Synthesis of ethyl (<Emphasis Type="Italic">S</Emphasis>)-4-chloro-3-hydroxybutanoate using <Emphasis Type="Italic">fabG</Emphasis>-homologues

This paper is a report on the successful application of bioinformatics to enzyme screening. The synthesis of ethyl ( S)-4-chloro-3-hydroxybutanoate (ECHB) by asymmetric reduction of ethyl 4-chloroacetoacetate (ECAA) using fabG-homologues was studied. beta-Ketoacyl-acyl carrier protein reductases from both Escherichia coli and Bacillus subtilis, which are components of type II fatty acid synthase, could reduce ECAA to ( S)-ECHB with 94-98% ee. Furthermore, acetoacetyl-CoA reductases (ARs) from both Ralstonia eutropha and Zoogloea ramigera, whose genes are significantly similar to fabG genes and play a physiological role in the biosynthesis of poly-beta-3-hydroxybutyrate, could also catalyze the asymmetric reduction of ECAA to ( S)-ECHB with >99% ee. ( S)-ECHB was synthesized to 48.7 g/l with an optical purity of 99.8% ee, using recombinant E. coli cells coexpressing AR from R. eutropha and glucose dehydrogenase from B. subtilis for the regeneration of NADPH.     

A novel NADH-dependent carbonyl reductase from Kluyveromyces aestuarii and comparison of NADH-regeneration system for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate

To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.     

Stereoselective Reduction of Ethyl 4-chloro-3-oxobutanoate by Fungi

The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).     

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by fungi

The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).     

Robust NADH-regenerator: improved α-haloketone-resistant formate dehydrogenase

Formate dehydrogenases (FDH) are useful for the regeneration of NADH, which is required for asymmetric reduction by several dehydrogenases and reductases. FDHs have relatively low activity and are labile, especially to -haloketones, thus FDH cannot be applied to the industrial manufacture of optically active -haloalcohols. To stabilize a FDH from Mycobacterium vaccae (McFDH) against the -haloketone ethyl 4-chloroacetoacetate (ECAA), a set of cysteine-mutant enzymes was constructed. Sensitivity to ECAA of mutant C6S was similar to that of the wild-type enzyme, and mutants C249S and C355S showed little activity. In contrast, mutant C256S exhibited remarkable tolerance to ECAA. Surprisingly, mutant C146S was activated by several organic compounds such as ethyl acetate. An optimized mutant, C6A/C146S/C256V (McFDH-26), was obtained by combining several effective mutations. Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-ECHB] was synthesized from ECAA to 49.9 g/l with an optical purity of more than 99% e.e. using recombinant Escherichia coli cells coexpressing McFDH-26 and a carbonyl reductase (KaCR1) from Kluyveromyces aestuarii.     

Stereoselective hydrolysis of racemic ethyl 4-chloro-3-hydroxybutyrate by a lipase

The stereoselective hydrolysis of racemic ethyl 4-chloro-3-hydroxybutyrate (ECHB) was performed by using Novozym 435 lipase in an aqueous phase. It was found that racemic ECHB was hydrolysed to (R)-ECHB and (S)-3-hydroxy-gamma-butyrolactone (HGBL) via (S)-4-chloro-3-hydroxybutyric acid. From this result, (R)-ECHB (99%ee) was produced in a good yield on a preparative scale.     

Synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis. Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.     

Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD⁺ and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD⁺ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k _cat/K _m) with NADP⁺. The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00?±?0.13 U?mg^?1 and a K _{m, NADP} ⁺ of 0.147?±?0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP⁺-accepting FDH (v _max, 10.25?±?1.63 U?mg^?1). However, the half-saturation constant for NADP⁺ (K _{m, NADP} ⁺ , 0.92?±?0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.     

Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake

Point mutation of Gly1250Ser (1250S) of the yeast fatty acid synthase gene FAS2 confers cerulenin resistance. This mutation also results in a higher production of the apple-like flavor component ethyl caproate in Japanese sake. We mutated the 1250th codon by in vitro site-directed mutagenesis to encode Ala (1250A) or Cys (1250C) and examined cerulenin resistance and ethyl caproate production. The mutated FAS2 genes were inserted into a binary plasmid vector containing a drug-resistance marker and a counter-selectable marker, GALp-GIN11M86. The plasmids were integrated into the wild-type FAS2 locus of a sake yeast strain, and the loss of the plasmid sequences from the integrants was done by growth on galactose plates, which is permissive for loss of GALp-GIN11M86. These counter-selected strains contained either the wild type or the mutated FAS2 allele but not the plasmid sequences, from which FAS2 mutant strains were selected by allele-specific PCR. The FAS2-1250C mutant produced a higher amount of ethyl caproate in sake than FAS2-1250S, while FAS2-1250A produced an ethyl caproate level intermediate between FAS2-1250S and the parental Kyokai no. 7 strain. Interestingly, these mutants only showed detectable cerulenin resistance. These 'self-cloning' yeast strains should be acceptable to the public because they can improve sake quality without the presence of extraneous DNA sequences.     

A key role for orphan nuclear receptor liver receptor homologue-1 in activation of fatty acid synthase promoter by liver X receptor

Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others.     

