Metabolic engineering to produce palmitic acid or palmitoleic acid in an oleic acid-producing Corynebacterium glutamicum strain.

Focusing on the differences in the catalytic properties of two type I fatty acid synthases FasA and FasB, the fasA gene was disrupted in an oleic acid-producing Corynebacterium glutamicum strain. The resulting oleic acid-requiring strain whose fatty acid synthesis depends only on FasB exhibited almost exclusive production (217 mg/L) of palmitic acid (C16:0) from 1% glucose under the conditions supplemented with the minimum concentration of sodium oleate for growth. Plasmid-mediated amplification of fasB led to a 1.47-fold increase in palmitic acid production (320 mg/L), while fasB disruption resulted in no fatty acid production, with excretion of malonic acid (30 mg/L). Next, aiming at conversion of the palmitic acid producer to a producer of palmitoleic acid (POA, C16:1Δ9), we introduced the Pseudomonas nitroreducens Δ9-desaturase genes desBC into the palmitic acid producer. Although this resulted in failure, we noticed the emergence of suppressor mutants that exhibited the oleic acid-non-requiring phenotype. Production experiments revealed that one such mutant M-1 undoubtedly produced POA (17 mg/L) together with palmitic acid (173 mg/L). Whole genomic analysis and subsequent genetic analysis identified the suppressor mutation of strain M-1 as a loss-of-function mutation for the DtxR protein, a global regulator of iron metabolism. Considering that DesBC are both iron-containing enzymes, we investigated the conditions for increased iron availability to improve the DesBC-dependent conversion ratio of palmitic acid to POA. Eventually, supplementation of both hemin and the iron chelator protocatechuic acid in the engineered strain dramatically enhanced POA production to 161 mg/L with a conversion ratio of 80.1%. Cellular fatty acid analysis revealed that the POA-producing cells were really equipped with unnatural membrane lipids comprised predominantly of palmitic acid (85.1% of total cellular fatty acids), followed by non-native POA (12.4%).

A Futile Metabolic Cycle of Fatty Acyl-CoA Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production.

Fatty acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD) catalyze opposing reactions between acyl-CoAs and free fatty acids. Within the genome of Corynebacterium glutamicum, several candidate genes for each enzyme are present, although their functions remain unknown. Modified expressions of the candidate genes in the fatty acid producer WTΔfasR led to identification of one tes gene (tesA) and two fadD genes (fadD5 and fadD15), which functioned positively and negatively in fatty acid production, respectively. Genetic analysis showed that fadD5 and fadD15 are responsible for utilization of exogenous fatty acids and that tesA plays a role in supplying fatty acids for synthesis of the outer layer components mycolic acids. Enzyme assays and expression analysis revealed that tesA, fadD5, and fadD15 were co-expressed to create a cyclic route between acyl-CoAs and fatty acids. When fadD5 or fadD15 was disrupted in wild-type C. glutamicum, both disruptants excreted fatty acids during growth. Double disruptions of them resulted in a synergistic increase in production. Additional disruption of tesA revealed a canceling effect on production. These results indicate that the FadDs normally shunt the surplus of TesA-generated fatty acids back to acyl-CoAs for lipid biosynthesis and that interception of this shunt provokes cells to overproduce fatty acids. When this strategy was applied to a fatty acid high-producer, the resulting fadDs-disrupted and tesA-amplified strain exhibited a 72% yield increase relative to its parent and produced fatty acids, which consisted mainly of oleic acid, palmitic acid, and stearic acid, on the gram scale per liter from 1% glucose.IMPORTANCE The industrial amino acid producer Corynebacterium glutamicum has currently evolved into a potential workhorse for fatty acid production. In this organism, we obtained evidence showing the presence of a unique mechanism of lipid homeostasis, namely, a formation of a futile cycle of acyl-CoA hydrolysis and resynthesis mediated by acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD), respectively. The biological role of the coupling of Tes and FadD would be to supply free fatty acids for synthesis of the outer layer components mycolic acids and to recycle their surplusage to acyl-CoAs for membrane lipid synthesis. We further demonstrated that engineering of the cycle in a fatty acid high-producer led to dramatically improved production, which provides a useful engineering strategy for fatty acid production in this industrially important microorganism.

The accD3 gene for mycolic acid biosynthesis as a target for improving fatty acid production by fatty acid-producing Corynebacterium glutamicum strains.

