Yeast metabolic engineering for hemicellulosic ethanol production.

Efficient fermentation of hemicellulosic sugars is critical for the bioconversion of lignocellulosics to ethanol. Efficient sugar uptake through the heterologous expression of yeast and fungal xylose/glucose transporters can improve fermentation if other metabolic steps are not rate limiting. Rectification of cofactor imbalances through heterologous expression of fungal xylose isomerase or modification of cofactor requirements in the yeast oxidoreductase pathway can reduce xylitol production while increasing ethanol yields, but these changes often occur at the expense of xylose utilization rates. Genetic engineering and evolutionary adaptation to increase glycolytic flux coupled with transcriptomic and proteomic studies have identified targets for further modification, as have genomic and metabolic engineering studies in native xylose fermenting yeasts.

Metabolic engineering for improved fermentation of pentoses by yeasts.

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g(-1) sugar consumed, so commercialization seems feasible for some applications.

Bacteria engineered for fuel ethanol production: current status.

The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.

Ethanol and thermotolerance in the bioconversion of xylose by yeasts.

The mechanisms underlying ethanol and heat tolerance are complex. Many different genes are involved, and the exact basis is not fully understood. The integrity of cytoplasmic and mitochondrial membranes is critical to maintain proton gradients for metabolic energy and nutrient uptake. Heat and ethanol stress adversely affect membrane integrity. These factors are particularly detrimental to xylose-fermenting yeasts because they require oxygen for biosynthesis of essential cell membrane and nucleic acid constituents, and they depend on respiration for the generation of ATP. Physiological responses to ethanol and heat shock have been studied most extensively in S. cerevisiae. However, comparative biochemical studies with other organisms suggest that similar mechanisms will be important in xylose-fermenting yeasts. The composition of a cell's membrane lipids shifts with temperature, ethanol concentration, and stage of cultivation. Levels of unsaturated fatty acids and ergosterol increase in response to temperature and ethanol stress. Inositol is involved in phospholipid biosynthesis, and it can increase ethanol tolerance when provided as a supplement. Membrane integrity determines the cell's ability to maintain proton gradients for nutrient uptake. Plasma membrane ATPase generates the proton gradient, and the biochemical characteristics of this enzyme contribute to ethanol tolerance. Organisms with higher ethanol tolerance have ATPase activities with low pH optima and high affinity for ATP. Likewise, organisms with ATPase activities that resist ethanol inhibition also function better at high ethanol concentrations. ATPase consumes a significant fraction of the total cellular ATP, and under stress conditions when membrane gradients are compromised the activity of ATPase is regulated. In xylose-fermenting yeasts, the carbon source used for growth affects both ATPase activity and ethanol tolerance. Cells can adapt to heat and ethanol stress by synthesizing trehalose and heat-shock proteins, which stabilize and repair denatured proteins. The capacity of cells to produce trehalose and induce HSPs correlate with their thermotolerance. Both heat and ethanol increase the frequency of petite mutations and kill cells. This might be attributable to membrane effects, but it could also arise from oxidative damage. Cytoplasmic and mitochondrial superoxide dismutases can destroy oxidative radicals and thereby maintain cell viability. Improved knowledge of the mechanisms underlying ethanol and thermotolerance in S. cerevisiae should enable the genetic engineering of these traits in xylose-fermenting yeasts.

Genetic engineering for improved xylose fermentation by yeasts.

Xylose utilization is essential for the efficient conversion of lignocellulosic materials to fuels and chemicals. A few yeasts are known to ferment xylose directly to ethanol. However, the rates and yields need to be improved for commercialization. Xylose utilization is repressed by glucose which is usually present in lignocellulosic hydrolysates, so glucose regulation should be altered in order to maximize xylose conversion. Xylose utilization also requires low amounts of oxygen for optimal production. Respiration can reduce ethanol yields, so the role of oxygen must be better understood and respiration must be reduced in order to improve ethanol production. This paper reviews the central pathways for glucose and xylose metabolism, the principal respiratory pathways, the factors determining partitioning of pyruvate between respiration and fermentation, the known genetic mechanisms for glucose and oxygen regulation, and progress to date in improving xylose fermentations by yeasts.

Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production.

