Application of metabolic engineering to improve both the production and use of biotech indigo.

A fermentation process was developed for production of indigo from glucose using recombinant Escherichia coli. This was achieved by modifying the tryptophan pathway to cause high-level indole production and adding the Pseudomonas putida genes encoding naphthalene dioxygenase (NDO). In comparison to a tryptophan-over-producing strain, the first indigo-producing strain made less than half of the expected amount of indigo. Severe inactivation of the first enzyme of aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (the aroGfbr gene product), was observed in cells collected from indigo fermentations. Subsequent in vitro experiments revealed that DAHP synthase was inactivated by exposure to the spontaneous chemical conversion of indoxyl to indigo. Indigo production was thereafter improved by increasing the gene dosage of aroGfbr or by increasing substrate availability to DAHP synthase in vivo by either amplifying the tktA (transketolase) gene or inactivating both isozymes of pyruvate kinase. By combining all three strategies for enhancing DAHP formation in the cell, a 60% increase in indigo production was achieved. Metabolic engineering was then further applied to eliminate a byproduct of the spontaneous conversion of indoxyl to indigo, thereby solving a serious problem with the use of bio-indigo in the final denim dyeing application.

Correlation between Bacillus subtilis scoC phenotype and gene expression determined using microarrays for transcriptome analysis.

The availability of the complete sequence of the Bacillus subtilis chromosome (F. Kunst et al., Nature 390:249-256, 1997) makes possible the construction of genome-wide DNA arrays and the study of this organism on a global scale. Because we have a long-standing interest in the effects of scoC on late-stage developmental phenomena as they relate to aprE expression, we studied the genome-wide effects of a scoC null mutant with the goal of furthering the understanding of the role of scoC in growth and developmental processes. In the present work we compared the expression patterns of isogenic B. subtilis strains, one of which carries a null mutation in the scoC locus (scoC4). The results obtained indicate that scoC regulates, either directly or indirectly, the expression of at least 560 genes in the B. subtilis genome. ScoC appeared to repress as well as activate gene expression. Changes in expression were observed in genes encoding transport and binding proteins, those involved in amino acid, carbohydrate, and nucleotide and/or nucleoside metabolism, and those associated with motility, sporulation, and adaptation to atypical conditions. Changes in gene expression were also observed for transcriptional regulators, along with sigma factors, regulatory phosphatases and kinases, and members of sensor regulator systems. In this report, we discuss some of the phenotypes associated with the scoC mutant in light of the transcriptome changes observed.

Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses.

Previous studies have shown that the CcpA protein of Bacillus subtilis is a major transcription factor mediating catabolite repression. We report here whole-transcriptome analyses that characterize CcpA-dependent, glucose-dependent gene expression and correlate the results with full-genome computer analyses of DNA binding (CRE) sites for CcpA. The data obtained using traditional approaches show good agreement with those obtained using the transcriptome approach. About 10% of all genes in B. subtilis are regulated > 3x by glucose, with repressed genes outnumbering activated genes three to one. Eighty per cent of these genes depend on CcpA for regulation. Classical approaches have provided only evidence for CcpA-mediated, glucose-dependent activation or repression. We show here that CcpA also mediates glucose-independent activation or repression, and that glucose may alter either the direction or the intensity of either effect. Computer analyses revealed the presence of CRE sites in most operons subject to CcpA-mediated glucose repression, but not in those subject to glucose activation, suggesting that either secondary transcription factors regulate the latter genes or activation by CcpA involves a dissimilar binding site. Operons encoding the constituents of ABC-type transporters that are subject to CcpA-mediated glucose regulation show two distinct patterns: either all genes in the operon are regulated in parallel (the minor class) or the gene encoding the extracytoplasmic solute-binding receptor is preferentially regulated (the major class). Genes subject to CcpA-independent catabolite repression are primarily concerned with sporulation. Several transcription factors were identified that are themselves regulated by CcpA at the transcriptional level. Representative data with functionally characterized genes are presented to illustrate the novel findings. The comprehensive transcriptome data are available on our website: www.biology.uesd.edu/~MSAIER/regulation/ and also on http://www.blackwell-science.com/ products/journals/suppmat/MMI/MMI2328/MMI2328sm.htm

Indoxyl-UDPG-glucosyltransferase from Baphicacanthus cusia.

