Shaunita H. Rose

Progress and challenges in the engineering of non-cellulolytic microorganisms for consolidated bioprocessing

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.

Cellulase Production from Spent Lignocellulose Hydrolysates by Recombinant Aspergillus niger

ABSTRACT A recombinant Aspergillus niger strain expressing the Hypocrea jecorina endoglucanase Cel7B was grown on spent hydrolysates (stillage) from sugarcane bagasse and spruce wood. The spent hydrolysates served as excellent growth media for the Cel7B-producing strain, A. niger D15[egI], which displayed higher endoglucanase activities in the spent hydrolysates than in standard medium with a comparable monosaccharide content (e.g., 2,100 nkat/ml in spent bagasse hydrolysate compared to 480 nkat/ml in standard glucose-based medium). In addition, A. niger D15[egI] was also able to consume or convert other lignocellulose-derived compounds, such as acetic acid, furan aldehydes, and phenolic compounds, which are recognized as inhibitors of yeast during ethanolic fermentation. The results indicate that enzymes can be produced from the stillage stream as a high-value coproduct in second-generation bioethanol plants in a way that also facilitates recirculation of process water.

Exploring improved endoglucanase expression in Saccharomyces cerevisiae strains

The endoglucanase I and II genes (egI or Cel7B and egII or Cel5A) of Trichoderma reesei QM6a were successfully cloned and expressed in Saccharomyces cerevisiae under the transcriptional control of the yeast ENO1 promoter and terminator sequences. Random mutagenesis of the egI-bearing plasmid resulted in a twofold increase in extracellular EGI activity. Both endoglucanase genes were co-expressed with the synthetic, codon-optimised cellobiohydrolase gene (s-cbhI) from T. reesei as well as the β-glucosidase gene (bgl1) from Saccharomycopsis fibuligera in S. cerevisiae. Extracellular endoglucanase activity was lower when co-expressed with s-cbhI or bgl1. Recombinant strains were able to hydrolyse phosphoric acid swollen cellulose, generating mainly cellotriose, cellobiose and glucose. Cellobiose accumulated, suggesting the β-glucosidase activity to be the rate-limiting factor. As a consequence, the recombinant strains were unable to produce enough glucose for growth on amorphous cellulose. The results of this study provide insight into further optimisation of recombinantly expressed cellulase combinations for saccharification and fermentation of cellulose to ethanol.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases

The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch‐based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon‐optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α‐amylase (SFA1) genes were δ‐integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1‐SFA1] and MEL2[TLG1‐SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch‐to‐ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre‐treatment or commercial enzyme addition) using industrial yeast strains co‐secreting both a glucoamylase and α‐amylase. Biotechnol. Bioeng. 2015;112: 1751–1760.

Designing industrial yeasts for the consolidated bioprocessing of starchy biomass to ethanol.

Consolidated bioprocessing (CBP), which integrates enzyme production, saccharification and fermentation into a one step process, is a promising strategy for the effective ethanol production from cheap lignocellulosic and starchy materials. CBP requires a highly engineered microbial strain able to both hydrolyze biomass with enzymes produced on its own and convert the resulting simple sugars into high-titer ethanol. Recently, heterologous production of cellulose and starch-degrading enzymes has been achieved in yeast hosts, which has realized direct processing of biomass to ethanol. However, essentially all efforts aimed at the efficient heterologous expression of saccharolytic enzymes in yeast have involved laboratory strains and much of this work has to be transferred to industrial yeasts that provide the fermentation capacity and robustness desired for large scale bioethanol production. Specifically, the development of an industrial CBP amylolytic yeast would allow the one-step processing of low-cost starchy substrates into ethanol. This article gives insight in the current knowledge and achievements on bioethanol production from starchy materials with industrial engineered S. cerevisiae strains.

Exploitation of Aspergillus niger for the Heterologous Production of Cellulases and Hemicellulases

Filamentous fungi of the Aspergillus niger group are native soil saprophytic fungi. Industrial strains of this group have been extensively used for the production of plant degrading enzymes for the food and beverage, animal feed and paper-and-pulp industries. Recombinant DNA technology allows for the overproduction of these enzymes in copious amounts. The advantages and limitations of A. niger as recombinant host for enzyme production are briefly discussed. Specific attention is devoted to the overproduction of several cellulases and hemicellulases to high homogeneity in the protease-deficient strain A. niger D15. The size, temperature and pH optima of the heterologous enzymes were shown to be similar to that of their natively produced counter parts. The optimization of enzyme production in dilute sugar cane molasses, using a recombinant strain producing the xylanase II of Trichoderma reesei as example, was also demonstrated.

Consolidated bioprocessing of raw starch with Saccharomyces cerevisiae strains expressing fungal alpha-amylase and glucoamylase combinations

&NA; Cost‐effective consolidated bioprocessing (CBP) of raw starch for biofuel production requires recombinant Saccharomyces cerevisiae strains expressing &agr;‐amylases and glucoamylases. Native Aureobasidium pullulans apuA, Aspergillus terreus ateA, Cryptococcus sp. S‐2 cryA and Saccharomycopsis fibuligera sfiA genes encoding raw‐starch &agr;‐amylases were cloned and expressed in the S. cerevisiae Y294 laboratory strain. Recombinant S. cerevisiae Y294[ApuA] and Y294[AteA] strains produced the highest extracellular &agr;‐amylase activities (2.17 U mL‐1 and 2.98 U mL‐1, respectively). Both the ApuA and AteA &agr;‐amylases displayed a preference for pH 4 to 5 and retained more than 75% activity after 5 days at 30°C. When ateA was co‐expressed with the previously reported Aspergillus. tubingensis glucoamylase gene (glaA), the amylolytic S. cerevisiae Y294[AteA‐GlaA] strain produced 45.77 g L‐1 ethanol after 6 days. Ethanol production by this strain was improved with the addition of either 2.83 &mgr;L STARGEN 002 (54.54 g L‐1 ethanol and 70.44% carbon conversion) or 20 &mgr;L commercial glucoamylase from Sigma‐Aldrich (73.80 g L‐1 ethanol and 90.19% carbon conversion). This is the first report of an engineered yeast strain that can replace up to 90% of the enzymes required for raw starch hydrolysis, and thus contributes to the realisation of a CBP yeast for starch‐based biofuel production.

Expression of Fungal Hydrolases in Saccharomyces cerevisiae

Abstract Conversion of plant biomass to ethanol or other commodities through direct microbial conversion could be economically feasible if microbes that have optimal processing and product formation properties could be identified or engineered. The yeast Saccharomyces cerevisiae holds several advantages over other candidate organisms for this development, such as its process robustness, long industrial history, and genetic malleability. Because this yeast cannot use polymeric sugar substrates, the heterologous production of the hydrolytic activities required for the degradation of the cellulose and hemicellulose components of plant biomass is imperative. Several researchers have attempted the expression of genes encoding lignocellulolytic hydrolases in S. cerevisiae over the past three decades. This chapter will assess the progress made in this field and will highlight some of the successes and future challenges.