5-Ketofructose (5-KF) has recently been identified as a promising non-nutritive natural sweetener. Gluconobacter oxydans strains have been developed that allow efficient production of 5-KF from fructose by plasmid-based expression of the fructose dehydrogenase genes fdhSCL of Gluconobacter japonicus. As plasmid-free strains are preferred for industrial production of food additives, we aimed at the construction of efficient 5-KF production strains with the fdhSCL genes chromosomally integrated. For plasmid-free 5-KF production, we selected four sites in the genome of G. oxydans IK003.1 and inserted the fdhSCL genes under control of the strong P264 promoter into each of these sites. All four recombinant strains expressed fdhSCL and oxidized fructose to 5-KF, but site-specific differences were observed suggesting that the genomic vicinity influenced gene expression. For further improvement, a second copy of the fdhSCL genes under control of P264 was inserted into the second-best insertion site to obtain strain IK003.1::fdhSCL2. The 5-KF production rate and the 5-KF yield obtained with this double-integration strain were considerably higher than for the single integration strains and approached the values of IK003.1 with plasmid-based fdhSCL expression. We identified four sites in the genome of G. oxydans suitable for expression of heterologous genes and constructed a strain with two genomic copies of the fdhSCL genes enabling efficient plasmid-free 5-KF production. This strain will serve as basis for further metabolic engineering strategies aiming at the use of alternative carbon sources for 5-KF production and for bioprocess optimization.

中文翻译：

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用于生产天然甜味剂5-酮果糖的新型无质粒氧化葡糖杆菌。

5-果糖（5-KF）最近被确定为一种很有前途的非营养性天然甜味剂。已经开发了氧化葡糖杆菌菌株，其可以通过基于质粒的日本葡糖杆菌脱氢酶基因fdhSCL的质粒表达有效地从果糖生产5-KF。由于无质粒菌株优选用于食品添加剂的工业生产，因此我们的目标是构建具有染色体整合的fdhSCL基因的高效5-KF生产菌株。对于无质粒的5-KF产生，我们在氧化羟色胺IK003.1的基因组中选择了四个位点，并在强P264启动子的控制下将fdhSCL基因插入到这些位点中。所有四种重组菌株均表达fdhSCL并将果糖氧化为5-KF，但观察到位点特异性差异，表明基因组附近影响基因表达。为了进一步改善，将在P264控制下的fdhSCL基因的第二个拷贝插入第二好的插入位点以获得菌株IK003.1 :: fdhSCL2。用这种双整合菌株获得的5-KF产率和5-KF产率明显高于单整合菌株，并接近基于质粒的fdhSCL表达的IK003.1的值。我们鉴定了适合于异源基因表达的氧化G. oxydans基因组中的四个位点，并构建了带有两个基因组拷贝的fdhSCL基因的菌株，可实现高效的无质粒5-KF生产。