Metabolic engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for linalool production

Objectives

To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.

Results

Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l^?1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l^?1.

Conclusions

Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

     

Enhanced production of 2,3-butanediol by engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> through fine-tuning of pyruvate decarboxylase and NADH oxidase activities

Background

2,3-Butanediol (2,3-BD) is a promising compound for various applications in chemical, cosmetic, and agricultural industries. Pyruvate decarboxylase (Pdc)-deficient Saccharomyces cerevisiae is an attractive host strain for producing 2,3-BD because a large amount of pyruvate could be shunted to 2,3-BD production instead of ethanol synthesis. However, 2,3-BD yield, productivity, and titer by engineered yeast were inferior to native bacterial producers because of the following metabolic limitations. First, the Pdc-deficient yeast showed growth defect due to a shortage of C₂-compounds. Second, redox imbalance during the 2,3-BD production led to glycerol formation that lowered the yield.

Results

To overcome these problems, the expression levels of Pdc from a Crabtree-negative yeast were optimized in S. cerevisiae. Specifically, Candida tropicalis PDC1 (CtPDC1) was used to minimize the production of ethanol but maximize cell growth and 2,3-BD productivity. As a result, productivity of the BD5_G1CtPDC1 strain expressing an optimal level of Pdc was 2.3 folds higher than that of the control strain in flask cultivation. Through a fed-batch fermentation, 121.8 g/L 2,3-BD was produced in 80 h. NADH oxidase from Lactococcus lactis (noxE) was additionally expressed in the engineered yeast with an optimal activity of Pdc. The fed-batch fermentation with the optimized 2-stage aeration control led to production of 154.3 g/L 2,3-BD in 78 h. The overall yield of 2,3-BD was 0.404 g 2,3-BD/g glucose which corresponds to 80.7% of theoretical yield.

Conclusions

A massive metabolic shift in the engineered S. cerevisiae (BD5_G1CtPDC1_nox) expressing NADH oxidase was observed, suggesting that redox imbalance was a major bottleneck for efficient production of 2,3-BD by engineered yeast. Maximum 2,3-BD titer in this study was close to the highest among the reported microbial production studies. The results demonstrate that resolving both C₂-compound limitation and redox imbalance is critical to increase 2,3-BD production in the Pdc-deficient S. cerevisiae. Our strategy to express fine-tuned PDC and noxE could be applicable not only to 2,3-BD production, but also other chemical production systems using Pdc-deficient S. cerevisiae.

     

Engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase,xylosidase and a bacterial xylose isomerase

Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes.     

Rationally designed perturbation factor drives evolution in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for industrial application

Saccharomyces cerevisiae strains with favorable characteristics are preferred for application in industries. However, the current ability to reprogram a yeast cell on the genome scale is limited due to the complexity of yeast ploids. In this study, a method named genome replication engineering-assisted continuous evolution (GREACE) was proved efficient in engineering S. cerevisiae with different ploids. Through iterative cycles of culture coupled with selection, GREACE could continuously improve the target traits of yeast by accumulating beneficial genetic modification in genome. The application of GREACE greatly improved the tolerance of yeast against acetic acid compared with their parent strain. This method could also be employed to improve yeast aroma profile and the phenotype could be stably inherited to the offspring. Therefore, GREACE method was efficient in S. cerevisiae engineering and it could be further used to evolve yeast with other specific characteristics.     

Increasing n-butanol production with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by optimizing acetyl-CoA synthesis,NADH levels and trans-2-enoyl-CoA reductase expression

Background

n-Butanol can serve as an excellent gasoline substitute. Naturally, it is produced by some Clostridia species which, however, exhibit only limited suitability for industrial n-butanol production. The yeast Saccharomyces cerevisiae would be an ideal host due to its high robustness in fermentation processes. Nevertheless, n-butanol yields and titers obtained so far with genetically engineered yeast strains are only low.

