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.

The feasibility of using a single vector to clone a cytochrome P450 monooxygenase (P450) in different yeasts and then compare whole-cell hydroxylase activity was investigated. A broad-range yeast expression vector using the ylTEFp to drive expression of the cloned gene and the scTEFp to drive the hygromycin resistance marker gene was used to clone the genes encoding two self-sufficient P450s, CYP102A1 and CYP505A1. Both genes were cloned into Saccharomyces cerevisiae, Kluyveromyces marxianus, Yarrowia lipolytica (two strains) and Arxula adeninivorans. 4-Hexylbenzoic acid (HBA), which is subterminally hydroxylated by both CYP102A1 and CYP505A1, was used to compare whole-cell hydroxylase activity of transformants. Kluyveromyces marxianus and A. adeninivorans exhibited activity with both CYP102A1 and CYP505A1, while S. cerevisiae only displayed CYP102A1 activity and Y. lipolytica only CYP505A1 activity. The highest CYP102A1 activity (0.8 mM HBA converted in 24 h) was observed with concentrated resting-cell suspensions of S. cerevisiae. The CYP505A1 activity observed with growing cultures of A. adeninivorans was however at least 12 times higher than the CYP102A1 activity of S. cerevisiae with up to 2 mM HBA converted within 6 h. The use of K. marxianus and A. adeninivorans for P450 expression has not previously been reported.

Summary Cytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large‐scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self‐sufficient P450s for their potential as whole‐cell biocatalysts. Most P450s are, however, not self‐sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450‐reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450‐CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad‐range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole‐cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450‐based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole‐cell biocatalyst was emphasized, supporting earlier findings.

Pichia pastoris is a very popular yeast for recombinant protein production, mainly due to the strong, methanol-inducible P AOX1 promoter. Methanol induction however poses several drawbacks. One approach to improve processes is to use MutS strains with reduced methanol catabolic ability. Various reports claim that MutS allows higher recombinant protein production levels than Mut+ but scarcely elaborate on reasons for differences. In this study, enhanced green fluorescent protein was used as a P AOX1 -driven reporter for the investigation of expression differences between Mut+ and MutS strains. Mut+ exhibited higher responses to methanol, with faster growth (0.07 vs. 0.01 hr −1 ) and higher consumption of methanol (2.25 vs.1.81 mmol/g DCW .hr) and oxygen (2.2 vs. 0.66 mmol/g DCW .hr) than MutS. Mut+ yielded more biomass than MutS (2.3 vs. 1.3 g DCW /L), and carbon dioxide analysis of bioreactor off-gas suggested that considerable amounts of methanol were consumed by Mut+ via the dissimilatory pathway. In contrast, it was demonstrated that the MutS population switched to an induced state more rapidly than Mut+. In addition, MutS exhibited 3.4-fold higher fluorescence levels per cell (77,509 vs. 23,783 SFU) indicative of higher recombinant protein production. The findings were verified by similar results obtained during the expression of a lipase. Based on the differences in response to methanol versus recombinant protein production, it was proposed that higher energy availability occurs in MutS for recombinant protein synthesis, contrary to Mut+ that uses the energy to maintain high levels of methanol catabolic pathways and biomass production.

The methylotrophic yeast Komagataella (Pichia) pastoris has become one of the most utilized cell factories for the production of recombinant proteins over the last three decades. This success story is linked to its specific physiological traits, i.e., the ability to grow at high cell density in inexpensive culture medium and to secrete proteins at high yield. Exploiting methanol metabolism is at the core of most P. pastoris-based processes but comes with its own challenges. Co-feeding cultures with glycerol/sorbitol and methanol is a promising approach, which can benefit from improved understanding and prediction of metabolic response. The development of profitable processes relies on the construction and selection of efficient producing strains from less efficient ones but also depends on the ability to master the bioreactor process itself. More specifically, how a bioreactor processes could be monitored and controlled to obtain high yield of production. In this review, new perspectives are detailed regarding a multi-faceted approach to recombinant protein production processes by P. pastoris; including gaining improved understanding of the metabolic pathways involved, accounting for variations in transcriptional and translational efficiency at the single cell level and efficient monitoring and control of methanol levels at the bioreactor level.

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