代谢工程技术构建产均聚物聚3-巯基丙酯的Advenella mimigardefordensis菌株
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摘要
PTE (polythioester)是一类人工改造的在碳骨架上含有硫的新型化合物。聚3-巯基丙酸酯(poly(3-mercaptopropionate), PMP)是PTE聚酯的一种。和结构类似物聚羟基脂肪酸酯(PHA)相比,PTE在主链上将氧原子换成了硫原子。因为这种改变,该聚合物具有了一些更有价值的性质,比如其熔点更高,溶解度更低,热稳定性也更高。同时另一个吸引人的性质是其不可降解性,这些性质让这类聚合物有着很多潜在的用途。随着不可再生的化石能源的日益枯竭,原本来源于化石原料的聚合物都需要找到合适的替代产品。现代化房屋建造行业,汽车制造行业以及其他许多行业都需要不可降解的聚合物材料,这是PHA这类可降解材料不能满足的。因此开拓PTE的生物合成方法具有重要意义。
PTE于1951年通过化学方法首次合成,其后又发展了多种不同的化学合成方法,但是化学方法的难制备,高成本以及低产率的问题制约了PTE的商业化生产；更重要的是这些方法原料基本来源于石油基,面临着原料日益短缺及环境污染的诸多问题。使用重组有BPEC代谢途径(由Clostridium acetobutylicum的丁酸激酶(Buk)和磷酸丁酰转移酶(Ptb)以及Thiocapsa pfennigii的PHA合成酶PhaEC组成)的大肠杆菌菌株实现了合成均聚物PMP。这种生物法合成的PTE材料相比较于化学法合成的材料具有单体均一并且聚合度高的优点。但该大肠杆菌重组菌只能利用毒性较高的3MP(3-mercaptopropioante,3-巯基丙酸)为原料合成产物,而不能利用TDP或DTDP等低毒性原料合成PMP,这阻碍了其后期工业化应用。
为了找到一株可以使用DTDP这种低毒性,低成本原料合成PMP的菌种,我们研究组进行了大规模的筛选,最终得到了若干株不同的可以降解DTDP并利用其作为唯一碳源进行生长的菌株。对其中首次鉴定的A. mimigardfordensis DPN7T进行了Tn5转座子的突变筛选,全基因组测序和相关酶的表达与酶活测定,最终发现和鉴定了一条完整的DTDP降解途径。该菌对DTDP降解途径的第一步是通过硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase, LpdA)将其催化变为3MP,而3MP又是合成PMP的一个直接前体。
本研究正是利用这株菌作为构建合成PMP的基础菌株。我们首先寻找到了可以在该菌株中稳定自主复制的质粒pBBR1MCS5,并尝试着将BPEC这条唯一已知的可以合成PMP的代谢途径构建到质粒pBBR1MCS5上得到了pBBRlMCS5::BPEC。但这个重组质粒不能稳定的在菌株A. mimigardefordensis SHX1中复制。出现了抑制菌体生长和质粒传代结构不稳定等现象。通过进一步的质粒构建和培养实验,最终确定重组buk-ptb操纵子过表达是导致这些问题的根源。之后我们发展了一套适用于该菌株的,利用自杀质粒同源双交换原理进行基因敲除或基因替代的方法。利用此方法,将buk-ptb操纵子成功的插入A. mimigardefordensis菌株的基因组中,得到了生长正常的菌株和稳定遗传的重组质粒。并利用反转录PCR的方法定性的证实了buk-ptb操纵子在被插入到基因组后都可以进行转录。3-巯基丙酸双加氧酶(3-mercaptopropionate dioxygenase, Mdo)是DTDP降解途径中的第二个催化酶,该酶催化3MP生成3-亚磺酸基丙酸(3-sulfinopropionate,3SP),因此该代谢支路会导致3MP前体底物的减少,是影响PMP产量的关键因素之一。而利用mdo：：Tn5突变株进行阻断研究时,我们发现Tn5转座子不稳定,并且观察到因转座导致质粒变大的现象。于是我们改用等位替换方法将mdo基因进行敲除,发酵结果表明敲除了mdo的菌株PMP产量进一步提高。本文研究同时表明A. mimigardefordesis菌株的常规碳源葡萄糖酸并不适合用于PMP合成的发酵碳源。与之相比,琥珀酸作为发酵碳源可以提高菌体量并同时提高PMP产量。综合以上的工作,使用琥珀酸发酵的重组菌SHX5胞内的PMP含量可以稳定在5%-6%。我们初步实现了利用DTDP合成PMP的目的。
尽管如此,但该菌株的产量较低,并且使用的琥珀酸碳源价格昂贵。为了进一步提高PMP产量和降低成本,从发酵水平分别对碳源进行比较,并对发酵具体方式进行优化以及发酵中各组分随时间的改变进行分析。结果表明当以甘油为碳源时,细胞虽然生长缓慢,但可以积累到和以琥珀酸为碳源时相似的菌体量,并且PMP的胞内含量几乎提高了一倍,达到了10%,这使它成为替代琥珀酸的廉价碳源。一个出乎意料的结果表明在使用甘油和丙酸的混合碳源培养基中(丙酸浓度设为0.2%(w/v)),丙酸作为一种‘刺激物’进一步提高了聚合物产量(聚合物占细胞总重量的18%左右),并提高了菌体的生长速率。但这种发酵方法使含有3MP单体的PTE中混入了少量的3HB和3HV单体(占PTE总质量分数少于10%),并得到了一种含有三种单体的未见报道的新化合物poly(3MP-co-3HB-co-3HV)。之后对该类聚合物组成在发酵时间上的变化进行了分析,最终发现很难通过控制发酵时间的方法将3HB和3HV单体去除。这说明这种混合碳源的发酵模式无法很好的用于PMP的生产。
