重组嗜水气单胞菌Aeromonas hydrophila 4AK4中3-羟基丁酸3-羟基己酸共聚酯PHBHHx的发酵生产

3-羟基丁酸和3-羟基己酸共聚酯（PHBHHx）是一种性能优良的新型生物可降解材料,其机械和加工性能与3-羟基己酸（3HHx）在共聚物中的含量密切相关。在嗜水气单孢菌Aeromonas hydrophila 4AK4中引入了编码β酮基硫解酶 (β-ketothiolase)的phbA基因和编码乙酰乙酰辅酶A还原酶(AcetoacetylCoA reductase)的phbB基因,使重组菌增加了一条利用乙酰辅酶A合成3羟基丁酸-CoA的代谢途径,这使得利用非相关性碳源调控PHBHHx的单体组成比例成为可能。利用葡萄糖酸钠和月桂酸作为碳源,对重组Aeromonas hydrophila 4AK4进行了摇瓶培养及5 L发酵罐培养的研究。在摇瓶实验中,通过改变碳源中两种组分的比例,可以使A. hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量由原来的15%左右降低到3%～12 %,成功地实现了对PHBHHx单体组成的调控;当以月桂酸为唯一碳源时,在5 L发酵罐中,经过56 h的培养,获得了51.5 g/L的细胞干重（CDW）,其中62 %为PHBHHx,3HHx在PHBHHx中的摩尔含量为9.7 %;当以1:1的葡萄糖酸钠和月桂酸为碳源时,48 h的5 L发酵罐培养获得了32.8 g/L的CDW和52 %的PHBHHx含量,其中3HHx在PHBHHx中的摩尔含量为6.7 %。结果证明了该重组菌在大规模生产单体组成可控PHBHHx方面具有很大的应用潜力。     

代谢工程技术调控3-羟基丁酸与3-羟基己酸共聚酯PHBHHx的微生物合成

3-羟基丁酸和3-羟基己酸共聚酯(PHBHHx)是一种新型生物可降解材料,其性能与3-羟基己酸(3HHx)在共聚物中的摩尔百分含量密切相关。本研究在两株嗜水性气单孢菌Aeromonas hydrophila WQ和Aeromonas hydrophila 4AK4中分别引入了编码酯酰辅酶A脱氢酶的yafH基因和编码合成3-羟基丁酸-CoA的phbA和phbB基因,将A.hydrophila WQ合成的PHBHHx中的3HHx的摩尔含量由3％—5％提高到20％以上;而A.hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量则由15％左右降低到3％-12％。成功地实现了对PHBHHx单体组成的调控。     

丁醇对发酵生产3-羟基丁酸与3-羟基己酸共聚酯(PHBHHx)单体组成的影响

3羟基丁酸与3羟基己酸共聚酯(PHBHHx)是由微生物合成的完全可降解高分子材料,其材料性能与3羟基己酸(3HHx)在共聚物中的含量有关。嗜水性气单孢菌A.hydrophila4AK4合成的PHBHHx中,3HHx含量通常都在12～15mol%之间。通过在培养基中添加正丁醇,降低了PHBHHx中3HHx的含量。在摇瓶培养中获得了含3HHx为58mol%的PHBHHx;在6L发酵罐中54h的发酵培养,获得40gL的细胞干重(CDW),并将3HHx的含量在发酵过程中有效地降低到5～10mol%。     

