High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Engineering the oleaginous yeast Yarrowia lipolytica for β-farnesene overproduction

β-farnesene is a sesquiterpenoid with various industrial applications which is now commercially produced by a Saccharomyces cerevisiae strain obtained by random mutagenesis and genetic engineering. We rationally designed a genetically defined Yarrowia lipolytica through recovery of L-leucine biosynthetic route, gene dosage optimization of β-farnesene synthase and disruption of the competition pathway. The resulting β-farnesene titer was improved from 8 to 345 mg L^-1. Finally, the strategy for decreasing the lipid accumulation by individually and iteratively knocking out four acyltransferases encoding genes was adopted. The result displayed that β-farnesene titer in the engineered strain CIBT6304 in which acyltransferases (DGA1 and DGA2) were deleted increased by 45% and reached 539 mg L^-1 (88 mg g^-1 DCW). Using fed-batch fermentation, CIBT6304 could produce the highest β-farnesene titer (22.8 g L^-1) among the genetically defined strains. This study will provide the foundation of engineering Y. lipolytica to produce other terpenoids more cost-efficiently.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016     

Production of α-linolenic acid in Yarrowia lipolytica using low-temperature fermentation

α-Linolenic acid (ALA) is an essential ω-3 fatty with reported health benefits. However, this molecule is naturally found in plants such as flaxseed and canola which currently limits production. Here, we demonstrate the potential to sustainably produce ALA using the oleaginous yeast Yarrowia lipolytica. Through the use of a recently identified Δ12–15 desaturase (Rk Δ12–15), we were able to enable production in Y. lipolytica. When combined with a previously engineered lipid-overproducing strain with high precursor availability, further improvements of ALA production were achieved. Finally, the cultivation of this strain at lower temperatures significantly increased ALA content, with cells fermented at 20 °C accumulating nearly 30% ALA of the total lipids in this cell. This low-temperature fermentation represents improved ALA titer up to 3.2-fold compared to standard growth conditions. Scale-up into a fed-batch bioreactor produced ALA at 1.4 g/L, representing the highest published titer of this ω-3 fatty acid in a yeast host.

     

Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Herein, we report the development of a microbial bioprocess for high‐level production of 5‐aminolevulinic acid (5‐ALA), a valuable non‐proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5‐ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5‐ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl‐CoA for enhanced 5‐ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high‐level 5‐ALA biosynthesis from glycerol (~30 g L^?1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L^?1 (36.9% of the theoretical maximum yield) and 6.93 g L^?1 (50.9% of the theoretical maximum yield) 5‐ALA, respectively. This study represents one of the most effective bio‐based production of 5‐ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio‐based production.     

Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)₃ as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L⁻¹). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)₃ as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L⁻¹. Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L⁻¹ (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L⁻¹ for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

     

Engineering of Klebsiella oxytoca for production of 2,3‐butanediol using mixed sugars derived from lignocellulosic hydrolysates

2,3‐Butanediol (2,3‐BDO) is a promising bulk chemical owing to its high potential in industrial applications. Here, we engineered Klebsiella oxytoca for the economic production of 2,3‐BDO using mixed sugars from renewable biomass. First, to improve xylose consumption, the xylose transporter gene (xylE) was integrated into the methylglyoxal synthase A (mgsA)‐coding gene loci, and the engineered CHA004 strain showed much faster consumption of xylose than wild‐type (WT) strain with 1.4‐fold increase of overall sugar consumption rate. To further improve sugar utilization, we performed adaptive laboratory evolution for 90 days. The evolved strain (CHA006) was evaluated by cultivating it in the media containing single‐ or mixed‐sugars, and it was clearly observed that CHA006 has improved sugar consumption and 2,3‐BDO production than those of the parental strain. Finally, we demonstrated the superiority of CHA006 by culturing in two lignocellulosic hydrolysates derived from sunflower or pine tree. Particularly, in the pine tree hydrolysate containing xylose, glucose, galactose, and mannose, the CHA006 strain showed much improved consumption rates for all sugars, and 2,3‐BDO productivity (0.73 g L^?1 hr^?1) increased by 3.2‐fold compared to WT strain. We believe that the engineered CHA006 strain can be a potential host in the development of economic bioprocess for 2,3‐BDO through efficient utilization of mixed sugars derived from lignocellulosic biomass.     

