酮异戊酸脱羧酶V461的饱和诱变使得能够通过酮酸延长周期特异性合成1-戊醇。,Scientific Reports

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酮异戊酸脱羧酶V461的饱和诱变使得能够通过酮酸延长周期特异性合成1-戊醇。
Scientific Reports ( IF 3.8 ) Pub Date : 2017-Sep-12 , DOI: 10.1038/s41598-017-11624-z
Grey S. Chen , Siang Wun Siao , Claire R. Shen

迭代酮酸延长一直是工程化人工代谢特别是合成醇中的必不可少的工具。然而，对产物特异性的精确控制仍然受到酮酸脱羧酶的底物混杂性的极大挑战，所述底物的混杂性与目标酮酸一起无选择地劫持了来自延伸周期的酮酸中间体。在这项工作中，通过对关键残基V461进行饱和诱变，然后筛选所得的醇谱，实现了乳酸乳球菌酮异戊酸酯脱羧酶（Kivd）对1-戊醇合成的特异性的优先调节。用低极性极性氨基酸甘氨酸或丝氨酸取代V461，可通过降低其对上游酮酸2-酮丁酸和2-酮戊酸的催化效率，显着提高其对1-戊醇前体2-酮丙酸的Kivd选择性。相反，用笨重或带电的侧链代替V461表现出严重的不利影响。通过乙酸酯进料增加迭代加成单元乙酰基-CoA的供应进一步将2-酮酸通量带入延长周期，并提高了1-戊醇的生产率。Kivd V461G变体在不使用油醇提取的情况下，使1-戊醇的生产特异性约为总醇含量的90％。这项工作增加了对Kivd活动站点选择性的见识。用笨重的或带电的侧链代替V461表现出严重的不利影响。通过乙酸酯进料增加迭代加成单元乙酰基-CoA的供应进一步将2-酮酸通量带入延长周期，并提高了1-戊醇的生产率。Kivd V461G变体在不使用油醇提取的情况下，使1-戊醇的生产特异性约为总醇含量的90％。这项工作增加了对Kivd活动站点选择性的见识。用笨重的或带电的侧链代替V461表现出严重的不利影响。通过乙酸酯进料增加迭代加成单元乙酰基-CoA的供应进一步将2-酮酸通量带入延长周期，并提高了1-戊醇的生产率。Kivd V461G变体在不使用油醇提取的情况下，使1-戊醇的生产特异性约为总醇含量的90％。这项工作增加了对Kivd活动站点选择性的见识。

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Saturated mutagenesis of ketoisovalerate decarboxylase V461 enabled specific synthesis of 1-pentanol via the ketoacid elongation cycle.

Iterative ketoacid elongation has been an essential tool in engineering artificial metabolism, in particular the synthetic alcohols. However, precise control of product specificity is still greatly challenged by the substrate promiscuity of the ketoacid decarboxylase, which unselectively hijacks ketoacid intermediates from the elongation cycle along with the target ketoacid. In this work, preferential tuning of the Lactococcus lactis ketoisovalerate decarboxylase (Kivd) specificity toward 1-pentanol synthesis was achieved via saturated mutagenesis of the key residue V461 followed by screening of the resulting alcohol spectrum. Substitution of V461 with the small and polar amino acid glycine or serine significantly improved the Kivd selectivity toward the 1-pentanol precursor 2-ketocaproate by lowering its catalytic efficiency for the upstream ketoacid 2-ketobutyrate and 2-ketovalerate. Conversely, replacing V461 with bulky or charged side chains displayed severely adverse effect. Increasing supply of the iterative addition unit acetyl-CoA by acetate feeding further drove 2-ketoacid flux into the elongation cycle and enhanced 1-pentanol productivity. The Kivd V461G variant enabled a 1-pentanol production specificity around 90% of the total alcohol content with or without oleyl alcohol extraction. This work adds insight to the selectivity of Kivd active site.

更新日期：2017-09-12

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