Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic Engineering Communications Pub Date : 2024-05-10 DOI:10.1016/j.mec.2024.e00236
Matan Gabay , Inbar Stern , Nadya Gruzdev , Adi Cohen , Lucia Adriana-Lifshits , Tamar Ansbacher , Itamar Yadid , Maayan Gal
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Abstract

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichia coli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

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通过并行进化谷氨酸棒状杆菌直接硫化途径中的两种酶来工程化蛋氨酸辅助营养大肠杆菌,使其能够在最小培养基中复原
蛋氨酸的生物合成依赖于多种酶的顺序催化。大肠杆菌是科研和工业中用于蛋白质生产和工程的主要细菌,它利用由 L-高丝氨酸 O-琥珀酰转移酶、胱硫醚 gamma 合成酶和胱硫醚 beta 裂解酶催化的三步反式硫化途径将 L-高丝氨酸转化为 L-高半胱氨酸。然而,大多数细菌采用两步直接硫化途径,涉及 L-高丝氨酸 O-乙酰转移酶和 O-乙酰高丝氨酸巯基酶。我们以前曾发现,大肠埃希氏菌株(MG1655)缺失了编码 L-高丝氨酸 O-琥珀酰转移酶的 metA 和编码胱硫醚γ合成酶的 metB,但可以通过引入编码 L-高丝氨酸 O-琥珀酰转移酶的 metX、编码 L-高丝氨酸 O-乙酰转移酶的基因 metX 和编码 O-乙酰高丝氨酸巯基酶的基因 metY,从而改变大肠杆菌蛋氨酸生物合成代谢途径,使其直接硫化。然而,从谷氨酸棒杆菌(Corynebacterium glutamicum)中引入 metX 和 metY 无法补充蛋氨酸辅助营养。在此,我们以谷氨酸棒杆菌的 metX 和 metY 为基础生成了一个随机基因文库,并将其转化到缺乏 metA 和 metB 基因的蛋氨酸辅助营养大肠杆菌菌株中。通过多次富集循环,我们成功分离出能够在 M9 最小培养基中生长的活性克隆。在进化的蛋氨酸自养型大肠埃希菌中,显性的 metX 突变为 L315P 和 H46R。有趣的是,我们发现只编码野生型 MetY 酶 438 个氨基酸中 106 个 N 端的 metY 基因具有功能,并能支持蛋氨酸辅助营养体的生长。将新基因重新克隆到原始质粒中并转化到蛋氨酸辅助营养大肠杆菌中验证了其功能。这些结果表明,定向酶进化能够在大肠杆菌蛋氨酸直接硫化途径中快速、同步地设计出新的活性变体,从而实现高效互补。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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