Results

In our recent work, we showed that n-butanol production via a clostridial acetoacetyl-CoA-derived pathway in engineered yeast was limited by the availability of coenzyme A (CoA) and cytosolic acetyl-CoA. Increasing their levels resulted in a strain producing up to 130 mg/L n-butanol under anaerobic conditions. Here, we show that under aerobic conditions. this strain can even produce up to 235 mg/L n-butanol probably due to a more efficient NADH re-oxidation. Nevertheless, expression of a bacterial water-forming NADH oxidase (nox) significantly reduced n-butanol production although it showed a positive effect on growth and glucose consumption. Screening for an improved version of an acetyl-CoA forming NAD⁺-dependent acetylating acetaldehyde dehydrogenase, adhE^{A267T/E568K/R577S}, and its integration into n-butanol-producing strain further improved n-butanol production. Moreover, deletion of the competing NADP⁺-dependent acetaldehyde dehydrogenase Ald6 had a superior effect on n-butanol formation. To increase the endogenous supply of CoA, amine oxidase Fms1 was overexpressed together with pantothenate kinase coaA from Escherichia coli, and could completely compensate the beneficial effect on n-butanol synthesis of addition of pantothenate to the medium. By overexpression of each of the enzymes of n-butanol pathway in the n-butanol-producing yeast strain, it turned out that trans-2-enoyl-CoA reductase (ter) was limiting n-butanol production. Additional overexpression of ter finally resulted in a yeast strain producing n-butanol up to a titer of 0.86 g/L and a yield of 0.071 g/g glucose.

Conclusions

By further optimizing substrate supply and redox power in the form of coenzyme A, acetyl-CoA and NADH, n-butanol production with engineered yeast cells could be improved to levels never reached before with S. cerevisiae via an acetoacetyl-CoA-derived pathway in synthetic medium. Moreover, our results indicate that the NAD⁺/NADH redox balance and the trans-2-enoyl-CoA reductase reaction seem to be bottlenecks for n-butanol production with yeast.

     

Improving isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with over-expressing NADPH-specific glucose-6-phosphate dehydrogenase (Zwf1)

Isobutanol is a more promising biofuel than ethanol due to its higher energy density and lower hygroscopicity. Saccharomyces cerevisiae, as a model eukaryote, has the potential advantage to produce isobutanol because of its greater tolerance to higher alcohols. NADPH is a key cofactor for isobutanol synthesis, and glucose-6-phosphate dehydrogenase (Zwf1) is one of the main NADPH-supplying sources in S. cerevisiae. In this study, we investigated the effects of over-expressing ZWF1 on isobutanol titers. Our results showed that engineered strain HZAL-7023 produced 6.22 mg isobutanol per g glucose, which increased by 6.64-fold compared with the parent strain, while engineered strain HZAL-7023 22-ZWF1 produced 11.46 mg isobutanol per g glucose, which increased by 1.82-fold compared with engineered strain HZAL-7023. These results suggested that improvement of NADPH supply through over-expressing ZWF1 contributed to isobutanol biosynthesis in S. cerevisiae. These results also verified the proposed concept of increasing isobutanol titers in S. cerevisiae by resolving cofactor imbalance. Finally, this study provides a new strategy for enhancing isobutanol biosynthesis.     

Heterologous expression of mitochondrial nicotinamide adenine dinucleotide transporter (Ndt1) from <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> rescues impaired growth in <Emphasis Type="Italic">Δndt1Δndt2 Saccharomyces cerevisiae</Emphasis> strain

Our understanding of nicotinamide adenine dinucleotide mitochondrial transporter 1 (Ndt1A) in Aspergillus fumigatus remains poor. Thus, we investigated whether Ndt1A could alter fungi survival. To this end, we engineered the expression of an Ndt1A-encoding region in a Δndt1Δndt2 yeast strain. The resulting cloned Ndt1A protein promoted the mitochondrial uptake of nicotinamide adenine dinucleotide (NAD⁺), generating a large mitochondrial membrane potential. The NAD⁺ carrier utilized the electrochemical proton gradient to drive NAD⁺ entrance into mitochondria when the mitochondrial membrane potential was sustained by succinate. Its uptake has no impact on oxidative stress, and Ndt1A expression improved growth and survival of the Δndt1Δndt2 Saccharomyces cerevisiae strain.     

Metabolic engineering for high glycerol production by the anaerobic cultures of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.     

