PDF(Pubmed)
Abstract：
BACKGROUND: Mycosporine-like amino acids (MAAs) are a class of strongly UV-absorbing compounds produced by cyanobacteria, algae and corals and are promising candidates for natural sunscreen components. Low MAA yields from natural sources, coupled with difficulties in culturing its native producers, have catalyzed synthetic biology-guided approaches to produce MAAs in tractable microbial hosts like Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, the MAA titres obtained in these hosts are still low, necessitating a thorough understanding of cellular factors regulating MAA production.
RESULTS: To delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells.
CONCLUSIONS: Reversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.
RESULTS: To delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells.
CONCLUSIONS: Reversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.
摘要：
背景：分枝杆菌素样氨基酸（MAAs）是一类由蓝细菌产生的强紫外线吸收化合物，藻类和珊瑚，是天然防晒成分的有希望的候选者。来自天然来源的低MAA产量，再加上培养本土生产者的困难，已经催化了合成生物学指导的方法，在易于处理的微生物宿主如大肠杆菌中生产MAAs，酿酒酵母和谷氨酸棒杆菌。然而,在这些宿主中获得的MAA滴度仍然很低，需要彻底了解调节MAA产生的细胞因子。
结果：为了描述调节MAA生产的因素，我们通过在酿酒酵母中表达来自点状Nostoc的四种MAA生物合成酶，构建了一种产生shinorine（分枝杆菌素-甘氨酸-丝氨酸）的酵母菌株。我们表明，shinorine是由磷酸戊糖途径中间的sedo庚酮糖7-磷酸（S7P）产生的，而不是像以前建议的那样来自莽草酸途径中间体3-脱氢奎因（3DHQ）。转醛缩酶（TAL1）和磷酸果糖激酶（PFK1/PFK2）基因的缺失通过独立的机制促进了S7P/shinorine的产生。出乎意料的是,PFK突变体中S7P/shinorine产生的增强并不完全是由于朝向磷酸戊糖途径的通量增加。我们提供了多条证据来支持糖酵解与非氧化戊糖磷酸途径（NOPPP）之间的逆转途径，该途径可促进磷酸果糖激酶突变细胞中S7P/shinorine的产生。
结论：逆转糖酵解和NOPPP之间的流动方向为酿酒酵母提供了一种新的代谢工程策略。
结果：为了描述调节MAA生产的因素，我们通过在酿酒酵母中表达来自点状Nostoc的四种MAA生物合成酶，构建了一种产生shinorine（分枝杆菌素-甘氨酸-丝氨酸）的酵母菌株。我们表明，shinorine是由磷酸戊糖途径中间的sedo庚酮糖7-磷酸（S7P）产生的，而不是像以前建议的那样来自莽草酸途径中间体3-脱氢奎因（3DHQ）。转醛缩酶（TAL1）和磷酸果糖激酶（PFK1/PFK2）基因的缺失通过独立的机制促进了S7P/shinorine的产生。出乎意料的是,PFK突变体中S7P/shinorine产生的增强并不完全是由于朝向磷酸戊糖途径的通量增加。我们提供了多条证据来支持糖酵解与非氧化戊糖磷酸途径（NOPPP）之间的逆转途径，该途径可促进磷酸果糖激酶突变细胞中S7P/shinorine的产生。
结论：逆转糖酵解和NOPPP之间的流动方向为酿酒酵母提供了一种新的代谢工程策略。
