Corynebacterium glutamicum: A Platform For Studying Actinobacterial Protein-O-Mannosylation And High-Yield Heterologous Protein Production

• Author / Creator

  Saxena, Hirak

• Corynebacterium glutamicum ATCC 13032 is a generally regarded as safe (GRAS) soil actinobacterium that is widely utilized in industry and was originally used for the large-scale production of amino acids. Through both genetic and metabolic engineering, the organism has been developed into a biorefinery, producing value-added products such as polyamines, food-grade chemicals, medicines, branched-chain amino acids, and many more. In addition to its industrial applications, C. glutamicum has even been engineered to function as a “biocontainer” for the bioremediation of arsenic from contaminated soils and waters. With a well-developed molecular toolbox available for the organism, C. glutamicum has only recently begun to be investigated for its potential application as a recombinant expression host.

  As C. glutamicum is capable of protein-O-mannosylation (POM), the organism serves as an ideal host system for the investigation of this process in actinobacteria. POM is a form of O-glycosylation that is ubiquitous throughout all domains of life and has been extensively characterized in eukaryotic systems. However, in prokaryotes, this process has only been investigated in the context of pathogenicity (in Mycobacterium tuberculosis) even though there are many non-pathogenic bacteria that are known to regularly carry out POM. To date, there is no consensus on what benefit POM imparts to the non-pathogenic bacteria that can perform it. Through the expression and native-like mannosylation of known actinobacterial mannoproteins produced recombinantly in C. glutamicum, this work shows that this bacterium can be utilized as a host system for the study of actinobacterial POM. The complementation of a POM deficient mutant of C. glutamicum (via knockout of the native GT-39, the enzyme responsible for the initiation of POM) by other actinobacterial GT-39s provides evidence that these closely related enzymes may have different activities and substrate specificities for targets of POM. Moreover, evidence is presented suggesting POM does not only occur in a general secretory pathway (SEC)-dependent manner; it also occurs with twin-arginine translocase (TAT) and non-SEC secreted substrates in a specific and tightly regulated manner. These results highlight the need for further biochemical characterization of POM in these and other bacterial species to help elucidate the true nature of its biological functions.
  There are several inherent benefits to using C. glutamicum as a protein production platform: low levels of cytoplasmic and extracellular proteases, secretion via well-characterized pathways (SEC and TAT), and its status as an endo-toxin free strain. These factors make C. glutamicum an attractive host system for the production of Carbohydrate Active EnZymes (CAZymes) that can be used to modify the glycan profiles of therapeutic proteins. The accurate glycosylation of protein therapeutics is currently one of the various stratagems to increase both their efficacy and serum half-life. It has been repeatedly demonstrated that the capping of N-linked glycans by terminal sialic (N-acetylneuraminic) acids significantly increases the in-vivo half-life of a given glycoprotein. Terminal glycosylation has proven to be difficult to reproduce in mammalian cell culture systems due to large variations in the efficiency of native sialyltransferases (STs) in various production strains, in addition to non-human cell lines incorporating a non-human analogue of sialic acid N-glycolylneuraminic acid (Neu5Gc) which is highly antigenic to humans; the incorporation of these antigenic epitopes into recombinant therapeutics must be avoided. To circumvent these complications, STs produced in prokaryotic expression systems have been employed to perform the final glycosylation step of N-linked glycans in vitro. Unlike enzymes produced in E. coli, STs produced in C. glutamicum would be essentially endotoxin free – facilitating the final polishing of therapeutic proteins and decreasing associated costs. This work details the development of C. glutamicum as a recombinant host for the production of STs, showing that with minimal engineering and the co-expression of folding chaperones, the strain is capable of producing both prokaryotic and eukaryotic STs with comparable activities and yields to the traditional recombinant host E. coli. Overall, this work demonstrates that C. glutamicum shows great potential as a recombinant expression platform for many applications.

• Subjects / Keywords

  • Corynebacterium glutamicum
  • Protein-O-mannosylation
  • Actinobacteria
  • Cellulomonas fimi
  • Cellulomonas flavigena
  • O-glycosylation
  • Protein-O-mannosyltransferase
  • Glycosyltransferase family 39
  • Secretion
  • General secretory pathway (SEC)
  • Twin arginie translocon (TAT)
  • Recombinant host engineering
  • Recombinant protein production
  • Sialyltransferase
  • Glycosyltransferase family 42
  • Glycosyltransferase family 29
  • Glycosyltransferase family 80
  • Co-expression
  • Glycomodification
  • Therapeutic glycoproteins

• Graduation date

  Spring 2024

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-xmjf-bk57

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.