An enhanced electron transport chain improved astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a carotenoid pigment with antioxidant properties widely used as a feed supplement. Wild-type strains of Phaffia rhodozyma naturally produce low AX yields, but we increased AX yields 50-fold in previous research using random mutagenesis of P. rhodozyma CBS6938 and fermentation optimization. On that study, genome changes were linked with phenotype, but relevant metabolic changes were not resolved. In this study, the wild-type and the superior P. rhodozyma mutant strains were grown in chemically defined media and instrumented fermenters. Differential kinetic, metabolomics, and transcriptomics data were collected. Our results suggest that carotenoid production was mainly associated with cell growth and had a positive regulation of central carbon metabolism metabolites, amino acids, and fatty acids. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites were in abundance and media supplementation of citrate, malate, α-ketoglutarate, succinate, or fumarate increased AX production in the mutant strain. Transcriptomic data correlated with the metabolic and genomic data and found a positive regulation of genes associated with the electron transport chain suggesting this to be the main driver for improved AX production in the mutant strain.

Towards commercial levels of astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a potent antioxidant with increasing biotechnological and commercial potential as a feed supplement, and gives salmonids and crustaceans their attractive characteristic pink color. The red yeast Phaffia rhodozyma naturally produces AX as its main fermentation product but wild-type strains and those previously generated through classical random mutagenesis produce low yields of AX. Existing strains do not meet commercial economic requirements, fundamentally due to a lack of understanding of the underlying mechanisms and genotype-phenotype associations regarding AX production in P. rhodozyma. In the present study, screening of P. rhodozyma CBS 6938 mutant strains generated through chemical and ultra violet radiation mutagenesis delivered increased AX production yields that were then maximized using culture media optimization and fed-batch culture kinetic modeling. The whole genomes of the wild-type and eight increased production strains were sequenced to identify genomic changes. The selected strains produced 50-fold more AX than the wild-type strain with a total biomass of around 100 gDCW/L and a carotenoid production of 1 g/L. Genomic variant analyses found 368 conserved mutations across the selected strains with important mutations found in protein coding regions associated with regulators and catalysts of AX precursors in the mevalonate pathway, the electron transport chain, oxidative stress mechanisms, and carotenogenesis.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains. RESULTS: Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter. DISCUSSION: The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.