A universal gene expression system for fungi.

Biotechnological production of fuels, chemicals and proteins is dependent on efficient production systems, typically genetically engineered microorganisms. New genome editing methods are making it increasingly easy to introduce new genes and functionalities in a broad range of organisms. However, engineering of all these organisms is hampered by the lack of suitable gene expression tools. Here, we describe a synthetic expression system (SES) that is functional in a broad spectrum of fungal species without the need for host-dependent optimization. The SES consists of two expression cassettes, the first providing a weak, but constitutive level of a synthetic transcription factor (sTF), and the second enabling strong, at will tunable expression of the target gene via an sTF-dependent promoter. We validated the SES functionality in six yeast and two filamentous fungi species in which high (levels beyond organism-specific promoters) as well as adjustable expression levels of heterologous and native genes was demonstrated. The SES is an unprecedentedly broadly functional gene expression regulation method that enables significantly improved engineering of fungi. Importantly, the SES system makes it possible to take in use novel eukaryotic microbes for basic research and various biotechnological applications.

Biotechnological production of glycolic acid and ethylene glycol: current state and perspectives.

Glycolic acid (GA) and ethylene glycol (EG) are versatile two-carbon organic chemicals used in multiple daily applications. GA and EG are currently produced by chemical synthesis, but their biotechnological production from renewable resources has received a substantial interest. Several different metabolic pathways by using genetically modified microorganisms, such as Escherichia coli, Corynebacterium glutamicum and yeast have been established for their production. As a result, the yield of GA and EG produced from sugars has been significantly improved. Here, we describe the recent advancement in metabolic engineering efforts focusing on metabolic pathways and engineering strategies used for GA and EG production.

Production of ethylene glycol or glycolic acid from D-xylose in Saccharomyces cerevisiae.

The important platform chemicals ethylene glycol and glycolic acid were produced via the oxidative D-xylose pathway in the yeast Saccharomyces cerevisiae. The expression of genes encoding D-xylose dehydrogenase (XylB) and D-xylonate dehydratase (XylD) from Caulobacter crescentus and YagE or YjhH aldolase and aldehyde dehydrogenase AldA from Escherichia coli enabled glycolic acid production from D-xylose up to 150 mg/L. In strains expressing only xylB and xylD, 29 mg/L 2-keto-3-deoxyxylonic acid [(S)-4,5-dihydroxy-2-oxopentanoic acid] (2K3DXA) was produced and D-xylonic acid accumulated to ca. 9 g/L. A significant amount of D-xylonic acid (ca. 14%) was converted to 3-deoxypentonic acid (3DPA), and also, 3,4-dihydroxybutyric acid was formed. 2K3DXA was further converted to glycolaldehyde when genes encoding by either YagE or YjhH aldolase from E. coli were expressed. Reduction of glycolaldehyde to ethylene glycol by an endogenous aldo-keto reductase activity resulted further in accumulation of ethylene glycol of 14 mg/L. The possibility of simultaneous production of lactic and glycolic acids was evaluated by expression of gene encoding lactate dehydrogenase ldhL from Lactobacillus helveticus together with aldA. Interestingly, this increased the accumulation of glycolic acid to 1 g/L. The D-xylonate dehydratase activity in yeast was notably low, possibly due to inefficient Fe-S cluster synthesis in the yeast cytosol, and leading to D-xylonic acid accumulation. The dehydratase activity was significantly improved by targeting its expression to mitochondria or by altering the Fe-S cluster metabolism of the cells with FRA2 deletion.

Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis.

BACKGROUND: Glycolic acid is a C2 hydroxy acid that is a widely used chemical compound. It can be polymerised to produce biodegradable polymers with excellent gas barrier properties. Currently, glycolic acid is produced in a chemical process using fossil resources and toxic chemicals. Biotechnological production of glycolic acid using renewable resources is a desirable alternative. RESULTS: The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are suitable organisms for glycolic acid production since they are acid tolerant and can grow in the presence of up to 50 g l(-1) glycolic acid. We engineered S. cerevisiae and K. lactis for glycolic acid production using the reactions of the glyoxylate cycle to produce glyoxylic acid and then reducing it to glycolic acid. The expression of a high affinity glyoxylate reductase alone already led to glycolic acid production. The production was further improved by deleting genes encoding malate synthase and the cytosolic form of isocitrate dehydrogenase. The engineered S. cerevisiae strain produced up to about 1 g l(-1) of glycolic acid in a medium containing d-xylose and ethanol. Similar modifications in K. lactis resulted in a much higher glycolic acid titer. In a bioreactor cultivation with D-xylose and ethanol up to 15 g l(-1) of glycolic acid was obtained. CONCLUSIONS: This is the first demonstration of engineering yeast to produce glycolic acid. Prior to this work glycolic acid production through the glyoxylate cycle has only been reported in bacteria. The benefit of a yeast host is the possibility for glycolic acid production also at low pH, which was demonstrated in flask cultivations. Production of glycolic acid was first shown in S. cerevisiae. To test whether a Crabtree negative yeast would be better suited for glycolic acid production we engineered K. lactis in the same way and demonstrated it to be a better host for glycolic acid production.

Identification of the galactitol dehydrogenase, LadB, that is part of the oxido-reductive D-galactose catabolic pathway in Aspergillus niger.