为了进一步提高PMP的产量,我们对之前的结果进行了总结,发现在使用甘油为碳源后,作为对照的不含有BPEC途径的菌株SHX12也可以积累不显著的PMP(胞内含量低于2%),多次重复结果相同。这提示该菌株自身含有一条内在的PMP途径。通过与基因组的序列比对分析,找到了PhaEC的同工酶：由内源的phaCAm基因编码的PHA合成酶(PhaCAm)。将phaCAm基因进行敲除,比较其对内源PMP合成途径和重组的BPEC代谢途径合成PMP的影响,结果说明了三点：第一,起关键作用的酶是其自身的phaCAm基因编码的PHA合成酶PhaCAm。第二,尽管phaE和phaC基因的转录本可以通过逆转录PCR被检测到,但是异源表达的同工酶PhaEC在该菌株中没有活性,也没有对PMP合成有任何贡献。第三,验证了之前的结论,即插入基因组的buk-ptb操纵子所表达的Buk和Ptb对PMP合成有比较重要的影响,显著提高了菌株合成PMP的能力。从而最终发现并鉴定了编码内源PMP合成途径上的PHA合成酶基因phaCAm。为了寻找更优的PHA合成酶,我们比较了三种不同来源的phaC基因。结果表明其自身来源的phaCAm是最有效的。
进一步对内源phaC进行了过量表达来提高PMP的含量,并优化得到了最佳的PHA合成酶的表达量来满足最大化的PMP合成。我们得到了PMP产量最高的菌株SHX22。产量达到20%,最高可以得到25%。对菌株SHX22的菌体干重、PMP含量和培养基中各组分进行了随发酵时间的变化分析,最终发现3MP的“相对剩余”的现象。在A. mimigardefordensis菌株的细胞中,3MP分子只能快速的被转运到胞外而不能成功的穿越细胞膜再次进入细胞。我们判断这是导致PMP产量无法继续提高的一个重要限制因素。据此提出了三种解决方案：第一种通过降低DTDP的浓度进而降低胞内DTDP的降解速度来协调其和PMP合成速度的策略,但该方法最终被证明是不可行的。第二种方法寻找一个转运蛋白将3MP转入胞内比较困难,第三种策略是进一步提高3MP到3MP-CoA的转化速率。为此我们寻找到了合适的酶,即来自Clostridium kluyveri的cat2,并在大肠杆菌中成功的验证了其功能。进一步验证工作正在进行。
最后我们建立了一种适合于重组菌的PMP纯化方法,获得了纯的PMP产物,并通过GC/MS的分析确认了得到的PMP产物的均一性。尼罗红染色证实该菌株中的PMP是以常规的PMP颗粒的形式存在。而这种具有生物活性PMP颗粒可以使我们对其和完整PHA酶系之间的关系进行深入研究。为此我们优化并建立了一套适合于该菌株的活性PMP颗粒的甘油密度梯度超速离心的纯化方法并试着对其蛋白成分组成进行了分析。
PMP材料是用生物方法人工合成的新型聚合物材料。因为材料在性质上的优越性,具有较高的实用价值。但其生产目前受限于前体底物3MP的毒性,采用低毒性的DTDP进行PMP合成无疑具有很大的优势。本论文通过对一株可以利用DTDP进行生长的菌株A. mimigardefordensis进行异源PMP合成途径BPEC的构建和优化,以及之后的一系列发酵优化改造,成功实现了利用低毒性DTDP为底物,在不能生产PMP的菌株细胞中积累到10%的PMP含量。之后,通过内源的新的PMP合成途径的发现以及之后的关键酶phaCAm的鉴定和优化,将PMP胞内含量进一步提高到了20%,最高可以达到25%.之后对合成的PMP进行了纯化和一些性质鉴定的工作。本研究在成功构建新的PMP合成途径的基础上,首次实现了利用低毒性DTDP合成PMP的目的,为PMP材料的生物合成提供了一个新的研究思路和产业化的可行性。
PTE (polythioester) is a kind of polymer containing sulfur in its backbone which are artificially transformed. Poly(3-mercaptopropionate)(PMP) is one kind of PTE. It has similar structure with its analogues, polyoxoesters. The only difference is the substitution of oxygen with sulfur in the chain backbone. Because of this alteration, this kind of polymer shows valuable characters, such as higher melting points, slower solubility and increased heat stability. Whereas the most attracting property is its non-biodegradability. This