Enzymatic hydrolysis of single-use bioplastic items by improved recombinant yeast strains.

Single-use bioplastic items pose new challenges for a circular plastics economy as they require different processing than petroleum-based plastics items. Microbial and enzymatic recycling approaches could address some of the pitfalls created by the influx of bioplastic waste. In this study, the recombinant expression of a cutinase-like-enzyme (CLE1) was improved in the yeast Saccharomyces cerevisiae to efficiently hydrolyse several commercial single-use bioplastic items constituting blends of poly(lactic acid), poly(1,4-butylene adipate-co-terephthalate), poly(butylene succinate) and mineral fillers. The hydrolysis process was optimised in controlled bioreactor configurations to deliver substantial monomer concentrations and, ultimately, 29 to 78% weight loss. Product inhibition studies and molecular docking provided insights into potential bottlenecks of the enzymatic hydrolysis process, while FT-IR analysis showed the preferential breakdown of specific polymers in blended commercial bioplastic items. This work constitutes a step towards implementing enzymatic hydrolysis as a circular economy approach for the valorisation of end-of-life single-use bioplastic items.

Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Engineered yeast for the efficient hydrolysis of polylactic acid.

Polylactic acid (PLA) is a major contributor to the global bioplastic production capacity. However, post-consumer PLA waste is not fully degraded during non-optimal traditional organic waste treatment processes and can persist in nature for many years. Efficient enzymatic hydrolysis of PLA would contribute to cleaner, more energy-efficient, environmentally friendly waste management processes. However, high costs and a lack of effective enzyme producers curtail the large-scale application of such enzymatic systems. This study reports the recombinant expression of a fungal cutinase-like enzyme (CLE1) in the yeast Saccharomyces cerevisiae, which produced a crude supernatant that efficiently hydrolyses different types of PLA materials. The codon-optimised Y294[CLEns] strain delivered the best enzyme production and hydrolysis capabilities, releasing up to 9.44 g/L lactic acid from 10 g/L PLA films with more than 40% loss in film weight. This work highlights the potential of fungal hosts producing PLA hydrolases for future commercial applications in PLA recycling.

Heterologous production of cellulose- and starch-degrading hydrolases to expand Saccharomyces cerevisiae substrate utilization: Lessons learnt.

Selected strains of Saccharomyces cerevisiae are used for commercial bioethanol production from cellulose and starch, but the high cost of exogenous enzymes for substrate hydrolysis remains a challenge. This can be addressed through consolidated bioprocessing (CBP) where S. cerevisiae strains are engineered to express recombinant glycoside hydrolases during fermentation. Looking back at numerous strategies undertaken over the past four decades to improve recombinant protein production in S. cerevisiae, it is evident that various steps in the protein production "pipeline" can be manipulated depending on the protein of interest and its anticipated application. In this review, we briefly introduce some of the strategies and highlight lessons learned with regards to improved transcription, translation, post-translational modification and protein secretion of heterologous hydrolases. We examine how host strain selection and modification, as well as enzyme compatibility, are crucial determinants for overall success. Finally, we discuss how lessons from heterologous hydrolase expression can inform modern synthetic biology and genome editing tools to provide process-ready yeast strains in future. However, it is clear that the successful expression of any particular enzyme is still unpredictable and requires a trial-and-error approach.

Evaluating and engineering Saccharomyces cerevisiae promoters for increased amylase expression and bioethanol production from raw starch.

Bioethanol production from starchy biomass via consolidated bioprocessing (CBP) will benefit from amylolytic Saccharomyces cerevisiae strains that produce high levels of recombinant amylases. This could be achieved by using strong promoters and modification thereof to improve gene expression under industrial conditions. This study evaluated eight endogenous S. cerevisiae promoters for the expression of a starch-hydrolysing α-amylase gene. A total of six of the native promoters were modified to contain a promoter-proximal intron directly downstream of the full-length promoter. Varying results were obtained; four native promoters outperformed the ENO1P benchmark under aerobic conditions and two promoters showed better expression under simulated CBP conditions. The addition of the RPS25A intron significantly improved the expression from most promoters, displaying increased transcript levels, protein concentrations and amylase activities. Raw starch-utilising strains were constructed through co-expression of selected α-amylase cassettes and a glucoamylase gene. The amylolytic strains displayed improved fermentation vigour on raw corn starch and broken rice, reaching 97% of the theoretical ethanol yield and converting 100% of the available carbon to products within 120 h in small-scale CBP fermentations on broken rice. This study showed that enhanced amylolytic strains for the conversion of raw starch to ethanol can be achieved through turnkey promoter selection and/or engineering.

Application of industrial amylolytic yeast strains for the production of bioethanol from broken rice.

Amylolytic Saccharomyces cerevisiae derivatives of Ethanol Red™ Version 1 (ER T12) and M2n (M2n T1) were assessed through enzyme assays, hydrolysis trials, electron microscopy and fermentation studies using broken rice. The heterologous enzymes hydrolysed broken rice at a similar rate compared to commercial granular starch-hydrolysing enzyme cocktail. During the fermentation of 20% dw/v broken rice, the amylolytic strains converted rice starch to ethanol in a single step and yielded high ethanol titers. The best-performing strain (ER T12) produced 93% of the theoretical ethanol yield after 96 h of consolidated bioprocessing (CBP) fermentation at 32 °C. Furthermore, the addition of commercial enzyme cocktail (10% of the recommended dosage) in combination with ER T12 did not significantly improve the maximum ethanol concentration, confirming the superior ability of ER T12 to hydrolyse raw starch. The ER T12 strain was therefore identified as an ideal candidate for the CBP of starch-rich waste streams.