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High-level production of tetraacetyl phytosphingosine (TAPS) by combined genetic engineering of sphingoid base biosynthesis and L-serine availability in the non-conventional yeast Pichia ciferrii

Paper ID Volume ID Publish Year Pages File Format Full-Text
31586 44821 2012 13 PDF Available
Title
High-level production of tetraacetyl phytosphingosine (TAPS) by combined genetic engineering of sphingoid base biosynthesis and L-serine availability in the non-conventional yeast Pichia ciferrii
Abstract

The non-conventional yeast Pichia ciferrii is known to secrete the sphingoid long-chain base phytosphingosine in a tetraacetylated form (TAPS). Sphingolipids are important ingredients in cosmetic applications as they play important roles in human skin. Our work aimed to improve TAPS production by genetic engineering of P. ciferrii. In the first step we improved precursor availability by blocking degradation of L-serine, which is condensed with palmitoyl-CoA by serine palmitoyltransferase in the first committed step of sphingolipid biosynthesis. Successive deletion of two genes, SHM1 and SHM2, encoding L-serine hydroxymethyltransferases, and of CHA1 encoding L-serine deaminase, resulted in a strain producing 65 mg(TAPS) g−1(cdw), which is a threefold increase in comparison with the parental strain. Attempts to increase the metabolic flux into and through the L-serine biosynthesis pathway did not improve TAPS production. However, genetic engineering of the sphingolipid pathway further increased secretion of TAPS. Blocking of sphingoid long-chain base phosphorylation by deletion of the LCB kinase gene PcLCB4 resulted in a further increase in TAPS production by 78% and significant secretion of the direct precursor of phytosphingosine, sphinganin, in a triacetylated form (TriASa). Overproduction of two serine palmitoyltransferase subunits, Lcb1 and Lcb2, together with a deletion of the gene ORM12 encoding a putative negative regulator of sphingolipid synthesis resulted in a strain producing 178 mg(TAPS) g−1(cdw). Additional overproduction of the C4-hydroxylase Syr2 converting sphinganine to phytosphingosine reduced TriASa production and further improved TAPS production. The final recombinant P. ciferrii strain produced up to 199 mg(TAPS) g−1(cdw) with a maximal production rate of 8.42 mg×OD600nm−1 h−1 and a titer of about 2 g L−1, and should be applicable for industrial TAPS production.

Research highlights► A Pichia ciferrii strain for high level production of tetraacetyl phytosphingosine (TAPS) has been constructed. ► Key steps in the TAPS production pathway have been investigated. ► Serine degradation has been blocked to increase TAPS production. ► Genetic engineering of the sphingolipid pathway has been performed.

Keywords
Non-conventional yeast; Sphingolipid metabolism; Sphingoid long-chain base; Genetic engineering; Metabolic engineering; Pichia ciferrii
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High-level production of tetraacetyl phytosphingosine (TAPS) by combined genetic engineering of sphingoid base biosynthesis and L-serine availability in the non-conventional yeast Pichia ciferrii
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 14, Issue 2, March 2012, Pages 172–184
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us