The enzyme catalyzing the transfer of glucose from uridine diphosphate glucose to indoxyl yielding the indoxyl glucoside indican was isolated from Baphicacanthus cusia Bremek (Acanthaceae). The indoxyl-uridine diphosphate glucose (UDPG)-glucosyltransferase was purified to homogeneity in six chromatographic steps. The decisive step for the recovery of a homogeneous enzyme was the application of immobilized metal affinity chromatography yielding an 863-fold purified enzyme. From a total of 60 substances tested, in addition to the natural substrate 3-OH-indole (indoxyl), only 4-OH-, 5-OH-, 6-OH-, and 7-OH-indole were accepted as substrates by the glucosyltransferase. However, the latter substrates were metabolized to varying extent. The optimum pH of the enzyme was 8.5, the optimum temperature was 30 degrees C and the isoelectric point was pH 6.5. The M(r) of the enzyme was determined to be 60 +/- 2 x 10(3). Indoxyl as substrate yielded a K(m) of 1.2 mM, while a K(m) of 1.7 mM was found for UDPG.

The commercial production of chemicals using pathway engineering.

Integration of metabolic pathway engineering and fermentation production technologies is necessary for the successful commercial production of chemicals. The 'toolbox' to do pathway engineering is ever expanding to enable mining of biodiversity, to maximize productivity, enhance carbon efficiency, improve product purity, expand product lines, and broaden markets. Functional genomics, proteomics, fluxomics, and physiomics are complementary to pathway engineering, and their successful applications are bound to multiply product turnover per cell, channel carbon efficiently, shrink the size of factories (i.e., reduce steel in the ground), and minimize product development cycle times to bring products to market.

Kinetic properties of cloned human liver monoamine oxidase A.

Monoamine oxidases deaminate many amines, including neurotransmitters, by oxidation followed by spontaneous breakdown of the imine product. The reduced enzyme is reoxidized slowly by oxygen, but in the presence of amines, the rate of reoxidation is markedly enhanced. The extent of enhancement depends on the amine substrate, kynuramine enhancing the rate 125-fold, but 5-hydroxytryptamine only 6-fold. Here we describe the properties of human liver monoamine oxidase A which has been cloned into and overexpressed in yeast. The purified enzyme has a higher Km for oxygen than does the placental enzyme, but the steady-state parameters for the endogenous amines are the same. Tertiary amines are oxidized at slightly different rates by the two enzymes. The consequences of the branched pathway mechanism with substrate-dependent enhancement of reoxidation for the steady-state levels of the various enzyme species is discussed.

Functional expression of C-terminally truncated human monoamine oxidase type A in Saccharomyces cerevisiae.

The deduced amino acid sequence of human liver monoamine oxidase type A was analyzed with secondary structure programs. These analyses and comparison to other flavoproteins identified a single potential transmembrane hydrophobic peptide at the C-terminus suggesting that this peptide is a membrane anchor and that the remainder of the protein constitutes a soluble domain. Truncation of the C-terminus by 24 amino acids which are inclusive of the putative transmembrane peptide, however, gave a protein which exhibited solubility properties substantially similar to the wild type enzyme. This result indicates that the hydrophobic behavior of monoamine oxidase type A is due to more complex features than a single transmembrane anchor. The mutant enzyme expressed in yeast appears to form a disulfide bond which reduces catalytic efficiency by up to 90%. Full activity, however, can be recovered by incubation with dithiothreitol, suggesting that in the wild type enzyme the amino acid residues deleted in the mutant protein protect two cysteine residues (those involved in the formation of the disulfide bond in the mutant) from oxidation and that the deleted residues are in close proximity to the active site. The activation experiments indicated that the deleted amino acids do not contribute any catalytic residues to the active site.

Differences in substrate specificities of monoamine oxidase A from human liver and placenta.

