Background Mannans represent the largest hemicellulosic fraction in softwoods and also
Background Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. from 1,4–d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. Conclusions We successfully displayed -mannanase and -mannosidase on the yeast cell surface. Our results clearly demonstrate the power of the strain co-displaying -mannanase and -mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering -mannanase and -mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials made up of mannan components. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0600-4) contains supplementary material, which Rabbit Polyclonal to GDF7 is available to authorized users. recombinant strain co-displaying -mannanase and -mannosidase; this yeast strain was expected to grant ethanol fermentation using mannan as a biomass resource (Fig.?1). We demonstrate that the designed yeast cells successfully hydrolyze the linear mannans and produce ethanol by assimilation 203849-91-6 IC50 of mannose generated by enzymatic degradation of 1,4–d-mannan or of ivory nut mannan. Fig.?1 Schematic overview of -mannanase and -mannosidase display on the cell wall of for ethanol production from 1,4–d-mannan. For the yeast cell surface display of the protein of interest, the genes encoding a secretion … Results and discussion Cell growth of using mannose as a carbon source To test whether yeast is usually able to assimilate mannose as a single carbon source, cell growth of YPH499 was investigated (Additional file 1: Physique H1). YPH499 cells were pre-cultured in synthetic dextrose (SD) minimal medium made 203849-91-6 IC50 up of 2?% glucose. After collecting and washing, the cells were separately and inoculated into fresh SD minimal glucose medium and synthetic mannose (SM) minimal medium (made up of 2?% mannose) to monitor cell growth. YPH499 yeast cells exhibited comparable growth kinetics in SD and SM media, although the growth in SM medium was delayed compared to that in SD medium (Additional file 1: Physique H1). The delay in SM medium might be attributed to the change of carbon sources from glucose to mannose. The specific growth rates () of YPH499 in early logarithmic phase were 0.327??0.006?h?1 in SD medium and 0.303??0.014?h?1 in SM medium (6C8?h), respectively (mean??standard deviation). Construction of -mannanase- and -mannosidase-co-displaying yeast In the most widely used yeast cell surface-display systems, target proteins are encoded with N-terminal secretion signal sequences and C-terminal anchor protein signal sequences [i.at the., cell wall proteins made up of glycosylphosphatidylinositol (GPI) anchor attachment sequences] (Fig.?1) [20, 24]. One such cell surface-display system using Flo1p (a lectin-like cell wall protein) permits the incorporation of an anchor ranging in length from 42 a.a. (Flo42) to 1326 a.a. (Flo1326), thereby accommodating convenience of large substrates to the target enzymes displayed on the yeast cell wall . Among the anchors, Flo428 (428 a.a.) is usually a well-balanced anchor protein that provides both substrate convenience and enzyme manifestation [25C27]. To make the mannan-fermenting yeast strains, we selected the -mannanase (Man5A) [14, 15] and -mannosidase (Mnd2A)  from as the mannan-degrading enzymes. The genes encoding Man5A and Mnd2A were amplified as open reading frames (ORFs) lacking the start codons and the stop codons; the and ORFs were amplified with downstream sequences encoding epitope tags (i.at the., to encode C-terminal FLAG HA or label label, respectively). The causing sequences had been 203849-91-6 IC50 cloned into the multiple cloning sites of pFGK426 and pFGK424 candida cell surface-display 2 multi-copy vectors, respectively (Desk?1). The causing plasmids (pFGK426-AaMan5A and pFGK424-AaMnd2A) had been designed to screen Man5A and Mnd2A on the candida cell surface area using a prepro–factor release sign and a Flo428 point proteins (Desk?1). In parallel buildings, and ORFs had been increased without begin codons but with end codons pursuing the particular epitope tag-encoding sequences. The ORFs had been cloned into pFGK426 and pFGK424 (respectively), containing plasmids (pFGK426-AaMan5A-TAA and pFGK424-AaMnd2A-TAA) that directed the extracellular release (without cell wall structure anchoring) of Man5A and Mnd2A (Desk?1). Paired and Individual plasmids, as well as the particular clear control (model) plasmids (pFGK426 and/or pFGK424), had been changed into YPH499 (Desk?2). Desk?1 Plasmids used in this scholarly research Desk?2 Transformants used in this research After cultivating the transformants in man made dextroseCcasamino acids (SDC) selection press, the relatives -mannanase actions and the -mannosidase actions of the candida cells had been measured using azo-galactomannan and Man5A-displaying candida cells. Guy5A-secreting candida … Next, neon immunostaining was performed to assess the existence of Guy5A and Mnd2A on the candida cell surface area (Fig.?3; Extra document 1: Shape S i90002). Cultured yeast cells had been impure with Alexa Fluor 488-tagged anti-HA and anti-FLAG.