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首页> 外文期刊>Biotechnology Progress >Whole cell biosynthetic activity of Komagataella phaffii (Pichia pastoris) GS115 strains engineered with transgenes encoding Chromobacterium violaceum omega-transaminase alone or combined with native transketolase
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Whole cell biosynthetic activity of Komagataella phaffii (Pichia pastoris) GS115 strains engineered with transgenes encoding Chromobacterium violaceum omega-transaminase alone or combined with native transketolase

机译：Komagataella Phaffii（Pichia Pastoris）的全细胞生物合成活性（Pichia Pastoris）GS115菌株用单独编码紫茎转氨酶的转基因或与天然转铁糖酶组合的转基因或与天然铁蛋白酶组合

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摘要

Whole cell biocatalysis is an ideal tool for biotransformations that demand enzyme regeneration or robustness to fluctuating pH, osmolarity and biocontaminant load in feedstocks. The methylotrophic yeast Komagataella phaffii is an attractive alternative to Escherichia coli for whole cell biocatalysis due to its genetic tractability and capacity to grow to up to 60% wet cell weight by volume. We sought to exploit high cell density K. phaffii to intensify whole-cell chiral amino-alcohol (CAA) biosynthesis. We engineered two novel K. phaffii GS115 strains: one by inserting a Chromobacterium violaceum omega-transaminase CV2025 transgene, for strain PpTAmCV708, and a second strain, PpTAm-TK16, by also inserting the same CV2025 transgene plus a second transgene for a native transketolase. At high cell density, both strains tolerated high substrate concentrations. When fed three low cost substrates, 200 mM glycolaldehyde, 200 mM hydroxypyruvate and 150 mM methylbenzylamine, PpTAm-TK16 whole cells achieved 0.29 g L-1 hr(-1) space-time yield of the acetophenone by-product, a 49-fold increase of the highest levels reported for E. coli whole cells harboring the equivalent pathway. When fed only the low-cost substrate, 150 mM methylbenzylamine, strain PpTAmCV708 achieved a 105-fold increase of reported E. coli whole cell biocatalysis performance, with a space-time yield of 0.62 g L-1 hr(-1) of the CAA, 2-amino-1,3,4-butanetriol (ABT). The rapid growth and high biomass characteristics of K. phaffii were successfully exploited for production of ABT by whole-cell biocatalysis at higher levels than the previously achieved with E. coli in the presence of the same substrates.
机译：全细胞生物分析是一种理想的生物转化工具，需要酶再生或鲁棒性，使原料中的pH，渗透压和生物污染载荷波动。由于其遗传途径和产量高达60％的湿细胞重量，对全细胞生物分析的大肠杆菌是一种有吸引力的替代性替代物的替代品。我们寻求利用高细胞密度K. phaffii加强全细胞手性氨基 - 醇（CAA）生物合成。我们设计了两种新型K. phaffii GS115菌株：通过插入菌株PPTAMCV708和第二菌株PPTAM-TK16，通过将相同的CV2025转基因加上天然转基金酶的第二转基因插入菌株PPTAMINA酶CV2025转基因的菌株。 。在高细胞密度下，两种菌株耐受高底物浓度。当喂3个低成本底物时，200mM甘醇醛，200mM羟基吡合他分和150mM甲基苄胺，PPTAM-TK16全细胞达到苯乙酮副产品的0.29g L-1 HR（-1）时空产率，49倍增加含有等同途径的大肠杆菌全细胞的最高水平的增加。当仅喂食低成本基质，150mM甲基苄胺时，菌株PPTAMCV708达到报告的大肠杆菌全细胞生物分析性能的105倍，节目时间率为0.62g L-1 HR（-1） CAA，2-氨基-1,3,4-丁醇（ABT）。 K.Phaffii的快速生长和高生物量特征是通过在相同底物存在下的预先通过大肠杆菌的预先实现的全细胞生物分析来生产ABT的快速生长和高生物量特征。

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