The xylose-utilizing yeast, Pichia stipitis, has a complex respiratory system that contains cytochrome and non-cytochrome alternative electron transport chains in its mitochondria. To gain primary insights into the alternative respiratory pathway, a cytochrome c gene (PsCYC1, Accession No. AF030426) was cloned from wild-type P. stipitis CBS 6054 by cross-hybridization to CYC1 from Saccharomyces cerevisiae. The 333 bp open reading frame of PsCYC1 showed 74% and 69% identity to ScCYC1 and ScCYC7, respectively, at the DNA level. Disruption of PsCYC1 resulted in a mutant that uses the salicylhydroxamic acid (SHAM)-sensitive respiratory pathway for aerobic energy production. Cytochrome spectra revealed that cytochromes c and a.a(3) both disappeared in the cyc1-Delta mutant, so no electron flow through the cytochrome c oxidase was possible. The cyc1-Delta mutant showed 50% lower growth rates than the parent when grown on fermentable sugars. The cyc1-Delta mutant was also found to be unable to grow on glycerol. Interestingly, the mutant produced 0.46 g/g ethanol from 8% xylose, which was 21% higher in yield than the parental strain (0.38 g/g). These results suggested that the alternative pathway might play an important role in supporting xylose conversion to ethanol under oxygen-limiting conditions.

Bioconversion of secondary fiber fines to ethanol using counter-current enzymatic saccharification and co-fermentation.

This research examined several enzymatic and microbial process for the conversion of waste cellulosic fibers into ethanol. The first was a one-stage process in which pulp fines were contacted with commercial enzyme solutions. The second process used sequential, multistage saccharification. The third used sequential enzyme addition in a countercurrent mode. Experiments compared the results with various feedstocks, different commercial enzymes, supplementation with beta-glucosidase, and saccharification combined with fermentation. The highest saccharification (65%) from a 4% consistency pulp and the highest sugar concentration (5.4%) from an 8% consistency pulp were attained when 5 FPU/g plus 10 IU/g of beta-glucosidase were used. Sequential addition of enzyme to the pulp in small aliquots produced a higher overall sugar yield/U enzyme than the addition of the same total amount of enzyme in a single dose. In the saccharification and fermentation experiments, we produced 2.12% ethanol from a 5.4% sugar solution. This represents 78% of the theoretical maximum. This yield could probably be increased through optimization of the fermentation step. Even when little saccharification occurred, the enzyme facilitated separation of water, fiber, and ash, so cellulase treatment could be an effective means for dewatering pulp sludges.

Transcriptional control of ADH genes in the xylose-fermenting yeast Pichia stipitis.

We studied the expression of the genes encoding group I alcohol dehydrogenases (PsADH1 and PsADH2) in the xylose-fermenting yeast Pichia stipitis CBS 6054. The cells expressed PsADH1 approximately 10 times higher under oxygen-limited conditions than under fully aerobic conditions when cultivated on xylose. Transcripts of PsADH2 were not detectable under either aeration condition. We used a PsADH1::lacZ fusion to monitor PsADH1 expression and found that expression increased as oxygen decreased. The level of PsADH1 transcript was repressed about 10-fold in cells grown in the presence of heme under oxygen-limited conditions. Concomitantly with the induction of PsADH1, PsCYC1 expression was repressed. These results indicate that oxygen availability regulates PsADH1 expression and that regulation may be mediated by heme. The regulation of PsADH2 expression was also examined in other genetic backgrounds. Disruption of PsADH1 dramatically increased PsADH2 expression on nonfermentable carbon sources under fully aerobic conditions, indicating that the expression of PsADH2 is subject to feedback regulation under these conditions.

Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae.

Respiratory and fermentative pathways coexist to support growth and product formation in Pichia stipitis. This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions. Expression of Saccharomyces cerevisiae URA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present. ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth. Initial P. stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose. Cells produced even more ethanol faster following two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism. This is the first report of genetic engineering to enable anaerobic growth of a eukaryote.

A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris.

In methylotrophic yeasts, glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required for the metabolism of methanol as a carbon source and certain alkylated amines such as methylamine as nitrogen sources. We describe the isolation and characterization of the FLD1 gene from the yeast Pichia pastoris. The gene contains a single short intron with typical yeast-splicing signals near its 5' end, the first intron to be demonstrated in this yeast. The predicted FLD1 product (Fld1p) is a protein of 379 amino acids (approx. 40 kDa) with 71% identity to the FLD protein sequence from the n-alkane-assimilating yeast Candida maltosa and 61-65% identity with dehydrogenase class III enzymes from humans and other higher eukaryotes. Using beta-lactamase as a reporter, we show that the FLD1 promoter (PFLD1) is strongly and independently induced by either methanol as sole carbon source (with ammonium sulfate as nitrogen source) or methylamine as sole nitrogen source (with glucose as carbon source). Furthermore, with either methanol or methylamine induction, levels of beta-lactamase produced under control of PFLD1 are comparable to those obtained with the commonly used alcohol oxidase I gene promoter (PAOX1). Thus, PFLD1 is an attractive alternative to PAOX1 for expression of foreign genes in P. pastoris, allowing the investigator a choice of carbon (methanol) or nitrogen source (methylamine) regulation with the same expression strain.

