Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes.

Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93 mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151 mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.

Interação dos isômeros todo-trans, 9-cis e 13-cis do "BETA"-caroteno na bioconversão desses em vitamina A / Interaction between the 9-cis, 13-cis and all-trans \"BETA\"-caroteno isomers on their bioconversion into vitamin A

Com o objetivo de se verificar a importância da interação entre os isômeros 9-cis e todo-trans, 13-cis e todo-trans do "BETA"-caroteno na bioconversão desse em vitamina A foi realizado um ensaio baseado no modelo de esgotamento das reservas hepáticas dessa vitamina em ratos. A ratos depletados em vitamina A hepática foram fornecidos diferentes formas de vitamina A (acetato de vitamina A, "BETA"-caroteno e isômeros). Os grupos formados foram: grupos água e óleo, ambos sem vitamina A, grupos vitamina A, "BETA"-caroteno todo-trans, e dois grupos que receberam mistura isomérica, uma do "BETA"-caroteno todo-trans com o isômero 9-cis e a outra do "BETA"-caroteno todo-trans com isômero 13-cis...

The yeast A kinases differentially regulate iron uptake and respiratory function.

Yeast has three A kinase catalytic subunits, which have greater than 75% identity and are encoded by the TPK genes (TPK1, TPK2, and TPK3) [Toda, T., Cameron, S., Sass, P., Zoller, M. & Wigler, M. (1987) Cell 50, 277-287]. Although they are redundant for viability, the three A kinases are not redundant for pseudohyphal growth [Robertson, L. S. & Fink, G. R. (1998) Proc. Natl. Acad. Sci. USA 95, 13783-13787; Pan, X. & Heitman, J. (1999) Mol. Cell. Biol. 19, 4874-4887]; Tpk2, but not Tpk1 or Tpk3, is required for pseudohyphal growth. Genome-wide transcriptional profiling has revealed unique signatures for each of the three A kinases leading to the identification of additional functional diversity among these proteins. Tpk2 negatively regulates genes involved in iron uptake and positively regulates genes involved in trehalose degradation and water homeostasis. Tpk1 is required for the derepression of branched chain amino acid biosynthesis genes that seem to have a second role in the maintenance of iron levels and DNA stability within mitochondria. The fact that TPK2 mutants grow better than wild types on nonfermentable carbon sources and on media deficient in iron supports the unique role of Tpk2 in respiratory growth and carbon source use.

Purification and characterization of a fusion protein of plant acetohydroxy acid synthase and acetohydroxy acid isomeroreductase.

The nucleotide sequence coding for the Arabidopsis thaliana acetohydroxy acid synthase was genetically fused in frame with the nucleotide sequence coding for the Spinacia oleracea acetohydroxy acid isomeroreductase and expressed in Escherichia coli. This construction allowed the production of large amounts of soluble fusion protein. The pure chimeric enzyme exhibits high acetohydroxy acid synthase and acetohydroxy acid isomeroreductase specific activities. Fusion and native enzymes exhibit similar Km values for their substrates and for most cofactors. Furthermore, whereas native plant acetohydroxy acid synthase is highly unstable, the stability of this enzyme in the fusion has been increased. Thus, the chimeric enzyme appears to be a useful tool for the determination of kinetic and structural properties of plant acetohydroxy acid synthase.

An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability.

The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA.

Cloning and molecular analysis of two different ILV5 genes from a brewing strain of Saccharomyces cerevisiae.

Two different ILV5 genes encoding acetohydroxy-acid isomeroreductases, and named ILV5G and ILV5X, were cloned and sequenced from a Saccharomyces cerevisiae brewing strain. The coding sequence of ILV5X shows a single nucleotide change with respect to that from the ILV5 gene of a S. cerevisiae laboratory strain. In addition, all promoter motifs which are, or are presumed to be, implicated in transcription regulatory functions are identical in ILV5 and ILV5X. In contrast, the coding sequence of ILV5G differs in 5.6% of its nucleotides from that of ILV5 and most of its promoter regulatory motifs show a single nucleotide change with respect to those from ILV5.

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana.

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.

Isolation and kinetic properties of acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) chloroplasts overexpressed in Escherichia coli.

