Heterologous expression of lytic polysaccharide monooxygenases (LPMOs)

LyGo: A platform for rapid screening of lytic polysaccharide monooxygenase production

“…Despite its biotechnological importance and wide use in industry, relatively few genetic tools are readily available. However, K. phaffii has proven to be a promising production host for carbohydrate active enzymes like LPMOs 16,56,57,58,59 and could be a valuable addition to the LyGo platform. Figure 2E).…”

“…Cytoplasmic expression is the most common strategy for the production of heterologous proteins in E. coli 62 , and is often advantageous over other expression strategies as it mitigates the need for screening signal peptides and has the potential to result in product yields exceeding 50% of the total cellular protein amounts 63 . Furthermore, cytoplasmic expression supports the use of several genetic tools such as co-expression of chaperones 64 Periplasmic expression is a popular strategy for expressing LPMOs in E. coli 16 , as it naturally provides the means for N-terminal processing and disulfide bond formation. However, secretion has previously been shown to constitute a bottleneck resulting in low yields 73 .…”

A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable His6-Tag cloning, expression, and purification

“…In order to yield an active LPMO through heterologous expression with a proper oxidative activity, a methodology addressing necessary features are required: (i) the presence of an appropriate signal peptide for the correct post-translational processing and secretion; (ii) avoidance of any N-terminal tag considering the importance of the His1, α-amino group and side-chain for the copper-binding and catalysis; (iii) appropriate copper saturation of the enzyme; (iv) suitable glycosylation and methylation for effective expression and stability of fungal LPMOs; and (v) codon optimization to improve the heterologous expression level [19,20]. These singular features have been recently reviewed in detail by Gaber et al [21], and must be taken into consideration all together during LPMO expression, representing therefore a challenge to its heterologous expression [19].…”

“…To fulfil this requirement, LPMOs can be produced in a phylogenetically close host expression system, such as bacterial LPMOs found in family AA10 expressed using E. coli, and fungal LPMOs from the other families in A. nidulans or P. pastoris. The methylotrophic yeast P. pastoris (Komagataella phaffi) is a highly robust cell factory that tolerates high-cell-density, usually produces high yields of target proteins [23], and has been the most widely used host for heterologous expression of fungal LPMOs [21]. Although the mentioned benefits, it must also be noted that when expressing fungal LPMOs in P. pastoris it results in a lack of methylation to His1 residue as instead occurring in their native form or when expressed in fungal hosts as A. nidulans.…”

“…Although the mentioned benefits, it must also be noted that when expressing fungal LPMOs in P. pastoris it results in a lack of methylation to His1 residue as instead occurring in their native form or when expressed in fungal hosts as A. nidulans. Fortunately has been shown that the absence of a methylation on the His1 it only impact the resistance of the enzymes towards environmental oxidation or self-oxidation due to presence or ROS species or hydrogen peroxide, these evidences were again reviewed by Eijsink et al [19] and by Gaber et al [21].…”