endows PTE plenty of potentials. In the foreseeable future, as the nonrenewable fossil energy consuming, more and more polymer products should be replaced by proper substitutes. But not all non-biodegardable polymers from fossil oil could be replaced by the biodegradable substitutes from renewable sources as we normally thought. Modern construction of houses, automobiles and other major developments would be unthinkable and impractical without the availability of persistent polymers. In considering of these industrial demands, the production of PTE from renewable feedstock by biotechnical method makes sense.
The chemical synthesis of PTE has already been reported in1951. A lot of other chemical methods to synthesize PTE have also been reported ever since. But all these methods have not been technically produced and commercialized due to difficult preparations, high costs and low yields from laborious synthesis. The key point is that its synthesis still needs petroleum base feedstock which is still in the influence of fossil oil. One recombinant E. coli strain which harbors the non-natural BPEC pathway was successfully used to produce PMP. It contains the following recombining enzymes:butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) from Clostridium acetobutylicum in addition to the PHA synthase from Thiocapsa pfennigii, PhaEC which is recognized as BPEC pathway. This PMP material has sole monomer and it has higher degree of polymerization. But this strain could not use low toxic TDP or DTDP to produce PMP which makes this method hard to be industrially promoted.
To find one strain which could use the low toxic and more stable precursor DTDP, our team screened plenty of strains in large scale. Finally, some strains that could use DTDP as the sole carbon source to grow were shown. One new identified strain A. mimigardefordensis DPN7T attracted our interests. After Tn5mutagenesis screening and relevant enzymes assay, a clear DTDP degradation pathway was figured out. The whole genome sequence has been got and the detailed annotation was processed. The first step of this degradation pathway is at the help of LpdA to form3MP which is just the start precursor for PMP production.
