Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa, United States of America; Center for Metabolic Biology, Iowa State University, Ames, Iowa, United States of America.
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa, United States of America; Center for Metabolic Biology, Iowa State University, Ames, Iowa, United States of America; Center for Biorenewable Chemicals, Iowa State University, Ames, Iowa, United States of America.
PLoS One. 2014 May 19;9(5):e96621. doi: 10.1371/journal.pone.0096621. eCollection 2014.
This study explored the potential of using Rhodosprillum rubrum as the biological vehicle to convert chemically simple carbon precursors to a value-added bio-based product, the biopolymer PHA. R. rubrum strains were bioengineered to overexpress individually or in various combinations, six PHA biosynthetic genes (phaC1, phaA, phaB, phaC2, phaC3, and phaJ), and the resulting nine over-expressing strains were evaluated to assess the effect on PHA content, and the effect on growth. These experiments were designed to genetically evaluate: 1) the role of each apparently redundant PHA polymerase in determining PHA productivity; 2) identify the key gene(s) within the pha biosynthetic operon that determines PHA productivity; and 3) the role of phaJ to support PHA productivity. The result of overexpressing each PHA polymerase-encoding gene indicates that phaC1 and phaC2 are significant contributors to PHA productivity, whereas phaC3 has little effect. Similarly, over-expressing individually or in combination the three PHA biosynthesis genes located in the pha operon indicates that phaB is the key determinant of PHA productivity. Finally, analogous experiments indicate that phaJ does not contribute significantly to PHA productivity. These bioengineering strains achieved PHA productivity of up to 30% of dry biomass, which is approximately 2.5-fold higher than the non-engineered control strain, indicating the feasibility of using this approach to produce value added bio-based products.
本研究探讨了利用红硫菌（Rhodosprillum rubrum）作为生物载体，将化学结构简单的碳前体转化为增值的生物基产品——聚羟基脂肪酸酯（PHA）的潜力。通过生物工程手段，过量表达了六个 PHA 生物合成基因（phaC1、phaA、phaB、phaC2、phaC3 和 phaJ），分别或组合过量表达，共构建了 9 株工程菌，并对其进行了评价，以评估对 PHA 含量的影响和对生长的影响。这些实验旨在从遗传学角度评估：1）每个似乎冗余的 PHA 聚合酶在决定 PHA 生产力中的作用；2）确定决定 PHA 生产力的pha 生物合成操纵子内的关键基因；3）phaJ 对支持 PHA 生产力的作用。过量表达每个 PHA 聚合酶编码基因的结果表明，phaC1 和 phaC2 对 PHA 生产力有重要贡献，而 phaC3 影响较小。同样，单独或组合过量表达位于 pha 操纵子中的三个 PHA 生物合成基因表明，phaB 是 PHA 生产力的关键决定因素。最后，类似的实验表明，phaJ 对 PHA 生产力没有显著贡献。这些工程菌实现了高达 30%干重生物量的 PHA 生产力，比未经工程改造的对照菌株高出约 2.5 倍，表明了利用这种方法生产增值生物基产品的可行性。