Selective up-regulation of LXR-regulated genes ABCA1, ABCG1, and APOE in macrophages through increased endogenous synthesis of 24(S),25-epoxycholesterol

Liver X receptor (LXR) activation represents a mechanism to prevent macrophage foam cell formation. Previously, we demonstrated that partial inhibition of oxidosqualene:lanosterol cyclase (OSC) stimulated synthesis of the LXR agonist 24(S),25-epoxycholesterol (24(S),25-epoxy) and enhanced ABCA1-mediated cholesterol efflux. In contrast to a synthetic, nonsteroidal LXR activator, TO-901317, triglyceride accumulation was not observed. In the present study, we determined whether endogenous 24(S),25-epoxy synthesis selectively enhanced expression of macrophage LXR-regulated cholesterol efflux genes but not genes that regulate fatty acid metabolism. THP-1 human macrophages incubated with the OSC inhibitor (OSCi) RO0714565 (15 nM) significantly reduced cholesterol synthesis and maximized synthesis of 24(S),25-epoxy. Endogenous 24(S),25-epoxy increased ABCA1, ABCG1, and APOE mRNA abundance and consequently increased cholesterol efflux to apoAI. In contrast, OSCi had no effect on LXR-regulated genes LPL (lipoprotein lipase) and FAS (fatty acid synthase). TO-901317 (>or=10 nM) significantly enhanced expression of all genes examined. OSCi and TO-901317 increased the mRNA and precursor form of SREBP-1c, a major regulator of fatty acid and triglyceride synthesis. However, conversion of the precursor to the active form (nSREBP-1c) was blocked by OSCi-induced 24(S),25-epoxy but not by TO-901317 (>or=10 nm), which instead markedly increased nSREBP-1c. Disruption of nSREBP-1c formation by 24(S),25-epoxy accounted for diminished FAS and LPL expression. In summary, endogenous synthesis of 24(S),25-epoxy selectively up-regulates expression of macrophage LXR-regulated cholesterol efflux genes without stimulating genes linked to fatty acid and triglyceride synthesis.     

Enantioselective bioconversion using Escherichia coli cells expressing Saccharomyces cerevisiae reductase and Bacillus subtilis glucose dehydrogenase

Ethyl (R, S)-4-chloro-3-hydroxybutanoate (ECHB) is a useful chiral building block for the synthesis of L-carnitine and hypercholesterolemia drugs. The yeast reductase, YOL151W (GenBank locus tag), exhibits an enantioselective reduction activity, converting ethyl-4-chlorooxobutanoate (ECOB) exclusively into (R)-ECHB. YOL151W was generated in Escherichia coli cells and purified via Ni- NTA and desalting column chromatography. It evidenced an optimum temperature of 45 degrees C and an optimum pH of 6.5-7.5. Bacillus subtilis glucose dehydrogenase (GDH) was also expressed in Escherichia coli, and was used for the recycling of NADPH, required for the reduction reaction. Thereafter, Escherichia coli cells co-expressing YOL151W and GDH were constructed. After permeablization treatment, the Escherichia coli whole cells were utilized for ECHB synthesis. Through the use of this system, the 30 mM ECOB substrate could be converted to (R)-ECHB.     

Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.     

Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes.

The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids. By immunological screening of a B. ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences. Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da. This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B. ammoniagenes FAS. A second reading frame encoding another B. ammoniagenes FAS variant (FasB) had been identified previously. Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively. By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein. Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth. Correspondingly, oleate-dependent B. ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA. The data indicate that B. ammoniagenes contains two related though differently expressed type I FASs. FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid.     

Characterization of the folic acid C9-N10-cleaving enzyme of Dictyostelium minutum V3

Folic acid is a chemoattractant for the slime mold Dictyostelium minutum V3. The activity of extracellular folic acid is regulated by a folic acid C9-N10 splitting enzyme (FAS). The products were identified as pterin-6-aldehyde and p-amino-benzoylglutamic acid. The enzyme was stabilized by EDTA. For the extracellular enzyme, the Km was 10(-7) M, and the optimal pH was 4.0. During starvation, FAS activity was mainly secreted into the medium; after 3 h, a plateau was reached. The membrane-bound activity was constant, but only 12% of the extracellular activity at 3 h. Intracellular activity also increased up to 3 h to a level of 23% of the extracellular FAS. The substrate recognition of FAS was found to be based on 4-O or N3 or both, N5 or N8 or both, N10, and the p-aminobenzoic acid moiety, whereas 2-NH2, N1, and the glutamic acid moiety were not recognized. Other slime mold species were found to secrete FAS with 20-fold or more reduced activity than D. minutum V3.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the α subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 μM, whereas the IC₅₀ value was 15 μM for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly → Ser) in the α subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.     

Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein.

We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.     

Improved ethyl caproate production of Chinese liquor yeast by overexpressing fatty acid synthesis genes with <Emphasis Type="Italic">OPI1</Emphasis> deletion

During yeast fermentation, ethyl esters play a key role in the development of the flavor profiles of Chinese liquor. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is the characteristic flavor of strong aromatic liquor, which is the best selling liquor in China. In the traditional fermentation process, ethyl caproate is mainly produced at the later fermentation stage by aroma-producing yeast, bacteria, and mold in a mud pit instead of Saccharomyces cerevisiae at the expense of grains and fermentation time. To improve the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae) with less food consumption and shorter fermentation time, we constructed three recombinant strains, namely, α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 by overexpressing acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid synthase 2 (FAS2) with OPI1 (an inositol/choline-mediated negative regulatory gene) deletion, respectively. In the liquid fermentation of corn hydrolysate, the contents of ethyl caproate produced by α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 increased by 0.40-, 1.75-, and 0.31-fold, correspondingly, compared with the initial strain α5. The contents of other fatty acid ethyl esters (FAEEs) (C8:0, C10:0, C12:0) also increased. In comparison, the content of FAEEs produced by α5-FAS1ΔOPI1 significantly improved. Meanwhile, the contents of acetyl-CoA and ethyl acetate were enhanced by α5-FAS1ΔOPI1. Overall, this study offers a promising platform for the development of pure yeast culture fermentation of Chinese strong aromatic liquor without the use of a mud pit.