Acetohydroxy acid isomeroreductase catalyses a two-step reaction, an alkyl migration and a NADPH-dependent reduction, in the assembly of the carbon skeletons of branched-chain amino acids. Detailed investigations of acetohydroxy acid isomeroreductase aimed at elucidating the biosynthetic pathway of branched-chain amino acids and at designing new inhibitors of the enzyme having herbicidal potency have so far been conducted with the enzymes isolated from bacteria. To gain more information on a plant system, the gene encoding the mature acetohydroxy acid isomeroreductase from spinach (Spinacia oleracea) leaf chloroplasts has been used to transform Escherichia coli cells and to overexpress the enzyme. A rapid protocol is described that allows the preparation of large quantities of pure spinach chloroplast acetohydroxy acid isomeroreductase. Kinetic and structural properties of the plant enzyme expressed in Escherichia coli are compared with those reported in our previous studies on the native enzymes purified from spinach chloroplasts and with those reported for the corresponding enzymes isolated from Escherichia coli and Salmonella typhimurium. Both the plant and the bacterial enzymes obey an ordered mechanism in which NADPH binds first, followed by substrate (either 2-acetolactate or 2-aceto-2-hydroxybutyrate). Inhibition studies employing an inactive substrate analogue, 2-hydroxy-2-methyl-3-oxopentanoate, showed, however, that the binding of 2-hydroxy-2-methyl-3-oxopentanoate and NADPH occurs randomly, suggestive of some flexibility of the plant enzyme active site. The observed preference of the enzyme for 2-aceto-2-hydroxybutyrate over 2-acetolactate is discussed with regard to the contribution of acetohydroxy acid isomeroreductase activity in the partitioning between isoleucine and valine biosyntheses. Moreover, the kinetic properties of the chloroplast enzyme support the notion that biosynthesis of branched-chain amino acids in plants is controlled by light. As judged by analytical-ultracentrifugation and gel-filtration analyses the overexpressed plant enzyme is a dimer of identical subunits.

Structure and expression of a cyanobacterial ilvC gene encoding acetohydroxyacid isomeroreductase.

Acetohydroxyacid isomeroreductase (AHAIR) is the shared second enzyme in the biosynthetic pathways leading to isoleucine and valine. AHAIR is encoded by the ilvC gene in bacteria. A 1,544-bp fragment of genomic DNA containing the ilvC gene was cloned from the cyanobacterium Synechocystis sp. strain PCC 6803, and the complete nucleotide sequence was determined. The identity of the gene was established by comparison of the nucleotide and derived peptide sequences with those of other ilvC genes. The highest degree of sequence similarity was found with the ilvC gene from Rhizobium meliloti. The isolated Synechocystis ilvC gene complemented an Escherichia coli ilvC mutant lacking AHAIR activity. The expressed Synechocystis gene encodes a protein that has a molecular mass of 35.7 kDa and that has AHAIR activity in an in vitro assay. Polyclonal antibodies raised against purified Synechocystis AHAIR produced a single band on a Western blot (immunoblot) of a Synechocystis cell extract and detected the protein in an extract of an E. coli ilvC mutant strain that was transformed with a plasmid containing the Synechocystis ilvC gene. The antibody did not react with an extract of an E. coli ilvC mutant strain that was transformed with a control plasmid lacking the Synechocystis ilvC gene or with an extract of an E. coli IlvC+ control strain.

Characterization of the ilv-2 gene from Neurospora crassa encoding alpha-keto-beta-hydroxylacyl reductoisomerase.

We have isolated the cDNA and corresponding genomic DNA for the ilv-2 locus of Neurospora crassa. This gene encodes alpha-keto-beta-hydroxylacyl reductoisomerase (Ilv-2), required for the synthesis of isoleucine and valine. The gene contains four introns, maps to the right arm of chromosome V, and encodes a protein of 400 amino acids (aa). Alignment of the aa sequence of Ilv-2 with the two other known eukaryotic sequences encoding this enzyme reveals two conserved regions.

Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts.

Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3-dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively. We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor. The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame. The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues. The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA. The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins. There are two blocks of conserved amino acid residues in these three proteins. One of these is a sequence similar to the 'fingerprint' region of the NAD(P)H-binding site found in a large number of NAD(P)H-dependent oxidoreductases. The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction. Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns.

Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp.