Pichia stipitis genes for alcohol dehydrogenase with fermentative and respiratory functions.

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designated PsADH1 and PsADH2, have been identified and isolated from Pichia stipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been characterized through disruption. The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74.7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lactis. The PsADH isozymes are presumably localized in the cytoplasm, as they do not possess the amino-terminal extension of mitochondrion-targeted ADHs. Gene disruption studies suggest that PsADH1 plays a major role in xylose fermentation because PsADH1 disruption results in a lower growth rate and profoundly greater accumulation of xylitol. Disruption of PsADH2 does not significantly affect ethanol production or aerobic growth on ethanol as long as PsADH1 is present. The PsADH1 and PsADH2 isozymes appear to be equivalent in the ability to convert ethanol to acetaldehyde, and either is sufficient to allow cell growth on ethanol. However, disruption of both genes blocks growth on ethanol. P. stipitis strains disrupted in either PsADH1 or PsADH2 still accumulate ethanol, although in different amounts, when grown on xylose under oxygen-limited conditions. The PsADH double disruptant, which is unable to grow on ethanol, still produces ethanol from xylose at about 13% of the rate seen in the parental strain. Thus, deletion of both PsADH1 and PsADH2 blocks ethanol respiration but not production, implying a separate path for fermentation.

Cloning and disruption of the beta-isopropylmalate dehydrogenase gene (LEU2) of Pichia stipitis with URA3 and recovery of the double auxotroph.

Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5'-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ "pop-out" cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu- Ura+ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5'-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu- Ura- phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.

Cloning and characterization of two pyruvate decarboxylase genes from Pichia stipitis CBS 6054.

In Pichia stipitis, fermentative and pyruvate decarboxylase (PDC) activities increase with diminished oxygen rather than in response to fermentable sugars. To better characterize PDC expression and regulation, two genes for PDC (PsPDC1 and PsPDC2) were cloned and sequenced from P. stipitis CBS 6054. Aside from Saccharomyces cerevisiae, from which three PDC genes have been characterized, P. stipitis is the only organism from which multiple genes for PDC have been identified and characterized. PsPDC1 and PsPDC2 have diverged almost as far from one another as they have from the next most closely related known yeast gene. PsPDC1 contains an open reading frame of 1,791 nucleotides encoding 597 amino acids. PsPDC2 contains a reading frame of 1,710 nucleotides encoding 570 amino acids. An 81-nucleotide segment in the middle of the beta domain of PsPDC1 codes for a unique segment of 27 amino acids, which may play a role in allosteric regulation. The 5' regions of both P. stipitis genes include two putative TATA elements that make them similar to the PDC genes from S. cerevisiae, Kluyveromyces marxianus, and Hanseniaspora uvarum.

Comparative study of xylanase kinetics using dinitrosalicylic, arsenomolybdate, and ion chromatographic assays.

Xylanases are commonly assayed by the dinitrosalicylic acid (DNS) or the arsenomolybdate (ARS) method. However, specific activities are many times higher with DNS than with ARS. This is because the DNS assay is more reactive and the ARS assay is less reactive with xylooligosaccharides than with xylose. Xylose is often used as a standard, even though oligosaccharides are prevalent, so the DNS method overestimates and the ARS method underestimates specific activity. Ion chromatography, with pulsed amperometric detection, separates and measures all products and intermediates, but quantitation on a molar basis is difficult, because few xylooligosaccharide response factors are known. This report directly compares these three assay methods for the assay of xylanase activities.

Regulation of phosphotransferases in glucose- and xylose-fermenting yeasts.

This research examined the titers of hexokinase (HK), phosphofructokinase (PFK), and xylulokinase (XUK) in Saccharomyces cerevisiae and two xylose fermenting yeasts, Pachysolen tannophilus and Candida shehatae, following shifts in carbon source and aeration. Xylose-grown C. shehatae, glucose-grown P. tannophilus, and glucose-grown S. cerevisiae, had the highest specific activities of XUK, HK, and PFK, respectively. XUK was induced by xylose to moderate levels in both P. tannophilus and C. shehatae, but was present only in trace levels in S. cerevisiae. HK activities in P. tannophilus were two to three fold higher when cells were grown on glucose than when grown on xylose, but HK levels were less inducible in C. shehatae. The PFK activities in S. cerevisiae were 1.5 to 2 times higher than in the two xylose-fermenting yeasts. Transfer from glucose to xylose rapidly inactivated HK in P. tannophilus, and transfer from xylose to glucose inactivated XUK in C. shehatae. The patterns of induction and inactivation indicate that the basic regulatory mechanisms differ in the two xylose fermenting yeasts.

