Physiological and fermentation properties of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> and a mutant lacking fermentative lactate dehydrogenase activity

Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50–55°C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55°C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35°C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55°C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.     

Heterologous expression and characterization of <Emphasis Type="Italic">Bacillus coagulans</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn²⁺ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K _m, V _max and k _cat/K _m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM⁻¹min⁻¹, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L⁻¹ h⁻¹.     

Efficient production of lactic acid from sucrose and corncob hydrolysate by a newly isolated <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> GY18

The aim of this study is to investigate production of l-lactic acid from sucrose and corncob hydrolysate by the newly isolated R. oryzae GY18. R. oryzae GY18 was capable of utilizing sucrose as a sole source, producing 97.5 g l⁻¹ l-lactic acid from 120 g l⁻¹ sucrose. In addition, the strain was also efficiently able to utilize glucose and/or xylose to produce high yields of l-lactic acid. It was capable of producing up to 115 and 54.2 g l⁻¹ lactic acid with yields of up to 0.81 g g⁻¹ glucose and 0.90 g g⁻¹ xylose, respectively. Corncob hydrolysates obtained by dilute acid hydrolysis and enzymatic hydrolysis of the cellulose-enriched residue were used for lactic acid production by R. oryzae GY18. A yield of 355 g lactic acid per kg corncobs was obtained after 72 h incubation. Therefore, sucrose and corncobs could serve as potential sources of raw materials for efficient production of lactic acid by R. oryzae GY18.     

Substrate specificity of a recombinant ribose-5-phosphate isomerase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> and its application in the production of <Emphasis Type="SmallCaps">l</Emphasis>-lyxose and <Emphasis Type="SmallCaps">l</Emphasis>-tagatose

A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg⁻¹ by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l-lyxose, l-talose, d-gulose, d-ribose, l-mannose, d-allose, l-xylulose, l-tagatose, d-sorbose, d-ribulose, l-fructose, and d-psicose and exhibited particularly high activity with l-form monosaccharides such as l-lyxose, l-xylulose, l-talose, and l-tagatose. With l-xylulose (500 g l⁻¹) and l-talose (500 g l⁻¹) substrates, the optimum concentrations of RpiB were 300 and 600 U ml⁻¹, respectively. The enzyme converted 500 g l⁻¹ l-xylulose to 350 g l⁻¹ l-lyxose after 3 h, and yielded 450 g l⁻¹ l-tagatose from 500 g l⁻¹ l-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l-form monosaccharides.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

Enhanced <Emphasis Type="SmallCaps">d</Emphasis>-ribose biosynthesis in batch culture of a transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain by citrate

In this study, the effects of citrate addition on d-ribose production were investigated in batch culture of a transketolase-deficient strain, Bacillus subtilis EC2, in shake flasks and bioreactors. Batch cultures in shake flasks and a 5-l reactor indicated that supplementation with 0.2–0.5 g l⁻¹ of citrate enhanced d-ribose production. When B. subtilis EC2 was cultivated in a 15-l reactor in a complex medium, the d-ribose concentration was 70.9 g l⁻¹ with a ribose yield of 0.497 mol mol⁻¹. When this strain was grown in the same medium supplemented with 0.3 g l⁻¹ of citrate, 83.4 g l⁻¹ of d-ribose were obtained, and the ribose yield was increased to 0.587 mol mol⁻¹. Addition of citrate reduced the activities of pyruvate kinase and phosphofructokinase, while it increased those of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Metabolic flux distribution in the stationary phase indicated that citrate addition resulted in increased fluxes in the pentose phosphate pathway and TCA cycle, and decreased fluxes in the glycolysis and acetate pathways.     

<Emphasis Type="SmallCaps">l</Emphasis>-Arabinose pathway engineering for arabitol-free xylitol production in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in biological xylitol production, and most XRs have broad substrate specificities. During xylitol production from biomass hydrolysate, non-specific XRs can reduce l-arabinose, which is the second-most abundant hemicellulosic sugar, to the undesirable byproduct arabitol, which interferes with xylitol crystallization in downstream processing. To minimize the flux from l-arabinose to arabitol, the l-arabinose-preferring, endogenous XR was replaced by a d-xylose-preferring heterologous XR in Candida tropicalis. Then, Bacillus licheniformis araA and Escherichia coli araB and araD were codon-optimized and expressed functionally in C. tropicalis for the efficient assimilation of l-arabinose. During xylitol fermentation, the control strains BSXDH-3 and KNV converted 9.9 g l-arabinose l⁻¹ into 9.5 and 8.3 g arabitol l⁻¹, respectively, whereas the recombinant strain JY consumed 10.5 g l-arabinose l⁻¹ for cell growth without forming arabitol. Moreover, JY produced xylitol with 42 and 16% higher productivity than BSXDH-3 and KNV, respectively.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Fermentative production of<Emphasis Type="SmallCaps"> l</Emphasis>-(+)-lactic acid by an alkaliphilic marine microorganism

Of six strains of lactic acid-producing alkaliphilic microorganisms, Halolactibacillus halophilus was most efficient. It produced the highest concentration and yield of lactic acid, with minimal amounts of acetic and formic acid when sucrose and glucose were used as substrate. Mannose and xylose were poorly utilized. In batch fermentation at 30°C, pH 9 with 4 and 8% (w/v) sucrose, lactic acid was produced at 37.7 and 65.8 g l⁻¹, with yields of 95 and 83%, respectively. Likewise, when 4 and 8% (w/v) glucose were used, 33.4 and 59.6 g lactic acid l⁻¹ were produced with 85 and 76% yields, respectively. l-(+)-lactic acid had an optical purity of 98.8% (from sucrose) and 98.3% (from glucose).     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) synthesis from glucose

l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS⁻ gluc⁺ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q _l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q _l-Tyr in the PTS⁺ and the PTS⁻ gluc⁺ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS⁻ gluc⁺ tyrR ⁻ strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS⁻ gluc⁺ tyrR ⁻ phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

A strain of Bacillus coagulans that converted mixed sugars of glucose, xylose, and arabinose to l-lactic acid with 85% yield at 50°C was isolated from composted dairy manure. The strain was tolerant to aldehyde growth inhibitors at 2.5 g furfural/l, 2.5 g 5-hydroxymethylfurfural/l, 2.5 g vanillin/l, and 1.2 g p-hydroxybenzaldehyde/l. In a simultaneous saccharification and fermentation process, the strain converted a dilute-acid hydrolysate of 100 g corn fiber/l to 39 g lactic acid/l in 72 h at 50°C. Because of its inhibitor tolerance and ability to fully utilize pentose sugars, this strain has potential to be developed as a biocatalyst for the conversion of agricultural residues into valuable chemicals.     

Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. l-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and l-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with l-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and l-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 g l⁻¹ glucose and 7.5 g l⁻¹ l-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 g l⁻¹, 1,350 mg l⁻¹, 32 mg g cells⁻¹, and 40 mg l⁻¹ h⁻¹, respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene.     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Engineering <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for the production of pyruvate

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD⁺-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN produced about 190 mM pyruvate with a Y _P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g_(CDW)⁻¹ h⁻¹ (i.e., 0.08 g g_(CDW) ⁻¹ h⁻¹) in the production phase.