Ertesvåg Helga, Sletta Håvard, Senneset Mona, Sun Yi-Qian, Klinkenberg Geir, Konradsen Therese Aursand, Ellingsen Trond E, Valla Svein
Department of Biotechnology, NTNU-Norwegian University of Science and Technology, Trondheim, Norway.
SINTEF Materials and Chemistry, Trondheim, Norway.
BMC Genomics. 2017 Jan 3;18(1):11. doi: 10.1186/s12864-016-3467-7.
Polysaccharides often are necessary components of bacterial biofilms and capsules. Production of these biopolymers constitutes a drain on key components in the central carbon metabolism, but so far little is known concerning if and how the cells divide their resources between cell growth and production of exopolysaccharides. Alginate is an industrially important linear polysaccharide synthesized from fructose 6-phosphate by several bacterial species. The aim of this study was to identify genes that are necessary for obtaining a normal level of alginate production in alginate-producing Pseudomonas fluorescens.
Polysaccharide biosynthesis is costly, since it utilizes nucleotide sugars and sequesters carbon. Consequently, transcription of the genes necessary for polysaccharide biosynthesis is usually tightly regulated. In this study we used an engineered P. fluorescens SBW25 derivative where all genes encoding the proteins needed for biosynthesis of alginate from fructose 6-phosphate and export of the polymer are expressed from inducible Pm promoters. In this way we would avoid identification of genes merely involved in regulating the expression of the alginate biosynthetic genes. The engineered strain was subjected to random transposon mutagenesis and a library of about 11500 mutants was screened for strains with altered alginate production. Identified inactivated genes were mainly found to encode proteins involved in metabolic pathways related to uptake and utilization of carbon, nitrogen and phosphor sources, biosynthesis of purine and tryptophan and peptidoglycan recycling.
The majority of the identified mutants resulted in diminished alginate biosynthesis while cell yield in most cases were less affected. In some cases, however, a higher final cell yield were measured. The data indicate that when the supplies of fructose 6-phosphate or GTP are diminished, less alginate is produced. This should be taken into account when bacterial strains are designed for industrial polysaccharide production.
多糖通常是细菌生物膜和荚膜的必要组成成分。这些生物聚合物的产生会消耗中心碳代谢中的关键成分，但到目前为止，关于细胞是否以及如何在细胞生长和胞外多糖产生之间分配资源，人们知之甚少。藻酸盐是一种在工业上具有重要意义的线性多糖，由几种细菌从6-磷酸果糖合成。本研究的目的是鉴定在产藻酸盐的荧光假单胞菌中获得正常水平藻酸盐产生所必需的基因。
多糖生物合成成本高昂，因为它利用核苷酸糖并封存碳。因此，多糖生物合成所需基因的转录通常受到严格调控。在本研究中，我们使用了一种工程改造的荧光假单胞菌SBW25衍生物，其中所有编码从6-磷酸果糖生物合成藻酸盐所需蛋白质以及聚合物输出所需蛋白质的基因都由可诱导的Pm启动子表达。通过这种方式，我们可以避免鉴定仅仅参与调控藻酸盐生物合成基因表达的基因。对该工程菌株进行随机转座子诱变，并筛选了一个约11500个突变体的文库，以寻找藻酸盐产生改变的菌株。鉴定出的失活基因主要编码参与与碳、氮和磷源的摄取和利用、嘌呤和色氨酸生物合成以及肽聚糖循环相关代谢途径的蛋白质。
大多数鉴定出的突变体导致藻酸盐生物合成减少，而在大多数情况下细胞产量受影响较小。然而，在某些情况下，最终细胞产量更高。数据表明，当6-磷酸果糖或GTP的供应减少时，藻酸盐的产生也会减少。在设计用于工业多糖生产的细菌菌株时应考虑到这一点。
