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首页> 外文期刊>Applied Microbiology >Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli
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Identification of Homophenylalanine Biosynthetic Genes from the Cyanobacterium Nostoc punctiforme PCC73102 and Application to Its Microbial Production by Escherichia coli

机译：点状蓝细菌PCC73102同型苯丙氨酸生物合成基因的鉴定及其在大肠杆菌生产中的应用

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l-Homophenylalanine (l-Hph) is a useful chiral building block for synthesis of several drugs, including angiotensin-converting enzyme inhibitors and the novel proteasome inhibitor carfilzomib. While the chemoenzymatic route of synthesis is fully developed, we investigated microbial production of l-Hph to explore the possibility of a more efficient and sustainable approach to l-Hph production. We hypothesized that l-Hph is synthesized from l-Phe via a mechanism homologous to 3-methyl-2-oxobutanoic acid conversion to 4-methyl-2-oxopentanoic acid during leucine biosynthesis. Based on bioinformatics analysis, we found three putative homophenylalanine biosynthesis genes, hphA (Npun_F2464), hphB (Npun_F2457), and hphCD (Npun_F2458), in the cyanobacterium Nostoc punctiforme {"type":"entrez-protein","attrs":{"text":"PCC73102","term_id":"1245706357","term_text":"PCC73102"}}PCC73102, located around the gene cluster responsible for anabaenopeptin biosynthesis. We constructed Escherichia coli strains harboring hphABCD -expressing plasmids and achieved the fermentative production of l-Hph from l-Phe. To our knowledge, this is the first identification of the genes responsible for homophenylalanine synthesis in any organism. Furthermore, to improve the low conversion efficiency of the initial strain, we optimized the expression of hphA , hphB , and hphCD , which increased the yield to ～630 mg/liter. The l-Hph biosynthesis and l-Leu biosynthesis genes from E. coli were also compared. This analysis revealed that HphB has comparatively relaxed substrate specificity and can perform the function of LeuB, but HphA and HphCD show tight substrate specificity and cannot complement the LeuA and LeuC/LeuD functions, and vice versa. Finally, the range of substrate tolerance of the l-Hph-producing strain was examined, which showed that m -fluorophenylalanine, o -fluorophenylalanine, and l-tyrosine were accepted as substrates and that the corresponding homoamino acids were generated.
机译：1-高苯丙氨酸（1-Hph）是合成几种药物的有用手性构件，包括血管紧张素转化酶抑制剂和新型蛋白酶体抑制剂卡非佐米。当化学酶的合成途径得到全面发展时，我们研究了L-Hph的微生物生产，以探索生产L-Hph的更有效和可持续方法的可能性。我们假设1-Hph是通过亮氨酸生物合成过程中与3-甲基-2-氧代丁酸转化为4-甲基-2-氧代戊酸的机制从1-Phe合成的。根据生物信息学分析，我们在蓝点点菜细菌{“ type”：“ entrez-protein”，“ attrs”：{}中发现了三个推定的高苯丙氨酸生物合成基因hphA（Npun_F2464），hphB（Npun_F2457）和hphCD（Npun_F2458）。 “ text”：“ PCC73102”，“ term_id”：“ 1245706357”，“ term_text”：“ PCC73102”}} PCC73102，位于负责鱼腥肽素生物合成的基因簇周围。我们构建了带有表达hphABCD的质粒的大肠杆菌菌株，并实现了从L-Phe发酵生产L-Hph。据我们所知，这是任何生物中高苯丙氨酸合成基因的首次鉴定。此外，为了提高初始菌株的低转化效率，我们优化了hphA，hphB和hphCD的表达，使产量提高至约630 mg / L。还比较了大肠杆菌的L-Hph生物合成和L-Leu生物合成基因。该分析表明，HphB具有相对宽松的底物特异性，并且可以执行LeuB的功能，但是HphA和HphCD显示出紧密的底物特异性，不能补充LeuA和LeuC / LeuD的功能，反之亦然。最后，检查了产生I-Hph的菌株的底物耐受性范围，这表明间-氟苯丙氨酸，邻-氟苯丙氨酸和1-酪氨酸被接受为底物，并且产生了相应的均氨基酸。

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