Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol

Abstract

We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch® SuperMan strain to prevent hyperglycosylation. The two (His)-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was ManGlcNAc with, however, a significant contribution of ManGlcNAc. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.

Molecular Characterization, Evolution, and Expression Profiling of the Dirigent (DIR) Family Genes in Chinese White Pear (Pyrus bretschneideri)

“…PbDIR4, 10, 12, 13 , and 14 were up-regulated at 47 and 55 DAF, but only PbDIR4 was up-regulated at 47 and 55 DAF, and the expression of these five genes was low at 15, 39, 63, 79, 102, and 145 DAF. In addition, there are some genes that are up-regulated at maturity, such as the four genes PbDIR18, 27, 31 , and 33 , and they may be involved in the synthesis of terpenoids of pear fruit to produce aroma ( He et al, 2015 ; Effenberger et al, 2017 ; Paniagua et al, 2017 ).…”

Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison

“…Glyco-engineered strains often show a decrease in productivity over time [ 18 , 21 , 25 ], which might be linked to their altered glycosylation machinery and therefore a stressed metabolism. Based on our recent findings with the ∆ OCH1 strain [ 12 , 26 ], we found that cellular agglomeration and therefore an altered morphology, affected process performance over time.…”

“…In literature, environmental stressors are known to affect a protein’s post-translational processing [ 24 ], which highlights the importance of analyzing a protein’s properties during the controlled production in bioreactors. To our knowledge, only a few studies exist, which analyzed the behavior of Man 5 GlcNAc 2 glycosylating P. pastoris strains in the controlled environment of a bioreactor [ 18 , 21 , 25 ]. Jacobs et al [ 25 ] were able to produce a maximum of 760 mg L −1 of a murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) at high cell densities upon MeOH induction of alcohol oxidase 1 promoter (P AOX1 ).…”

“…During their glycerol fed-batch cultivation, they detected chymase-like proteolytic activity after 72 h of induction, which might have resulted from physiological stress. Recently, the cotton plant proteins GbDIR2 and GhDIR3 were produced with > 90% Man 5 GlcNAc 2 glycosylation homogeneity, but the authors observed an increased MeOH toxicity for their glyco-engineered strain compared to conventional wild-type strains [ 21 ]. Concluding, all of these research groups experienced a decrease in Pichia’s process performance over time.…”

An Update on Jacalin-Like Lectins and Their Role in Plant Defense

“…This step is crucial, because the optical activity determines the properties of most lignans [ 53 ]. An example is gossypol, where both isomers are involved in plant defense but only the (-)-gossypol has antispermatogenic and antiviral activities, whereas the (+)-isomer is toxic to non-ruminant animals [ 54 ]. There are numerous examples that demonstrate involvement of dirigent proteins in the response of plants to pathogen attack (reviewed in [ 51 ]).…”