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Genetic engineering of Pyrococcus furiosus to use chitin as a carbon source

机译：激烈热球菌的基因工程，以几丁质为碳源

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Background Bioinformatic analysis of the genes coding for the chitinase in Pyrococcus furiosus and Thermococcus kodakarensis revealed that most likely a one nucleotide insertion in Pyrococcus caused a frame shift in the chitinase gene. This splits the enzyme into two separate genes, PF1233 and PF1234, in comparison to Thermococcus kodakarensis . Furthermore, our attempts to grow the wild type strain of Pyrococcus furiosus on chitin were negative. From these data we assume that Pyrococcus furiosus is most likely unable to use chitin as a carbon source. The aim of this study was to analyze in vivo if the one nucleotide insertion is responsible for the inability to grow on chitin, using a recently described genetic system for Pyrococcus furiosus . Results A marker-less genetic system for Pyrococcus furiosus was developed using simvastatin for positive selection and 6-methylpurine for negative selection. Resistance against simvastatin was achieved by overexpression of the hydroxymethylglutaryl coenzyme A reductase gene. For the resistance to 6-methylpurine the hypoxanthine-guanine phosphoribosyltransferase gene was deleted. This system was used to delete the additional nucleotide at position 1006 in PF1234. The resulting chitinase in the mutant strain was a single subunit enzyme and aligns perfectly to the enzyme from Thermococcus kodakarensis . A detailed analysis of the wild type and the mutant using counted cell numbers as well as ATP and acetate production as growth indicators revealed that only the mutant is able to use chitin as a carbon source. An additional mutant strain containing a reduced chitinase version containing just one catalytic and one chitin-binding domain showed diminished growth on chitin in comparison to the mutant containing the single large enzyme. Conclusions Wild type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. The overall high sequence identity of the two chitinases between P. furiosus and T. kodakarensis indicates that this mutation occurred very recently or there is still some kind of selection pressure for a functional enzyme using programmed +/?1 frameshifting.
机译：背景对激烈热球菌和柯达卡尔热球菌的几丁质酶编码基因的生物信息学分析表明，在热球菌中插入一个核苷酸很可能导致几丁质酶基因发生移码。与柯达氏嗜热球菌相比，这将酶分为两个独立的基因PF1233和PF1234。此外，我们在几丁质上生长激烈热球菌野生型菌株的尝试是负面的。根据这些数据，我们假设激烈热球菌很可能无法使用几丁质作为碳源。这项研究的目的是使用最近描述的激烈热球菌遗传系统，分析体内的一个核苷酸插入是否导致不能在几丁质上生长。结果使用辛伐他汀进行阳性选择，使用6-甲基嘌呤进行阴性选择，开发了激烈热球菌无标记遗传系统。对辛伐他汀的抗性通过羟甲基戊二酰辅酶A还原酶基因的过表达实现。为了抵抗6-甲基嘌呤，删除了次黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因。该系统用于删除PF1234中1006位的额外核苷酸。突变菌株中产生的几丁质酶是一种亚基酶，与柯达球菌的酶完全一致。使用计数的细胞数以及ATP和乙酸盐的产生作为生长指标对野生型和突变体进行的详细分析显示，只有突变体才能使用几丁质作为碳源。与仅含有一种大酶的突变体相比，另一种含有降低的几丁质酶版本的突变体菌株仅包含一个催化和一个几丁质结合域，表现出在几丁质上的生长减少。结论由于核苷酸插入将几丁质酶基因分成两个ORF，野生型激烈热球菌最有可能无法在几丁质上在自然表位上生长，而具有缺失核苷酸的基因工程菌株能够在几丁质上生长。 P. furiosus和T. kodakarensis之间的两个几丁质酶的总体高序列同一性表明，这种突变是最近才发生的，或者使用程序化的+/- 1移码对功能性酶仍然存在某种选择压力。

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