Xie Dongming, Shao Zengyi, Achkar Jihane, Zha Wenjuan, Frost John W, Zhao Huimin
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Biotechnol Bioeng. 2006 Mar 5;93(4):727-36. doi: 10.1002/bit.20759.
Native g2ps1-encoded 2-pyrone synthase (2-PS) from Gerbera hybrida, a mutant Brevibacterium ammoniagenes fatty acid synthase B (FAS-B) and two different mutants of Penicillium patulum 6-methylsalycilic acid synthase (6-MSAS) are examined to identify the best enzyme to recruit for the microbial synthesis of triacetic acid lactone (TAL). To identify the best microbial host for these evaluations, the native TAL-synthesizing activity of g2ps1-encoded 2-PS is expressed in recombinant Escherichia coli and Saccharomyces cerevisiae constructs. Five-fold higher expression levels of 2-PS are observed in S. cerevisiae. Consequently, microbial synthesis of TAL focuses on S. cerevisiae constructs. Comparison of different promoters for the expression of g2ps1 in S. cerevisiae indicates that the alcohol dehydrogenase II promoter (P(ADH2)) affords the highest expression levels of 2-PS. As a result, the genes encoding the various TAL-synthesizing enzyme activities are expressed in S. cerevisiae from a P(ADH2) promoter. To extend TAL-synthesizing activity beyond g2ps1-encoded 2-PS, the ketoreductase domains of fasB-encoded FAS-B and 6-MSAS-encoded 6-MSAS are modified using a single mutation. Modification of the nicotinamide cofactor-binding site of 6-MSAS with a triple mutation is also examined. Separate S. cerevisiae constructs expressing native g2ps1, mutant Y2226F fasB, mutant Y1572F 6-MSAS, and mutant G1419A-G1421P-G1424A 6-MSAS are cultured under the same fermentor-controlled conditions. The highest concentration (1.8 g/L) and yield (6%) of TAL are synthesized from glucose by S. cerevisiae expressing the Y1572F mutant of 6-MSAS.
对来自非洲菊的天然g2ps1编码的2-吡喃酮合酶（2-PS）、突变型产氨短杆菌脂肪酸合酶B（FAS-B）以及两种不同的展青霉6-甲基水杨酸合酶（6-MSAS）突变体进行了研究，以确定用于微生物合成三乙酸内酯（TAL）的最佳酶。为了确定进行这些评估的最佳微生物宿主，在重组大肠杆菌和酿酒酵母构建体中表达了g2ps1编码的2-PS的天然TAL合成活性。在酿酒酵母中观察到2-PS的表达水平高五倍。因此，TAL的微生物合成集中在酿酒酵母构建体上。对酿酒酵母中g2ps1表达的不同启动子进行比较表明，乙醇脱氢酶II启动子（P(ADH2)）可使2-PS的表达水平最高。结果，编码各种TAL合成酶活性的基因在酿酒酵母中由P(ADH2)启动子表达。为了将TAL合成活性扩展到g2ps1编码的2-PS之外，使用单一突变对fasB编码的FAS-B和6-MSAS编码的6-MSAS的酮还原酶结构域进行了修饰。还研究了用三重突变对6-MSAS的烟酰胺辅因子结合位点进行修饰。分别表达天然g2ps1、突变体Y2226F fasB、突变体Y1572F 6-MSAS和突变体G1419A-G1421P-G1424A 6-MSAS的酿酒酵母构建体在相同的发酵罐控制条件下培养。表达6-MSAS的Y1572F突变体的酿酒酵母从葡萄糖合成的TAL浓度最高（1.8 g/L），产量最高（6%）。
