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

详细信息	查看全文 | 推荐本文 |

• 作者：Christoph Schorsch^a ; ^{schorsch@bio.uni-frankfurt.de} ; Tim Kö ; hler^b ; ^{tim.koehler@evonik.com} ; Heiko Andrea^c ; ^{heiko.andrea@evonik.com} ; Eckhard Boles^a ; ^{e.boles@bio.uni-frankfurt.de}
• 关键词：Non-conventional yeast ; Sphingolipid metabolism ; Sphingoid long-chain base ; Genetic engineering ; Metabolic engineering ; Pichia ciferrii
• 刊名：Metabolic Engineering
• 出版年：2012
• 期刊代码：98_10967176
• 类别：ce
• 出版时间：March, 2012
• 卷：14
• 期：2
• 页码：172-184
• 文件大小：614 K

摘要

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^鈭?_(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^鈭?_(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^鈭?_(cdw) with a maximal production rate of 8.42 mg脳OD_600nm^鈭? h^鈭? and a titer of about 2 g L^鈭?, and should be applicable for industrial TAPS production.