嗜水气单胞菌合成含3-羟基戊酸单体的聚羟基脂肪酸共聚酯的研究

分别利用葡萄糖或葡萄糖酸钠与十一碳酸、月桂酸与十一碳酸为混合碳源进行嗜水气单孢菌 (Aeromonashydrophila)菌株 4AK4的摇瓶培养 ,实现了含有 3 羟基戊酸 (3HV)单体的聚羟基脂肪酸酯的微生物合成。当使用葡萄糖或葡萄糖酸钠与十一碳酸为混合碳源时 ,野生型A .hydrophila 4AK4及含有 3 羟基丁酸辅酶A合成基因phaA和phaB的重组A .hydrophila 4AK4 (pTG01)能够合成-3-羟基丁酸(3HB)与-3HV的共聚物 ,且葡萄糖或葡萄糖酸钠与十一碳酸比例为 1∶1时最利于细胞生长和PHA的积累。当使用月桂酸和十一碳酸为混合碳源时 ,A .hydrophila4AK4能够合成-3HB、3HV与 β-羟基己酸 (3HHx)的共聚物 ,且随着混合碳源中十一碳酸的含量增加 ,A .hydrophila4AK4合成的PHA中-3HV的比例增加 ,而-3HB和-3HHx的比例降低.     

嗜水气单胞菌WQ中PHBHHx的合成及其分子基础研究

聚羟基脂肪酸酯(Polyhydroxyalkanoate,PHA)是一系列生物合成的高分子材料,其单体可由多种3-羟基脂肪酸(3-hydroxyalkanoate,3HA)构成^[1]。PHA物理和机械性能的变化很大,从高脆性到弹性体,这跟它们的单体成分有很大关系^[2]。短链和中长链单体共聚的PHA比短链单体或中长链单体聚合得到的PHA有着更好的性能^[3]。在1994年,豚鼠气单胞菌(Aeromonas caviae)FA440被发现能以偶数碳原子数脂肪酸或植物油作为碳源在体内积累PHBHHx^[4]其PHA生物合成基因被成功克隆^[5]。根据亚基数目和底物特异性,PHA合成的关键酶,即PHA合酶或PhaC,被分成了3种类型。A．caviae的PHA合酶属于第1类PHA合酶^[6]。PHA合酶的一些类型含有一些保守的基因序列,该特征可被用于克隆,特别是第Ⅱ类PHA合酶^[2,8]。嗜水气单胞菌(Aeromonas hydrophila)WQ和A．hydrophila 4AK4是能够合成PHBHHx的另外两种菌株,其中A．hydrophila 4AK4已被用作大规模生产PHBHHx。就目前来说,不管生长条件怎么改变,其合成的PHBHHx中3羟基己酸单体(3-hydroxyhexanoate,3HHx)的含量始终在12％～17％之间变化^[9]。而A．hydrophila WQ合成的PHBHHx中则含有6％～14％ 3HHx。本论文研究了A．hydrophila WQ的PHA生物合成及其分子基础。     

高效启动子在微生物生产4-羟基丁酸中的应用

4-羟基丁酸(4HB)是一种精神类药物,还可用于合成聚-4-羟基丁酸酯(P4HB)、聚(3-羟基丁酸酯-co-4-羟基丁酸酯)(P3HB4HB)等聚合物。在醇脱氢酶(DhaT)和醛脱氢酶(AldD)的共同作用下,1,4-丁二醇(BD)可转化为4-羟基丁酸。通过引入T7和PRe两种高效启动子,加强了dhaT和aldD基因的表达,促进合成4-羟基丁酸的反应进行。同时还研究了底物1,4-丁二醇的浓度对4HB生产的影响。结果表明:提供10 g/L的1,4-丁二醇,受PRe启动子调控的重组菌A.hydrophila 4AK4(pZQ01)可生产6.00 g/L的4-羟基丁酸,比对照组提高43.20%;而受T7启动子调控的重组菌A.hydrophila 4AK4(pZQ04)可生产4.87 g/L 4-羟基丁酸,比对照组提高16.23%。意味着T7和PRe这两种启动子确实发挥了提高基因表达水平的作用,加速了4-羟基丁酸的生物合成。     

