Excessive by-product formation: A key contributor to low isobutanol yields of engineered Saccharomyces cerevisiae strains.

It is theoretically possible to engineer Saccharomyces cerevisiae strains in which isobutanol is the predominant catabolic product and high-yielding isobutanol-producing strains are already reported by industry. Conversely, isobutanol yields of engineered S. cerevisiae strains reported in the scientific literature typically remain far below 10% of the theoretical maximum. This study explores possible reasons for these suboptimal yields by a mass-balancing approach. A cytosolically located, cofactor-balanced isobutanol pathway, consisting of a mosaic of bacterial enzymes whose in vivo functionality was confirmed by complementation of null mutations in branched-chain amino acid metabolism, was expressed in S. cerevisiae. Product formation by the engineered strain was analysed in shake flasks and bioreactors. In aerobic cultures, the pathway intermediate isobutyraldehyde was oxidized to isobutyrate rather than reduced to isobutanol. Moreover, significant concentrations of the pathway intermediates 2,3-dihydroxyisovalerate and α-ketoisovalerate, as well as diacetyl and acetoin, accumulated extracellularly. While the engineered strain could not grow anaerobically, micro-aerobic cultivation resulted in isobutanol formation at a yield of 0.018±0.003 mol/mol glucose. Simultaneously, 2,3-butanediol was produced at a yield of 0.649±0.067 mol/mol glucose. These results identify massive accumulation of pathway intermediates, as well as overflow metabolites derived from acetolactate, as an important, previously underestimated contributor to the suboptimal yields of 'academic' isobutanol strains. The observed patterns of by-product formation is consistent with the notion that in vivo activity of the iron-sulphur-cluster-requiring enzyme dihydroxyacid dehydratase is a key bottleneck in the present and previously described 'academic' isobutanol-producing yeast strains.

Comparative assessment of native and heterologous 2-oxo acid decarboxylases for application in isobutanol production by Saccharomyces cerevisiae.

BACKGROUND: Decarboxylation of α-ketoisovalerate to isobutyraldehyde is a key reaction in metabolic engineering of Saccharomyces cerevisiae for isobutanol production with published studies relying on overexpression of either the native ARO10 gene or of the Lactococcus lactis kivD decarboxylase gene resulting in low enzymatic activities. Here, we compare relevant properties for isobutanol production of Aro10, KivD and an additional, less studied, L. lactis decarboxylase KdcA. RESULTS: To eliminate interference by native decarboxylases, each 2-oxo acid decarboxylase was overexpressed in a 'decarboxylase-negative' (pdc1Δ pdc5Δ pdc6Δ aro10Δ) S. cerevisiae background. Kinetic analyses in cell extracts revealed a superior V max/K m ratio of KdcA for α-ketoisovalerate and a wide range of linear and branched-chain 2-oxo acids. However, KdcA also showed the highest activity with pyruvate which, in engineered strains, can contribute to formation of ethanol as a by-product. Removal of native decarboxylase genes eliminated growth on valine as sole nitrogen source and subsequent complementation of this growth impairment by expression of each decarboxylase indicated that based on the increased growth rate, the in vivo activity of KdcA with α-ketoisovalerate was higher than that of KivD and Aro10. Moreover, during oxygen-limited incubation in the presence of glucose, strains expressing kdcA or kivD showed a ca. twofold higher in vivo rate of conversion of α-ketoisovalerate into isobutanol than an ARO10-expressing strain. Finally, cell extracts from cultures grown on different nitrogen sources revealed increased activity of constitutively expressed KdcA after growth on both valine and phenylalanine, while KivD and Aro10 activity was only increased after growth on phenylalanine suggesting a difference in the regulation of these enzymes. CONCLUSIONS: This study illustrates important differences in substrate specificity, enzyme kinetics and functional expression between different decarboxylases in the context of isobutanol production and identifies KdcA as a promising alternative decarboxylase not only for isobutanol production but also for other branched-chain and linear alcohols.