Overexpression of <Emphasis Type="Italic">ARO10</Emphasis> in <Emphasis Type="Italic">pdc5</Emphasis>Δmutant resulted in higher isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To investigate effects of different pyruvate decarboxylases on isobutanol titers in Saccharomyces cerevisiae, single-gene deletion of the three PDCs genes encoding pyruvate decarboxylases were constructed in this study. In addition, we over-expressed Ilv2, which catalyzed the first step in the valine synthetic pathway, and Bat2, which was the cytoplasmic branched-chain amino-acid aminotransferase that catalyzed L-valine to 2-ketoisovalerate, to increase isobutanol production in the genetically modified strains. Our results showed that knockout of PDC5 were one of the main factors among the three PDC genes for improving isobutanol titers in S. cerevisiae. Additionally, we found that deletion of PDC5 in strain carrying overexpressed ILV2 and ARO10 resulted in 8-fold higher isobutanol productivity as compared to the control strain in micro-aerobic fermentations. Our results also suggested that engineered strain pdc5ΔpILV2 pARO10 generated lower ethanol titers and higher acetate acid titers than the control strain, while the growth rate and glucose consumption rate of engineered strain pdc5ΔpILV2 pARO10 were slightly lower than that of the control strain. Meanwhile, the biomass concentration of pdc5ΔpILV2 pARO10 decreased dramatically than that of the control strain.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Engineering of an endogenous hexose transporter into a specific D-xylose transporter facilitates glucose-xylose co-consumption in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background

Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized.

Results

We have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose.

Conclusions

Engineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.

     

<Superscript>13</Superscript>C metabolite profiling to compare the central metabolic flux in two yeast strains

¹³C metabolite profiling to quantify the dynamic changes of central carbon metabolites was attempted using mass isotopomer distribution analysis in two yeast strains, Saccharomyces cerevisiae and Kluyveromyces marxianus. Mass and isotopomer balances of the intermediates were examined and calculated in both yeast species and central carbon metabolic fluxes were successfully determined. Metabolic fluxes of pentose phosphate pathway in K. marxianus were 1.66 times higher than S. cerevisiae. The flux difference was also supported by relatively high abundance of partially labeled fructose 6-phosphate and 3-phosphoglycerate as well as an increased concentration of labeled L-valine in K. marxianus. Metabolic flux analysis combined with dynamic metabolite profiling has provided better understanding in the central carbon metabolic pathways of two model organisms and can be applied as a method to analyze more complicated metabolic networks in other organisms.     

Cloning and Functional Analysis of <Emphasis Type="Italic">SaCLCc1</Emphasis>, a Gene Belonging to the Chloride Channel Family (<Emphasis Type="Italic">CLC</Emphasis>), from the Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe²⁺-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn²⁺. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl^– ions.     

Cloning,deletion, and overexpression of a glucose oxidase gene in <Emphasis Type="Italic">Aureobasidium</Emphasis> sp. P6 for Ca<Superscript>2+</Superscript>-gluconic acid overproduction

This study aimed to overexpress a glucose oxidase gene (GOD1) in Aureobasidium sp. P6 to achieve Ca²⁺-gluconic acid (GA) overproduction. The GOD1 gene was cloned, deleted, and overexpressed. A protein deduced from the GOD1 gene of Aureobasidium sp. P6 strain had 1824 bp that encoded a protein with 606 amino acids, with a conserved NADB-ROSSMAN domain and a GMC-oxred domain. Deleting the GOD1 gene made the disruptant GOK1 completely lose the ability to produce GA and GOD1 activity, whereas overexpressing the GOD1 gene rendered the transformant GOEX8 to produce considerably more Ca²⁺-GA (160.5?±?5.6 g/L) and higher GOD1 activity (1438.6?±?73.2 U/mg of protein) than its parent P6 strain (118.7?±?4.3 g/L of Ca²⁺-GA and 1100.0?±?23.6 U/mg of GOD1 protein). During a 10-L fermentation, the transformant GOEX8 grown in the medium containing 160.0 g/L of glucose produced 186.8?±?6.0 g/L of Ca²⁺-GA, the yield was 1.2 g/g of glucose, and the volumetric productivity was 1.7 g/L/h. Most of the produced GOD1 were located in the yeast cell wall. The purified product was identified to be a GA. The transformant GOEX8 overexpressing the GOD1 gene could produce considerably more Ca²⁺-GA (186.8?±?6.0 g/L) than its wild-type strain P6.     

Electron Microscopy of Cells of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> infected with Double Stranded RNA Viruses from <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium stoloniferum</Emphasis>

LHOAS¹ has demonstrated the infection of mating pairs of Saccharomyces cerevisiae with double stranded RNA viruses from Aspergillus niger and Penicillium stoloniferum (preceding communication). I wish to give details of the appearance of the virus particles within the infected yeast cells as determined by electron microscopy.     

Rapid and stable production of 2,3-butanediol by an engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain in a continuous airlift bioreactor

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.     

Evolution of copper arsenate resistance for enhanced enargite bioleaching using the extreme thermoacidophile <Emphasis Type="Italic">Metallosphaera sedula</Emphasis>

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu²⁺ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.