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The enzymatic production of formate from CO2 with the immobilized NADH-dependent formate dehydrogenase (FDH) on the activated electrospun polystyrene nanofibers (EPSNF) was investigated to develop a sustainable process for CO2 reduction. Direct electrochemical regeneration on a Cu foam electrode was employed to supply the reaction with the reduced cofactor (NADH). The formate production was studied in two modes of batch and semi-continuous operations with the cofactor recycle. Results indicated that the regenerated cofactor concentrations in both systems were nearly identical (0.5 mM) which ensured the desirable activity of the immobilized enzyme. This showed that the electrochemical regeneration system was effective even in the semi-continuous operation. Although the cumulative formate concentration in the batch operation was higher, the total amounts of the produced formate were higher for the semi-continuous mode for more than 42% which was justified by the fact that the lower formate concentration in the semi-continuous mode would be favorable to the progress of the enzymatic CO2 to formate conversion. Finally, it was concluded that the proposed semi-continuous process in this work could be considered as a promising process for the enzymatic CO2 conversion.

Pretreatment is the key factor for producing fermentable sugars and biofuels from lignocellulosic biomass. However, during pretreatment lignin repolymerization occurs which reduces the efficiency of carbohydrates hydrolysis. In the present study, three additives (2-naphthol-7-sulfonate, 2-naphthol, and mannitol) were evaluated for boosting the effectiveness of combined pretreatment (liquid hot water + green liquor) of pine-wood biomass. The results revealed that supplied additives into combined pretreatment changed the physicochemical structure of lignin, therefore, alleviating the inhibitory effect of lignin repolymerization and notably raised sugars yield (glucose, xylose) ranging from 85% to 93%. XPS and FTIR analyses confirmed that −OH groups of additives led to lower formation of phenolic hydroxyl (PhOH) and higher hydrophilic groups of lignin, accounting for reduced non-specific binding of lignin into cellulase enzymes. Furthermore, additives increased surface lignin coverage, biomass porosity, and fiber swelling by 1.96–2.59, 1.66–1.86, and 1.13–1.29 folds respectively, comparing to corresponding sample without additives caused more liquified and extractable lignin, which notably reduced the surface lignin barrier. Hence, both factors including non-specific binding effect and surface lignin barrier, contributed to reducing lignin repolymerization, thereby increased cellulose accessibility for high production of fermentable sugars and bioethanol in softwood biomass.

Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2.

Sweet sorghum plant, a widely grown energy crop, was utilized through a biorefinery process, by which its grains were hydrolyzed by the crude amylases produced from its bagasse. The hemicellulosic part of the bagasse was hydrolyzed with 0.5–1.0% sulfuric acid at 140–180 °C for 30–60 min and applied for amylase production using halotolerant bacterium Nesterenkonia sp. strain F. In the hydrolysate obtained at 140 °C for 60 min using 1% acid, Nesterenkonia showed 73.3 U/mL amylase activity by the consumption of 16.2 g/L xylose and 8.3 g/L other sugars. Supplementation of the hydrolysates with sorghum grain resulted in 38–67% higher amylase production. Furthermore, addition of biocompatible surfactants of Tween 20 and Tween 80 (0.1 g/L) increased the activity to 93 and 97 U/mL, respectively. The resulting crude enzyme was used in the process of ethanol production from both tannin-containing and tannin-free sorghum grains (6%), leading to 17.7 and 17.0 g/L bioethanol production, respectively. Through the cultivation of Nesterenkonia on the hemicellulosic hydrolysates, 5–10 g/L volatile fatty acids (VFA), 0.36–0.69 g/L acetone-butanol-ethanol (ABE), and 468–721 mg/L single cell protein (SCP) were also produced. The obtained SCP contained most of the essential amino acids and relatively high amounts of phenylalanine (8%), threonine (7%), methionine (6%), and lysine (6%).

