ONE EXAMPLE OF THE VALUE OF GENOMIC INFORMATION FROM NEUROSPORA
Contributed by Robert L. Metzenberg, Stanford University
When wood is made into paper, the useful portion (cellulose) must be separated from two other major high-molecular weight constituents: hemicellulose, and lignin. The removal of these, whether by acid treatment and sulfiting, or biopulping, or a combination, create major economic and ecological problems. Disposal of (for instance) acid-solublized hemicellulose liquors in wastewater can add massively to the biological oxygen demand and turn forest streams into foul-smelling watercourses devoid of fish and of recreational value.
While considerable activity has centered around the removal of lignin, especially involving the basidiomycete Phanerochaete, rather less attention seems to have been paid to hemicellulose though it makes up variously 1/4 to 1/3 of the mass of woodpulp, sugarcane bagasse, corn stover, etc. However, there are some dozens of small studies. Deshpande et al. (1986) reported the direct conversion of hemicellulose and cellulose to ethanol by Neurospora, and Phadtare et al. (1997) speak of Neurospora's"unique ability to directly convert biomass to ethanol".
We believe hemicellulose has been slighted for at least two reasons. First, microbiologists typically think first of yeasts and bacteria rather than of filamentous fungi, and yeasts and bacteria generally handle this class of compounds poorly. Second, the literature suggests to us that the enzyme kinetics applicable to D-xylose utilization are often poorly understood by workers in this field, with the result that data acquired under slightly different conditions in different laboratories are not easily compared. Furthermore, because of this fragility of understanding, the possible strategies for strain improvement, where they have been used at all, have been far from optimal.
It is necessary first to consider the fungal pathway for bringing D-xylose, the main sugar of hemicellulose, into the mainstream of metabolism.
1. b-D-xylanase depolymerizes hemicellulose to give b-D-xylofuranose (a hemiacetal, not an aldehyde).
2. b-D-xylofuranose spontaneously comes into equilibrium with a-D-xylofuranose and the two D-xylopyranoses, the intermediate being the D-xylose aldehyde. The key point here is that spontaneous mutarotation is relatively slow, with a first-order k-value of about 0.094/min at biological temperatures (Bailey et al., 1969), and the equilibrium concentration of D-xylose aldehyde, the substrate for the next reaction, is only of the order of 0.1% of the total xylose.
3. The D-xylose aldehyde is acted upon in a typical reduction by NADPH to give D-xylitol.
The D-xylitol is reoxidized by NAD+ to give D-xylulose.
4. D-xylulose is phosphorylated by ATP to give D-xylulose-5-P.
5. D-xylulose-5-P goes through pentose phosphate shunt, into glycolysis, and to ethanol and CO2.
Reactions 2 and 3, taken together, are the steps that results in erroneous (and highly variable) literature values of the kinetic constants Vmax and Km. One learns in beginning biochemistry that these measurements are only valid if the fraction of the substrate disappearing during the reaction is small compared to the total amount present. On the surface, this might seem to be the case with D-xylose reduction because the total amount of D-xylose, in all its forms, is barely lowered during the course of a typical measurement. This disguises the fact that, unless only a very small amount of enzyme is present, Reaction 3 will outstrip (the spontaneous) Reaction 2, and the real substrate of Reaction 3 quickly drops to a new steady state that may be much lower, even, than the equilibrium concentration. This means that, for example, doubling the amount of enzyme may produce little increase in reaction rate, so that Vmax values are likely to be meaningless. Likewise, the Km can only be understood in terms of the form that the enzyme "sees" -- D-xylose aldehyde -- so that even if Reaction 3 is conducted extremely slowly, the "real" Km is about 1000 times lower than the apparent Km. This, in itself, merely distorts the scale of the measurements, not the relative rates. But if Reaction 3 is run at a substantial rate, not a snailŐs pace, this thousandfold overestimation of the Km becomes much greater still because the aldehyde is much depleted compared to the anomeric forms. Under these conditions, doubling the amount of total D-xylose can be expected to double the amount of D-xylose aldehyde, so that the Km will appear to be much greater than a thousand times the "real" Km. All of this is only to say that fungi, and more to the point, Neurospora, can degrade hemicelluloses to useful products, but the existing data need to be greatly refined.
