Gerold Barth

Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand

The 1,2-dicarbonyl compounds 3-deoxyglucosulose (3-DG), glyoxal (GO), and methylglyoxal (MGO) were measured as the corresponding quinoxalines after derivatization with orthophenylendiamine using RP-HPLC and UV-detection in commercially available honey samples. Whereas for most of the samples values for 3-DG, MGO, and GO were comparable to previously published data, for six samples of New Zealand Manuka (Leptospermum scoparium) honey very high amounts of MGO were found, ranging from 38 to 761 mg/kg, which is up to 100-fold higher compared to conventional honeys. MGO was unambigously identified as the corresponding quinoxaline via photodiodearry detection as well as by means of mass spectroscopy. Antibacterial activity of honey and solutions of 1,2-dicarbonyl towards Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were analyzed using an agar well diffusion assay. Minimum concentrations needed for inhibition of bacterial growth (minimum inhibitory concentration, MIC) of MGO were 1.1 mM for both types of bacteria. MIC for GO was 6.9 mM (E. coli) or 4.3 mM (S. aureus), respectively. 3-DG showed no inhibition in concentrations up to 60 mM. Whereas most of the honey samples investigated showed no inhibition in dilutions of 80% (v/v with water) or below, the samples of Manuka honey exhibited antibacterial activity when diluted to 15-30%, which corresponded to MGO concentrations of 1.1-1.8 mM. This clearly demonstrates that the pronounced antibacterial activity of New Zealand Manuka honey directly originates from MGO.

Vectors for gene expression and amplification in the yeast Yarrowia lipolytica

New vector systems were developed for gene expression in Y. lipolytica. These plasmids contain: (a) as integration target sequences, either a rDNA region or the long terminal repeat zeta of the Y. lipolytica retrotransposon Ylt1; (b) the YlURA3 gene as selection marker for Y. lipolytica, either as the non‐defective ura3d1 allele for single integration or the promotor truncated ura3d4 allele for multiple integration; (c) the inducible ICL1 or XPR2 promoters for gene expression; and (d) unique restriction sites for gene insertion. Multiple plasmid integration occurred as inserted tandem‐repeats, which are present at 3–39 copies per cell. A correlation between gene copy number and the expressed enzyme activity was demonstrated with Escherichia coli lacZ as reporter gene under the control of the regulated ICL1 promoter. Increases in copy numbers from 5 to 13 for the lacZ expression cassettes resulted in an up to 10–11‐fold linear increase of the β‐galactosidase activity in multicopy transformants during their growth on ethanol or glucose, compared with the low‐copy replicative plasmid transformants (1.6 plasmid copies). These new tools will enhance the interest in Y. lipolytica as an alternative host for heterologous protein production. Copyright

Insertional Mutagenesis in the n-Alkane-Assimilating Yeast Yarrowia lipolytica: Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization

Tagged mutants affected in the degradation of hydrophobic compounds (HC) were generated by insertion of a zeta-URA3 mutagenesis cassette (MTC) into the genome of a zeta-free and ura3 deletion-containing strain of Yarrowia lipolytica. MTC integration occurred predominantly at random by nonhomologous recombination. A total of 8,600 Ura(+) transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain lengths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethanol as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas). A total of 257 mutants were obtained, of which about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk(-)) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants. Among Alk(-) mutants, growth defects depending on the alkane chain length were observed (alkAa to alkAc). Furthermore, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated. Flanking borders of the integrated MTC were sequenced to identify the disrupted genes. Sequence analysis indicated that the MTC was integrated in the LEU1 locus in N083, a leucine-auxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gene (PEX14) in N078 (alkD). This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in Y. lipolytica.

Production of Lycopene in the Non-Carotenoid-Producing Yeast Yarrowia lipolytica

ABSTRACT The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g−1 (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.

Overproduction and secretion of α-ketoglutaric acid by microorganisms

This mini-review presents a summary of research results of biotechnological production of alpha-ketoglutaric acid (KGA) by bacteria and yeasts. KGA is of particular industrial interest due to its broad application scope, e.g., as building block chemical for the chemical synthesis of heterocycles, dietary supplement, component of infusion solutions and wound healing compounds, or as main component of new elastomers with a wide range of interesting mechanical and chemical properties. Currently KGA is produced via different chemical pathways, which have a lot of disadvantages. As an alternative several bacteria and yeasts have already been studied for their ability to produce KGA as well as for conditions of overproduction and secretion of this intermediate of the tricarboxylic acid cycle. The aim of this mini-review was to summarize the known data and to discuss the potentials of biotechnological processes of KGA production.

Overexpression of the ICL1 gene changes the product ratio of citric acid production by Yarrowia lipolytica

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric (CA) and isocitric (ICA) acids, triggered by growth limitation caused by different factors and an excess of carbon source. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether the CA/ICA product ratio can be influenced by gene-dose-dependent overexpression or by disruption of the isocitrate lyase (ICL)-encoding gene ICL1, recombinant Y. lipolytica strains were constructed, which harbour multiple ICL1 copies or a defective icl1 allele. The high-level expression of ICL in ICL1 multicopy integrative transformants resulted in a strong shift of the CA/ICA ratio into direction of CA. On glycerol, glucose and sucrose, the ICA proportion decreased from 10–12% to 3–6%, on sunflower oil or hexadecane even from 37–45% to 4–7% without influencing the total amount of acids (CA and ICA) produced. In contrast, the loss of ICL activity in icl1-defective strains resulted in a moderate 2–5% increase in the ICA proportion compared to ICL wild-type strains on glucose or glycerol.