Industrial scale production of biobutanol has been hampered by substrate cost and availability. Sweet sorghum grain is an inexpensive substrate for acetone-butanol-ethanol (ABE) production by Clostridium acetobutylicum. Amylolytic activity of C. acetobutylicum eliminates the need for the hydrolysis of starchy grain prior to fermentation. However, untreated grain contains phenolic compounds, i.e. tannins, which exhibit inhibitory effects against amylolytic activity and ABE fermentation. Less than 3 g/L ABE was obtained from untreated sweet sorghum grain at different substrate concentrations. Concentration of 0.2 mM gallic acid equivalent (GAE) of sorghum tannins was detected as the critical concentration which inhibits severely ABE fermentation. Applying a multi-stage hot water treatment resulted in tannins removal and significant enhancement in total ABE production up to 18 g/L. For efficient butanol production from 40, 60, and 80 g/L sorghum grain, hot water treatment with two, five, and six stages were found to be essential for efficient butanol production, respectively. Moreover, the amylolytic activity of C. acetobutylicum was inhibited by sorghum grain tannins, more than twice as high as the effects on the ABE fermentation pathway. Furthermore, unlike most substrates, sweet sorghum grain could provide all nutrients required for ABE fermentation, eliminating the need for supplementing expensive additional nutrients.

The yeast cells were coated with Fe3O4 magnetic nanoparticles and employed as biocatalyst for the microbial biotransformation of benzaldehyde into l-phenylacetylcarbinol (l-PAC). Saccharomyces cerevisiae CEN.PK113-7D yeast cells were coated with magnetic nanoparticles to facilitate the cells separation process. Transmission electron microscopy, powder XRD diffraction, and vibrating sample magnetometer were used to characterize magnetic nanoparticles and magnetic nanoparticle-coated yeast cells. Then the reusability of magnetically recoverable cells in production of l-PAC was investigated. Results show that coating yeast cells with magnetic nanoparticles does not affect their size and structure. Coated cells were also used in seven consecutive batch cycles and no significant reduction for l-PAC titer was observed in any of the cycles. Coating yeast cells with magnetic nanoparticles enabled rapid separation and reuse of cells in several successive batch cycle without affecting their ability to produce l-PAC.

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone-butanol-ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful "coculture" for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.

L-Phenylacetylcarbinol (L-PAC) which is used as a precursor for the production of ephedrine and pseudoephedrine is the first reported biologically produced α-hydroxy ketone compound. l-PAC is commercially produced by the yeast Saccharomyces cerevisiae. Yeast cells transform exogenously added benzaldehyde into l-PAC by using the action of pyruvate decarboxylase (PDC) enzyme. In this work, genome-scale model and flux balance analysis were used to identify novel target genes for the enhancement of l-PAC production in yeast. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene and minimization of metabolic adjustments. Six single gene deletion strains, namely Δrpe1, Δpda1, Δadh3, Δadh1, Δzwf1 and Δpdc1, were predicted in silico and further tested in vivo by using knock-out strains cultivated semi-anaerobically on glucose and benzaldehyde as substrates. Δzwf1 mutant exhibited the highest l-PAC formation (2.48 g/L) by using 2 g/L of benzaldehyde which is equivalent to 88 % of the theoretical yield.

The γ-decalactone is one of the lactones with peachy aroma which has been approved as food additive by FDA. The aim of this study was to optimize media composition and conditions for microbial biotransformation of ricinoleic acid and castor oil as substrates to γ-decalactone using the yeast Yarrowia lipolytica. The Y. lipolytica DSM 3286 strain was used as biotransformation agent in different trails designed by Taguchi method. The highest concentration of γ-decalactone was 62.4 and 52.9 mg/L from 1.5% ricinoleic acid and 2.5% castor oil, respectively. Nitrogen sources exhibited significant effect on the biotransformation. The maximum γ-decalactone production occurred at pH 6. The results showed that the composition of biotransformation medium composition is important for γ-decalactone production.