Conversion of glucose‐xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose‐xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed‐batch process, both sugars in a glucose‐xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g.     

Engineering industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for xylose fermentation and comparison for switchgrass conversion

Saccharomyces’ physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04–0.17 h⁻¹. Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17–0.31 g/l/h, with ethanol yields 0.18–0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13–17% more ethanol than the parental control strains because of their ability to ferment xylose.     

Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of L-lactic acid

Conversion of lignocellulose to lactic acid requires strains capable of fermenting sugar mixtures of glucose and xylose. Recombinant Escherichia coli strains were engineered to selectively produce L-lactic acid and then used to ferment sugar mixtures. Three of these strains were catabolite repression mutants (ptsG ⁻) that have the ability to simultaneously ferment glucose and xylose. The best results were obtained for ptsG ⁻ strain FBR19. FBR19 cultures had a yield of 0.77 (g lactic acid/g added sugar) when used to ferment a 100 g/l total equal mixture of glucose and xylose. The strain also consumed 75% of the xylose. In comparison, the ptsG ⁺ strains had yields of 0.47–0.48 g/g and consumed 18–22% of the xylose. FBR19 was subsequently used to ferment a variety of glucose (0–40 g/l) and xylose (40 g/l) mixtures. The lactic acid yields ranged from 0.74 to 1.00 g/g. Further experiments were conducted to discover the mechanism leading to the poor yields for ptsG ⁺ strains. Xylose isomerase (XI) activity, a marker for induction of xylose metabolism, was monitored for FBR19 and a ptsG ⁺ control during fermentations of a sugar mixture. Crude protein extracts prepared from FBR19 had 10–12 times the specific XI activity of comparable samples from ptsG ⁺ strains. Therefore, higher expression of xylose metabolic genes in the ptsG ⁻ strain may be responsible for superior conversion of xylose to product compared to the ptsG ⁺ fermentations. Received 14 December 2000/ Accepted in revised form 28 June 2002     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

Metabolic engineering of Yarrowia lipolytica to produce chemicals and fuels from xylose

Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content.Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials.     

Production of enriched in B vitamins biomass of Yarrowia lipolytica grown in biofuel waste

Yarrowia lipolytica as an oleaginous yeast is capable of growing in various non-conventional hydrophobic substrate types, especially industrial wastes. In this study, the content of thiamine (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), biotin (vitamin B7) and folic acid (vitamin B9) in the wet biomass of Y. lipolytica strains cultivated in biofuel waste (SK medium), compared to the standard laboratory YPD medium, was assessed. Additionally, the biomass of Y. lipolytica A-101 grown in biofuel waste (SK medium) was dried and examined for B vitamins concentration according to the recommended microbial methods by AOAC Official Methods. The mean values of these vitamins per 100 g of dry weight of Y. lipolytica grown in biofuel waste (SK medium) were as follows: thiamine 1.3 mg/100 g, riboflavin 5.3 mg/100 g, pyridoxine 4.9 mg/100 g, biotin 20.0 µg/100 g, and folic acid 249 µg/100 g. We have demonstrated that the dried biomass is a good source of B vitamins which can be used as nutraceuticals to supplement human diet, especially for people at risk of B vitamin deficiencies in developed countries. Moreover, the biodegradation of biofuel waste by Y. lipolytica is desired for environmental protection.     

Hexokinase—A limiting factor in lipid production from fructose in Yarrowia lipolytica

Microbial biolipid production has become an important part of making biofuel production economically feasible. Genetic engineering has been used to improve the ability of Yarrowia lipolytica, an oleaginous yeast, to produce lipids using glucose-based media. However, few studies have examined lipid accumulation by Y. lipolytica׳s ability to utilize other hexose sugars, and as of yet, the rate-limiting steps in this process are unidentified. In this study, we investigated the de novo accumulation of lipids by Y. lipolytica when grown in glucose, fructose, and sucrose. Three Y. lipolytica wild-type (WT) strains of varied origin differed significantly in their lipid production, growth, and fructose utilization. Hexokinase (ylHXK1p) activity partially explained these differences. Overexpression of the ylHXK1 gene led to increased hexokinase activity (6.5–12 times higher) in the mutants versus the WT strains; a pronounced reduction in cell filamentation in mutants grown in fructose-based media; and improved biomass production, particularly in the mutant whose parent had shown the lowest growth capacity in fructose (French strain W29). All mutants showed improved lipid yield and production when grown on fructose, although the effect was strain dependent (23–55% improvement). Finally, we overexpressed ylHXK1 in a highly modified strain of Y. lipolytica W29 engineered to optimize oil production. This modification was combined with Saccharomyces cerevisiae invertase gene expression to evaluate the resulting mutant׳s ability to produce lipids using cheap industrial substrates, namely sucrose (a major component of molasses). Sucrose turned out to be a better substrate than either of its building blocks, glucose or fructose. Over its 96 h of growth in the bioreactors, this highly modified strain produced 9.15 g L⁻¹ of lipids, yielding 0.262 g g⁻¹ of biomass.     