Diminished respirative growth and enhanced assimilative sugar uptake result in higher specific fermentation rates by the mutant Pichia stipitis FPL-061.

A mutant strain of Pichia stipitis, FPL-061, was obtained by selecting for growth on L-xylose in the presence of respiratory inhibitors. The specific fermentation rate of FPL-061, was higher than that of the parent,Pichia stipitis CBS 6054, because of its lower cell yield and growth rate and higher specific substrate uptake rate. With a mixture of glucose and xylose, the mutant strain FPL-061 produced 29.4 g ethanol/L with a yield of 0.42 g ethanol/g sugar consumed. By comparison, CBS 6054 produced 25.7 g ethanol/L with a yield of 0.35 gJg. The fermentation was most efficient at an aeration rate of 9.2 mmoles O2 L-1 h-1. At high aeration rates (22 mmoles O2 L-1 h-1), the mutant cell yield was less than that of the parent. At low aeration rates, (1.1 to 2.5 O2 L-1 h-1), cell yields were similar, the ethanol formation rates were low, and xylitol accumulation was observed in both the strains. Both strains respired the ethanol once sugar was exhausted. We infer from the results that the mutant, P. stipitis FPL-061, diverts a larger fraction of its metabolic energy from cell growth into ethanol production.

Biochemistry and genetics of microbial xylanases.

Xylanases are classified into two major families (10 or F and 11 or G) of glycosyl hydrolases. Both use ion pair catalytic mechanisms and both retain anomeric configuration following hydrolysis. Family 10 xylanases are larger, more complex and produce smaller oligosaccharides; Family 11 xylanases are more specific for xylan. Alkaline-active and extreme-thermophilic enzymes are of particular interest. Such xylanases are being commercialized for bleaching pulps and other applications.

Increased xylose reductase activity in the xylose-fermenting yeast Pichia stipitis by overexpression of XYL1.

The Pichia stipitis xylose reductase gene (XYL1) was inserted into an autonomous plasmid that P. stipitis maintains in multicopy. The plasmid pXOR with the XYL1 insert or a control plasmid pJM6 without XYL1 was introduced into P. stipitis. When grown on xylose under aerobic conditions, the strain with pXOR had up to 1.8-fold higher xylose reductase (XOR) activity than the control strain. Oxygen limitation led to higher XOR activity in both experimental and control strains grown on xylose. However, the XOR activities of the two strains grown on xylose were similar under oxygen limitation. When grown on glucose under aerobic or oxygen-limited conditions, the experimental strain had XOR activity up to 10 times higher than that of the control strain. Ethanol production was not improved, but rather it decreased with the introduction of pXOR compared to the control, and this was attributed to nonspecific effects of the plasmid.

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.

Purification, Characterization, and Substrate Specificities of Multiple Xylanases from Streptomyces sp. Strain B-12-2.

The endoxylanase complex from Streptomyces sp. strain B-12-2 was purified and characterized. The organism forms five distinct xylanases in the absence of significant cellulase activity when grown on oat spelt xylan. This is the largest number of endoxylanases yet reported for a streptomycete. On the basis of their physiochemical characteristics, they can be divided into two groups: the first group (xyl 1a and xyl 1b) consists of low-molecular-mass (26.4 and 23.8 kDa, respectively) neutral- to high-pI (6.5 and 8.3, respectively) endoxylanases. Group 1 endoxylanases are unable to hydrolyze aryl-beta-d-cellobioside, have low levels of activity against xylotetraose (X(4)) and limited activity against xylopentaose, produce little or no xylose, and form products having a higher degree of polymerization with complex substrates. These enzymes apparently carry out transglycosylation. The second group (xyl 2, xyl 3, and xyl 4) consists of high-molecular-mass (36.2, 36.2, and 40.5 kDa, respectively), low-pI (5.4, 5.0, and 4.8, respectively) xylanases. Group 2 endoxylanases are able to hydrolyze aryl-beta-d-cellobioside, show higher levels of activity against X(4), and hydrolyze xylopentaose completely with the formation of xylobiose and xylotriose plus limited amounts of X(4) and xylose. The enzymes display intergroup synergism when acting on kraft pulp. Despite intragroup similarities, each enzyme exhibited a unique action pattern and physiochemical characteristic. xyl 2 was highly glycosylated, and xyl 1b (but no other enzyme) was completely inhibited by p-hydroxymercuribenzoate.