The remedy for making meaningful analyses, and also probably for bioengineering an organism to convert hemicelluloses efficiently to ethanol, is to greatly accelerate Reaction 2 with the enzyme mutarotase rather than relying on spontaneous conversion. Mutarotase was actually first reported in Penicillium notatum and it is probably present at some level in all fungi. D-galactose mutarotase is necessary for any appreciable rate of growth of E. coli on galactose, and this enzyme has been cloned and carefully studied (Beebe and Frey, 1998). It is reported to act quite generally on both hexoses and pentoses, including D-xylose and D-arabinose, constituents of hemicellulose. The aldehydes have been shown to be intermediates in the mutarotation.
We wish to argue that Neurospora is a most favorable organism for the purposes outlined above. We suggest that it could be genetically modified into a valuable organism for the removal of hemicellulose from wood during paper production, and also for the conversion of biomass to ethanol. This engineering could only be done optimally if the DNA sequence of the organism were known. The reasons are as follows:
1. For removing hemicellulose without also attacking cellulose, one would want to knock out the gene for cellulase, and obviously this would require knowledge of the gene and its flanking sequence. We say "the gene" instead of "genes" because in Neurospora this is at least a reasonable hope. Neurospora seems to stand alone among fungi in typically not having duplicated genes, probably because of the phenomenon of RIP during the early stages of the sexual cycle. This would "strangle such duplicate genes in their cribs", and that is the probable reason for their scarcity. (There do appear to be two D-xylose reductases in Neurospora, however -- Zhao, Gao, and Wang, 1998)
2. The enzymes of D-xylose metabolism would need to be cloned, and the rate-limiting ones put under the control of more active promoters. Clearly this is best done in a sexual organism that is amenable to recombining various manipulated genes into a single excellently-performing strain rather than being stuck with dead-end asexual strains. Optimizing an organism that cannot be easily modified further by recombining desirable traits is likely to be very cumbersome.
3. Clearly one would want to know if Neurospora already contains a mutarotase, and if so, one would want to get high-expression mutants of it. The importance of a genome sequence is obvious. In addition, one might wish to clone in one or more copies of the E. coli mutarotase, or the comparable enzymes that are active in Penicillium or in bovine kidney cortex, with suitable regulatory regions, into Neurospora. Again, a sequence would be most valuable.
4. One of the many problems with current technology for removal and degradation of hemicelluloses to ethanol is that furfural, a by-product of the acid extraction of hemicelluloses, is toxic and inhibits the bioprocessing of these wastes (Jeffries, 1983). But it should be possible to select a mutant that is resistant to this compound. Furfural is a quite specific chemical activator or pheromone in Neurospora, causing germination of its very dormant ascospores, so in a sense, it is already not "foreign". This is hardly surprising when we recall that the niche of Neurospora is one of very rapid growth on heat-killed plant material after brush fires.
5. If a filamentous fungus is to be used in as massive an enterprise as biopulping or ethanol production, there is sure to be heavy exposure of thousands of workers and their families to the organism chosen for the work. Neurospora is peculiarly non-pathogenic, perhaps because it normally has the luxury of growing in a fire-scoured landscape, without competitors. To our knowledge, no infection or intoxication by Neurospora has ever been reported in human beings, including immune compromised people, nor in any live animal or plant. The extraordinary safety of this organism alone would be enough to commend it for careful consideration.
REFERENCES
Bailey, J. M., Fishman, P. H., and Pentchev, P. G. (1969). J. Biol. Chem 244: 781-788.
Beebe, J. A. and Frey, P. A. (1998). Biochemistry 37: 14989-14997.
Deshpande, V., Keskar, S., Mishra, C., and Rao, M. (1986). Enzyme and Microbial Technology 8: 149-152.
Jeffries, T. W. (1983). Adv. Biochem. Eng./Biotechnol. 27: 1-32.
Phadtare, S. U., Rawat, U. B., and Rao, M. B. (1997). FEMS Microbiology Letters 146: 79-83.
Zhao, X., Gao, P. J., and Wang, Z. N. (1998). Applied Biochemistry and Biotechnology Vol. 70-72: 405-414.