NATURE BIOTECHNOLOGY ADVANCE ONLINE PUBLICATION  ARTICLES Dwindling fossil resources and concerns about greenhouse gas emissions are driving the development of alternative feedstocks for producing fuels and chemicals 1 . Cellulosic feedstocks are among the leading alternatives because they are abundant, low cost and are not used as human food. Unfortunately, producing fuels and chemicals from cellulosic biomass suffers from high production costs, par- ticularly those associated with biomass pretreatment and enzymatic hydrolysis 2,3 . High enzyme requirements and limited enzyme pro- duction capacity remain a significant challenge for the cellulosic processing industry 4,5 . In contrast to microbial consolidated bio- processing 6 , which relies on the availability of fermentable sugars for co-production of enzymes and biofuels, in planta consolidated bioprocessing, which consolidates enzyme expression in the feed- stock, is predicted to be more cost effective because it does not require fermentable sugars for microbial enzyme production 7 and eliminates the mass transfer resistance of the enzyme diffusing to its substrate, which may decrease pretreatment requirements and costs. Despite these potential advantages, in planta expression of CWD enzymes can have developmental repercussions, including stunted plant stature, poor seed set and germination, reduced fertility, increased susceptibility to disease 8–11 and lower yield. To overcome these adverse effects while retaining the potential benefits, we engi- neered thermoregulated intein splicing to control the activity of a hydrolytic xylanase in maize. Inteins are self-splicing peptides, present in proteins in some lower Eucarya, Eubacteria and Archaea 12 . An intein may disrupt the function- ality of the host protein until it is excised. Upon excision, inteins ligate the bordering ‘extein’ polypeptide sequences with a peptide bond in a splicing reaction 13 , thereby restoring host protein function. Inteins that self-splice in response to temperature or other stimuli, have been described 14–18 , but have not been used to engineer a CWD enzyme in plants. Intein techno- logy has found applications in controlling gene flow in dicotyledonous plants and herbicide tolerance 19–23 , and intein-mediated regulation of in planta protein activity promises to make substantial contributions in fiber conversion for biofuels and animal feed production 24 . Xylanases, which hydrolyze hemicellulose polymers and make cellulose more accessible to enzymatic hydrolysis 25–27 , are required for complete hydrolysis of plant cell walls into fermentable sugars, such as glucose and xylose. Cellulases and xylanases that function over a wide pH range and at high temperatures are well-suited for processing lignocellulosic feedstocks because of their potential for enzymatic activity under desirable operating conditions 28 . Process consolidation using in planta xylanase production could affect biomass pretreatment and potentially reduce costs and demand for fermentation-derived enzymes, if crop yields are maintained at economically competitive levels 6,8,29 . By engineering thermoregulated intein splicing to control xylanase catalytic activity, greenhouse- grown transgenic maize plants did not develop the infertility or seed shriveling that is observed in plants expressing the unmodified xylanase, and had improved process performance in laboratory-scale evaluations. Intein modification of xylanase ensures that its CWD activity in maize plants only occurs after a pretreatment process (60–70 °C) that was subsequently followed by enzymatic hydrolysis with a commercial enzyme preparation. Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing Binzhang Shen, Xueguang Sun, Xiao Zuo, Taran Shilling, James Apgar, Mary Ross, Oleg Bougri, Vladimir Samoylov, Matthew Parker, Elaina Hancock, Hector Lucero, Benjamin Gray, Nathan A Ekborg, Dongcheng Zhang, Jeremy C Schley Johnson, Gabor Lazar & R Michael Raab Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall–degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields. Agrivida, Inc., Medford, Massachusetts, USA. Correspondence should be addressed to R.M.R. (rmraab@agrivida.com). Received 31 May; accepted 28 September; published online 21 October 2012; doi:10.1038/nbt.2402