The yeast Saccharomyces cerevisiae is able to biotransform benzaldehyde into L-phenylacetylcarbinol (L-PAC), a key intermediate in the production of ephedrine and pseudoephedrine, by the action of pyruvate decarobxylase (PDC) enzyme. This biotransformation can alternatively be performed by acetohydroxyacid synthase (AHAS) which is a mitochondrial enzyme. In the yeast petite mutants, AHAS accumulates in the cytosol. In the current study, wild-type yeast cells and yeast petite mutants were examined for L-PAC biosynthesis. The results showed higher L-PAC titers in the yeast petite mutants. In addition, the effect of cell immobilization and carbon source (glucose or molasses) on L-PAC production was investigated. It was found that cell immobilization enhances L-PAC formation. The highest L-PAC concentration (2.4 g/l) was obtained at 2 g/l of benzaldehyde using the immobilized petite mutants grown on molasses.

Substrate cost and availability are the major bottlenecks that limit the commercialization of next generation biofuels. The possibility of using waste acorn as an abundant and inexpensive feedstock in Iran for acetone–butanol–ethanol (ABE) production with Clostridium acetobutylicum was investigated. No solvent was produced using untreated acorn due to its high contents of growth-inhibitory tannic acid. To circumvent this problem, a simple extraction in close-to-boiling water was employed to remove tannic acid prior to using acorn powder in fermentation. This pretreatment significantly improved the fermentability of acorn powder by C. acetobutylicum. The maximum yields of solvents (g ABE per g substrate) achieved in serum-bottle experiments were 0.43 ± 0.02, 0.40 ± 0.02, and 0.34 ± 0.02 for glucose, pure starch, and treated acorn starch, respectively. Volumetric ABE productivity with tannin-free acorn powder was 0.21 ± 0.01 g L−1 h−1, slightly higher than that achieved with pure starch (0.18 ± 0.05 g L−1 h−1). When scaled up in 5 L fermentors, ABE production using pretreated acorn powder and pure starch resulted in solvent profiles and yields similar to those observed in the serum bottle experiments. This study demonstrates that acorn powder can be a viable feedstock for biobutanol production in Iran. Future work warrants optimization of a two-step process including extraction of tannic acid as a valuable by-product followed by fermentation of tannin-free acorn powder to improve the economic feasibility of acorn to biobutanol conversion process.

The microbial pathway of butanol biosynthesis is a unique route for the production of a biomass-derived advanced biofuel with high potential to be utilized in place of the fossil fuels. In the present study, Nesterenkonia sp. strain F was applied as a beneficial partner for Clostridium acetobutylicum in “starch-to-butanol process”. The capability of Nesterenkonia sp. strain F in secreting organic solvent-tolerant amylase was utilized for upgrading the yield of solvent, i.e., acetone, butanol, and ethanol (ABE), production under aerobic conditions. Monitoring the amylolytic activity and glucose concentration throughout the micro-aerobic co-culture revealed higher amylase activity and glucose concentration in comparison with the monoculture. The co-culture led to 63% higher amylase activity through the microaerobic cultivation on starch. After optimizing the conditions with response surface methodology (RSM), 10.6 g/L butanol was produced from untreated potato starch (UPS) with a high yield of 0.23 g ABE/g starch, leading to 30% improvement in ABE production. To assess its performance at larger scale, the fermentation was conducted in a 5 L fermenter continually aerated with a rate of 0.05 vvm and 150 rpm. This led to production of 9.7 g/L butanol, 5.0 g/L acetone, and 0.3 g/L ethanol with a yield and productivity of 0.20 g/g and 0.21 g/L.h, respectively. Furthermore, ABE production from tannin-containing sorghum grain was improved by about 3-fold.

