Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria −42; fungi −19; plant −28; animal −22; plant and animal −1 and common P450 family −1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study’s results offer new understanding of the dynamic structural nature of P450s.
The large-scale production of recombinant proteins (rProt) is becoming increasingly economically important. Among the different hosts used for rProt production, yeasts are gaining popularity. The socalled non-conventional yeasts, such as the methylotrophic Pichia pastoris and the dimorphic Yarrowia lipolytica, are popular choices due to their favorable characteristics and well-established expression systems. Nevertheless, a direct comparison of the two systems for rProt production and secretion was lacking. This study therefore aimed to directly compare Y. lipolytica and P. pastoris for the production and secretion of lipase CalB in bioreactor. Y. lipolytica produced more than double the biomass and more than 5-fold higher extracellular lipase than P. pastoris. Furthermore, maximal CalB production levels were reached by Y. lipolytica in half the cultivation time required for maximal production by P. pastoris. Conversely, P. pastoris was found to express 7-fold higher levels of CalB mRNA. Secreted enhanced green fluorescent protein -in isolation and fused to CalB-and protease inhibitor MG-132 were used in P. pastoris to further investigate the reasons behind such discrepancy. The most likely explanation was ultimately found to be protein degradation by endoplasmic reticulum-associated protein degradation preceding successful secretion. This study highlighted the multifaceted nature of rProt production, prompting a global outlook for selection of rProt production systems.The production of recombinant proteins (rProt) at industrial scale is of increasing economic importance. Among the different microbial chassis that have been developed for that purpose, yeasts are emerging as the preferred option for the production of recombinant enzymes and therapeutic proteins. The main advantage of yeasts over bacterial systems such as Escherichia coli is the possibility to obtain post-translational modified proteins in the culture supernatant at a gram per liter scale. Historically, Saccharomyces cerevisiae has been used as the reference eukaryotic chassis, however it suffers several drawbacks such as low protein productivity, overflow metabolism and hyperglycosylation of rProt. Moreover, it is less metabolically adapted to catabolize raw carbon and nitrogen sources, which are nowadays increasingly considered as feedstocks in bioprocesses, with the intention of reducing the process costs. Currently, non-conventional yeasts such as Pichia pastoris and Yarrowia lipolytica are considered as realistic alternatives to S. cerevisiae for rProt synthesis. Both species combine the advantages of growth at high cell density and production and secretion of rProt at high yields, with low nutritional requirements, thus allowing growth on raw materials or industrial by-products 1,2 .In most cases, the processes developed for rProt production are two-step systems involving a first phase of biomass generation under repressive or non-inducing conditions, followed by an induction phase during which rProt are synthetized and secreted into the cult...
Non-conventional yeasts are efficient cell factories for the synthesis of value-added compounds such as recombinant proteins, intracellular metabolites, and/or metabolic by-products. Most bioprocess, however, are still designed to use pure, ideal sugars, especially glucose. In the quest for the development of more sustainable processes amid concerns over the future availability of resources for the ever-growing global population, the utilization of organic wastes or industrial by-products as feedstocks to support cell growth is a crucial approach. Indeed, vast amounts of industrial and commercial waste simultaneously represent an environmental burden and an important reservoir for recyclable or reusable material. These alternative feedstocks can provide microbial cell factories with the required metabolic building blocks and energy to synthesize value-added compounds, further representing a potential means of reduction of process costs as well. This review highlights recent strategies in this regard, encompassing knowledge on catabolic pathways and metabolic engineering solutions developed to endow cells with the required metabolic capabilities, and the connection of these to the synthesis of value-added compounds. This review focuses primarily, but not exclusively, on Yarrowia lipolytica as a yeast cell factory, owing to its broad range of naturally metabolizable carbon sources, together with its popularity as a non-conventional yeast.
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