PHB production from cellobiose with Saccharomyces cerevisiae

Abstract

Replacement of petrochemical-based materials with microbially produced biodegradable alternatives calls for industrially attractive fermentation processes. Lignocellulosic materials offer non-edible alternatives for cultivated sugars, but require often use of expensive sugar releasing enzymes, such as β-glucosidases. These cellulose treatment costs could be reduced if microbial production hosts could use short cellodextrins such as cellobiose directly as their substrates. In this study, we demonstrate production of poly(hydroxybutyrate) (PHB) in yeast Saccharomyces cerevisiae using cellobiose as a sole carbon source. Yeast strains expressing PHB pathway genes from Cupriavidus necator and cellodextrin transporter gene CDT-1 from Neurospora crassa were complemented either with β-glucosidase gene GH1-1 from N. crassa or with cellobiose phosphorylase gene cbp from Ruminococcus flavefaciens. These cellobiose utilization routes either with Gh1-1 or Cbp enzymes differ in energetics and dynamics. However, both routes enabled higher PHB production per consumed sugar and higher PHB accumulation % of cell dry weight (CDW) than use of glucose as a carbon source. As expected, the strains with Gh1-1 consumed cellobiose faster than the strains with Cbp, both in flask and bioreactor batch cultures. In shake flasks, higher final PHB accumulation % of CDW was reached with Cbp route (10.0 ± 0.3%) than with Gh1-1 route (8.1 ± 0.2%). However, a higher PHB accumulation was achieved in better aerated and pH-controlled bioreactors, in comparison to shake flasks, and the relative performance of strains switched. In bioreactors, notable PHB accumulation levels per CDW of 13.4 ± 0.9% and 18.5 ± 3.9% were achieved with Cbp and Gh1-1 routes, respectively. The average molecular weights of accumulated PHB were similar using both routes; approximately 500 kDa and 450 kDa for strains expressing either cbp or GH1-1 genes, respectively. The formation of PHB with high molecular weights, combined with efficient cellobiose conversion, demonstrates a highly potential solution for improving attractiveness of sustainable polymer production using microbial cells.

Co-utilization of glucose and xylose for the production of poly-β-hydroxybutyrate (PHB) by Sphingomonas sanxanigenens NX02

“…Then 1.0 ml of fermentation broth samples were added into the weighed tubes and centrifuged at 12,000 rpm for 2 min. Tubes with samples were dried for 24 h at 60 °C and weighed [ 50 ].…”

“…Each sample was subjected to methanolysis by heating at 100 ℃ for 300 min in a solution containing 1.0 ml chloroform, 30 µl sulfuric acid, 2 µl internal standard (benzoic acid), and 968 µl methanol. In addition, PHB purchased was treated similarly as a reference sample [ 50 ]. After the samples were cooled to room temperature, 1.0 ml aliquot of distilled water was added to the methyl ester solution and the mixtures were vortexed for 5 min.…”

Saccharomyces cerevisiae cell surface display technology: Strategies for improvement and applications

“…2) Glucose-induced carbon catabolite repression affects the co-utilization of other sugars. Reconstitution of the cellodextrin transport system in S. cerevisiae has been reported ( Galazka Jonathan et al, 2010 ; Choi et al, 2022 ; Ylinen et al, 2022 ). This system promotes the efficient utilization of cellulose and mitigates the inhibitory effect of glucose ( Fan et al, 2016 ; Li et al, 2017 ).…”

Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model

“…Partial cellulose hydrolysis and intracellular assimilation of resulting oligomers emerge as an alternative strategy to standard hydrolysis protocols aiming at maximized glucose yields (Oh and Jin, 2020;Parisutham et al, 2017). The adoption of microorganisms capable of efficient processing of both glucose and cellooligosaccharides is thus a rational option that can reduce the capital costs of lignocellulose biotechnologies (Parisutham et al, 2017;Taha et al, 2016;Ylinen et al, 2022;Zhang et al, 2022). In our J o u r n a l P r e -p r o o f previous work, we enriched the EM42 strain with bglC gene which encodes cytoplasmic glucosidase from Thermobifida fusca (Dvořák and de Lorenzo, 2018).…”