Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering

Background: The production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production. Results: An expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate. Conclusions: An industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates

Effect of Temperature on Simultaneous Saccharification and Fermentation of Pretreated Spruce and Arundo

A critical factor in simultaneous saccharification and fermentation (SSF) is the selection of process temperature, which needs to be a compromise between the optimal temperature for enzymatic hydrolysis and that for fermentation. In the present work, isothermal and nonisothermal SSF was experimentally studied using pretreated spruce and arundo materials at a loading of 10% water insoluble solids, with an industrial strain of Saccharomyces cerevisiae as the fermenting organism. In the nonisothermal experiments, the temperatures were linearly increased during the batch time of 96 h, and the results were compared to isothermal SSF operation. The final ethanol concentrations obtained for the arundo material was 16.5 g/L in the nonisothermal process using an initial temperature of 32 degrees C and a linearly increasing temperature of 0.135 degrees C/h. As a comparison, the final ethanol concentration obtained was 13.4 g/L for an isothermal operation at 32 degrees C and 15.3 g/L for an isothermal process running at 39 degrees C. The corresponding values for the spruce material were 33.1 g/L, and 29.1 g/L and 32.2 g/L, for nonisothermal and isothermal operation at 32 and 39 degrees C, respectively. The obtained ethanol yields, in particular for the case of arundo, demonstrate that nonisothermal SSF operation can give increased ethanol yields in comparison to isothermal SSF

Progress in terpene synthesis strategies through engineering of <i>Saccharomyces cerevisiae</i>

<p>Terpenes are natural products with a remarkable diversity in their chemical structures and they hold a significant market share commercially owing to their distinct applications. These potential molecules are usually derived from terrestrial plants, marine and microbial sources. <i>In vitro</i> production of terpenes using plant tissue culture and plant metabolic engineering, although receiving some success, the complexity in downstream processing because of the interference of phenolics and product commercialization due to regulations that are significant concerns. Industrial workhorses’ viz., <i>Escherichia coli</i> and <i>Saccharomyces cerevisiae</i> have become microorganisms to produce non-native terpenes in order to address critical issues such as demand-supply imbalance, sustainability and commercial viability. <i>S. cerevisiae</i> enjoys several advantages for synthesizing non-native terpenes with the most significant being the compatibility for expressing cytochrome P450 enzymes from plant origin. Moreover, achievement of high titers such as 40 g/l of amorphadiene, a sesquiterpene, boosts commercial interest and encourages the researchers to envisage both molecular and process strategies for developing yeast cell factories to produce these compounds. This review contains a brief consideration of existing strategies to engineer <i>S. cerevisiae</i> toward the synthesis of terpene molecules. Some of the common targets for synthesis of terpenes in <i>S. cerevisiae</i> are as follows: overexpression of <i>tHMG1</i>, <i>ERG20</i>, <i>upc2-1</i> in case of all classes of terpenes; repression of ERG9 by replacement of the native promoter with a repressive methionine promoter in case of mono-, di- and sesquiterpenes; overexpression of BTS1 in case of di- and tetraterpenes. Site-directed mutagenesis such as Upc2p (G888A) in case of all classes of terpenes, ERG20p (K197G) in case of monoterpenes, HMG2p (K6R) in case of mono-, di- and sesquiterpenes could be some generic targets. Efforts are made to consolidate various studies (including patents) on this subject to understand the similarities, to identify novel strategies and to contemplate potential possibilities to build a robust yeast cell factory for terpene or terpenoid production. Emphasis is not restricted to metabolic engineering strategies pertaining to sterol and mevalonate pathway, but also other holistic approaches for elsewhere exploitation in the <i>S. cerevisiae</i> genome are discussed. This review also focuses on process considerations and challenges during the mass production of these potential compounds from the engineered strain for commercial exploitation.</p

Regeneration of NADPH Coupled with HMG-CoA Reductase Activity Increases Squalene Synthesis in <i>Saccharomyces cerevisiae</i>

Although overexpression of the <i>tHMG1</i> gene is a well-known strategy for terpene synthesis in <i>Saccharomyces cerevisiae</i>, the optimal level for tHMG1p has not been established. In the present study, it was observed that two copies of the <i>tHMG1</i> gene on a dual gene expression cassette improved squalene synthesis in laboratory strain by 16.8-fold in comparison to single-copy expression. It was also observed that tHMG1p is limited by its cofactor (NADPH), as the overexpression of NADPH regenerating genes’, viz., <i>ZWF1</i> and <i>POS5</i> (full length and without mitochondrial presequence), has led to its increased enzyme activity. Further, it was demonstrated that overexpression of full-length <i>POS5</i> has improved squalene synthesis in cytosol. Finally, when <i>tHMG1</i> and full-length <i>POS5</i> were co-overexpressed there was a net 27.5-fold increase in squalene when compared to control strain. These results suggest novel strategies to increase squalene accumulation in <i>S. cerevisiae</i>

Effect of Temperature on Simultaneous Saccharification and Fermentation of Pretreated Spruce and Arundo

A critical factor in simultaneous saccharification and fermentation (SSF) is the selection of process temperature, which needs to be a compromise between the optimal temperature for enzymatic hydrolysis and that for fermentation. In the present work, isothermal and nonisothermal SSF was experimentally studied using pretreated spruce and arundo materials at a loading of 10% water insoluble solids, with an industrial strain of <i>Saccharomyces cerevisiae</i> as the fermenting organism. In the nonisothermal experiments, the temperatures were linearly increased during the batch time of 96 h, and the results were compared to isothermal SSF operation. The final ethanol concentrations obtained for the arundo material was 16.5 g/L in the nonisothermal process using an initial temperature of 32 °C and a linearly increasing temperature of 0.135 °C/h. As a comparison, the final ethanol concentration obtained was 13.4 g/L for an isothermal operation at 32 °C and 15.3 g/L for an isothermal process running at 39 °C. The corresponding values for the spruce material were 33.1 g/L, and 29.1 g/L and 32.2 g/L, for nonisothermal and isothermal operation at 32 and 39 °C, respectively. The obtained ethanol yields, in particular for the case of arundo, demonstrate that nonisothermal SSF operation can give increased ethanol yields in comparison to isothermal SSF