Cell wall and cell membrane defects contribute to reduced cell growth at 28 °C in Phaffia rhodozyma mutant strain MK19
Authors: Miao Lili , Chi Shuang , Hou Ting-Ting , Liu Zhi-Pei , Li Ying 
Published: August 2020

Background
The yeast Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The optimal temperature range for cell growth and astaxanthin synthesis in this species is low (17–21 °C), which presents an obstacle to efficient industrial-scale astaxanthin production. The molecular regulatory mechanisms of cell growth and astaxanthin synthesis in P. rhodozyma remain poorly understood, and the regulatory effects of temperature have not been investigated.
Results
MK19, a mutant P. rhodozyma strain that grows well at moderate temperatures, was generated from wild-type (WT) strain JCM9042, which is unable to grow at 28 °C. MK19 cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and biomass production reached 6 g/L after 100 h culture. Transcriptome analysis indicated that low viability at 28 °C resulted from strong suppression of DNA synthesis. Growth inhibition at 28 °C was due to low mRNA content of fatty acid (f.a.) pathway genes (acc, fas1, fas2), and consequent low f.a. content. Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C. In MK19 cultured for 48 h at 28 °C, levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. Downregulation of crtI mRNA, whose product catalyzes four dehydrogenation steps in the phytoene-to-lycopene process, contributed to inhibition of astaxanthin synthesis in WT at 25 °C. High crtI mRNA content in MK19 (> 10-fold higher than that in WT) counteracted the inhibitory effect of high temperature on crtI transcription, making possible the efficient synthesis of lycopene which was subsequently converted to astaxanthin.
Conclusion
Inhibition of growth of WT P. rhodozyma at 28 °C results from blocking of DNA, f.a., and cell wall biosynthesis. Growth at this temperature could be increased by enhancement of such biosynthesis. In moderate-temperature strain MK19, precursor molecules accumulated from f.a. pathway are diverted to astaxanthin and ergosterol biosynthesis, making possible biomass production 6 g/L at 28 °C. Strengthening of the MVA pathway to make available more precursors from f.a. pathway is a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in MK19 or other moderate-temperature producer strains. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of transcription regulatory networks and feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.