Hemicellulose is a cheap and abundant substrate for biofuel production. However, industrial scale production of biofuels from hemicellulose is relatively inefficient because of expensive pretreatment processes and poor pentoses utilization by most microorganisms. In this study, a cost-effective autohydrolysis process using water as the only reagent to hydrolyze lignocellulose was exploited. Sweet sorghum stalk was also utilized as an economic source of hemicellulose. The autohydrolyzed lignocellulosic compounds from sweet sorghum were used as substrate in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum which is able to ferment pentoses and hexoses into ABE. Separation of 79 % of hemicellulose and roughly 20 % of cellulose from sweet sorghum stalk was detected as a desirable result for autohydrolysis at 210 °C; however, over-production of inhibitors made it an inappropriate pretreatment for ABE fermentation. No butanol production was detected in autohydrolysates of 210 °C, even after using detoxification methods for inhibitory compounds removal. On the other hand, only 20 % of sweet sorghum’s bagasse hemicellulose was separated at 150 °C; yet, it was detected as the most desirable hydrolysate for ABE fermentation. Nevertheless, inherent inefficiency of C. acetobutylicum to ferment xylo-oligomers (as the sole carbon source) led to less than 1 g/L of ABE production in autohydrolysates at 150 °C. Co-fermentation of these hydrolysates with sorghum grain starch was investigated as a solution and it significantly increased the ABE production up to 8.3 g/L. Furthermore, the synergistic effect of co-fermentation was investigated where 35 % improvement in ABE production was detected. Accordingly, xylose utilization increased from 45 % to 80 %.

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

Cheese whey lactose was used as a carbon source for xanthan gum production with Xanthomonas campestris and Xanthomonas pelargonii. Proteins were precipitated and removed from whey prior to fermentation. Box-Behnken response surface methodology was used for optimization of the carbon, magnesium, and phosphate source concentrations in the culture medium to maximize xanthan gum production. After 48 h of fermentation using X. campestris, the highest xanthan concentration (16.4 g/L) was achieved at 65.2 g/L of cheese whey (39.1 g/L of lactose), 14.8 g/L of phosphate (K H2PO4), and 1.1 g/L of magnesium (MgSO4·7H2O). The corresponding optimum cheese whey, phosphate, and magnesium concentrations in cultures of X. pelargonii were 80.0, 6.7, and 0.8 g/L, respectively, which resulted in a xanthan production of 12.8 g/L. The xanthan gum yield (g of xanthan/g of lactose) was 0.42 for X. campestris and 0.27 for X. pelargonii.

Production of acetone–butanol–ethanol (ABE) from potato waste starch using free and immobilized cells of Clostridium acetobutylicum was investigated. The starch was directly fermented to ABE by C. acetobutylicum without hydrolysis, and the results were compared with those obtained from glucose as a reference. Maximum butanol yields from waste starch and glucose were 0.21 and 0.26 g/g of initial carbon source (20 g/L), respectively. Batch fermentation of 60 g/L of waste starch in a 5 L bioreactor with free cells resulted in production of 9.9 g/L butanol. In a repeated batch fermentation, using immobilized cells of the bacterium in calcium alginate–polyvinyl alcohol (PVA)–boric acid beads and 60 g/L of the waste starch, a final butanol concentration of 15.3 g/L was achieved.

γ-Decalactone is an industrially important flavor compound with a peachy aroma which has been approved by FDA as a food additive. The aim of this study was to compare batch and fed-batch cultivation for production of γ-decalactone using Yarrowia lipolytica and castor oil as substrate. Microbial production of γ-decalactone from castor oil using the obligate aerobic yeast Y. lipolytica was investigated in a 3 l bioreactor. Batch and fed-batch fermentations were compared for the production of γ-decalactone. Also the effect of enhancing oxygen transfer rate by using higher agitation rates or pure oxygen for aeration was investigated. The highest γ-decalactone concentration (220 mg/l) was obtained in the fed-batch fermentation using pure oxygen which was 3-fold more compared to the batch cultivation. Using pure oxygen instead of atmospheric air in the fed-batch fermentation also resulted in 60% increase in γ-decalactone production.

A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using Opt Gene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase en coded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain. (C) 2009 Elsevier Inc. All rights reserved.

