Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose
Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose
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DOI：
10.1128/aem.71.12.8221-8227.2005
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发表时间：
2005-12-01
影响因子：
4.4
通讯作者：
Wery, J
中科院分区：
文献类型：
--
作者：
Wierckx, NJP;Ballerstedt, H;Wery, J
在耐溶剂菌株恶臭假单胞菌 S12 中，通过中心代谢物酪氨酸实现了葡萄糖向苯酚的高效生物转化。将编码酪氨酸苯酚裂解酶的成团泛菌 tpl 基因引入恶臭假单胞菌 S12 中以实现苯酚生产。酪氨酸的可用性是高效生产的瓶颈。通过过表达aroF-1基因（编码酪氨酸生物合成途径中的第一种酶）和随机诱变程序（涉及有毒抗代谢物间氟-DL-苯丙氨酸和间氟-L-酪氨酸的选择）来优化生产宿主。对以此方式获得的类似物抗性突变体的高通量筛选产生了恶臭假单胞菌S12衍生物，其能够在摇瓶培养物中产生1.5mM苯酚，产率为6.7%(mol/mol)。在分批补料工艺中，生产率受到培养基中 5 mM 苯酚积累的限制。通过在双相介质辛醇系统中使用辛醇作为苯酚的萃取剂克服了这种毒性。这种方法导致辛醇相中积累了 58 mM 苯酚，与单相补料批次相比，总产量增加了一倍。
Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-DL-phenylalanine and m-fluoro-L-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.