Progress and challenges in the engineering of non-cellulolytic microorganisms for consolidated bioprocessing Riaan den Haan 1 , Euge ´ ne van Rensburg 2 , Shaunita H Rose 3 , Johann F Go ¨ rgens 2 and Willem H van Zyl 3 Lignocellulosic biomass is an abundant, renewable feedstock for the production of fuels and chemicals, if an efficient and affordable conversion technology can be established to overcome its recalcitrance. Consolidated bioprocessing (CBP) featuring enzyme production, substrate hydrolysis and fermentation in a single step is a biologically mediated conversion approach with outstanding potential if a fit-for- purpose microorganism(s) can be developed. Progress in developing CBP-enabling microorganisms is ongoing by engineering (i) naturally cellulolytic microorganisms for improved product-related properties or (ii) non-cellulolytic organisms exhibiting high product yields to heterologously produce different combinations of cellulase enzymes. We discuss progress on developing yeast and bacteria for the latter strategy and consider further challenges that require attention to bring this technology to market. Addresses 1 Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville 7530, South Africa 2 Department of Process Engineering, Stellenbosch University, Private Bag XI, Matieland 7602, South Africa 3 Department of Microbiology, Stellenbosch University, Private Bag XI, Matieland 7602, South Africa Corresponding author: van Zyl, Willem H (whvz@sun.ac.za) Current Opinion in Biotechnology 2015, 33:32–38 This review comes from a themed issue on Energy biotechnology Edited by E Terry Papoutsakis and Jack T Pronk http://dx.doi.org/10.1016/j.copbio.2014.10.003 0958-1669/# 2014 Elsevier Ltd. All right reserved. Introduction Incorporation of biologically produced alcohols and chemicals into existing energy-based infrastructure is gain- ing increasing prominence worldwide. However, several barriers remain as evident from pressure on the 2022 Renew- able Fuel Standard (RFS) target due to paucity in com- mercial scale cellulosic ethanol production. Insufficiency in cellulosic ethanol production cannot be attributed solely to feedstock supply, given estimates of approximately 1368 million tonnes of feedstock available in the US alone [1  ]. This capacity could amount to approximately 76 billion gallons ethanol, assuming 85% sugar recovery from pretreat- ment-hydrolysis and 96% of the theoretical ethanol yield (Y p/s = 0.49) from fermentation, far exceeding the RFS target of 36 billion gallons ethanol, incorporating 16 billion gallons of cellulosic ethanol (Alternative Fuels Data Center; URL: http://www.afdc.energy.gov/laws/eisa). A key attribu- table factor responsible for the cellulosic ethanol shortfall resides in barriers to cost-effective cellulosic ethanol pro- duction, where substrate recalcitrance and enzyme cost remain key elements. To avoid viscosity and mass transfer constraints, separate hydrolysis and fermentation (SHF, Figure 1) or simul- taneous saccharification and fermentation (SSF) are stan- dard methodologies for lignocellulose fermentation [2]. Whereas the former approach allows improved pro- ductivity rates from enzyme hydrolysis at optimal temperature, the latter limits feedback inhibition through product removal of glucose by direct fermentation. Nevertheless, exogenous enzyme addition is required in both instances. Simultaneous saccharification and co- fermentation (SSCF) further combines the hexose and pentose sugar fermentation steps. Consolidated biopro- cessing (CBP), the biological conversion of suitably pre- treated lignocellulose by one or more organisms into desired products in a single unit operation without added enzymes, could offer recourse to bringing lignocellulosic ethanol closer to market [3,4  ]. The main challenges for CBP technology include (i) suitably high levels of enzyme production, without compromising ethanol fer- mentation capacity and (ii) co-fermentation of hexose and pentose sugars, while (iii) tolerating harsh conditions such as high levels of the desired product (e.g. ethanol) and lignocellulose degradation products. No natural organism with such capabilities has been isolated to date. CBP organism development efforts fall into two categories: (i) engineering cellulase producers to produce ethanol or other desirable products or (ii) engineering organisms with favourable product forming attributes (such as an ethanologen) to produce cellulases during SSCF of pre- treated lignocellulose (Table 1) [3]. The status quo on the latter topic is the main subject of this review. Engineering cellulase production into Saccharomyces cerevisiae Ethanol produced by S. cerevisiae fermentation remains the dominant biofuel in the global context, due to the Available online at www.sciencedirect.com ScienceDirect Current Opinion in Biotechnology 2015, 33:32–38 www.sciencedirect.com