Steffens Tim, Vorhölter Frank-Jörg, Giampà Marco, Hublik Gerd, Pühler Alfred, Niehaus Karsten
Proteom- und Metabolomforschung, Fakultät für Biologie, Centrum für Biotechnologie (CeBiTec), Universität Bielefeld, Universitätsstraße 27, 33615, Bielefeld, Germany.
Genomforschung industrieller Mikroorganismen, Centrum für Biotechnologie (CeBiTec), Universität Bielefeld, Universitätsstraße 27, 33615, Bielefeld, Germany.
BMC Microbiol. 2016 May 23;16:93. doi: 10.1186/s12866-016-0710-y.
The exopolysaccharide xanthan is a natural product which is extensively used in industry. It is a thickening agent in many fields, from oil recovery to the food sector. Xanthan is produced by the Gram negative bacterium Xanthomonas campestris pv. campestris (Xcc). We analyzed the lipopolysaccharide (LPS) of three mutant strains of the Xcc wild type B100 to distinguish if the xanthan production can be increased when LPS biosynthesis is affected.
The Xcc B100 O-antigen (OA) is composed of a linear main chain of rhamnose residues with N-acetylfucosamine (FucNAc) side branches at every second rhamnose. It is the major LPS constituent. The O-antigen was missing completely in the mutant strain H21012 (deficient in wxcB), since neither rhamnose nor FucNAc could be detected as part of the LPS by MALDI-TOF-MS, and only a slight amount of rhamnose and no FucNAc was found by GC analysis. The LPS of two other mutants was analyzed, Xcc H28110 (deficient in wxcK) and H20110 (wxcN). In both of them no FucNAc could be detected in the LPS fraction, while the rhamnose moieties were more abundant than in wild type LPS. The measurements were carried out by GC and confirmed by MALDI-TOF-MS analyses that indicated an altered OA in which the branches are missing, while the rhamnan main chain seemed longer than in the wild type. Quantification of xanthan confirmed our hypothesis that a missing OA can lead to an increased production of the extracellular polysaccharide. About 6.3 g xanthan per g biomass were produced by the Xcc mutant H21012 (wxcB), as compared to the wild type production of approximately 5 g xanthan per g biomass. In the two mutant strains with modified OA however, Xcc H28110 (wxcK) and Xcc H20110 (wxcN), the xanthan production of 5.5 g and 5.3 g, respectively, was not significantly increased.
Mutations affecting LPS biosynthesis can be beneficial for the production of the extracellular polysaccharide xanthan. However, only complete inhibition of the OA resulted in increased xanthan production. The inhibition of the FucNAc side branches did not lead to increased production, but provoked a novel LPS phenotype. The data suggests an elongation of the linear rhamnan main chain of the LPS OA in both the Xcc H28110 (wxcK) and Xcc H20110 (wxcN) mutant strains.
胞外多糖黄原胶是一种广泛应用于工业的天然产物。它在从石油开采到食品行业的许多领域都是一种增稠剂。黄原胶由革兰氏阴性菌野油菜黄单胞菌野油菜致病变种（Xcc）产生。我们分析了Xcc野生型B100的三个突变株的脂多糖（LPS），以确定当LPS生物合成受到影响时黄原胶产量是否会增加。
Xcc B100 O抗原（OA）由鼠李糖残基的线性主链组成，每隔一个鼠李糖带有N - 乙酰岩藻糖胺（FucNAc）侧链。它是主要的LPS成分。突变株H21012（wxcB缺陷）中O抗原完全缺失，因为基质辅助激光解吸电离飞行时间质谱（MALDI - TOF - MS）未检测到鼠李糖和FucNAc作为LPS的组成部分，气相色谱（GC）分析仅发现少量鼠李糖且未发现FucNAc。分析了另外两个突变株Xcc H28110（wxcK缺陷）和H20110（wxcN缺陷）的LPS。在这两个突变株的LPS组分中均未检测到FucNAc，而鼠李糖部分比野生型LPS中更丰富。通过GC进行测量，并经MALDI - TOF - MS分析证实，表明OA发生改变，其中分支缺失，而鼠李聚糖主链似乎比野生型更长。黄原胶的定量分析证实了我们的假设，即缺失OA可导致细胞外多糖产量增加。Xcc突变株H21012（wxcB）每克生物质产生约6.3克黄原胶，而野生型每克生物质产生约5克黄原胶。然而，在OA发生改变的两个突变株Xcc H28110（wxcK）和Xcc H20110（wxcN）中，黄原胶产量分别为5.5克和5.3克，未显著增加。
影响LPS生物合成的突变可能有利于细胞外多糖黄原胶的生产。然而，只有OA的完全抑制导致黄原胶产量增加。FucNAc侧链的抑制并未导致产量增加，而是引发了一种新的LPS表型。数据表明在Xcc H28110（wxcK）和Xcc H20110（wxcN）突变株中LPS OA的线性鼠李聚糖主链延长。