Basing on these facts, the first option is to use DTDP degradation strain A. mimigardfordensis DPN7T to produce PMP. First, we find a proper vector pBBR1MCS5which could be autonomously replicated in this strain. The entire BPEC pathway is inserted into the vector pBBR1MCS5and mobilized into the strain. However, the recombinant vector could not stably replicate in this strain SHX1. Finally, the plasmid structural instability is found to be related with the buk-ptb operon which could lead to growth repression of this strain. One gene deletion or gene exchange method is established basing on the suicide vector by using of allelic exchange. The integration of buk-ptb operon into the genome relieve the repression affection and stable the plasmid structure in the cell. Mdo is the second enzyme in the DTDP degradation pathway which could catalyze3MP into3SP and decrease the3MP amount in the cell, so it is thought as one key factor to affect PMP accumulation in the cells. Unfortunately, it is not possible to use mdo::Tn5mutant to carry out this strategy due to the transposition events of Tn5transposon in A. mimigardefordensis strains. In some cases, the plasmid size in our strain SHX1is observed in bigger size due to this affection. So the mdo gene is deleted instead of mdo::Tn5mutation in the same method. The PMP production is increased further after the mdo deletion procedure. Gluconate is one common used carbon source for cultivation of A. mimigardefordensis strains. But our results proves that it is not suitable for PMP production, so succinate is chosen instead. It improved the cell growth and the PMP content in the cells synchronously. After all these metabolic engineering modifications, the strain SHX5could produce PMP in5%-6%repeatedly and stably. It ultimately verified that the method to modified A. mimigardefordesis DPN7T for PMP production is feasible.
Although we have achieved so much achievements, the production ability of this strain which was around5%is still low and the utilization of costly succinate as carbon source is also limited. To further improve the PMP production and decrease the cost, more different carbon sources, the cultivation styles and the changes of different components along the time course in the medium were all tested. Although cells in glycerol grew slower, it could help the strains to accumulate similar biomass with double PMP content comparing with succinate. It was finally chosen as one cheaper alternative carbon source for PMP production. The addition of propionate increased the polymer production to around18%and recovered the growth of cells. Whereas few3HB and3HV monomers were also incorporated into this polymer (less than10%). The analysis of this polymer composition according to time scale indicated that the appearance of three different kinds of monomers were almost at the same time. It was hard to get rid of the3HB and3HV monomers by changing the cultivation methods.
The supernatant analysis demonstrated the increasing of3MP in the medium. It expelled it from one limited factor. The PMP production pathway itself was locked as the critical point for further PMP production. The previous data also implied that the strain itself contained one inherent PMP production pathway which is independent of the BPEC pathway. After genome blast analysis by using a phaC database, its own phaC gene which encodes PhaC Am as the isozyme of PhaEC was revealed. After the comparison of these two different genes encoding PHA synthase, the PHA synthase encoded by its own phaC was proved as the only functional enzyme. PhaEC was proved to be deficient although their transcripts were positive by reverse-transcript PCR assay. The buk-ptb operon was proved to improve the PMP production in the same experiment. Three different origins of PHA synthase were compared afterward and its own PhaCAm was proved as the most efficient. The affection of lac promoter was excluded at the same time. PHA synthase encoded by phaCl from R. eutropha H16also showed its activity to some extent.
The improved expression of phaC in the cells increased the PMP production further. And the optimal phaC expression amount was defined. The best PMP production strain SHX22was got finally. To further increase the PMP content, the components in the medium were analyzed according to time scale,'relative surplus'of3MP was thought as one key factor. More experiments proved that the3MP molecule could only be exported and could not be imported by the A. mimigardefordensis strains. It was thought to be the next obstacle for further PMP production. Three different solutions were proposed afterwards. The decrease of DTDP concentration was proved not applicable at last. The second strategy was to find a proper enzyme that could accelerate the catalysis speed from3MP to3MP-CoA. For that purpose, a candidate enzyme was proved to be active for PMP accumulation in E. coli strain. Further attempts are arranged at this moment.
Through the modification of PMP purification method, one method was established to purify PMP polymer from this strain. After GC/MS analysis, the PMP was confirmed as homo-polymer. One proper native PMP granules purification method was developed and its protein components were also analyzed by SDS-PAGE which could do great benefit for further research.
