Differences in the Fatty Acid Profile, Morphology, and Tetraacetylphytosphingosine-Forming Capability Between Wild-Type and Mutant Wickerhamomyces ciferrii.

Jun Young Choi,Hee Jin Hwang,Woo Yeon Cho,Pyung Cheon Lee,Jong-Il Choi

doi:10.3389/fbioe.2021.662979

Jun Young Choi, Hee Jin Hwang + Show 3 more

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https://doi.org/10.3389/fbioe.2021.662979

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Abstract

One tetraacetylphytosphingosine (TAPS)-producing Wickerhamomyces ciferrii mutant was obtained by exposing wild-type W. ciferrii to γ-ray irradiation. The mutant named 736 produced up to 9.1 g/L of TAPS (218.7 mg-TAPS/g-DCW) during batch fermentation in comparison with 1.7 g/L of TAPS (52.2 mg-TAPS/g-DCW) for the wild type. The highest production, 17.7 g/L of TAPS (259.6 mg-TAPS/g-DCW), was obtained during fed-batch fermentation by mutant 736. Fatty acid (FA) analysis revealed an altered cellular FA profile of mutant 736: decrease in C16:0 and C16:1 FA levels, and increase in C18:1 and C18:2 FA levels. Although a significant change in the cellular FA profile was observed, scanning electron micrographs showed that morphology of wild-type and mutant 736 cells was similar. Genetic alteration analysis of eight TAPS biosynthesis-related genes revealed that there are no mutations in these genes in mutant 736; however, mRNA expression analysis indicated 30% higher mRNA expression of TCS10 among the eight genes in mutant 736 than that in the wild-type. Collectively, these results imply that the enhancement of TAPS biosynthesis in mutant 736 may be a consequence of system-level genetic and physiological alterations of a complicated metabolic network. Reverse metabolic engineering based on system-level omics analysis of mutant 736 can make the mutant more suitable for commercial production of TAPS.

Highlights

Sphingolipids are a class of lipids containing a long-chain base (LCB) backbone and are essential components of eukaryotic cellular membranes (Gault et al, 2010; Börgel et al, 2012; Singh and Del Poeta, 2016)
Mutant 736 produced up to 9.1 glycerol consumed (g/L) of TAPS (218.7 mgTAPS/g-Dry cell weight (DCW)) during bioreactor fermentation at 130 g/L of glycerol with a higher conversion yield and faster growth rate when compared with the wild-type strain (1.7 g/L; 52.2 mgTAPS/g-DCW)
Fed-batch bioreactor fermentation by mutant 736 yielded even higher production of TAPS (17.7 g/L; 259.6 mgTAPS/g-DCW), which is the highest titer reported in W. ciferrii (Schorsch et al, 2012) and in a recombinant Y. lipolytica (Han et al, 2020)

Summary

Introduction

Sphingolipids are a class of lipids containing a long-chain base (LCB) backbone (known as a sphingoid base) and are essential components of eukaryotic cellular membranes (Gault et al, 2010; Börgel et al, 2012; Singh and Del Poeta, 2016). The simple structures of LCBs, including those of sphingosine, dihydrosphingosine, and phytosphingosine, form diverse sphingolipids via modifications such as phosphorylation or acylation. Aside from their key role as a component of cellular membranes, sphingolipids perform other important functions in eukaryotes, including. The rate-determining reaction step is the condensation of serine and palmitoyl-CoA by heterodimeric serine C-palmitoyltransferase (encoded by genes LCB1 and LCB2), resulting in 3-keto-sphinganine. The latter is reduced to sphinganine by 3-keto-sphinganine reductase (encoded by the TSC10 gene). Phytosphingosine is acetylated, meaning that TAPS is formed at this point by sphingoid base N/Oacetyltransferases (encoded by the genes Slli and Atf2) (Börgel et al, 2012; Schorsch et al, 2012; Ter Veld et al, 2013)

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