For the catabolism of D-galactose three different metabolic pathways have been described in filamentous fungi. Apart from the Leloir pathway and the oxidative pathway, there is an alternative oxido-reductive pathway. This oxido-reductive pathway has similarities to the metabolic pathway of L-arabinose, and in Trichoderma reesei (Hypocrea jecorina) and Aspergillus nidulans the same enzyme is employed for the oxidation of L-arabitol and galactitol. Here we show evidence that in Aspergillus niger L-arabitol dehydrogenase (LadA) is not involved in the D-galactose metabolism; instead another dehydrogenase encoding gene, ladB, is induced in response to D-galactose and galactitol and functions as a galactitol dehydrogenase. Deletion of ladB in A. niger results in growth arrest on galactitol and significantly slower growth on D-galactose supplemented with a small amount of D-xylose. D-galactose alone cannot be utilised by A. niger and the addition of D-xylose stimulates growth on D-galactose via transcriptional activation of the D-xylose-inducible reductase gene, xyrA. XyrA catalyses the first step of the D-galactose oxido-reductive pathway, the reduction to galactitol, which in turn seems to be an inducer of the downstream genes such as LadB. The deletion of xyrA results in reduced growth on D-galactose. The ladB gene was expressed in the heterologous host Saccharomyces cerevisiae and the tagged and purified enzyme characterised. LadB and LadA have similar in vitro activity with galactitol. It was confirmed that the reaction product of the LadB reaction from galactitol is L-xylo-3-hexulose as in the case of the T. reesei Lad1.

Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene.

There are two distinctly different pathways for the catabolism of l-rhamnose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. The other pathway has no phosphorylated intermediates and has only been described in eukaryotic microorganisms. For this pathway, the enzyme activities have been described but not the corresponding gene sequences. The first enzyme in this catabolic pathway is the NAD-utilizing L-rhamnose 1-dehydrogenase. The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI-TOF MS. This enabled the identification of the corresponding gene, RHA1. It codes for a protein with 258 amino acids belonging to the protein family of short-chain alcohol dehydrogenases. The ORF was expressed in Saccharomyces cerevisiae. As the gene contained a CUG codon that codes for serine in P. stipitis but for leucine in S. cerevisiae, this codon has changed so that the same amino acid was expressed in S. cerevisiae. The heterologous protein showed the highest activity and affinity with L-rhamnose and a lower activity and affinity with L-mannose and L-lyxose. The enzyme was specific for NAD. A northern blot analysis revealed that transcription in P. stipitis is induced during growth on L-rhamnose but not on other carbon sources.

Sorbitol dehydrogenase of Aspergillus niger, SdhA, is part of the oxido-reductive D-galactose pathway and essential for D-sorbitol catabolism.

In filamentous fungi D-galactose can be catabolised through the oxido-reductive and/or the Leloir pathway. In the oxido-reductive pathway D-galactose is converted to d-fructose in a series of steps where the last step is the oxidation of d-sorbitol by an NAD-dependent dehydrogenase. We identified a sorbitol dehydrogenase gene, sdhA (JGI53356), in Aspergillus niger encoding a medium chain dehydrogenase which is involved in D-galactose and D-sorbitol catabolism. The gene is upregulated in the presence of D-galactose, galactitol and D-sorbitol. An sdhA deletion strain showed reduced growth on galactitol and growth on D-sorbitol was completely abolished. The purified enzyme converted D-sorbitol to D-fructose with K(m) of 50±5 mM and v(max) of 80±10 U/mg.

Characterisation of the gene cluster for l-rhamnose catabolism in the yeast Scheffersomyces (Pichia) stipitis.

In Scheffersomyces (Pichia) stipitis and related fungal species the genes for L-rhamnose catabolism RHA1, LRA2, LRA3 and LRA4 but not LADH are clustered. We find that located next to the cluster is a transcription factor, TRC1, which is conserved among related species. Our transcriptome analysis shows that all the catabolic genes and all genes of the cluster are up-regulated on L-rhamnose. Among genes that were also up-regulated on L-rhamnose were two transcription factors including the TRC1. In addition, in 16 out of the 32 analysed fungal species only RHA1, LRA2 and LRA3 are physically clustered. The clustering of RHA1, LRA3 and TRC1 is also conserved in species not closely related to S. stipitis. Since the LRA4 is often not part of the cluster and it has several paralogues in L-rhamnose utilising yeasts we analysed the function of one of the paralogues, LRA41 by heterologous expression and biochemical characterization. Lra41p has similar catalytic properties as the Lra4p but the transcript was not up-regulated on L-rhamnose. The RHA1, LRA2, LRA4 and LADH genes were previously characterised in S. stipitis. We expressed the L-rhamnonate dehydratase, Lra3p, in Saccharomyces cerevisiae, estimated the kinetic constants of the protein and showed that it indeed has activity with L-rhamnonate.

The 'true' L-xylulose reductase of filamentous fungi identified in Aspergillus niger.

L-Xylulose reductase is part of the eukaryotic pathway for l-arabinose catabolism. A previously identified L-xylulose reductase in Hypocrea jecorina turned out to be not the 'true' one since it was not upregulated during growth on L-arabinose and the deletion strain showed no reduced L-xylulose reductase activity but instead lost the D-mannitol dehydrogenase activity. In this communication we identified the 'TRUE'L-xylulose reductase in Aspergillus niger. The gene, lxrA (JGI177736), is upregulated on L-arabinose and the deletion results in a strain lacking the NADPH-specific L-xylulose reductase activity and having reduced growth on l-arabinose. The purified enzyme had a K(m) for L-xylulose of 25 mM and a nu(max) of 650 U/mg.