PMP materials is one new valuable artificial polymer which could be produced through biotechnical method. However its production is limited to the high toxic precursor3MP. Thus the PMP production through lower toxic DTDP is more practical. Through the construction and optimization of BPEC pathway in a DTDP degradation strain A. mimigardedfordensis DPN7T and a serial of fermentation condition optimizations, the native strain which could not accumulate any PMP could accumulated up to10%PMP in its cells after these modifications. Soon after, its own native PMP production pathway was revealed and the key enzyme phaCAm was identified and its expression was optimized. The PMP production in the cells could arrived to around20%with the highest amount to25%. The purification and identification of this PMP material was studied basing on this system. This work achieved the purpose that the PMP was accumulated by using of low toxic DTDP. A new PMP production pathway was constructed in a new developed strain A. mimigardefordensis. It offers a new research thought and potentials of industrialization for biotechnical PMP production.
PTE于1951年通过化学方法首次合成,其后又发展了多种不同的化学合成方法,但是化学方法的难制备,高成本以及低产率的问题制约了PTE的商业化生产；更重要的是这些方法原料基本来源于石油基,面临着原料日益短缺及环境污染的诸多问题。使用重组有BPEC代谢途径(由Clostridium acetobutylicum的丁酸激酶(Buk)和磷酸丁酰转移酶(Ptb)以及Thiocapsa pfennigii的PHA合成酶PhaEC组成)的大肠杆菌菌株实现了合成均聚物PMP。这种生物法合成的PTE材料相比较于化学法合成的材料具有单体均一并且聚合度高的优点。但该大肠杆菌重组菌只能利用毒性较高的3MP(3-mercaptopropioante,3-巯基丙酸)为原料合成产物,而不能利用TDP或DTDP等低毒性原料合成PMP,这阻碍了其后期工业化应用。
为了找到一株可以使用DTDP这种低毒性,低成本原料合成PMP的菌种,我们研究组进行了大规模的筛选,最终得到了若干株不同的可以降解DTDP并利用其作为唯一碳源进行生长的菌株。对其中首次鉴定的A. mimigardfordensis DPN7T进行了Tn5转座子的突变筛选,全基因组测序和相关酶的表达与酶活测定,最终发现和鉴定了一条完整的DTDP降解途径。该菌对DTDP降解途径的第一步是通过硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase, LpdA)将其催化变为3MP,而3MP又是合成PMP的一个直接前体。
本研究正是利用这株菌作为构建合成PMP的基础菌株。我们首先寻找到了可以在该菌株中稳定自主复制的质粒pBBR1MCS5,并尝试着将BPEC这条唯一已知的可以合成PMP的代谢途径构建到质粒pBBR1MCS5上得到了pBBRlMCS5::BPEC。但这个重组质粒不能稳定的在菌株A. mimigardefordensis SHX1中复制。出现了抑制菌体生长和质粒传代结构不稳定等现象。通过进一步的质粒构建和培养实验,最终确定重组buk-ptb操纵子过表达是导致这些问题的根源。之后我们发展了一套适用于该菌株的,利用自杀质粒同源双交换原理进行基因敲除或基因替代的方法。利用此方法,将buk-ptb操纵子成功的插入A. mimigardefordensis菌株的基因组中,得到了生长正常的菌株和稳定遗传的重组质粒。并利用反转录PCR的方法定性的证实了buk-ptb操纵子在被插入到基因组后都可以进行转录。3-巯基丙酸双加氧酶(3-mercaptopropionate dioxygenase, Mdo)是DTDP降解途径中的第二个催化酶,该酶催化3MP生成3-亚磺酸基丙酸(3-sulfinopropionate,3SP),因此该代谢支路会导致3MP前体底物的减少,是影响PMP产量的关键因素之一。而利用mdo：：Tn5突变株进行阻断研究时,我们发现Tn5转座子不稳定,并且观察到因转座导致质粒变大的现象。于是我们改用等位替换方法将mdo基因进行敲除,发酵结果表明敲除了mdo的菌株PMP产量进一步提高。本文研究同时表明A. mimigardefordesis菌株的常规碳源葡萄糖酸并不适合用于PMP合成的发酵碳源。与之相比,琥珀酸作为发酵碳源可以提高菌体量并同时提高PMP产量。综合以上的工作,使用琥珀酸发酵的重组菌SHX5胞内的PMP含量可以稳定在5%-6%。我们初步实现了利用DTDP合成PMP的目的。
尽管如此,但该菌株的产量较低,并且使用的琥珀酸碳源价格昂贵。为了进一步提高PMP产量和降低成本,从发酵水平分别对碳源进行比较,并对发酵具体方式进行优化以及发酵中各组分随时间的改变进行分析。结果表明当以甘油为碳源时,细胞虽然生长缓慢,但可以积累到和以琥珀酸为碳源时相似的菌体量,并且PMP的胞内含量几乎提高了一倍,达到了10%,这使它成为替代琥珀酸的廉价碳源。一个出乎意料的结果表明在使用甘油和丙酸的混合碳源培养基中(丙酸浓度设为0.2%(w/v)),丙酸作为一种‘刺激物’进一步提高了聚合物产量(聚合物占细胞总重量的18%左右),并提高了菌体的生长速率。但这种发酵方法使含有3MP单体的PTE中混入了少量的3HB和3HV单体(占PTE总质量分数少于10%),并得到了一种含有三种单体的未见报道的新化合物poly(3MP-co-3HB-co-3HV)。之后对该类聚合物组成在发酵时间上的变化进行了分析,最终发现很难通过控制发酵时间的方法将3HB和3HV单体去除。这说明这种混合碳源的发酵模式无法很好的用于PMP的生产。
