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Grantee Research Project Results

Final Report: Development of a Fermentation Compatible Xylose Isomerase

EPA Contract Number: EPD11037
Title: Development of a Fermentation Compatible Xylose Isomerase
Investigators: Potochnik, Stephen
Small Business: Trillium FiberFuels Inc.
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2011 through August 31, 2011
Project Amount: $79,885
RFA: Small Business Innovation Research (SBIR) - Phase I (2011) RFA Text |  Recipients Lists
Research Category: SBIR - Biofuels , Small Business Innovation Research (SBIR)

Description:

The core of this investigation is the development of a fermentation compatible xylose isomerase (XI) enzyme. The primary attributes required are operation at a lower pH than is typical for this enzyme (<6.0 versus >7.0) and tolerance of ubiquitous impurities such as calcium. Previous reports (Vongsuvanlert and Tani, 1988, and Chandrakant and Bisaria, 2000) describe an acid tolerant XI in the yeast Candida boidinii. This project outlined tasks to characterize more fully the biochemical performance of the enzyme, determine the gene for its expression, and perform a bench-top Simultaneous Isomerization and Fermentation (SIF) using the desired enzyme with a conventional ethanol yeast.

Summary/Accomplishments (Outputs/Outcomes):

All of the originally proposed experiments plus additional work have been completed. Activities for deliverables #1 and #2 focused on the biochemical performance of the fermentation compatible xylose isomerase previously described. Trillium FiberFuels' data are weakly supportive of the previous work where they overlap, but appear to suffer from an extremely low abundance of the desired activity. Supplemental experiments showed that it is possible to generate what appears to be xylose isomerase activity via an alternative pathway, which raises suspicion about the veracity of the previous findings.
 
Productivity experiments (deliverable #3) did not deliver the desired results. Attempts to immobilize the enzyme on sub-micron glass fiber, a technique Trillium FiberFuels has used successfully with other xylose isomerases, yielded no activity over a week’s time. An attempt to use the enzyme in solution was contaminated with wild yeast but did produce a modest amount of ethanol and may have fermented through the xylulose pathway.
 
Additional funds from Oregon’s BEST Center allowed the investigators to sequence the entire genome of Candida boidinii in support of deliverable #4 (finding the XI gene). The microorganism’s genome does not contain the conserved regions that appear in all known XIs ranging from bacteria to higher plants. No gene emerged with strong similarity to sequenced xylose isomerases. Of course, this raised further concern about whether the proposed XI really exists. Investigators' attention turned to two genes whose primary function is not xylose isomerization, but may have significant collateral XI activity. The first gene is an endonuclease. The annotation of the genome shows that it contains a “TIM xylose isomerase-like barrel,” which is a configuration of the protein seen in XIs and several other classes of proteins. A second gene that may have secondary XI activity also has been identified. Although beyond the scope defined for this project, both genes are being expressed in E. coli to elucidate the identity and/or existence of the desired xylose isomerase activity. The putative genes then will be expressed in Pichia pastoris in case there are post-translational modification (i.e., folding) issues with bacterial expression. These experiments are in process at the time this report was prepared.
 
The final deliverable for this Phase I is the SIF of xylose to ethanol using the xylose isomerase activity from Candida boidinii in solution with a conventional Saccharomyces cerevisiae yeast. The serendipitous result was that this appears to have occurred because of wild yeast contamination of one of the assays. The yeast was morphologically similar to Saccharomyces and quite likely to be Saccharomyces because of its widespread use in the Trillium FiberFuels lab. This small experiment yielded 2.8 g/l of ethanol with an estimated yield of 86 percent on consumed sugar. A deliberate attempt at SIF yielded poor results due to the low isomerase activity. After 48 hours, the ethanol concentration was negligible.

Conclusions:

A somewhat surprising primary conclusion is that Candida boidinii probably does not have a conventional xylose isomerase. In retrospect, this may be less surprising because the reported activity is quite different than all known xylose isomerases. Trillium FiberFuels has identified two candidate genes that may produce proteins that have substantial xylose isomerase activity even though it is not the primary function of the protein in vivo. These genes will be heterologously expressed and tested for xylose isomerase activity. A secondary conclusion is that under the growth conditions tested, the XI activity from any expressed protein is very low. Therefore, Trillium FiberFuels believes that the native host is unsuitable for production of the enzyme even if the source of the activity can be determined. This is in accord with the investigators' original plan to express the protein in a prolific host such as Pichia pastoris.

Commercialization: There is a strong business need at Trillium FiberFuels to develop a fermentation compatible xylose isomerase. To make its business case competitive, Trillium FiberFuels needs to reduce the operating expense and capital barriers to entry, and this innovation would provide those benefits. However, the evidence for the existence of the desired enzyme is mixed at best. Two potential commercialization paths are being considered at this time. The first is to continue with CBXI. Trillium FiberFuels has several key experiments under way that may lead to the conclusion that there is an enzyme present with useful XI activity, although it is probably not a conventional xylose isomerase. If this turns out to be true, this could fill Trillium FiberFuels' original business need. Another choice is to re-focus efforts on another xylose isomerase that is showing much more consistent results, albeit at a pH that is not as desirable as was reported for Candida boidinii. Work on this new microorganism currently is under way through a Phase I SBIR from the National Science Foundtion. Trillium FiberFuels will continue to gather evidence on each of these cases and present its best case in its Phase II proposal.

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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