The substrate specificities of monoamine oxidase (MAO) A isolated from human placenta and of human liver expressed in yeast have been compared in homogeneous preparations with respect to Vmax and Km values for natural and synthetic substrates and Ki values for competitive inhibitors. MAO A from these two sources is known to differ in at least 5 amino acid residues. While the Km and Ki values were found to be nearly identical in the enzymes from these two sources, the Vmax differed significantly on bulky synthetic substrates.

Catalytically active monoamine oxidase type A from human liver expressed in Saccharomyces cerevisiae contains covalent FAD.

Monoamine oxidase type A from human liver cDNA was expressed in Saccharomyces cerevisiae. This enzyme's properties with respect to Km and Ki values for kynuramine and amphetamine, respectively, were similar to values for human placental enzyme. As expected, clorgyline inhibited the yeast enzyme at lower concentrations than deprenyl. Interestingly, the FAD cofactor was covalently attached and fluorescence properties of the enzyme bound prosthetic group indicate that it is attached to a cysteine residue, the same linkage observed in other monoamine oxidases. The yield of expressed enzyme is about 15 mg/l of culture with an A600 of 15. It is suggested that covalent flavin attachment proceeds by an autoflavination mechanism.

Biochemistry and genetics of monoamine oxidase.

This chapter reviews the two mitochondrial flavin containing isozymes of monoamine oxidase. Section 1, "Biochemistry" discusses assays, substrates and inhibitors, phylogenic and tissue distribution, interactions with lipids, nutritional studies, protein structure, kinetic and chemical mechanistic proposals, and biosynthesis. Section 2, "Inheritance" discusses possible genes involved in expression, genetic studies of platelet MAO-B and fibroblast MAO-A, and chromosomal location. Section 3, "Molecular Genetics" reviews the cloning of their cDNAs, their intra- and interspecies homology and structural inferences made from deduced amino acid sequences. Section 4, "Regulation" gives an overview of levels in development and aging, and effect of drugs. The final section 5, "Role in Human Disease" discusses physiological function and effects of altered levels in humans and animal models including complete absence due to a submicroscopic chromosomal deletion in several human patients.

Monoamine oxidase A from human placenta and monoamine oxidase B from bovine liver both have one FAD per subunit.

I present the first clear evidence that the protein: FAD ratio in human monoamine oxidase A and bovine monoamine oxidase B has an upper limit of 65 kDa and 57 kDa per FAD, respectively. To now it had been assumed that the protein: FAD ratio was 100-120 kDa to 1 FAD and that there was one FAD per two subunits which were assumed to be of the same size. For the present work the purity of monoamine oxidase A and monoamine oxidase B was improved over that previously achieved. Protein was determined by quantitative amino acid analysis and FAD content was measured by spectrophotometric titration of SDS-denatured enzyme with NaS2O4 standardized against riboflavin. The cause of the previous misassignment of the protein: FAD ratio was judged as having been due to the use of impure enzyme preparations. Knowledge of the correct protein: FAD ratio is important in devising cloning strategies for this enzyme, in understanding its structure, function, mechanism, and in the studies of its biosynthesis.

The primary structure of bovine monoamine oxidase type A. Comparison with peptide sequences of bovine monoamine oxidase type B and other flavoenzymes.

We have isolated cDNA clones believed to encompass the full-length coding sequences for a subunit of bovine monoamine oxidase type A (MAO-A). The clones code for an apoprotein of 527 amino acid residues corresponding to a molecular mass of 59,806 Da. The inferred protein sequences show an overall similarity of 68% with partial amino acid sequences of bovine type B MAO (about 41% of the total sequence), as well as a greater similarity (greater than 90%) with some regions including that for the published sequence of the flavin-binding region. Sequence comparisons indicate that these two forms of MAO are encoded by distinct genes. Comparison of this sequence with other flavoenzymes showed similarity with regions associated with non-covalent flavin-binding sites. Analysis of mRNAs coding for MAO enzymes showed a heterogeneity of transcripts consistent with several different forms of monoamine oxidase.

Structural features of human monoamine oxidase A elucidated from cDNA and peptide sequences.

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.

Partial amino acid sequence analysis of human placenta monoamine oxidase A and bovine liver monoamine oxidase B.

