiation because mitochondria are susceptible to ROS, the response to which triggers an apoptotic anticancer effect. Consequently, these hypotheses are demonstrated by in vitro and in vivo studies, and the results indicate strong potential for use of MitDts as efficient single-molecule-based PDT agents for cancer treatment.

Synthesis of Fe3O4@nickel-silicate core-shell nanoparticles for His-tagged enzyme immobilizing agents.

Immobilizing enzymes on artificially fabricated carriers for their efficient use and easy removal from reactants has attracted enormous interest for decades. Specifically, binding platforms using inorganic nanoparticles have been widely explored because of the benefits of their large surface area, easy surface modification, and high stability in various pH and temperatures. Herein, we fabricated Fe3O4 encapsulated 'sea-urchin' shaped nickel-silicate nanoparticles with a facile synthetic route. The enzymes were then rapidly and easily immobilized with poly-histidine tags (His-tags) and nickel ion affinity. Porous nickel silicate covered nanoparticles achieved a high immobilization capacity (85 µg mg-1) of His-tagged tobacco etch virus (TEV) protease. To investigate immobilized TEV protease enzymatic activity, we analyzed the cleaved quantity of maltose binding protein-exendin-fused immunoglobulin fusion protein, which connected with the TEV protease-specific cleavage peptide sequence. Moreover, TEV protease immobilized nanocomplexes conveniently removed and recollected from the reactant by applying an external magnetic field, maintained their enzymatic activity after reuse. Therefore, our newly developed nanoplatform for His-tagged enzyme immobilization provides advantageous features for biotechnological industries including recombinant protein processing.

Artificial de novo biosynthesis of hydroxystyrene derivatives in a tyrosine overproducing Escherichia coli strain.

BACKGROUND: Styrene and its derivatives as monomers and petroleum-based feedstocks are valuable as raw materials in industrial processes. The chemical reaction for styrene production uses harsh reaction conditions such as high temperatures or pressures, or requires base catalysis with microwave heating. On the other hand, production of styrene and its derivatives in Escherichia coli is an environmental friendly process to produce conventional petroleum-based feedstocks. RESULTS: An artificial biosynthetic pathway was developed in E. coli that yields 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene from simple carbon sources. This artificial biosynthetic pathway has a codon-optimized phenolic acid decarboxylase (pad) gene from Bacillus and some of the phenolic acid biosynthetic genes. E. coli strains with the tal and pad genes, the tal, sam5, and pad genes, and the tal, sam5, com, and pad genes produced 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene, respectively. Furthermore, these pathways were expressed in a tyrosine overproducing E. coli. The yields for 4-hydroxystyrene, 3,4-dihydroxystyrene and 4-hydorxy-3-methoxystyrene reached 355, 63, and 64 mg/L, respectively, in shaking flasks after 36 h of cultivation. CONCLUSIONS: Our system is the first to use E. coli with artificial biosynthetic pathways for the de novo synthesis of 3,4-dihydroxystyrene and 4-hydroxy-3-methoxystyrene in a simple glucose medium. Similar approaches using microbial synthesis from simple sugar could be useful in the synthesis of plant-based aromatic chemicals.

Isolation and characterization of a novel ε-caprolactam-degrading microbe, Acinetobacter calcoaceticus, from industrial wastewater by chemostat-enrichment.

For the isolation of a ε-caprolactam-degrading microbe from wastewaters of a factory producing caprolactam, we applied a chemostat-enrichment technique with a selective medium containing caprolactam as sole source of carbon and nitrogen. This allowed for the isolation of a novel caprolactam-degrading microbe, identified as Acinetobacter calcoaceticus. The strain had a critical tolerance of 19 g caprolactam l(-1) in minimal medium, which is higher than any previously reported caprolactam-degrading microbe. A. calcoaceticus also decreased the caprolactam content in medium by 65 % within 72 h despite the high caprolactam content (10 g l(-1)). This study highlights the potential use of A. calcoaceticus strain for the bioremediation of recalcitrant synthetic polymers, such as caprolactam.

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein.

BACKGROUND: The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. RESULTS: An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q(Ab)) than that of the unsorted pool. The q(Ab) was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q(Ab) in individual selected clones. CONCLUSIONS: This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q(Ab) with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

Exploring the effects of carbon sources on the metabolic capacity for shikimic acid production in Escherichia coli using in silico metabolic predictions.

Effects of various industrially important carbon sources (glucose, sucrose, xylose, gluconate, and glycerol) on shikimic acid (SA) biosynthesis in Escherichia coli were investigated to gain new insight into the metabolic capability for overproducing SA. At the outset, constraints-based flux analysis using the genome-scale in silico model of E. coli was conducted to quantify the theoretical maximum SA yield. The corresponding flux distributions fueled by different carbon sources under investigation were compared with respect to theoretical yield and energy utilization, thereby identifying the indispensable pathways for achieving optimal SA production on each carbon source. Subsequently, a shikimate-kinase-deficient E. coli mutant was developed by blocking the aromatic amino acid pathway, and the production of SA on various carbon sources was experimentally examined during 5 l batch culture. As a result, the highest production rate, 1.92 mmol SA/h, was obtained when glucose was utilized as a carbon source, whereas the efficient SA production from glycerol was obtained with the highest yield, 0.21 mol SA formed per mol carbon atom of carbon source consumed. The current strain can be further improved to satisfy the theoretically achievable SA production that was predicted by in silico analysis.

Improved L-threonine production of Escherichia coli mutant by optimization of culture conditions.

L-threonine production was investigated in a minimal salt medium using L-threonine-overproducing Escherichia coli MT201, derived from E. coli K-12. It was observed that dry cell weight reached 12.5 g/l with 15.9 g/lL-threonine. To increase dry cell weight and L-threonine production, the fermentation process was optimized. When biotin was added as growth factor, L-threonine production reached 52.0 g/l from 15.9 g/l without biotin. Dry cell weight and L-threonine production were further increased by continuous feeding of the feed media with an optimized L-methionine concentration (5.0 g/l). However, high-cell-density culture caused oxygen-limited condition, which resulted in the accumulation of organic acids. To overcome this problem, oxygen-enriched air was supplied to the fermentor with the minimal salt medium. Under these optimal conditions, we achieved an L-threonine production of 80.2 g/l in the minimal salt medium.