We have recently developed Corynebacterium glutamicum strains that produce free fatty acids in culture supernatant due to enhanced fatty acid biosynthesis. Of these producing strains, the basic producer PAS-15 has a defect in the gene for a fatty acid biosynthesis repressor protein, and the advanced producer PCC-6 has two additional mutations to augment the production by strain PAS-15. The aim of the present study was to obtain novel genetic traits for improving fatty acid production by these producers. A new mutant with increased production derived from strain PAS-15 had a missense mutation in the accD3 gene (mutation accD3A433T), which is involved in the biosynthesis of mycolic acids that are cell envelope lipids of C. glutamicum, as the causal mutation. Mutation accD3A433T was verified to reduce the AccD3 enzymatic activity and increase fatty acid production in strain PAS-15 by 1.8-fold. Deletion of the accD3 gene in strain PAS-15, which was motivated by the characteristic of mutation accD3A433T, increased fatty acid production by 3.2-fold. Susceptibility of strain PAS-15 to vancomycin was significantly increased by accD3 gene deletion and by mutation accD3A433T to the intermediate level, suggesting that the cell envelope permeability barrier by mycolic acids is weakened by this engineering. Furthermore, mutation accD3A433T also increased fatty acid production in strain PCC-6 by 1.3-fold. These increased production levels were suggested to be involved not only in the redirection of carbon flux from mycolic acid biosynthesis to fatty acid production but also in the permeability of the cell envelope.

In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicumIMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty acid synthase (FAS-II) system. In this study, we reported genetic evidence demonstrating that the FAS-I system is the source of the biotin precursor in vivo in the engineered biotin-prototrophic C. glutamicum strain. This study also uncovered the important physiological role of FasB in lipoic acid biosynthesis. Here, we present an FAS-I enzyme that functions in supplying the lipoic acid precursor, although its biosynthesis has been believed to exclusively depend on FAS-II in organisms. The findings obtained here provide new insights into the metabolic engineering of this industrially important microorganism to produce these compounds effectively.

l-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene.

We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2A(iol) was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect l-lysine production in the engineered GapN strains, while a drastic decrease in l-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP(+) ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient l-lysine production independent of the oxidative pentose phosphate pathway.

A third glucose uptake bypass in Corynebacterium glutamicum ATCC 31833.

In Corynebacterium glutamicum, the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) has long been the only known glucose uptake system, but we recently found suppressor mutants emerging from a PTS-negative strain of C. glutamicum ATCC 31833 on glucose agar plates, and identified two alternative potential glucose uptake systems, the myo-inositol transporters encoded by iolT1 and iolT2. The expression of either gene renders the PTS-negative strain WTΔptsH capable of growing on glucose. In the present study, we found a suppressor strain that still grew on glucose even after the iolT1 and iolT2 genes were both disrupted under the PTS-negative background. Whole-genome sequencing of the suppressor strain SPH1 identified a G-to-T exchange at 134 bp upstream of the bglF gene encoding an EII component of the ß-glucoside-PTS, which is found in limited wild-type strains of C. glutamicum. Introduction of the mutation into strain WTΔptsH allowed the PTS-negative strain to grow on glucose. Reverse transcription-quantitative PCR analysis revealed that the mutation upregulates the bglF gene by approximately 11-fold. Overexpression of bglF under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, and deletion of bglF in strain SPH1 abolished the growth again, proving that bglF is responsible for glucose uptake in the suppressor strain. Simultaneous disruption of three glucokinase genes, glk (Cgl2185, NCgl2105), ppgK (Cgl1910, NCgl1835), and Cgl2647 (NCgl2558), in strain SPH1 resulted in no growth on glucose. Plasmid-mediated expression of any of the three genes in the triple-knockout mutant restored the growth on glucose. These results indicate that C. glutamicum ATCC 31833 has an additional non-PTS glucose uptake route consisting of the bglF-specified EII permease and native glucokinases.

Development of fatty acid-producing Corynebacterium glutamicum strains.

To date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63(up), and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ± 0.11-fold and 1.29-fold ± 0.12-fold, respectively, and of the accD1 gene encoding the ß-subunit of acetyl-CoA carboxylase by 3.56-fold ± 0.97-fold. On the other hand, the fasA63(up) mutation upregulated the fasA gene by 2.67-fold ± 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63(up) fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic acid (208 mg/liter) and palmitic acid (47 mg/liter).

Development of biotin-prototrophic and -hyperauxotrophic Corynebacterium glutamicum strains.

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 µg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 µg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 µg per liter).

Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum.