Comparison of promoters suitable for regulated overexpression of β-galactosidase in the alkane-utilizing yeastYarrowia lipolytica

Promoters of the genesG3P, ICL1, POT1, POX1, POX2 andPOX5 of the yeastY. lipolytica were studied in respect to their regulations and activities during growth on different carbon sources. The aim of this study was to select suitable promoters for high expression of heterologous genes in this yeast. For this purpose the promoters were fused with the reporter genelacZ ofE. coli and integrated as single copies into the genome ofY. lipolytica strain PO1d. The measurement of expressed activities of β-galactosidase revealed thatpICL1, pPOX2 andpPOT1 are the strongest regulable promoters available forY. lipolytica, at present.pPOX2 andpPOT4 were highly induced during growth on oleic acid and were completely repressed by glucose and glycerol.pICL1 was strongly inducible by ethanol besides alkanes and fatty acids, however, not completely repressible by glucose or glycerol. Ricinoleic acid methyl ester appeared as a very strong inducer forpPOT1 andpPOX2, in spite of that it inhibited growth ofY. lipolytica transformants.

Aconitase overexpression changes the product ratio of citric acid production by Yarrowia lipolytica

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric acid (CA) and isocitric acid (ICA) under an excess of carbon source and several conditions of growth limitation. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether this CA/ICA product ratio can be influenced by gene–dose-dependent overexpression of aconitase (ACO)-encoding gene ACO1, a recombinant Y. lipolytica strain was constructed containing multiple copies of ACO1. The high-level expression of ACO in the ACO1 multicopy integrative transformant resulted in a shift of the CA/ICA product pattern into the direction of ICA. On sunflower oil, a striking increase of the ICA proportion from 35–49% to 66–71% was observed compared to wild-type strains without influencing the total amount of acids (CA and ICA) produced. On glycerol, glucose or sucrose, the ICA proportion increased only moderately from 10–12% to 13–17%. This moderate shift into the direction of ICA was also observed in an icl1-defective strain.

Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric, isocitric, pyruvic (PA), and α-ketoglutaric (KGA) acids, triggered by growth limitation and excess of carbon source. This is leading to an increased interest in this non-conventional yeast for biotechnological applications. To improve the KGA production by Y. lipolytica for an industrial application, it is necessary to reduce the amounts of by-products, e.g., fumarate (FU) and PA, because production of by-products is a main disadvantage of the KGA production by this yeast. We have examined whether the concentration of secreted organic acids (main product KGA and PA as major by-product and FU, malate (MA), and succinate (SU) as minor by-products) can be influenced by a gene–dose-dependent overexpression of fumarase (FUM) or pyruvate carboxylase (PYC) genes under KGA production conditions. Recombinant Y. lipolytica strains were constructed, which harbor multiple copies of the respective FUM1, PYC1 or FUM1, and PYC1 genes. Overexpression of the genes FUM1 and PYC1 resulted in strongly increased specific enzyme activities during cultivation of these strains on raw glycerol as carbon source in bioreactors. The recombinant Y. lipolytica strains showed different product selectivity of the secreted organic acids KGA, PA, FU, MA, and SU. Concentrations of the by-products FU, MA, SU, and PA decreased significantly at overproduction of FUM and increased at overproduction of PYC and also of FUM and PYC simultaneously. In contrast, the production of KGA with the multicopy strains H355A(FUM1) and H355A(FUM1-PYC1) was comparable with the wild-type strain H355 or slightly lower in case of H355(PYC1). KGA productivity was not changed significantly compared with strain H355 whereas product selectivity of the main product KGA was increased in H355A(FUM1).

Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids

The yeast Yarrowia lipolytica is one of the most intensively studied “non-conventional” yeast species. Its ability to secrete various organic acids, like pyruvic (PA), citric, isocitric, and alpha-ketoglutaric (KGA) acid, in large amounts is of interest for biotechnological applications. We have studied the effect of the alpha-ketoglutarate dehydrogenase (KGDH) complex on the production process of KGA. Being well studied in Saccharomyces cerevisiae this enzyme complex consists of three subunits: alpha-ketoglutarate dehydrogenase, dihydrolipoyl transsuccinylase, and lipoamide dehydrogenase. Here we report the effect of overexpression of these subunits encoding genes and resulting increase of specific KGDH activity on organic acid production under several conditions of growth limitation and an excess of carbon source in Y. lipolytica. The constructed strain containing multiple copies of all three KGDH genes showed a reduced production of KGA and an elevated production of PA under conditions of KGA production. However, an increased activity of the KGDH complex had no influence on organic acid production under citric acid production conditions.