We have prepared peptide maps from human placenta monoamine oxidase type A (MAO-A) and bovine monoamine oxidase type B (MAO-B) and determined the amino acid sequences of 21 of these peptides. These sequences have been compared to the cDNA deduced amino acid sequences of human MAO-A and -B. A result of special interest is the identification of two sets of MAO-A peptides which have sequences different from those deduced from cDNA sequences. This observation is consistent with the notion that MAO-A may be composed of at two subunits which are similar but not identical in primary amino acid sequence.

Human monoamine oxidase gene (MAOA): chromosome position (Xp21-p11) and DNA polymorphism.

An essentially full-length cDNA clone for the human enzyme monoamine oxidase type A (MAO-A) has been used to determine the chromosomal location of a gene encoding it. This enzyme is important in the degradative metabolism of biogenic amines throughout the body and is located in the outer mitochondrial membrane of many cell types. Southern blot analysis of PstI-digested human DNA revealed multiple fragments that hybridized to this probe. Using rodent-human somatic cell hybrids containing all or part of the human X chromosome, we have mapped these fragments to the region Xp21-p11. A restriction fragment length polymorphism (RFLP) for this MAOA gene was identified and used to evaluate linkage distances between this locus and several other loci on Xp. The MAOA locus lies between DXS14 and OTC, about 29 cM from the former.

Factors affecting the production of pyrroloquinoline quinone by the methylotrophic bacterium W3A1.

Two variants of the methylotrophic bacterium W3A1, designated W3A1-S (slimy) and W3A1-NS (nonslimy), were compared with respect to their ability to grow in batch culture on the C1 substrates methylamine, methanol, and trimethylamine. Substrate utilization, cell density, pH, cellular and soluble polysaccharide production, and concentrations of the enzymes methylamine dehydrogenase, trimethylamine dehydrogenase, and methanol dehydrogenase produced were measured as a function of growth. The ability of the two bacterial variants to excrete the redox cofactor pyrroloquinoline quinone into the growth medium was also investigated. The two variants were similar with respect to all properties measured, except that W3A1-S produced significantly more capsular polysaccharides than variant W3A1-NS. Pyrroloquinoline quinone was excreted when either variant was grown on any of the C1 substrates investigated but was maximally produced when the methylamine concentration was 0.45% (wt/vol). This cofactor is excreted only as bacterial growth enters the stationary phase, a time when the levels of trimethylamine dehydrogenase and the quinoproteins methanol dehydrogenase and methylamine dehydrogenase begin to decline. It is not known whether the pyrroloquinoline quinone found in the medium is made de novo for excretion, derived from the quinoprotein pool, or both. Pyrroloquinoline quinone excretion has been observed with other methylotrophs, but this is the first instance where the excretion was observed with substrates other than methanol.

2-Chloro-2-phenylethylamine as a mechanistic probe and active site-directed inhibitor of monoamine oxidase from bovine liver mitochondria.

The reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B was investigated to study the mechanism of this enzyme and its inactivation by this compound. 2-Chloro-2-phenylethylamine is a substrate with a Km of 30 microM and a turnover number of 80 min-1 at pH 6.5 at 30 degrees C. Incubation of 2-chloro-2-phenylethylamine with the enzyme led to the normal oxidation product, 2-chloro-2-phenylacetaldehyde, but only traces (0.25 mol%) of 2-phenylacetaldehyde, the product anticipated if the oxidation of substrate involved a stabilized carbanion at C-1 and elimination of chloride ion. These data suggest that a carbanion is not a likely intermediate in the oxidation of amines by monoamine oxidase. During the mechanistic studies we noted time-dependent inactivation of monoamine oxidase B by 2-chloro-2-phenylethylamine under both aerobic and anaerobic conditions. Inactivation was not reversible. Aerobically 2-chloro-2-phenylethylamine is oxidized to 2-chloro-2-phenylacetaldehyde which covalently modifies the enzyme (tau 1/2 = 40 min). Benzyl alcohol, a substrate analog, gives substantial protection against inactivation under aerobic conditions (tau 1/2 = 320 min), suggesting that an active site residue is modified. Anaerobic reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B probably proceeds by direct alkylation of an enzyme residue (tau 1/2 = 140 min). Reduction with [3H]NaBH4 of the inactivated enzyme gave from 0 to 0.7 and from 4.5 to 5.6 mol of hydride incorporation for enzyme inactivated anaerobically and aerobically, respectively. The latter results are in agreement with inactivation by unmodified inhibitor and inactivation by oxidized inhibitor for the anaerobic and aerobic reactions, respectively. It is suggested that 2-chloro-2-phenylethylamine or its oxidation product 2-chloro-2-phenylacetaldehyde may serve as an active site affinity reagent for monoamine oxidase.