The Cgl1427 gene was previously found to be relevant to the microaerobic growth of Corynebacterium glutamicum (Ikeda et al. Biosci Biotechnol Biochem 73:2806-2808, 2009). In the present work, Cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (CMP kinases) and on the basis of findings that deletion of Cgl1427 results in loss of CMP kinase activity. Deletion of the cmk gene significantly impaired the growth of C. glutamicum in oxygen-limiting static culture, and the impaired growth was restored by introducing a plasmid containing the cmk gene, suggesting that this gene plays an important role in the microaerobic growth of C. glutamicum. On the other hand, in the main culture with aerobic shaking, a prolonged lag phase was observed in the cmk disruptant, despite an unchanged growth rate, compared to the behavior of the wild-type strain. The prolongation was observed when using seed culture grown to later growth stages in which oxygen limitation occurred, but it was not observed when using seed culture grown to an earlier growth stage in which oxygen remained relatively plentiful. Since nucleotide biosynthesis in C. glutamicum requires oxygen, we hypothesized that the ability of the cmk disruptant to synthesize nucleotides was influenced by oxygen limitation in the later growth stages of the seed culture, which caused the prolongation of the lag phase in the following shaken culture. To verify this hypothesis, a plasmid containing genes encoding all components of a homologous ribonucleotide reductase, a key enzyme for nucleotide synthesis that requires oxygen for its reaction, was introduced into the cmk disruptant, which significantly ameliorated the lag phase prolongation. Furthermore, this experimental setup almost completely restored the growth of the cmk disruptant in the oxygen-limiting static culture. These results indicate that CMP kinase plays an important role in normal nucleotide biosynthesis under an oxygen-limiting environment.

Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum.

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to uptake and phosphorylate glucose; no other route has yet been identified. Disruption of the ptsH gene in wild-type C. glutamicum resulted, as expected, in a phenotype exhibiting little growth on any of the PTS sugars: glucose, fructose, and sucrose. However, a suppressor mutant that grew on glucose but not on the other two sugars was spontaneously isolated from the PTS-negative strain WTΔptsH. The suppressor strain SPH2, unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose which is known to be a toxic substrate for the glucose-PTS of this microbe, suggesting that strain SPH2 utilizes glucose via a different system involving a permease and native glucokinases. Analysis of the C. glutamicum genome sequence using Escherichia coli galactose permease, which can transport glucose, led to the identification of two candidate genes, iolT1 and iolT2, both of which have been reported as myo-inositol transporters. When cultured on glucose medium supplemented with myo-inositol, strain WTΔptsH was able to consume glucose, suggesting that glucose uptake was mediated by one or more myo-inositol-induced transporters. Overexpression of iolT1 alone and that of iolT2 alone under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, proving that each transporter played a role in glucose uptake. Disruption of iolT1 in strain SPH2 abolished growth on glucose, whereas disruption of iolT2 did not, revealing that iolT1 was responsible for glucose uptake in strain SPH2. Sequence analysis of the iol gene cluster and its surrounding region identified a single-base deletion in the putative transcriptional regulator gene Cgl0157 of strain SPH2. Introduction of the frameshift mutation allowed strain WTΔptsH to grow on glucose, and further deletion of iolT1 abolished the growth again, indicating that inactivation of Cgl0157 under a PTS-negative background can be a means by which to express the iolT1-specified glucose uptake bypass instead of the native PTS. When this strategy was applied to a defined lysine producer, the engineered strain displayed increased lysine production from glucose.

Engineering of Corynebacterium glutamicum with an NADPH-generating glycolytic pathway for L-lysine production.

A sufficient supply of NADPH is a critical factor in l-lysine production by Corynebacterium glutamicum. Endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of C. glutamicum was replaced with nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) of Streptococcus mutans, which catalyzes the reaction of glyceraldehyde 3-phosphate to 3-phosphoglycerate with the reduction of NADP(+) to NADPH, resulting in the reconstruction of the functional glycolytic pathway. Although the growth of the engineered strain on glucose was significantly retarded, a suppressor mutant with an increased ability to utilize sugars was spontaneously isolated from the engineered strain. The suppressor mutant was characterized by the properties of GapN as well as the nucleotide sequence of the gene, confirming that no change occurred in either the activity or the basic properties of GapN. The suppressor mutant was engineered into an l-lysine-producing strain by plasmid-mediated expression of the desensitized lysC gene, and the performance of the mutant as an l-lysine producer was evaluated. The amounts of l-lysine produced by the suppressor mutant were larger than those produced by the reference strain (which was created by replacement of the preexisting gapN gene in the suppressor mutant with the original gapA gene) by ∼70% on glucose, ∼120% on fructose, and ∼100% on sucrose, indicating that the increased l-lysine production was attributed to GapN. These results demonstrate effective l-lysine production by C. glutamicum with an additional source of NADPH during glycolysis.

Elucidation of genes relevant to the microaerobic growth of Corynebacterium glutamicum.