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three- to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to O2. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH. The partially purified enzyme was not sensitive to O2. The dihydroxy acid dehydratase is extremely sensitive to O2, and it has a half-life under 5% O2 of 6 min at 25 degrees C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.

Purification and characterization of acetohydroxyacid reductoisomerase from spinach chloroplasts.

Acetohydroxyacid reductoisomerase was purified over 400-fold to a specific activity of 62 nkat.mg-1, with 2-aceto-2-hydroxybutyrate as substrate, from the stroma of spinach leaf chloroplasts. The enzyme was not intrinsically membrane bound. The native enzyme was a tetramer with a subunit Mr of 59,000. The activity was optimum between pH 7.5 and 8.5. The apparent Km for 2-acetolactate was 25 microM and for 2-aceto-2-hydroxybutyrate was 37 microM. The enzyme required Mg2+ and the Vmax. was attained at physiological Mg2+ concentrations. NADP+ competitively inhibited the reaction when NADPH was the varied substrate. The native enzyme eluted from Mono-Q ion-exchange resins as three distinct peaks of activity. This elution pattern was preserved when the peaks were combined, dialysed and re-chromatographed. Each form exhibited identical Mr of 59,000 after SDS/polyacrylamide gel electrophoresis (PAGE), whereas they were easily distinguishable from each other after PAGE under non-denaturing conditions. These results provide evidence for the existence of multiple forms of acetohydroxyacid reductoisomerase in chloroplasts isolated from spinach leaves.

The herbicidally active experimental compound Hoe 704 is a potent inhibitor of the enzyme acetolactate reductoisomerase.

Growth inhibition of plants and bacteria by the experimental herbicide Hoe 704 (2-methylphosphinoyl-2-hydroxyacetic acid) was alleviated by the addition of the branched-chain amino acids to growth media. Hoe 704 caused a massive accumulation of acetoin and acetolactate, indicating its direct interference with the branched-chain amino acid biosynthetic pathway. The second enzyme of this pathway, acetolactate reductoisomerase (EC 1.1.1.86), was found to be subject to strong inhibition by Hoe 704. The inhibition was time-dependent and competitive with the enzyme's substrate, acetolactate. This report establishes acetolactate reductoisomerase as a new target for a herbicidal compound.

Transcriptional activation at adjacent operators in the divergent-overlapping ilvY and ilvC promoters of Escherichia coli.

The ilvC gene encodes acetohydroxy acid isomeroreductase (EC 1.1.1.89), the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Expression of the ilvC gene is induced by acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate. This substrate induction is mediated by a positive activator encoded by an adjacent gene, ilvY. The ilvY and ilvC genes are transcribed in opposite directions from promoters that are overlapping. In this paper we characterize the in vitro DNA binding properties of the ilvY-encoded activator protein. The ilvY product binds to two adjacent operator sites located in the divergent-overlapping ilvY and ilvC promoter region. One of these operators, designated O1 contains regions of dyad symmetry centered at position +17 relative to the ilvY transcriptional start site, and the second site, designated O2, contains an homologous inverted repeat sequence centered about the -35 region of the ilvC promoter. Binding of the ilvY product at the O1 and O2 operator sites is co-operative and this ilvY protein-DNA complex in the presence of acetohydroxy acid isomeroreductase substrate is a prerequisite for RNA polymerase binding to the ilvC promoter as detected by DNase I protection experiments. Additionally, chromosomal galK transcriptional fusion assays were performed to characterize the regulation of the ilvY and ilvC promoters in vivo. Transcription of the ilvC gene is maintained at a basal level of activity which is elevated as much as 15-fold in the presence of ilvY product and acetohydroxybutyrate. The ilvY product represses ilvY transcription in a manner that does not appear to be dependent on acetohydroxy acid isomeroreductase substrate. We discuss models in which activation of ilvC transcription results from a direct interaction of ilvY protein with RNA polymerase or an ilvY-mediated alteration of the DNA conformation of the ilvC -35 promoter region. Additionally, we discuss the role of acetohydroxybutyrate and acetolactate in ilvY transcriptional regulation.

The ILV5 gene of Saccharomyces cerevisiae is highly expressed.