3-羟基丁酸与3-羟基己酸共聚酯在心血管组织工程中的应用

为了研究可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯 (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)的血管内生物相容性, 采用脱细胞羊肺动脉为支架, 以PHBHHx涂层, 构建复合补片(Hybrid patch), 植入New Zealand兔腹主动脉内(12只), 以脱细胞未涂层羊肺动脉片(Uncoated patch)做为对照(12只)。分别于术后第1、4和12周处死动物, 取出移植补片进行组织学、免疫荧光染色、扫描电镜和钙含量测定。结果表明: hybrid patch管腔面光滑无血栓, 内膜增生适度, 再细胞化完全; 免疫荧光染色检测, 新生内膜组织中类内皮细胞呈CD31阳性反应, 单层连续排列, 间质细胞呈现SMA阳性反应; 钙含量测定, hybrid patch明显低于uncoated patch(P<0.05)。由此认为: PHBHHx的血管内生物相容性满意, 是心血管组织工程较为理想的腔内涂层材料。     

3-羟基丁酸与3-羟基己酸共聚酯在心血管组织工程中的应用

为了研究可降解聚合材料3-羟基丁酸与3-羟基己酸共聚酯 (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)的血管内生物相容性, 采用脱细胞羊肺动脉为支架, 以PHBHHx涂层, 构建复合补片(Hybrid patch), 植入New Zealand兔腹主动脉内(12只), 以脱细胞未涂层羊肺动脉片(Uncoated patch)做为对照(12只)。分别于术后第1、4和12周处死动物, 取出移植补片进行组织学、免疫荧光染色、扫描电镜和钙含量测定。结果表明: hybrid patch管腔面光滑无血栓, 内膜增生适度, 再细胞化完全; 免疫荧光染色检测, 新生内膜组织中类内皮细胞呈CD31阳性反应, 单层连续排列, 间质细胞呈现SMA阳性反应; 钙含量测定, hybrid patch明显低于uncoated patch(P<0.05)。由此认为: PHBHHx的血管内生物相容性满意, 是心血管组织工程较为理想的腔内涂层材料。     

大肠杆菌利用葡萄糖和木糖合成乙醇酸、乳酸和3-羟基丁酸共聚酯

以大肠杆菌为宿主,构建了以葡萄糖和木糖为底物获得乙醇酸、乳酸和3-羟基丁酸共聚酯的生物合成途径,包括过表达塔格糖-3-差向异构酶、核酮糖激酶、醛缩酶、醛脱氢酶、丙酰辅酶A转移酶、β-酮硫解酶、乙酰乙酰辅酶A还原酶和聚合酶等。在此基础上,表达聚羟基脂肪酸酯颗粒结合蛋白,提高了聚合物的合成,重组菌的细胞干重达到3.73g/L,含有38.72wt%的共聚酯。采用混菌共培养策略,实现以葡萄糖和木糖混合物为底物合成共聚酯,摇瓶实验中细胞干重达到4.01g/L,含有21.54wt%的聚合物。文中提供了一种以葡萄糖和木糖混合物为碳源合成聚合物的方法,为下一步纤维素水解物的有效利用提供了参考。     

羰基还原酶不对称还原（R）-6-氰基-5-羟基-3-羰基己酸叔丁酯

选择R-羰基还原酶和葡萄糖脱氢酶双酶,协同催化（R）-6-氰基5-羟基-3-羰基己酸叔丁酯不对称还原制备阿托伐他汀关键手性合成子6-氰基-（3R,5R）-二羟基已酸叔丁酯。转化条件优化结果显示：在不添加外源性辅酶NADP（H）、菌体用量15．0g/L、147．0g/L（R）-6-氰基-5-羟基-3-羰基己酸叔丁酯、128．2g／L葡萄糖,30℃、pH6．5条件下反应6h后,底物转化率达到100％,产物d．e．值大于99．5％。     

Enhanced production of D-(-)-3-hydroxybutyric acid by recombinant Escherichia coli

