The yeast Saccharomyces cerevisiae is the most intensively studied unicellular eukaryote and one of the main industrial microorganisms used in the production of biochemical. Apart from traditional applications in alcohol fermentations, baking processes and bioethanol production, S. cerevisiae is being used to produce many industrially relevant biochemical and for heterologous expression of proteins. Saccharomyces cerevisiae is a best production host for biotechnological process and there are two basic strategies for developing this production host. In the first, a suitable host can be selected from many species based on its performance regarding parameters such as product yield, productivity, and tolerance to the product or other environmental stressors (e.g., pH, temperature, salt). In many cases, targeted optimization of such a host is not possible because the tools for genetic analysis and engineering in those species are not available, leaving only evolutionary optimization or random mutagenesis to produce optimized strains. The second possible strategy is to start with a well-known species such as S. cerevisiae and optimize it for the desired product and required bioprocess conditions. Many examples for this strategy exist, but species-specific traits often hinder the development of hosts with high productivity and yields close to theoretical limits. Nevertheless, S. cerevisiae is the host of choice in many cases, due to the vast array of tools for genetic engineering and to the immense range of knowledge about all aspects of yeast biology.
Development of genome editing tools
Traditional DNA editing techniques, such as transformation and deletion of genes by homologous recombination, have been readily feasible for many years in S. cerevisiae. The use of Cre recombinase or other recombination-based approaches, like the 50:50 method, allow for marker recycling and performing delitti perfetti, leaving no foreign DNA in the yeast genome. Although such techniques are currently an important tool for synthetic biology, they are relatively time consuming and therefore not suitable for the introduction of completely heterologous metabolic pathways or deletions of several genes in a reasonably short time. In the last few years, new approaches like Zinc finger nucleases, Yeast Oligo-Mediated Genome Engineering (YOGE), transcription activator like (TAL) effector nucleases and the CRISPR-Cas system (clustered regularly interspaced short palindromic repeats) have been developed for deleting or inserting genes and for controlling gene expression. The main advantages of these tools over traditional techniques lie in their efficiency, accuracy and speed. The use of CRISPR, together with the site-specific Cas9 endonuclease, appears as the most promising tool for editing a genome at any number of different loci in a short time. Another approach make use of the high recombination efficiency of S. cerevisiae by simultaneous transformation of a recipient strain with several different integration cassettes. An alternative approach, the DNA assembler technique, is based on the in vivo recombination of overlapping DNA sequences. All genes of a pathway together with a marker were amplified by PCR with extension primers that resulted in overlapping sequences at the 3′-end of one gene and the 5′-end of the next one. The 5′-end of the first and the 3′-end of the last cassette bore sequences homologous to sequences in the chromosomal δ sites.
The most ambitious project in yeast synthetic biology is the complete de novo synthesis of all 16 chromosomes, Sc2.0. In this effort, all nonessential genes will be flanked by loxP sites, allowing for random deletion of genes upon expression of Cre recombinase and on screening for viable strains with improved characteristics for a selectable trait. Furthermore, one of the three stop codons will be eliminated from the genome in this project. In the future, an orthogonal codon could be used for the targeted incorporation of an alternative amino acid, thereby altering protein properties. Such a recoded genome will also enable the development of efficient biocontainment strategies as the free codon can be used to engineer orthogonal auxotrophies in cell factories to minimize risk in the case of accidental release and allow processes to be carried out in open bioreactors. Although this project is at its very beginning, with one synthetic chromosome completed, the consortium plans to finish all additional chromosomes until 2019. It is thus not yet clear whether replacement of all chromosomes with their synthetic analogues will be possible, but Sc2.0 will certainly provide new knowledge about the genetics of yeast and genome editing.
Development of orthogonal systems
One of the central aims of synthetic biology is to apply classical engineering principles to the development of strains. This includes the concept of orthogonality that requires a biological system to be divisible into modules that are independent from each other and can therefore be engineered individually, without consideration of other modules and with predictable outcome. In contrast, system wide approaches like systems biology and the various omics techniques teach us that virtually each part of a biological system could be responding to changes in another part, with spatial, temporal or functional causalities that are often difficult or impossible to predict with our current knowledge. Hence, absolute orthogonality may, soon at least, not be achievable for biological devices.
