A microalgal‐based preparation with synergistic cellulolytic and detoxifying action towards chemical‐treated lignocellulose

Abstract

High-temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the b-glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non-axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline-treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory byproducts released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose-derived sugars by C. vulgaris under mixotrophic growth.

Expression of a Hyperthermophilic Cellobiohydrolase in Transgenic Nicotiana tabacum by Protein Storage Vacuole Targeting

“…Unlike CBM3GH5-HA-VAC, the apoplastic version of CBM3GH5 was extracted as a single 90 kDa band by NaCl-supplemented buffer ( Figure 1 C). In these experiments, the chloroplast-expressed CBM3GH5-HA from C. reinhardtii was used as reference ( Figure 1 C) [ 31 ]. Both the CBM3GH5 isoforms expressed in tobacco showed a slightly higher molecular weight than the recombinant enzyme from C. reinhardtii , suggesting either an event of glycosylation or a partially processed protein.…”

“…In order to further characterize the expression of the recombinant enzyme, we purified CBM3GH5-HA-VAC from mature leaves of transgenic N. tabacum and performed a biochemical characterization. Purification was performed by a two-step procedure consisting of a heat-mediated enrichment of the total protein extract [ 40 ] followed by an anionic exchange chromatography (AEC) [ 22 , 31 ]. Extraction was carried out by using both the Tween 20-supplemented buffer and heat, following the optimized conditions of Figure 2 C. The elution of CBM3GH5-HA-VAC from AEC was performed by a stepwise NaCl gradient.…”

“…Alternatively, the recombinant enzyme could be recognized as a MAMP by tobacco PPRR, thus affecting plant development. According to this hypothesis, expression of GH5 cellulases resulted in plants with morphological defects or with low cellulolytic activities, despite the enzymes being compartmentalized or endowed with a temperature-inducible activity [ 17 , 31 , 56 ]. Alternatively, the lack of stable transformants accumulating CBM3GH5 in the apoplast could be a consequence of a reduced stability of CBM3GH5-HA with respect to CBM3GH5-HA-VAC.…”

“…Sequencing of the genes was performed in order to exclude the presence of undesired mutations using the primers Apo Fw, CBH785 Fw, CBH1565 Fw and CBH240 Rv ( Table S1 ). The CBM3GH5-HA was heterologously expressed in the C. reinhardtii chloroplast as reported in [ 31 ].…”

A novel Penicillium sumatraense isolate reveals an arsenal of degrading enzymes exploitable in algal bio-refinery processes

“…It is worth noting that Phi is only metabolizable by few chemolithotrophic bacteria possessing the phosphite dehydrogenase enzyme, such as Pseudomonas stutzeri [20], and therefore an eventual microbial growth in such medium could only be sustained at the expense of C. vulgaris biomass, used here as carbon and phosphate source. Moreover, Phi is characterized by a mild antimicrobial activity that made the selection in the algal trap more stringent [21][22][23]. In the absence of antimicrobial agents, several microorganisms could proliferate in the trap eating each other and exploiting the algal-supplemented medium as a mere basal salt source, hindering the discrimination of a real algivorous and algal saprophyte from the rest of contaminating microbes.…”