Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto-d-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing d-Gluconate Dehydrogenase
Screening of Thermotolerant Gluconobacter Strains for Production of 5-Keto-d-Gluconic Acid and Disruption of Flavin Adenine Dinucleotide-Containing d-Gluconate Dehydrogenase
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DOI：
10.1128/aem.00640-09
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发表时间：
2009-05
影响因子：
4.4
通讯作者：
Ittipon Saichana;D. Moonmangmee;O. Adachi;K. Matsushita;H. Toyama
中科院分区：
文献类型：
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作者：
Ittipon Saichana;D. Moonmangmee;O. Adachi;K. Matsushita;H. Toyama
摘要：我们分离出了耐热葡糖酸杆菌菌株，它们能够在37°C下产生5 - 酮 - D - 葡萄糖酸（5KGA），而在这个温度下，普通的中温产5KGA菌株的生长和5KGA产量要低得多。耐热菌株在30°C和37°C下均主要产生2 - 酮 - D - 葡萄糖酸（2KGA）作为主要产物。在37°C下产生的酮葡萄糖酸盐的量略少于在30°C下产生的量。为了提高这些菌株中5KGA的产量，我们破坏了负责2KGA生产的黄素腺嘌呤二核苷酸 - 葡萄糖酸脱氢酶（FAD - GADH）。通过反向聚合酶链式反应和体外克隆策略克隆了FAD - GADH基因。三个耐热菌株获得的序列相同，并且与氧化葡糖酸杆菌621H基因组中报道的FAD - GADH序列具有高度的一致性。使用卡那霉素抗性基因盒来破坏耐热菌株中的FAD - GADH基因。突变菌株仅产生5KGA，在30°C下最终产量超过90%，在37°C下为50%。我们发现，当细胞在37°C下生长时，在体外添加吡咯并喹啉醌（PQQ）和氯化钙会使负责5KGA生产的吡咯并喹啉醌（PQQ）依赖性甘油脱氢酶的活性增加。向突变菌株的培养基中添加5 mM氯化钙可使5KGA产量提高到初始底物转化率超过90%的程度。我们在本研究中分离出的耐热葡糖酸杆菌菌株为工业生产5KGA提供了一个有前景的新选择。
ABSTRACT We isolated thermotolerant Gluconobacter strains that are able to produce 5-keto-d-gluconic acid (5KGA) at 37°C, a temperature at which regular mesophilic 5KGA-producing strains showed much less growth and 5KGA production. The thermotolerant strains produced 2KGA as the major product at both 30 and 37°C. The amount of ketogluconates produced at 37°C was slightly less than the amount produced at 30°C. To improve the yield of 5KGA in these strains, we disrupted flavin adenine dinucleotide-gluconate dehydrogenase (FAD-GADH), which is responsible for 2KGA production. Genes for FAD-GADH were cloned by using inverse PCR and an in vitro cloning strategy. The sequences obtained for three thermotolerant strains were identical and showed high levels of identity to the FAD-GADH sequence reported for the genome of Gluconobacter oxydans 621 H. A kanamycin resistance gene cassette was used to disrupt the FAD-GADH genes in the thermotolerant strains. The mutant strains produced 5KGA exclusively, and the final yields were over 90% at 30°C and 50% at 37°C. We found that the activity of pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase, which is responsible for 5KGA production, increased in response to addition of PQQ and CaCl2 in vitro when cells were grown at 37°C. Addition of 5 mM CaCl2 to the culture medium of the mutant strains increased 5KGA production to the point where over 90% of the initial substrate was converted. The thermotolerant Gluconobacter strains that we isolated in this study provide a promising new option for industrial 5KGA production.