Chemical Engineering, School for Engineering of Matter, Transport, and Energy, Arizona State University, PO Box 876106, Tempe, Arizona 85287-6106, USA.
Biotechnol Bioeng. 2013 Jun;110(6):1726-34. doi: 10.1002/bit.24828. Epub 2013 Jan 17.
Through metabolic pathway engineering, novel microbial biocatalysts can be engineered to convert renewable resources into useful chemicals, including monomer building-blocks for bioplastics production. Here we describe the systematic engineering of Escherichia coli to produce, as individual products, two 5-carbon polyamide building blocks, namely 5-aminovalerate (AMV) and glutarate. The modular pathways were derived using "parts" from the natural lysine degradation pathway of Pseudomonas putida KT2440. Endogenous over-production of the required precursor, lysine, was first achieved through metabolic deregulation of its biosynthesis pathway by introducing feedback resistant mutants of aspartate kinase III and dihydrodipicolinate synthase. Further disruption of native lysine decarboxylase activity (by deleting cadA and ldcC) limited cadaverine by-product formation, enabling lysine production to 2.25 g/L at a glucose yield of 138 mmol/mol (18% of theoretical). Co-expression of lysine monooxygenase and 5-aminovaleramide amidohydrolase (encoded by davBA) then resulted in the production of 0.86 g/L AMV in 48 h. Finally, the additional co-expression of glutaric semialdehyde dehydrogenase and 5-aminovalerate aminotransferase (encoded by davDT) led to the production of 0.82 g/L glutarate under the same conditions. At this output, yields on glucose were 71 and 68 mmol/mol for AMV and glutarate (9.5 and 9.1% of theoretical), respectively. These findings further expand the number and diversity of polyamide monomers that can be derived directly from renewable resources.
通过代谢途径工程，可以对新型微生物生物催化剂进行工程改造，将可再生资源转化为有用的化学品，包括生产生物塑料用的单体构建块。在这里，我们描述了对大肠杆菌的系统工程改造，使其能够单独生产两种 5 碳聚酰胺构建块，即 5-氨基戊酸（AMV）和戊二酸。模块化途径是使用来自恶臭假单胞菌 KT2440 天然赖氨酸降解途径的“部件”衍生而来的。首先通过引入天冬氨酸激酶 III 和二氢二吡啶羧酸合酶的反馈抗性突变体，对其生物合成途径进行代谢去调控，从而实现必需前体赖氨酸的内源过表达。进一步破坏天然赖氨酸脱羧酶活性（通过删除 cadA 和 ldcC）限制腐胺副产物的形成，使赖氨酸产量达到 2.25 g/L，葡萄糖得率为 138 mmol/mol（18%理论值）。然后，共表达赖氨酸单加氧酶和 5-氨基戊酰胺酰胺水解酶（由 davBA 编码），导致在 48 h 内生产 0.86 g/L AMV。最后，额外共表达戊二醛半醛脱氢酶和 5-氨基戊酸氨基转移酶（由 davDT 编码），在相同条件下生产 0.82 g/L 戊二酸。在这种产量下，AMV 和戊二酸的葡萄糖得率分别为 71 和 68 mmol/mol（分别为理论值的 9.5%和 9.1%）。这些发现进一步扩展了可以直接从可再生资源中获得的聚酰胺单体的数量和多样性。