Chromosomal Copy Number Variation in Saccharomyces pastorianus Is Evidence for Extensive Genome Dynamics in Industrial Lager Brewing Strains.

Lager brewing strains of Saccharomyces pastorianus are natural interspecific hybrids originating from the spontaneous hybridization of Saccharomyces cerevisiae and Saccharomyces eubayanus. Over the past 500 years, S. pastorianus has been domesticated to become one of the most important industrial microorganisms. Production of lager-type beers requires a set of essential phenotypes, including the ability to ferment maltose and maltotriose at low temperature, the production of flavors and aromas, and the ability to flocculate. Understanding of the molecular basis of complex brewing-related phenotypic traits is a prerequisite for rational strain improvement. While genome sequences have been reported, the variability and dynamics of S. pastorianus genomes have not been investigated in detail. Here, using deep sequencing and chromosome copy number analysis, we showed that S. pastorianus strain CBS1483 exhibited extensive aneuploidy. This was confirmed by quantitative PCR and by flow cytometry. As a direct consequence of this aneuploidy, a massive number of sequence variants was identified, leading to at least 1,800 additional protein variants in S. pastorianus CBS1483. Analysis of eight additional S. pastorianus strains revealed that the previously defined group I strains showed comparable karyotypes, while group II strains showed large interstrain karyotypic variability. Comparison of three strains with nearly identical genome sequences revealed substantial chromosome copy number variation, which may contribute to strain-specific phenotypic traits. The observed variability of lager yeast genomes demonstrates that systematic linking of genotype to phenotype requires a three-dimensional genome analysis encompassing physical chromosomal structures, the copy number of individual chromosomes or chromosomal regions, and the allelic variation of copies of individual genes.

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae.

An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme.

Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and (13)C-(15)N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic (13)C(15)N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.

Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the ß-lactam producer Penicillium chrysogenum.

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of ß-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA.

Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies.

The homologous recombination mechanism for DNA-repair is not predominant in most filamentous fungi, resulting in extremely low targeting efficiencies for molecular engineering. To increase the gene targeting efficiency, it is becoming common practice to inactivate the non-homologous end-joining (NHEJ) pathway that causes random integration, by deleting the fungal homologs of the human KU70 and KU80 genes that encode proteins functioning in the NHEJ pathway. This has been described for several filamentous fungi, but limited knowledge on the physiological consequences is available. In this study we characterized targeting efficiency and physiology of penicillinG producing Penicillium chrysogenum strains, in which the KU70 or KU80 homologues hdfA and hdfB had been deleted. Targeting efficiency was increased from ca. 1% in the reference strain to 47% and 56% in the hdfA and hdfB mutant strains, respectively, using an ends-out construct. Physiological and transcriptome analysis of glucose-limited chemostat cultures of the hdfA deletion strain and the reference strain showed minimal differences. Although, in a direct competition experiment to assess strain fitness, the reference strain had a clear advantage over the deletion strain, the results demonstrate the potential of DeltahdfAP. chrysogenum strains for the functional analysis of the recently completed P. chrysogenum genome sequence and in further metabolic engineering of antibiotics production.

Alleviation of feedback inhibition in Saccharomyces cerevisiae aromatic amino acid biosynthesis: quantification of metabolic impact.

A quantitative analysis of the impact of feedback inhibition on aromatic amino acid biosynthesis was performed in chemostat cultures of Saccharomyces cerevisiae. Introduction of a tyrosine-insensitive allele of ARO4 (encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase) caused a three-fold increase of intracellular phenylalanine and tyrosine concentrations. These amino acids were not detected extracellularly. However, an over 100-fold increase of the extracellular levels of phenylacetate, phenylethanol and their para-hydroxyl analogues was observed. The total increase of the flux through the aromatic pathway was estimated to be over four-fold. Individual overexpression of either the wild-type or feedback insensitive allele of ARO7 (encoding chorismate mutase had no significant impact. However when they were combined with the Tyr-insensitive ARO4 allele in combination with the Tyr-insensitive ARO4 allele, extracellular concentrations of aromatic compounds were increased by over 200-fold relative to the reference strain, corresponding to a 4.5-fold increase of the flux through the aromatic amino acid biosynthesis pathway. Elimination of allosteric control on these two key reactions in aromatic amino acid metabolism significantly affected intracellular concentrations of several non-aromatic amino acids. This broader impact of amino acid biosynthesis presents a challenge in rational optimization of the production of specific amino acids and derived flavour compounds.