为了进一步提高PMP的产量,我们对之前的结果进行了总结,发现在使用甘油为碳源后,作为对照的不含有BPEC途径的菌株SHX12也可以积累不显著的PMP(胞内含量低于2%),多次重复结果相同。这提示该菌株自身含有一条内在的PMP途径。通过与基因组的序列比对分析,找到了PhaEC的同工酶：由内源的phaCAm基因编码的PHA合成酶(PhaCAm)。将phaCAm基因进行敲除,比较其对内源PMP合成途径和重组的BPEC代谢途径合成PMP的影响,结果说明了三点：第一,起关键作用的酶是其自身的phaCAm基因编码的PHA合成酶PhaCAm。第二,尽管phaE和phaC基因的转录本可以通过逆转录PCR被检测到,但是异源表达的同工酶PhaEC在该菌株中没有活性,也没有对PMP合成有任何贡献。第三,验证了之前的结论,即插入基因组的buk-ptb操纵子所表达的Buk和Ptb对PMP合成有比较重要的影响,显著提高了菌株合成PMP的能力。从而最终发现并鉴定了编码内源PMP合成途径上的PHA合成酶基因phaCAm。为了寻找更优的PHA合成酶,我们比较了三种不同来源的phaC基因。结果表明其自身来源的phaCAm是最有效的。
进一步对内源phaC进行了过量表达来提高PMP的含量,并优化得到了最佳的PHA合成酶的表达量来满足最大化的PMP合成。我们得到了PMP产量最高的菌株SHX22。产量达到20%,最高可以得到25%。对菌株SHX22的菌体干重、PMP含量和培养基中各组分进行了随发酵时间的变化分析,最终发现3MP的“相对剩余”的现象。在A. mimigardefordensis菌株的细胞中,3MP分子只能快速的被转运到胞外而不能成功的穿越细胞膜再次进入细胞。我们判断这是导致PMP产量无法继续提高的一个重要限制因素。据此提出了三种解决方案：第一种通过降低DTDP的浓度进而降低胞内DTDP的降解速度来协调其和PMP合成速度的策略,但该方法最终被证明是不可行的。第二种方法寻找一个转运蛋白将3MP转入胞内比较困难,第三种策略是进一步提高3MP到3MP-CoA的转化速率。为此我们寻找到了合适的酶,即来自Clostridium kluyveri的cat2,并在大肠杆菌中成功的验证了其功能。进一步验证工作正在进行。
最后我们建立了一种适合于重组菌的PMP纯化方法,获得了纯的PMP产物,并通过GC/MS的分析确认了得到的PMP产物的均一性。尼罗红染色证实该菌株中的PMP是以常规的PMP颗粒的形式存在。而这种具有生物活性PMP颗粒可以使我们对其和完整PHA酶系之间的关系进行深入研究。为此我们优化并建立了一套适合于该菌株的活性PMP颗粒的甘油密度梯度超速离心的纯化方法并试着对其蛋白成分组成进行了分析。
PMP材料是用生物方法人工合成的新型聚合物材料。因为材料在性质上的优越性,具有较高的实用价值。但其生产目前受限于前体底物3MP的毒性,采用低毒性的DTDP进行PMP合成无疑具有很大的优势。本论文通过对一株可以利用DTDP进行生长的菌株A. mimigardefordensis进行异源PMP合成途径BPEC的构建和优化,以及之后的一系列发酵优化改造,成功实现了利用低毒性DTDP为底物,在不能生产PMP的菌株细胞中积累到10%的PMP含量。之后,通过内源的新的PMP合成途径的发现以及之后的关键酶phaCAm的鉴定和优化,将PMP胞内含量进一步提高到了20%,最高可以达到25%.之后对合成的PMP进行了纯化和一些性质鉴定的工作。本研究在成功构建新的PMP合成途径的基础上,首次实现了利用低毒性DTDP合成PMP的目的,为PMP材料的生物合成提供了一个新的研究思路和产业化的可行性。
PTE (polythioester) is a kind of polymer containing sulfur in its backbone which are artificially transformed. Poly(3-mercaptopropionate)(PMP) is one kind of PTE. It has similar structure with its analogues, polyoxoesters. The only difference is the substitution of oxygen with sulfur in the chain backbone. Because of this alteration, this kind of polymer shows valuable characters, such as higher melting points, slower solubility and increased heat stability. Whereas the most attracting property is its non-biodegradability. This endows PTE plenty of potentials. In the foreseeable future, as the nonrenewable fossil energy consuming, more and more polymer products should be replaced by proper substitutes. But not all non-biodegardable polymers from fossil oil could be replaced by the biodegradable substitutes from renewable sources as we normally thought. Modern construction of houses, automobiles and other major developments would be unthinkable and impractical without the availability of persistent polymers. In considering of these industrial demands, the production of PTE from renewable feedstock by biotechnical method makes sense.