Wild-type bacteria including Escherichia coli normally do not produce extracellular D-(-)-3-hydroxybutyric acid (3HB). To produce extracellular chiral 3HB, a new pathway for synthesis of 3HB was constructed by simultaneous expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB), phosphor-transbutyrylase (ptb) and butyrate kinase (buk) in E. coli strain DH5alpha. E. coli DH5alpha containing any one of the four plasmids pBHR69, pUCAB, p68CM or pKKAB that harbor the phbA and phbB genes produced small amounts of 3HB, ranging from 75 to 400 mg l(-1), while E. coli DH5alpha harboring p68CMPTK containing genes of phbA, phbB, ptb and buk increased the 3HB concentration to 1.4 g l(-1) in shake flasks supplemented with LB broth and 20 g l(-1) glucose. 3HB production was further improved to over 2 g l(-1) in shake flasks when E. coli DH5alpha hosted two plasmids simultaneously that separately contained phbA and phbB in one plasmid while ptb and buk in the other. A batch fermentation run in a 5-l fermenter produced approximately 5 g l(-1) 3HB after 24 h. A fed-batch process increased 3HB production to 12 g l(-1) after 48 h of fermentation.     

Engineered Aeromonas hydrophila for enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers composition

Aeromonas hydrophila 4AK4 produces poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 3-hydroxybutyrate (3HB) and about 15 mol% 3-hydroxyhexanoate (3HHx) from dodecanoate. To study the factors affecting the monomer composition and PHBHHx content, genes encoding phasin (phaP), PHA synthase (phaC) and (R)-specific enoyl-CoA hydratase (phaJ) from Aeromonas punctata (formerly named Aeromonas caviae) were introduced individually or jointly into A. hydrophila 4AK4. The phaC gene increased 3HHx fraction more significantly than phaP, while phaJ had little effect. Expression of phaC alone increased the 3HHx fraction from 14 to 22 mol%. When phaC was co-expressed with phaP and phaJ, the 3HHx fraction increased from 14 to 34 mol%. Expression of phaP or phaC alone or with another gene enhanced PHBHHx content up to 64%, cell dry weight (CDW) as much as 4.4 gL(-1) and PHBHHx concentration to 2.7 gL(-1) after 48 h in shake flask culture. The results suggest that a higher PHA synthase activity could lead to a higher 3HHx fraction and PHBHHx content. Co-expression of phaJ with phaC or phaP would favor PHA accumulation, although over-expression of phaJ did not affect PHA synthesis much. In addition, inhibition of beta-oxidation by acrylate in A. hydrophila 4AK4 enhanced PHBHHx content. However, no monomers longer than 3HHx were detected. The results show that genetic modification of A. hydrophila 4AK4 enhanced PHBHHx production and altered monomer composition of the polymer.     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate) with flexible 3-hydroxyhexanoate content in<Emphasis Type="Italic"> Aeromonas hydrophila</Emphasis> CGMCC 0911

Aeromonas hydrophila CGMCC 0911 isolated from lake water was found to be able to synthesize a polyhydroxyalkanoate (PHA) copolymer (PHBHHx) consisting of 3-hydroxybutyrate (HB) and 4–6 mol% 3-hydroxyhexanoate (HHx). The wild-type bacterium accumulated 49% PHBHHx containing 6 mol% HHx in terms of cell dry weight (CDW) when grown on lauric acid for 48 h. When A. hydrophila CGMCC 0911 expressed the Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli, the recombinant strain could accumulate 47% PHBHHx, while the HHx content reached 17.4 mol%. The presence of changing glucose concentration in the culture changed the HHx content both in wild type and recombinant A. hydrophila CGMCC 0911. When 5 g l^–1 glucose was added to a culture containing 5 g l^–1 lauric acid as co-substrate, 45% PHBHHx/CDW consisting of 8.8 mol% HHx was produced by wild-type A. hydrophila CGMCC 0911 compared with only 5% in the absence of glucose. When the recombinant A. hydrophila CGMCC 0911 was grown on a mixed substrate containing lauric acid and 8–10 g l^–1 glucose, the HHx content could be further increased to 35.6 mol%. When the glucose concentration exceeded 10 g l^–1, cell growth, PHA content and mole percentages of HHx in PHBHHx were significantly reduced.     

Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp

Polyhydroxyalkanoate (PHA) synthase genes (phaC) were cloned from two Aeromonas hydrophila strains named WQ and 4AK5, respectively. Both strains are able to produce PHBHHx copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Sequence analysis showed that there was only 2 bp difference between these two PHA synthase genes, corresponding to two-amino acid difference at positions of 437 and 458 of the two synthases. PHA productivity and its monomer content produced by A. hydrophila WQ and A. hydrophila 4AK5 were quite different. A. hydrophila WQ accumulated 33% PHBHHx of its cell dry weight (CDW) with 5 mol% 3HHx in the copolyester when cultured in lauric acid for 48 h. Yet A. hydrophila 4AK5 was able to produce 43% PHBHHx of the CDW with 14 mol% 3HHx under the same condition. Hetero-expression of PHA synthase genes of A. hydrophila WQ and A. hydrophila 4AK5, respectively, in Escherichia coli XL1-Blue led to PHBHHx accumulation of 24% and 39% of the CDW and the 3HHx content in PHBHHx were 6 and 15 mol%, respectively. This indicated that the function of these two PHA synthases were different due to these two different residues at positions of 437 and 458. Site specific mutation was carried out to change these two amino acid residues. Results showed that the changes on either of the two amino acids negatively affected the PHA productivity.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains

The recombinant Escherichia coli strain, equipped with the newly cloned Aeromonas PHA biosynthesis genes, could produce a terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from dodecanoic acid plus odd carbon number fatty acid. In addition, the orf1 gene of Aeromonas hydrophila was found to play a critical role in assimilating the 3HV monomer and in regulating the monomer fraction in the terpolymer.     

Production of D-(--)-3-hydroxyalkanoic acid by recombinant Escherichia coli

Pathways for extracellular production of chiral D-(-)-3-hydroxybutyric acid (3HB) and D-(-)-3-hydroxyalkanoic acid (mcl-3HA) were constructed by co-expression of genes of beta-ketothiolase (phbA), acetoacetyl-CoA reductase (phbB) and 3-hydroxyacyl-ACP CoA transacylase (phaG), respectively, in Escherichia coli strain DH5alpha. The effect of acrylic acid and glucose on production of both 3HB and mcl-3HA was investigated. It was found that the addition of acrylic acid significantly increased production of 3HB and mcl-3HA consisting of 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid in a ratio of 1:3 from 199 mg x l(-1) to 661 mg x l(-1) and from 27 mg x l(-1) to 135 mg x l(-1), respectively, in shake flask studies when glucose was present in the medium at the very beginning of fermentation. The timing of glucose addition had no effect on 3HB production. In contrast, mcl-3HA production was affected by glucose addition, an mcl-3HA concentration of 193 mg x l(-1) was obtained when glucose was added to the culture at 12 h. A more than seven-fold increase was obtained when compared with that in medium containing glucose at the beginning of fermentation. However, a decrease in production of 3HB and mcl-3HA was found when glucose was added at 12 h to the culture containing acrylic acid. The repressive effect of acrylic acid on acetic acid production was also evaluated and discussed.     

需钠弧菌(Vibrio natriegens)合成聚羟基丁酸的研究

研究结果表明,V.natriegens可以利用葡萄糖,果糖,以及糖蜜为碳源合成聚羟基丁酸[Poly(3HB)] ,当以糖蜜为碳源时,积累的Poly(3HB)达到细胞干重的28．4％,实验结果还表明,Poly(3HB)的积累滞后于细胞生长,在培养前加入过量的碳源,不仅没有Poly(3HB)积累,还抑制细胞的生长,测定了与Poly(3HB)合成相关的PHA聚合酶,β-酮硫解酶和乙酰乙酰CoA还原酶的活性。结果表明,伴随Poly(3HB)合成,PHA聚合酶活性从无到有,β－酮硫解酶活性提高了10倍以上。进一步通过利用脂肪酸合成代谢抑制物－浅蓝菌素（cerulenin),研究了脂肪酸从头合成途径与Poly(3HB)合成途径的关系。发现浅蓝菌素能够明显降低细胞Poly(3HB)的累积。根据以上结果,推测在V.natrigens中可能存在两条代谢途径参与Poly(3HB)的合成。