When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation.

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average half-life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.

Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae.

Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGPI are not viable. Two strategies were developed to drastically reduce the intracellular concentration of UDP-Glc in order to study the consequences of this metabolic engineering on physiology and morphology. Firstly, UGP1 was placed under the strongly regulatable THI4 promoter. This resulted in a 95% reduction of UGPase activity in the presence of thiamine. The phenotypic effects of this reduction were slightly stronger than those of glucose on the GALI0/CYC1-UGP1 gene fusion [Daran et al. (1995) Eur. J. Biochem. 230, 520-530]. A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutase genes in the ugp1 conditional strain. The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell wall interfering drugs. Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and beta-glucans. Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears sufficient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the inability of yeast cells to properly form the cell wall.

Genetic and biochemical characterization of the UGP1 gene encoding the UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae.

We report here that the open reading frame YKL248, previously identified during the systematic sequencing of yeast chromosome XI [Purnelle B., Skala, J., Van Dijck, L. & Goffeau, A. (1992) Yeast 8, 977-986] encodes UDP-glucose pyrophosphorylase (UGPase), the enzyme which catalyses the reversible formation of UDP-Glc from glucose 1-phosphate and UTP. Proof for this function come from sequence alignment of the YKL248 product with UGPase of other species, from complementation studies of an Escherichia coli galU mutant deficient in UGPase activity, and from overexpression studies. In particular, the amino acid sequence motifs involved in the binding of glucose 1-phosphate and UDP-Glc are entirely conserved between the yeast, bovine, human and potato tuber UGPases, and multi-copy expression of YKL248 resulted in a 40-fold increase in UGPase activity. This gene was, therefore, renamed UGP1. Gene disruption at the UGP1 locus in a diploid strain, followed by tetrad analysis, showed that UGPase is essential for cell viability. Functional analysis of UGP1 was, therefore, carried out by generating strains in which UGPase could be either overexpressed or depleted. This was done by generating haploid strains carrying either UGP1 on a multicopy vector or the chromosomal deletion of UGP1, and rescued by a vector bearing the wild-type gene under the control of the glucose-repressible galactose-inducible promoter. The effects of overproducing UGPase on the cell metabolism and morphology were carbon-source dependent. On glucose medium, the 40-fold increase of UGPase activity was restricted to a twofold increase in the concentration of glycogen and UDP-Glc, with no significant effect on growth. In contrast, on galactose, the 40-fold increase in UGPase activity was accompanied by several effects, including a threefold reduction of the growth rate, a 3-5-fold increase in the concentrations of UDP-Glc, UDP-Gal and galactose 1-phosphate, a higher sensitivity to calcofluor white and an increase in the degree of protein glycosylation. Depletion of UGPase activity was performed by transferring the mutant strains from galactose to glucose medium. Unexpectedly, growth of these mutants on glucose was as efficient as that of the control, although the mutants contained only 5-10% wild-type UGPase activity, and a growth defect could never been obtained, even after serial transfers of the mutants to a 10% glucose medium. However, the 10-fold reduction of UGPase activity induced a multi-budding pattern, a higher resistance to zymolyase, a slight increase in the calcofluor sensitivity and a decrease in the cell-wall beta-glucan content. All these alterations, induced by manipulating the UGP1 gene, are discussed in the context of the strategic position of UDP-Glc in yeast metabolism.