The nucleotide sequence of the yeast ILV5 gene, which codes for the branched-chain amino acid biosynthesis enzyme acetohydroxyacid reductoisomerase, has been determined. The ILV5 coding region is 1,185 nucleotides, corresponding to a polypeptide with a molecular weight of 44,280. Transcription of the ILV5 mRNA initiates at position -81 upstream from the ATG translation start codon and terminates between 218 and 222 bases downstream from the stop codon. Consensus sequences have been identified for initiation and termination of transcription, and for general control of amino acid biosynthesis, as well as repression by leucine. The ILV5 gene is regulated slightly by general amino acid control. Codon usage of the ILV5 gene has the strong bias observed in yeast genes that are highly expressed. In agreement with this, the reductoisomerase monomer, with an apparent molecular weight of 40,000, has been identified in an SDS polyacrylamide gel pattern of total soluble yeast proteins as a gene dosage dependent band.

Role of acetohydroxy acid isomeroreductase in biosynthesis of pantothenic acid in Salmonella typhimurium.

Structural genes have been identified for all of the enzymes involved in the biosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coli K-12, with the exception of ketopantoic acid reductase, which catalyzes the conversion of alpha-ketopantoate to pantoate. The acetohydroxy acid isomeroreductase from S. typhimurium efficiently bound alpha-ketopantoate (K(m) = 0.25 mM) and catalyzed its reduction at 1/20 the rate at which alpha-acetolactate was reduced. Since two enzymes could apparently participate in the synthesis of pantoate, a S. typhimurium ilvC8 strain was mutagenized to derive strains completely blocked in the conversion of alpha-ketopantoate to pantoate. Several isolates were obtained that grew in isoleucine-valine medium supplemented with either pantoate or pantothenate, but not in the same medium supplemented with alpha-ketopantoate or beta-alanine. The mutations that conferred pantoate auxotrophy (designated panE) to these isolates appeared to be clustered, but were not linked to panB or panC. All panE strains tested had greatly reduced levels of ketopantoic acid reductase (3 to 12% of the activity present in DU201). The capacity of the isomeroreductase to synthesize pantoate in vivo was assessed by determining the growth requirements of ilvC(+) derivatives of panE ilvC8 strains. These strains required either alpha-ketopantoate, pantoate, or pantothenate when the isomeroreductase was present at low levels; when the synthesis of isomeroreductase was induced, panE ilvC(+) strains grew in unsupplemented medium. These phenotypes indicate that a high level of isomeroreductase is sufficient for the synthesis of pantoate. panE ilvC(+) strains also grew in medium supplemented with lysine and methionine. This phenotype resembles that of some S. typhimurium ilvG mutants (e.g., DU501) which are partially blocked in the biosynthesis of coenzyme A and are limited for succinyl coenzyme A. panE ilvC(+) strains which lack the acetohydroxy acid synthases required only methionine for growth (in the presence of leucine, isoleucine, and valine). This and other evidence suggested that the synthesis of pantoic acid by isomeroreductase was blocked by the alpha-acetohydroxy acids and that pantoic acid synthesis was enhanced in the absence of these intermediates, even when the isomeroreductase was at low levels. panE ilvC(+) strains reverted to pantothenate independence. Several of these revertants were shown to have elevated isomeroreductase levels under noninduced and induced conditions; the suppressing mutation in each revertant was shown to be closely linked to ilvC by P22 transduction. This procedure presents a means for obtaining mutants with altered regulation of isomeroreductase.

Mutations in the ilvY gene of Escherichia coli K-12 that cause constitutive expression of ilvC.

A derivative of Escherichia coli K-12 bearing an ilvC-lac fusion has been studied. beta-Galactosidase formation in this strain is under the control of the ilvC promoter and is therefore induced by the acetohydroxy acids. Derivatives of this fusion strain were isolated that constitutively expressed beta-galactosidase. When an ilvC-containing episome was introduced into these strains, acetohydroxy acid isomeroreductase was also constitutively expressed. The lesions are trans dominant and lie in ilvY, the structural gene specifying a positive control element, v, needed for induction of the isomeroreductase. It was concluded from measurements of beta-galactosidase levels in various diploid strains that, although wild-type v requires inducer to act as a positive control element, it does not act as a repressor in the absence of inducer.

Gene ilvY of Salmonella typhimurium.

Evidence is presented for the existence in Salmonella typhimurium LT2 of the regulatory gene ilv Y. The Escherichia coli K-12 ilvY gene product is shown to complement a S. typhimurium ilvY mutation in vivo.