Metabolic engineering of Escherichia coli for agmatine production

Agmatine is a kind of important biogenic amine. The chemical synthesis route is not a desirable choice for industrial production of agmatine. To date, there are no reports on the fermentative production of agmatine by microorganism. In this study, the base Escherichia coli strain AUX4 (JM109 ?speC ?speF ?speB ?argR) capable of excreting agmatine into the culture medium was first constructed by sequential deletions of the speC and speF genes encoding the ornithine decarboxylase isoenzymes, the speB gene encoding agmatine ureohydrolase and the regulation gene argR responsible for the negative control of the arg regulon. The speA gene encoding arginine decarboxylase harboured by the pKK223‐3 plasmid was overexpressed in AUX4, resulting in the engineered strain AUX5. The batch and fed‐batch fermentations of the AUX5 strain were conducted in a 3‐L bioreactor, and the results showed that the AUX5 strain was able to produce 1.13 g agmatine L^?1 with the yield of 0.11 g agmatine g^?1 glucose in the batch fermentation and the fed‐batch fermentation of AUX5 allowed the production of 15.32 g agmatine L^?1 with the productivity of 0.48 g agmatine L^?1 h^?1, demonstrating the potential of E. coli as an industrial producer of agmatine.     

Yarrowia lipolytica chassis strains engineered to produce aromatic amino acids via the shikimate pathway

Yarrowia lipolytica is widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast’s potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination of Y. lipolytica and heterologous feedback-insensitive enzyme variants. Our best chassis strain displayed high levels of de novo Ehrlich metabolite production (up to 0.14 g l⁻¹ in minimal growth medium), which represented a 93-fold increase compared to the wild-type strain (0.0015 g l⁻¹). Production was further boosted to 0.48 g l⁻¹ when glycerol, a low-cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l⁻¹). Among these metabolites, 2-phenylethanol is of particular interest due to its rose-like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan. We have thus demonstrated that Y. lipolytica can serve as a platform for the sustainable de novo bio-production of high-value aromatic compounds, and we have greatly improved our understanding of the potential feedback-based regulation of the shikimate pathway in this yeast.     

Comparison of citric acid production from glycerol and glucose by different strains of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

A wild type strain A-101 of Y. lipolytica and its three acetate-negative mutants (Wratislavia 1.31, Wratislavia AWG7, and Wratislavia K1) were compared for the production of citric acid from glucose and glycerol (pure and crude) in batch cultures. The substrates were used either as single carbon sources or as mixtures of glucose and pure or crude glycerol. The kinetic parameters, i.e., the volumetric citric acid production rate and yield obtained in the study show that the Wratislavia 1.31 and Wratislavia AWG7 strains produced the highest amount of citric acid from glycerol, with a yield from 0.40 to 0.53 g g⁻¹. This substrate was found to be a better carbon source for the biosynthesis of citric acid than glucose. The results obtained with the same strains have shown low content of isocitric acid and polyols, such as erythritol and mannitol. Y. lipolytica A-101 strain produced the highest amount of isocitric acid, from 13.8 to 21% isocitric acid in the sum of citric acids. However, the highest concentrations of erythritol were found in cultures with Y. lipolytica Wratislavia K1, from 18.1 to 30 g l⁻¹, for glucose and pure glycerol, respectively.     

Study of the persistence and dynamics of recombinant mCherry-producing Yarrowia lipolytica strains in the mouse intestine using fluorescence imaging

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.     

Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica

Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low‐pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis‐based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO₃. Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L^?1 in shaking flasks with buffering and more than 17 g L^?1 without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed. Biotechnol. Bioeng. 2010;107:673–682. © 2010 Wiley Periodicals, Inc.