The chemical synthesis of PTE has already been reported in1951. A lot of other chemical methods to synthesize PTE have also been reported ever since. But all these methods have not been technically produced and commercialized due to difficult preparations, high costs and low yields from laborious synthesis. The key point is that its synthesis still needs petroleum base feedstock which is still in the influence of fossil oil. One recombinant E. coli strain which harbors the non-natural BPEC pathway was successfully used to produce PMP. It contains the following recombining enzymes:butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) from Clostridium acetobutylicum in addition to the PHA synthase from Thiocapsa pfennigii, PhaEC which is recognized as BPEC pathway. This PMP material has sole monomer and it has higher degree of polymerization. But this strain could not use low toxic TDP or DTDP to produce PMP which makes this method hard to be industrially promoted.
To find one strain which could use the low toxic and more stable precursor DTDP, our team screened plenty of strains in large scale. Finally, some strains that could use DTDP as the sole carbon source to grow were shown. One new identified strain A. mimigardefordensis DPN7T attracted our interests. After Tn5mutagenesis screening and relevant enzymes assay, a clear DTDP degradation pathway was figured out. The whole genome sequence has been got and the detailed annotation was processed. The first step of this degradation pathway is at the help of LpdA to form3MP which is just the start precursor for PMP production.
Basing on these facts, the first option is to use DTDP degradation strain A. mimigardfordensis DPN7T to produce PMP. First, we find a proper vector pBBR1MCS5which could be autonomously replicated in this strain. The entire BPEC pathway is inserted into the vector pBBR1MCS5and mobilized into the strain. However, the recombinant vector could not stably replicate in this strain SHX1. Finally, the plasmid structural instability is found to be related with the buk-ptb operon which could lead to growth repression of this strain. One gene deletion or gene exchange method is established basing on the suicide vector by using of allelic exchange. The integration of buk-ptb operon into the genome relieve the repression affection and stable the plasmid structure in the cell. Mdo is the second enzyme in the DTDP degradation pathway which could catalyze3MP into3SP and decrease the3MP amount in the cell, so it is thought as one key factor to affect PMP accumulation in the cells. Unfortunately, it is not possible to use mdo::Tn5mutant to carry out this strategy due to the transposition events of Tn5transposon in A. mimigardefordensis strains. In some cases, the plasmid size in our strain SHX1is observed in bigger size due to this affection. So the mdo gene is deleted instead of mdo::Tn5mutation in the same method. The PMP production is increased further after the mdo deletion procedure. Gluconate is one common used carbon source for cultivation of A. mimigardefordensis strains. But our results proves that it is not suitable for PMP production, so succinate is chosen instead. It improved the cell growth and the PMP content in the cells synchronously. After all these metabolic engineering modifications, the strain SHX5could produce PMP in5%-6%repeatedly and stably. It ultimately verified that the method to modified A. mimigardefordesis DPN7T for PMP production is feasible.