Approaches in this field that go beyond theoretical considerations include the synthetic yeast strain with an orthogonal codon on the DNA level, the engineering of aminoacyl-tRNA synthetises, rib regulators, and orthogonal ribosomes on the translational level, and enzymes with specificity for orthogonal co-factors like xanthosine 5′-triphosphate on the level of enzyme activity. Such studies will undoubtedly contribute considerably to the implementation of orthogonality in synthetic biological systems. Transcriptional control orthogonality is the most common and promising because it regulates the flux through a pathway and examples for transcriptional orthogonality are the estradiol inducible chimeric TF, the retinoid X receptor, and the bacterial quorum sensing TF luxR, which has not yet been tested in S. cerevisiae.
Predicting improved robustness and stress tolerance
Biotechnological processes often require strains that are tolerant to one or several stress conditions from a broad spectrum, like extreme pH, high temperature, osmotic pressure, shearing forces, organic acids and toxic substances. Most of these properties are complex traits, encoded by several genes. Basic genetic analysis methods therefore fail to characterize the underlying genetic network, and efforts to optimize one of these traits traditionally rely on adaptive evolution or breeding strategies. The possibilities of whole genome sequencing at low cost and in a comparably short time have now opened the way for the use of advanced genome analysis tools like quantitative trait loci (QTL) analysis to identify, at least under some conditions, all causative genes for a certain trait or even several different genetic combinations giving rise to the same phenotype. Extreme QTL (X-QTL) analysis and intercrops QTL (iQTL), which have recently been improved greatly, provide sensitivity and detection of even modest changes in a trait to a single gene or even nucleotide level, simultaneously covering all causal loci contributing to heritability of the trait.
The aforementioned methods enable integration of data on mutations, environmental conditions and strain efficiency. As such, they will aid in the discovery of promising combinations of genetic manipulations, strains and environmental conditions to achieve multiple engineering objectives such as yield or breeding efficiency, as well as meeting productivity, efficiency or robustness constraints. Combining the knowledge of causative gene networks and metabolic models, it is possible to predict side effects and other trade-offs associated with manipulations. The main consequence of applying integrative mathematical modelling is, and will remain, the significant speed up gained by informed manipulations comparing over the traditional trial and error approach.
Improvement of the substrate spectrum
The metabolism of S. cerevisiae is specialized for the utilization of glucose, fructose and its disaccharide sucrose. In the emerging era of bio economy, however, microbial cell factories will have to efficiently utilize more sustainable, cheaper, and generally available carbon sources, especially lignocellulose. S. cerevisiae cannot directly utilize cellulose and therefore pre-treatment is required to release glucose. The second most abundant monosaccharide in plant biomass is xylose, but the rate of xylose metabolism in currently used laboratory and industrial yeast strains is too slow to be of use in a biotechnological process, especially because of too low xylitol dehydrogenase (XDH) activity. Adaptive evolution experiments resulted in strains with increased XDH activity and significantly shorter doubling times on xylose as the sole carbon source. Moreover, several wine strains have been found that harbour in their genomes a previously unknown XDH encoding gene named XDH1, indicating that it may be possible in the future to engineer an efficient endogenous xylose utilization pathway. Still, currently the most efficient utilization of xylose as the carbon source requires introduction of heterologous pathways, most often bacterial xylose isomerase. To construct the currently most efficient pentose fermenting strain published, a cassette of 13 genes, coding for enzymes of the xylose and arabinose utilization pathways and of the pentose phosphate pathway, was inserted into the genome of an industrial strain. Together with mutagenesis, genome shuffling and evolutionary engineering, the authors obtained a strain that produced 32% more ethanol from lignocellulosic hydrolysates than the parent strain. Although at lower consumption rates than for glucose, this synthetic strain fermented xylose to ethanol with yields close to the theoretical maximum.
Since direct utilization of lignocellulosic material as a feedstock for yeast is not yet possible, thermal, chemical and/or enzymatic pre-treatments are required to separate the polymers that constitute lignocellulose and release the sugar monomers. Subsequent detoxification is often necessary to remove pre-treatment derived inhibitory substances especially acetic acid, formic acid, furan derivatives and phenolic compounds. Mechanisms conferring tolerance to such inhibitory substances can be predicted and engineered in yeast, but development in this field has until now brought only limited success, although with promising predictions for the future. Therefore, despite the lower costs of the raw materials, the second-generation biofuels are currently still more expensive than the bioethanol produced from corn or sugar cane. To make cellulosic ethanol price-competitive, and to pave the way for the use of lignocellulose as raw material for other biotechnological processes, novel solutions will be required. The most promising ones aim at so-called third generation processes, enabled by consolidated bioprocessing (CBP). CBP requires a single organism capable of biomass hydrolysis and bio-product production. In terms of scientific approaches, development of such strains calls for merging of the fields of heterologous expression of celluloses and xylose fermentation.
