The study of Comamonas sp. L8U biosynthesized homopolymer poly(3-hydroxyvalerate)

• Main Authors: KUO, DING-CHUAN, 郭丁傳
• Other Authors: SHEU, DER-SHYAN
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/2eex8h

碩士 === 國立高雄海洋科技大學 === 海洋生物技術研究所 === 105 === Comamonas sp. L8U, a wild type bacterium isolated from soil in Taiwan. It grow and biosynthesized PHA under pH from 7.2 to 8.0. The optimal growth temperature is at 30oC (0.95 g/L of PHA yield). Levulinic acid is a renewable carbon source and can be transformed from cellulose catalyzed by acid. Cellulose is the component of agricultural wastes such as straws and bangasse. Levulinic acid is toxic for many bacteria generally. Comamonas sp. L8U accumulate high content of PHA from levulinic acid as sole carbon source. The polymer consisted of 96 mol% 3-hydroxyvalerate (3HV) and 4 mol% 3-hydroxybutyrate (3HB). In addition, Comamonas sp. L8U is able to grow from concentration 0.2% to 1.2% of levulinate (peaked at 0.8%). The molecular weight of thepolymer is about 2.88 × 106 Da analyzed by gel permeation chromatography (GPC). NMR spectrometry result exhibited the PHA structure consisted of 3HB and 3HV monomer. Comamonas sp. L8U biosyntheisized a high PHA content (78 dr wt%) and a homopolymer polyhydroxyvalerate in a ferrous ion free condition. Comamonas sp. L8U accumulated a 89 dry wt% PHA in a batch fermentation. The biomass reached 102 g/L from levulinate in a fed-batch fermentation. Comamonas sp. L8U possessed two PHA operons consisted of phaC1-phaA-phaB and phaC2-phaJ-ORF-pta-ack genes. The PhaC1 was the main PHA synthase of Comamonas sp. L8U for PHA polymerization. Deletion of phaC1 gene resulted in Comamonas sp. L8U accumulated no PHA and no influence while phaC2 gene was deleted. In addition, the phaB gene corresponds with 3-hydroxybutyryl-CoA providing. Deletion of phaB gene, Comamonas sp. L8U accumulated poly(3-hydroxyvalerate) polymer without 3HB monomer. Deletion of other genes in phaC2 operon did not impact the PHA accumulation. Insertion mutation with miniTn5 found 4 mutants accumulated respectively lower PHA content than wild type (M23, M30, M52, M66). The insertion site of the mutants M23, M30, M52, M66 was phaC1, molecular chaperone gene dnaK, MFS transporter gene and modulator of DNA gyrase, respectively.