Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose

Abstract

BackgroundA recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate.ResultsThe production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively.ConclusionsThis study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-0990-y) contains supplementary material, which is available to authorized users.

Developing cellulolytic Yarrowia lipolytica as a platform for the production of valuable products in consolidated bioprocessing of cellulose

“…However, we nevertheless observed definitive loss of one or several previously introduced heterologous genes during selection marker removal. This is probably caused by random recombination, which is due to the presence of multiple LoxP sites [ 18 ]. Therefore, to resolve this issue we set out to reduce the number of LoxP sites that are introduced during Y. lipolytica engineering.…”

“…To further engineer this strain, it is necessary to use Cre -recombinase to remove previously employed selection markers. However, this operation is complicated, because it often leads to the loss of previously introduced heterologous genes due to the presence of multiple LoxP sites [ 17 , 18 ]. Therefore, in this work, while further optimizing the expression of multiple cellulases, we have rescued selection makers to obtain an auxotrophic cellulolytic Y. lipolytica strain.…”

An artificial chromosome ylAC enables efficient assembly of multiple genes in Yarrowia lipolytica for biomanufacturing

“…Compared to conventional multi-step methods ( Supplementary Fig. 15), this method is no doubt much faster and provide more reliable results 21,27,49,50 .…”

“…Multiple gene integration is timeconsuming, since it requires repeated transformation-selection cycles and the rescue of selection markers 21 . Likewise, sequential genome insertions are generally hampered by decreasing transformation efficiencies and progressive loss of strain fitness 27 . Finally, non-homologous end-joining (NHEJ) is predominant over homologous recombination (HR) 28 .…”

Optimization of Yarrowia lipolytica-based consolidated biocatalyst through synthetic biology approach: transcription units and signal peptides shuffling

“…Due to unique native metabolic properties, like intrinsically high production of lipids [6], polyols [7], organic acids [8], and recently evidenced superior capacity for heterologous proteins production over a typical workhorse in this regard -Komagataella phaffi (P. pastoris) [9], Y. lipolytica stains are frequently subjected to genetic modifications broadening its scope of utilized substrates, with the aim to improve economic feasibility of production processes [10]. For example, Y. lipolytica strains has been endowed with artificial ability to grow on sucrose [11], inulin [12], galactose [13], starch [14] or even cellulose [15,16]. Utilization of renewable substrates, usually required introduction of several heterologous genes to Y. lipolytica cells, to either efficiently decompose biopolymer, or provide a link between new substrate and native metabolism of the host cell.…”