Enhancement of the product spectrum
The specialization of S. cerevisiae on fast fermentation of sugars is the basis for its use in the production of alcoholic beverages and biofuel and in the baking industry. At the same time, aerobic ethanol fermentation (also called the Crabtree effect) is one of the main obstacles to obtaining high yields in processes aimed at producing bulk products other than ethanol. Indeed, sustainable and cost-effective production of many commercially important metabolites cannot be achieved in S. cerevisiae as long as most of the carbon source is converted to ethanol. Hence, a stable conversion of S. cerevisiae physiology to respiration in the presence of high sugar levels, allowing efficient use of the substrate, is an important prerequisite for its use in high yield production processes.
Most attempts to eliminate the Crabtree effect in S. cerevisiae focus mainly on a reduction of the normally high glycolytic flux, because it is assumed that the degree of fermentative activity is a function of the rate of glucose catabolism. A promising approach towards this aim is deletion of the seven major hexose transporters and their replacement with a chimeric transporter. These modifications result in reduced growth rates, but increased biomass yields and absence of ethanol production at moderate glucose concentrations. Whether this strain is sufficiently robust to be used in biotechnological processes remains to be shown but its superior properties in heterologous protein production have already been demonstrated in the production of 2,3-butanediol or lactate.
Perspective: combining polygenic trait analysis with synthetic biology
Several genetic modules show that mutations in genes encoding regulatory proteins enable expression of the studied trait. Using synthetic biology tools to engineer trait specific genetic modules can thus be seen as a step towards synthetic regulatory circuits with the ability to drastically increase the productivity of yeast strains. Productive combination of genetic modules for tolerance to several stress factors will likewise remove some of the current bottlenecks in the development of new and more sophisticated cell factory based processes.
Improved substrate spectrum, enhanced product spectrum and increased stress tolerance and robustness are the main demands for the future cell factories that will be used in bio refineries and these traits are almost exclusively polygenic. Recently developed polygenic trait analysis methods, such as X-QTL and iQTL, enable identification of complete sets of causal alleles, i.e. genetic modules, for the desired traits. These traits are present in natural strains, and yeast biodiversity is therefore an attractive genetic pool for bio economy. The development of synthetic biology toolboxes, on the other hand, enables genetic modules to be inserted into platform strains. We foresee an approach in which the latest developments in complex genetics are combined with expertise in synthetic biology, with the aim of combining several genetic modules in single strains. This new approach will make it possible to combine multiple beneficial traits within a single organism, which is not possible in the current state of the art. Specific combinations of traits could result in strains custom-made for requirements of specific processes. Such cell factories should have a big potential for future bio refineries where several sources of feedstock and several different products will be used/produced within a relatively short time intervals. It is the ability to transform different molecules into pre-defined end products which makes the multi trait cell factories important within the value chain concept of bio economy. In addition, as multi trait cell factories will contain genetic modules comprising heterologous genes, the gap between biotechnological exploitation of S. cerevisiae and so-called non-conventional species will be diminished, since we can envision that some cell factories could make use of S. cerevisiae only as a chassis, whereas the specific biotechnologically relevant traits will come from several different organisms.
New technologies for the analysis of whole genomes and for large scale DNA editing have the potential to revolutionize biotechnology. The length or complexity of a pathway and the use of computational will no longer restrict the engineering of production strains and omics tools will enable more accurate prediction and prevention of undesirable side effects in the design phase. A well-developed toolbox for the analysis of yeast, both on the single gene level and in omics and systems biology techniques, is an important advantage of this organism. Combination of the recent developments in the fields of synthetic biology with polygenic trait analysis provides a means to engineer traits for increased stress tolerance and robustness, improved substrate spectrum, and enhanced product spectrum.
Dr. Pavan Kumar (R&D)