Although we have achieved so much achievements, the production ability of this strain which was around5%is still low and the utilization of costly succinate as carbon source is also limited. To further improve the PMP production and decrease the cost, more different carbon sources, the cultivation styles and the changes of different components along the time course in the medium were all tested. Although cells in glycerol grew slower, it could help the strains to accumulate similar biomass with double PMP content comparing with succinate. It was finally chosen as one cheaper alternative carbon source for PMP production. The addition of propionate increased the polymer production to around18%and recovered the growth of cells. Whereas few3HB and3HV monomers were also incorporated into this polymer (less than10%). The analysis of this polymer composition according to time scale indicated that the appearance of three different kinds of monomers were almost at the same time. It was hard to get rid of the3HB and3HV monomers by changing the cultivation methods.
The supernatant analysis demonstrated the increasing of3MP in the medium. It expelled it from one limited factor. The PMP production pathway itself was locked as the critical point for further PMP production. The previous data also implied that the strain itself contained one inherent PMP production pathway which is independent of the BPEC pathway. After genome blast analysis by using a phaC database, its own phaC gene which encodes PhaC Am as the isozyme of PhaEC was revealed. After the comparison of these two different genes encoding PHA synthase, the PHA synthase encoded by its own phaC was proved as the only functional enzyme. PhaEC was proved to be deficient although their transcripts were positive by reverse-transcript PCR assay. The buk-ptb operon was proved to improve the PMP production in the same experiment. Three different origins of PHA synthase were compared afterward and its own PhaCAm was proved as the most efficient. The affection of lac promoter was excluded at the same time. PHA synthase encoded by phaCl from R. eutropha H16also showed its activity to some extent.
The improved expression of phaC in the cells increased the PMP production further. And the optimal phaC expression amount was defined. The best PMP production strain SHX22was got finally. To further increase the PMP content, the components in the medium were analyzed according to time scale,'relative surplus'of3MP was thought as one key factor. More experiments proved that the3MP molecule could only be exported and could not be imported by the A. mimigardefordensis strains. It was thought to be the next obstacle for further PMP production. Three different solutions were proposed afterwards. The decrease of DTDP concentration was proved not applicable at last. The second strategy was to find a proper enzyme that could accelerate the catalysis speed from3MP to3MP-CoA. For that purpose, a candidate enzyme was proved to be active for PMP accumulation in E. coli strain. Further attempts are arranged at this moment.
Through the modification of PMP purification method, one method was established to purify PMP polymer from this strain. After GC/MS analysis, the PMP was confirmed as homo-polymer. One proper native PMP granules purification method was developed and its protein components were also analyzed by SDS-PAGE which could do great benefit for further research.
PMP materials is one new valuable artificial polymer which could be produced through biotechnical method. However its production is limited to the high toxic precursor3MP. Thus the PMP production through lower toxic DTDP is more practical. Through the construction and optimization of BPEC pathway in a DTDP degradation strain A. mimigardedfordensis DPN7T and a serial of fermentation condition optimizations, the native strain which could not accumulate any PMP could accumulated up to10%PMP in its cells after these modifications. Soon after, its own native PMP production pathway was revealed and the key enzyme phaCAm was identified and its expression was optimized. The PMP production in the cells could arrived to around20%with the highest amount to25%. The purification and identification of this PMP material was studied basing on this system. This work achieved the purpose that the PMP was accumulated by using of low toxic DTDP. A new PMP production pathway was constructed in a new developed strain A. mimigardefordensis. It offers a new research thought and potentials of industrialization for biotechnical PMP production.
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