Production of xanthan gum using immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii grown on glucose or hydrolyzed starch as carbon sources was investigated. Calcium alginate (CA) and calcium alginate-polyvinyl alcohol-boric acid (CA-PVA) beads were used for the immobilization of cells. Xanthan titers of 8.2 and 9.2g/L were obtained for X. campestris cells immobilized in CA-PVA beads using glucose and hydrolyzed starch, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively. Immobilized cells in CA-PVA beads were successfully employed in three consecutive cycles for xanthan production without any noticeable degradation of the beads whereas the CA beads were broken after the first cycle. The results of this study suggested that immobilized cells are advantageous over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells immobilized in CA beads for xanthan production.

Metallothioneins (MTs) are a family of Cys-rich, low molecular weight, cytoplasmic metal binding proteins. MTs are present in all eukaryotes as well as some prokaryotes. Plant MTs are divided into four types based on Cys distribution pattern in their amino acid sequences. In the present work, the gene encoding OsMTI-2b, a type 2 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into Escherichia coli strain Rosetta (DE3). Following the induction with Isopropyl β-d-1-thiogalactopyranoside the OsMTI-2b was expressed as carboxyl-terminal extensions of glutathione-S-transferase (GST-tag), a 6His-tag, and an S-tag. The expressed recombinant fusion protein was named GST-OsMTI-2b. As compared with control, transgenic E. coli cells expressing GST-OsMTI-2b accumulated more Pb(2+), Ni(2+), Cd(2+), Zn(2+) and Cu(2+) from culture medium and showed increased tolerance against these metals. Furthermore the E. coli cells expressing OsMTI-2b accumulated significantly higher Pb(2+) than previously made strains which expressing other rice OsMT isoforms. The recombinant GST-OsMTI-2b was purified using affinity chromatography. According to in vitro assays the protein GST-OsMTI-2b was able to form complexes with Pb(2+), Ni(2+), Cd(2+) and Zn(2+). However, the binding ability for the different metals differed in the order: Pb(2+)>Cd(2+)>Zn(2+)>Ni(2+).

Isoprenoids represent a wide group of chemically active compounds that can find a wide range of applications as flavors, perfumes, vitamins, nutraceuticals, and pharmaceuticals. Many isoprenoids are naturally produced in very low quantities by plants, which make their use in broader perspectives difficult. Microbial production of plant-originating isoprenoids quickly appeared as an alternative of choice. Metabolic engineering methods were applied and proved successful to improve the supply of precursors to derive toward isoprenoid compounds of interest. Combinations between metabolic engineering and the flourishing field of synthetic biology have also been observed with researchers attempting to reconstruct and optimize complex biosynthetic pathways in well-characterized and tractable microbial hosts. In this chapter, we review recent metabolic engineering studies for isoprenoid production in yeast and try to show, through key examples, that the fields of synthetic biology and metabolic engineering can go hand in hand to establish microbial cell factories for isoprenoid production.

Background The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. Results In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. Conclusions With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk.

The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Over-expression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous over-expression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids harboring cubebol synthase gene. This could be explained by a toxicity effect of cubebol, possibly resulting in higher transcription levels for the genes under control of MET3 promoter, which could lead to accumulation of squalene and ergosterol.

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.

A eukaryotic mevalonate pathway transferred and expressed in Escherichia coli, and a mammalian hydrocortisone biosynthetic pathway rebuilt in Saccharomyces cerevisiae are examples showing that transferring metabolic pathways from one organism to another can have a powerful impact on cell properties. In this study, we reconstructed the E. coli isoprenoid biosynthetic pathway in S. cerevisiae. Genes encoding the seven enzymatic steps of the pathway were cloned and expressed in S. cerevisiae. mRNA from the seven genes was detected, and the pathway was shown able to sustain growth of yeast in conditions of inhibition of its constitutive isoprenoid biosynthetic pathway.