New preprint on yeast bioengineering to produce modular plant hemicellulose

Despite rising interest in the engineering of polysaccharide-based biomaterials, relatively little is known about how plant hemicelluloses can be built and tailored in non-plant cell factories. Pichia pastoris is a promising host to study cellulose synthase-like A (CSLA; heteromannan synthases) and CSLC (xyloglucan synthase) activities but the functions of specific protein motifs and the impact of their products remain unclear. Here, we used a Golden Gate-based system to assemble chimeric CSL enzymes and optimize yeast cultivation to rapidly produce hemicellulose in mg scale.

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Part of Figure 1: Schematic of two plant CSLA enzymes, whose protein domains were exchanged in this study to generate a series of chimeric enzymes.

Interestingly, prolonged expression of a konjac AkCSLA3 glucomannan synthase was toxic to yeast cells, but this impairment was restored by swapping its C-terminal region with that of an Arabidopsis AtCSLA2 mannan synthase. Additional chimeras exceeded the yields of the native enzymes. Overall, cell size was increased or decreased depending on the CSLA sequences used.

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Part of Figure 2. Mannose (Man) content, representing insoluble heteromannans, is significantly increased in three of the eight chimeric enzymes tested compared to the parental controls.

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Part of Figure 5: The growth rate of the AkCSLA3 yeast strain is significantly lower than the rest. Prolonged glucomannan synthase expression also reduces cell integrity, evidenced by the uptake of the trypan blue strain (panel b), and size.

Yeast containing plant-like polysaccharides could be viewed as tunable biological capsules for further metabolic engineering studies. Sensitive macromolecules such as therapeutic proteins can be protected by encapsulation in non-toxic plant polysaccharides and Pichia cells are attractive hosts for recombinant protein production. Therefore, our strategy provides basic insight into matrix polysaccharide biosynthesis and could also be used to encapsulate valuable cargo.

Publication:

M Robert, J Waldhauer, F Stritt, B Yang, M Pauly, C Voiniciuc (2021) Rapid, modular biosynthesis of plant hemicellulose and its impact on yeast cells bioRxiv, https://doi.org/10.1101/2021.04.20.44061