ABSTRACT
BACKGROUND: Microbial expansins (EXLXs) are non-lytic proteins homologous to plant expansins involved in plant cell wall formation. Due to their non-lytic cell wall loosening properties and potential to disaggregate cellulosic structures, there is considerable interest in exploring the ability of microbial expansins (EXLX) to assist the processing of cellulosic biomass for broader biotechnological applications. Herein, EXLXs with different modular structure and from diverse phylogenetic origin were compared in terms of ability to bind cellulosic, xylosic, and chitinous substrates, to structurally modify cellulosic fibrils, and to boost enzymatic deconstruction of hardwood pulp. RESULTS: Five heterogeneously produced EXLXs (Clavibacter michiganensis; CmiEXLX2, Dickeya aquatica; DaqEXLX1, Xanthomonas sacchari; XsaEXLX1, Nothophytophthora sp.; NspEXLX1 and Phytophthora cactorum; PcaEXLX1) were shown to bind xylan and hardwood pulp at pH 5.5 and CmiEXLX2 (harboring a family-2 carbohydrate-binding module) also bound well to crystalline cellulose. Small-angle X-ray scattering revealed a 20-25% increase in interfibrillar distance between neighboring cellulose microfibrils following treatment with CmiEXLX2, DaqEXLX1, or NspEXLX1. Correspondingly, combining xylanase with CmiEXLX2 and DaqEXLX1 increased product yield from hardwood pulp by ~ 25%, while supplementing the TrAA9A LPMO from Trichoderma reesei with CmiEXLX2, DaqEXLX1, and NspEXLX1 increased total product yield by over 35%. CONCLUSION: This direct comparison of diverse EXLXs revealed consistent impacts on interfibrillar spacing of cellulose microfibers and performance of carbohydrate-active enzymes predicted to act on fiber surfaces. These findings uncover new possibilities to employ EXLXs in the creation of value-added materials from cellulosic biomass.
ABSTRACT
Mucic acid, a diacid with potential use in the food, cosmetic, chemical and pharmaceutical industries, can be produced by microbial conversion of D-galacturonic acid, which is abundant in pectin. Using the ambr®250 bioreactor system, we found that a recently generated transformant (D-221704, formerly referred to as T2) of a marine Trichoderma species produced up to 53 g L-1 mucic acid in glucose-limited fed-batch culture with D-galacturonic acid in the feed at pH 4, with a yield of 0.99 g mucic acid per g D-galacturonic acid consumed. Yeast extract was not essential for high production, but increased the initial production rate. Reducing the amount of glucose as the co-substrate reduced the amount of mucic acid produced to 31 g L-1. Mucic acid could also be produced at pH values less than 4.0 (3.5 and 3.0), but the amount produced was less than at pH 4.0. Furthermore, the yield of mucic acid on D-galacturonic acid at the end of the cultivations (0.5 to 0.7 g g-1) at these low pH levels suggested that recovery may be more difficult at lower pH on account of the high level of crystal formation. Another strain engineered to produce mucic acid, Trichoderma reesei D-161646, produced only 31 g L-1 mucic acid under the conditions used with D-221704.
