Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae
Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae
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DOI：
10.1128/aem.00405-15
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发表时间：
2015-07-01
影响因子：
4.4
通讯作者：
Nielsen, Alex Toftgaard
中科院分区：
文献类型：
--
作者：
Jendresen, Christian Bille;Stahlhut, Steen Gustav;Nielsen, Alex Toftgaard
苯丙氨酸和酪氨酸氨裂解酶形成肉桂酸和对香豆酸，它们是生物技术感兴趣的广泛芳香族化合物的前体。缺乏高活性和特异性的酪氨酸氨裂解酶以前一直是代谢工程方法的限制。因此，我们确定了22个序列的电脑使用同线性信息，并针对序列的分歧。我们进行了体内比较研究，在细菌和酵母中细胞内表达基因。当产生异源，一些酶导致显着更高的生产p-香豆酸在几个不同的工业上重要的生产生物。发现了三种新的酶专门对苯丙氨酸具有活性，包括来自低GC革兰氏阳性细菌侧孢短芽孢杆菌的酶，来自盘状网囊阿米巴的细菌型酶，和来自小立碗藓的苯丙氨酸解氨酶（在使用大肠杆菌作为异源宿主的培养基中，在600 nm [OD 600]处每单位光密度产生230 μ M肉桂酸）。新的酪氨酸氨裂解酶具有更高的底物特异性比以前表征的酶也被确定。从Herpetosiphon aurantiacus和Flavobacterium johnsoniae的酶导致在大肠杆菌（在培养基中产生440 μ M p-香豆酸OD 600单位（-1））和乳酸乳球菌中p-香豆酸的高产量。这些酶在酿酒酵母中也是有效的，其中p-香豆酸的积累比表达先前表征的酪氨酸氨裂解酶的菌株提高了5倍。
Phenylalanine and tyrosine ammonia-lyases form cinnamic acid and p-coumaric acid, which are precursors of a wide range of aromatic compounds of biotechnological interest. Lack of highly active and specific tyrosine ammonia-lyases has previously been a limitation in metabolic engineering approaches. We therefore identified 22 sequences in silico using synteny information and aiming for sequence divergence. We performed a comparative in vivo study, expressing the genes intra-cellularly in bacteria and yeast. When produced heterologously, some enzymes resulted in significantly higher production of p-coumaric acid in several different industrially important production organisms. Three novel enzymes were found to have activity exclusively for phenylalanine, including an enzyme from the low-GC Gram-positive bacterium Brevibacillus laterosporus, a bacterial-type enzyme from the amoeba Dictyostelium discoideum, and a phenylalanine ammonia-lyase from the moss Physcomitrella patens (producing 230 mu M cinnamic acid per unit of optical density at 600 nm [OD600]) in the medium using Escherichia coli as the heterologous host). Novel tyrosine ammonia-lyases having higher reported substrate specificity than previously characterized enzymes were also identified. Enzymes from Herpetosiphon aurantiacus and Flavobacterium johnsoniae resulted in high production of p-coumaric acid in Escherichia coli (producing 440 mu M p-coumaric acid OD600 unit(-1) in the medium) and in Lactococcus lactis. The enzymes were also efficient in Saccharomyces cerevisiae, where p-coumaric acid accumulation was improved 5-fold over that in strains expressing previously characterized tyrosine ammonia-lyases.