In the pursuit of economic bioproduct production from renewable lignocellulosic waste sources, consolidated bioprocessing is considered essential . Significant progress has been made over the last three decades by engineering yeast and other organisms with synthetic cellulolytic and hemi-cellulolytic capabilities (reviewed in ). With the major hydrolytic activities achieved in yeast independently, the focus in recent years has shifted to combinatorial cellulolytic enzyme expression and improving substrate unitisation efficiencies [10, 16, 22], with much interest in harmonising the synergistic action of the different enzymatic activities . In this study we aimed to develop a rapid cellulase expression optimisation system in yeast, allowing selection based on the highest synergistic enzymatic activities.
The generation of expression vectors with randomized copies of CEL3A and CEL5A was achieved through a simple in vitro assembly strategy where loxP-flanked expression cassettes were combined with a suitable acceptor vector. In our focussed screen within strains with high synergistic enzymatic activity, insertion of up to four expression cassettes was achieved (strain H4, data not shown). This is similar to a previous study which reported up to five insertions for β-carotene synthesis pathway assembly, using a similar methodology . In the previous study and this work, only a single round of in vitro SCRaMbLEing was performed; the potential of combinatorial assembly of a wider range of gene cassettes or subsequent rounds of in vitro SCRaMbLE holds the promise of achieving even higher insertion rates.
To facilitate the efficient screening of the combinatorial vector library, a rapid method was developed utilising the BPNPG5 substrate to evaluate the synergistic enzyme activity of Cel3A and Cel5A. While BPNPG5 has previously been used to measure the activity of endoglucanase activity , this is to our knowledge the first time BPNPG5 has been used to assess synergistic Cel3A and Cel5A activity. The use of BPNPG5 for the measurement of synergistic Cel3A and Cel5A activity has significant advantages over the more conventional use of phosphoric acid swollen cellulose (PASC) . Firstly, the production of PASC is considerably time consuming and hazardous as it involves the use of concentrated phosphoric acid. Furthermore, PASC is generally made in-house and is not standardized between different laboratories or even between different batches, limiting the comparative interpretation that can be made between results from other groups. As opposed to PASC, which is viscous and not amenable to microplate and other more automated systems, the simple BPNPG5 assay allows the rapid screening of many strains. Additionally, all components required for the enzymatic assay are safe, and the substrate is standardized allowing direct comparisons between different laboratories.
Our modelling results showed that maximum activity occurred at a Cel3A to Cel5A activity ratio of between 40:60 and 50:50 (Fig. 4), however not all library strains displaying this ideal activity ratio had high BPNPG5 hydrolysis. Up to a limit, the more total enzymatic activity that was present, in the ideal ratio, the higher the BPNPG5 hydrolysis was (Fig. 2). This observation is in line with the basic principles of enzyme kinetics. The impact of enzyme activity levels on BPNPG5 hydrolysis was clearly shown by the comparative BPNPG5 activities of the selected “high activity” and “intermediate activity” groups. Both groups had a similar Cel3A and Cel5A activity ratio (p > 0.1), however the high activity group had significantly higher Cel3A and Cel5A activities (p < 0.01, p < 0.02 respectively) and thus represented library strains with both the ideal gene ratio and optimized enzyme levels.
Reflecting the higher enzymatic activities of the “high activity” group, significantly higher numbers of cellulase expression cassettes were detected per cell, than the corresponding “intermediate activity” group. Strains with the highest BPNPG5 activity had on average 1.3 ± 0.6 CEL3A and 0.8 ± 0.2 CEL5A cassettes per cell. Between the two selected groups, vectors with one to four cellulase cassette inserts were observed (corresponding to up to 21,158 bp), however this did not have a detectable negative impact on plasmid copy numbers, with all strains having approximately two plasmids per cell (Fig. 5). Although no significant variation in plasmid copy numbers were observed between cells containing different vector sizes in our study, it is reasonable to expect that the incorporation of more DNA, and the metabolic burden of subsequent increased protein production, could impact the overall plasmid copy number per cell . This burden might have been minimized by the unexpected, but previously observed , low episomal plasmid copy numbers per cell.
Many factors are at play that influences the optimal ratio of enzyme-encoding genes. The strategy presented here harnessing in vitro SCRaMbLE efficiently produces diverse libraries of randomized gene-copies in a standardized fashion that can be rapidly screened to uncover optimal ratios for substrates of different compositions. Up to date, rational engineering approaches to optimize enzyme ratios of cellulolytic strains have been limited due to the complexity brought about by (1) gene expression levels, (2) the specific activity and mode of action of enzymes from different origins, (3) the consortium of enzymes used and their relevant synergistic enzyme kinetics, and (4) whether genes will be integrated or maintained on expression plasmids. In a semi-rational screening approach, different optimal gene ratios were found using the cocktail δ-integration method. A S. cerevisiae strain with high activity on PASC contained 1, 13 and 6 copies of Aspergillus aculeatus CEL3A, T. reesei CEL5A and T. reesei CEL6A (Cellobiohydrolase II-encoding gene) respectively . The differences in gene copy number (compared to the results described here) are not surprising, as a CEL3A from a different origin was used (from A. aculeatus), the ratio was optimized for activity on a different substrate (PASC) and the action of an addition enzyme was used (Cel6A). Additionally, δ-integration relies on targeting gene cassettes to random retro-transposon sequences throughout the yeast genome, allowing the insertion into genomic regions where expression might be limited or actively silenced (such as near telomeres) and thus might not indicate the ‘ideal’ cassette copy number required for re-engineering purposes. While two cellulase-encoding gene cassettes were used here in this proof-of-concept study, any enzyme-encoding genes could be added to the in vitro SCRaMbLE strategy where specific synergy optimisation between activities is required. In a CBP context, other genes such as those encoding cellobiohydrolases (CBH) or other supplementary activities could be added to enable the hydrolysis of more recalcitrant cellulose substrates such as Avicel. In the interest of uncovering precise ratios for efficient activity, the use of weaker promoters could allow smaller incremental changes and an even more gradual evaluation of expressed enzyme synergistic action.