Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts.

Microbial bioproduction of chemicals, proteins, and primary metabolites from cheap carbon sources is currently an advancing area in industrial research. The model yeast, Saccharomyces cerevisiae, is a well-established biorefinery host that has been used extensively for commercial manufacturing of bioethanol from myriad carbon sources. However, its Crabtree-positive nature often limits the use of this organism for the biosynthesis of commercial molecules that do not belong in the fermentative pathway. To avoid extensive strain engineering of S. cerevisiae for the production of metabolites other than ethanol, non-conventional yeasts can be selected as hosts based on their natural capacity to produce desired commodity chemicals. Non-conventional yeasts like Kluyveromyces marxianus, K. lactis, Yarrowia lipolytica, Pichia pastoris, Scheffersomyces stipitis, Hansenula polymorpha, and Rhodotorula toruloides have been considered as potential industrial eukaryotic hosts owing to their desirable phenotypes such as thermotolerance, assimilation of a wide range of carbon sources, as well as ability to secrete high titers of protein and lipid. However, the advanced metabolic engineering efforts in these organisms are still lacking due to the limited availability of systems and synthetic biology methods like in silico models, well-characterised genetic parts, and optimized genome engineering tools. This review provides an insight into the recent advances and challenges of systems and synthetic biology as well as metabolic engineering endeavours towards the commercial usage of non-conventional yeasts. Particularly, the approaches in emerging non-conventional yeasts for the production of enzymes, therapeutic proteins, lipids, and metabolites for commercial applications are extensively discussed here. Various attempts to address current limitations in designing novel cell factories have been highlighted that include the advances in the fields of genome-scale metabolic model reconstruction, flux balance analysis, 'omics'-data integration into models, genome-editing toolkit development, and rewiring of cellular metabolisms for desired chemical production. Additionally, the understanding of metabolic networks using C-labelling experiments as well as the utilization of metabolomics in deciphering intracellular fluxes and reactions have also been discussed here. Application of cutting-edge nuclease-based genome editing platforms like CRISPR/Cas9, and its optimization towards efficient strain engineering in non-conventional yeasts have also been described. Additionally, the impact of the advances in promising non-conventional yeasts for efficient commercial molecule synthesis has been meticulously reviewed. In the future, a cohesive approach involving systems and synthetic biology will help in widening the horizon of the use of unexplored non-conventional yeast species towards industrial biotechnology.