Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics
Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics
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用于增强聚羟基脂肪酸酯生物塑料可持续生产的无细胞原型设计策略
DOI：
10.1101/225144
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Kelwick R
中科院分区：
文献类型：
--
作者：
Kelwick R
聚羟基脂肪酸酯（PHA）是微生物产生的生物聚合物，有可能用作石油衍生塑料的可持续替代品。然而，PHA目前的生产成本比石油衍生塑料更高。因此，更有效的生产方法将是期望的。无细胞代谢工程策略已经用于优化几种生物合成途径，我们设想无细胞策略可以用于优化PHA生物合成途径。为此，我们开发了几种无大肠杆菌细胞系统，用于体外原型PHAs生物合成操纵子，并用于筛选相关代谢物回收酶。此外，我们通过添加乳清渗透物定制了我们的无细胞反应，乳清渗透物是一种以前用于优化体内PHA生产的工业废物。我们发现，包含最佳浓度的乳清渗透物使相对无细胞GFPmut 3b产量提高了约50%。在无细胞转录-翻译原型化反应中，无细胞3-羟基丁酸（3 HB）产量的气相色谱-质谱定量显示了测试的天然ΔPhaC_C319A（1.18 ± 0.39 µM）、C104ΔPhaC_C319A（4.62 ± 1.31 µM）和C101ΔPhaC_C319A（2.65 ± 1.27 µM）phaCAB操纵子活性之间的差异。有趣的是，在体外和体内测定中，最活跃的操纵子C104比NativephaCAB操纵子产生更高水平的PHA（或PHA单体）。耦合无细胞生物转化/转录-翻译反应产生了更高的3 HB产量（32.87 ± 6.58 µM），这些反应也用于表征丙酸梭菌乙酰辅酶A再循环酶。总之，这些数据表明，无细胞的方法补充vivoworkflow用于确定优化PHA生产的其他策略。
The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimize several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimizing PHAs biosynthetic pathways. To this end, we developed severalEscherichia colicell-free systems forin vitroprototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customized our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimizein vivoPHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by approximately 50%. In cell-free transcription–translation prototyping reactions, gas chromatography–mass spectrometry quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the NativeΔPhaC_C319A (1.18 ± 0.39 µM), C104ΔPhaC_C319A (4.62 ± 1.31 µM) and C101ΔPhaC_C319A (2.65 ± 1.27 µM)phaCABoperons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the NativephaCABoperon in bothin vitroandin vivoassays. Coupled cell-free biotransformation/transcription–translation reactions produced greater yields of 3HB (32.87 ± 6.58 µM), and these reactions were also used to characterize aClostridium propionicumAcetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complementin vivoworkflows for identifying additional strategies for optimizing PHAs production.
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影响因子：
2.8
作者：
Bosco, Francesca;Chiampo, Fulvia
通讯作者：
Chiampo, Fulvia
聚（3-羟基链烷酸酯）（PHA）组合物的分析方法
DOI：
10.1002/3527600035.bpol3b01
发表时间：
2002
期刊：
Biopolymers Online
影响因子：
--
作者：
T. Rijk;P. Meer;G. Eggink;R. Weusthuis
通讯作者：
R. Weusthuis
从负责任的研究到负责任的创新：实施中的挑战
影响因子：
3.8
作者：
J. Tait
通讯作者：
J. Tait
影响因子：
8.4
作者：
Hodgman, C. Eric;Jewett, Michael C.
通讯作者：
Jewett, Michael C.
影响因子：
4.4
作者：
Ahn, WS;Park, SJ;Lee, SY
通讯作者：
Lee, SY