Mutagenized cell libraries of Corynebacterium glutamicum were screened for mutants that lost the ability to grow under low oxygen concentrations. The resulting high-oxygen-requiring mutants were used to clone wild-type DNA fragments that could complement the phenotype. Sequencing and subcloning analyses identified six genes, Cgl0807, Cgl1102, Cgl0600, Cgl1427, Cgl2857, and Cgl2859, as the genes responsible for complementation. Some of these genes showed cross-complementation of the mutants in oxygen-limiting static culture, suggesting the utility of these genes for improved growth and production under oxygen limitation.

The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in Corynebacterium glutamicum.

Mutants of Corynebacterium glutamicum that were unable to grow under mild alkaline pH conditions were isolated by mutagenesis. Strain AL-43 exhibiting the highest sensitivity to alkaline pH among the mutants was selected and used to clone a DNA fragment that could complement the phenotype. Sequencing and subcloning of the cloned 4.0-kb EcoRI DNA fragment showed that the Cgl1281 gene was responsible for the complementation. The deduced amino acid sequence of Cgl1281 was found to show significant sequence similarity with CzcD, a Me2+/H+(K+) antiporter, from Bacillus subtilis and also possess the features of the cation diffusion facilitator (CDF) family: the presence of 6 putative transmembrane segments and a signature sequence, indicating that the gene product is a member of the CDF family. Chromosomal disruption of the Cgl1281 rendered C. glutamicum cells sensitive to alkaline pH as well as cobalt, while expression of the gene from a plasmid restored alkali-tolerance to the wild-type level and also led to increased cobalt resistance. These results demonstrated that the putative transporter of the CDF family mediates resistance to cobalt and also plays a physiological role in alkaline pH tolerance in C. glutamicum.

Characterization of mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine in an industrial Corynebacterium glutamicum strain.

Mutations induced by classical whole-cell mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG) were determined for all genes of pathways from glucose to L-lysine in an industrial L-lysine producer of Corynebacterium glutamicum. A total of 50 mutations with a genome-wide distribution were identified and characterized for mutational types and mutagenic specificities. Those mutations were all point mutations with single-base substitutions and no deletions, frame shifts, and insertions were found. Among six possible types of base substitutions, the mutations consisted of only two types: 47 G.C-->A.T transitions and three A.T-->G.C transitions with no transversion. The findings indicate a limited repertoire of amino acid substitutions by classical NTG mutagenesis and thus raise a new possibility of further improving industrial strains by optimizing key mutations through PCR-mediated site-directed mutagenesis.

Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum.

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O(2)), while no visible colonies were formed in the absence of O(2). However, in the presence of nitrate (NO3-), the organism exhibited limited growth anaerobically with production of nitrite (NO2-), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using L-lysine- and L-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.

Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with delta12-desaturase gene expression.

An oleaginous fungus, Mortierella alpina 1S-4, is used commercially for arachidonic acid production. Delta12-Desaturase, which desaturates oleic acid (18:1n-9) to linoleic acid (18:2n-6), is a key enzyme in the arachidonic acid biosynthetic pathway. To determine if RNA interference (RNAi) by double-stranded RNA occurs in M. alpina 1S-4, we silenced the Delta12-desaturase gene. The silenced strains accumulate 18:2n-9, 20:2n-9, and Mead acid (20:3n-9), which are not detected in either the control strain or wild type strain 1S-4. The fatty acid composition of stable transformants was similar to that of Delta12-desaturation-defective mutants previously identified. Thus, RNAi occurs in M. alpina and could be used to alter the types and relative amounts of fatty acids produced by commercial strains of this fungus without mutagenesis or other permanent changes in the genetic background of the producing strains.

Molecular evidence that the rate-limiting step for the biosynthesis of arachidonic acid in Mortierella alpina is at the level of an elongase.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain for arachidonic acid (AA) production. To determine its physiological properties and to clarify the biosynthetic pathways for PUFA, heterologous and homologous gene expression systems were established in this fungus. The first trial was performed with an enhanced green fluorescent protein gene to assess the transformation efficiency for heterologous gene expression. As a result, strong fluorescence was observed in the spores of the obtained transformant, suggesting that the foreign gene was inherited by the spores. The next trial was performed with a homologous PUFA elongase (GLELOp) gene, this enzyme having been reported to catalyze the elongation of GLA (18:3n-6) to dihomo-gamma-linolenic acid (20:3n-6), and to be the rate-limiting step of AA production. The FA composition of the transformant was different from that of the host strain: The GLA content was decreased whereas that of AA was increased. These data support the hypothesis that the GLELOp enzyme plays an important role in PUFA synthesis, and may indicate how to control PUFA biosynthesis.

Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production.

The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.

Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4.

Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5- mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.

Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4.

The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.