Immunological uniqueness of human monoamine oxidases A and B: new evidence from studies with monoclonal antibodies to human monoamine oxidase A.

Monoamine oxidase (EC 1.4.3.4; MAO) is the primary enzyme responsible for the intraneuronal degradation of biogenic amines in the central nervous system. An understanding of the physiological significance of the functional and regulatory differences between the two forms of the enzyme, MAOs A and B, would be facilitated by the availability of antibodies specific for the two forms of the enzyme. We previously isolated and characterized a monoclonal antibody (MAO B-1C2, previously designated MAO-1C2) which binds human MAO B but not A. We describe here four new monoclonal antibodies (designated MAO A-3C9, A-4F10, A-7B10, and A-7E10) which were elicited to highly purified MAO A from human placenta and which, in the presence of antimouse IgG and Staphylococcus aureus, immunoprecipitate greater than 90% of the catalytically active purified MAO A. MAO A-3C9 appears to have a lower affinity for purified MAO A than the other three antibodies and does not immunoprecipitate either MAO A or MAO B from human platelets or from Triton X-100 extracts of human placental and liver mitochondria. MAO A-4F10, A-7B10, and A-7E10 immunoprecipitate catalytically active MAO A from Triton X-100 extracts of human placental and liver mitochondria, but not catalytically active MAO B from either pletelets or from Triton X-100 extracts of human liver mitochondria. Collectively, these anti-MAO monoclonal antibodies reveal unique epitopes on human MAO A not shared by MAO B, and at least one epitope on MAO B not shared by MAO A. These immunochemical differences support the hypothesis that MAO A and MAO B are different proteins, presumably isozymes.

Purification and properties of mitochondrial monoamine oxidase type A from human placenta.

A high yield purification scheme for monoamine oxidase A from human placental mitochondria is described. The enzyme is solubilized by a combination of treatment with phospholipase A and C and extraction with Triton X-100 and further purified by partitioning between dextran and polyethylene glycol polymers. The enzyme was obtained in 35% yield and high purity on DEAE-Sepharose CL-6B chromatography. This product, 90% catalytically active, showed a single major and several minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further purification could be achieved by additional chromatography using Bio-Gel HTP, but concomitant loss of catalytic activity occurred (enzyme remained about 60% active). The difference extinction coefficient for flavinox--flavinred at 456 nm was 10,800 +/- 350 m-1 cm-1. A sulfhydryl to flavin ratio of 7.5 was obtained when enzyme was denatured with sodium dodecyl sulfate, reduced with 2-mercaptoethanol, and titrated with 2,2'-dipyridyl disulfide. Anaerobic titration with 0.5 eq of sodium dithionite gave rise to the red anionic flavin radical, and full reduction was observed on further addition of reagent. The Km value for kynuramine was essentially the same for mitochondria (0.12 mM) and enzyme after DEAE-Sepharose CL-6B chromatography (0.17 mM). The concentration of clorgyline and deprenyl required for 50% inactivation also remained essentially unchanged. Incubation of the enzyme with 2,2'-dipyridyl disulfide caused inactivation in a biphasic manner with apparent second-order rate constants of 1230 M-1 min-1 and 235 M-1 min-1 for the rapid and slow phase, respectively. This inactivation was largely abolished by the inclusion of the competitive inhibitor amphetamine (Ki = 20 microM) in the incubation mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit molecular mass of 60-64 kDa, about 1.5-2.5 kDa higher than human liver monoamine oxidase B.